Declines in some fish populations in the Upper Danube River, Germany, have been reported during the past decades despite extensive stocking efforts. Many theories exist for why such declines have occurred including habitat change, dams, invasive species, disease and pollution. One of the factors of concern in the Upper Danube River is pollution because a number of studies have shown that sediments collected from this area were acutely and/or chronically toxic to fish. Although it can be difficult to link bioassay results to direct effects on the population level, bioassays can give us insight into the potential of exposure of wildlife including fish to sediment. In combination with other researchers a large battery of sediment testing on the Upper Danube River is being performed. Testing includes sediment testing of estrogen receptor mediated processes, dioxin-like responses and genotoxic effects. In this study, four sediment extracts from the Upper Danube River in Germany were used with a novel fractionation technique to characterize the sediment extracts and fractions for their ability to disrupt steroidogenesis, for their mutagenic activities and their teratogenic effects. Fractionation of each of the four sediment samples was performed by separating compounds according to their polarity, planarity, and the size of the aromatic ring system in an on-line fractionation procedure on coupled high performance liquid chromatography columns.<p> Mutagenic activity was measured in the raw sediment extracts and all 18 fractions using the Ames fluctuation assay and the Danio rerio embryo assay was used to assess lethal endpoints. Furthermore, disruptions of steroidogenesis were assessed by first establishing methods and a proof of concept of the H295R assay by exposing H295R cells to 7 model chemicals and measuring changes from a control in estradiol, testosterone and aromatase activity. Once methods were established all sediments and their fractions were analyzed using the Assay.<p> Specifically, in the <i>Danio rerio</i> assay, two raw sediment extracts killed 100% of <i>Danio rerio</i> embryos at a concentration of 33.3 mg sediment equivalents (SEQ)/ml, but none of the 18 fractions of these samples produced any measured toxicity at a concentration of 100 mg SEQ/ml. In the Ames fluctuation assay, significant mutagenic activity was measured in raw sediment extracts and in the fractions. Fraction 10 produced a significant mutagenic response in all sediment samples measured only in S9 bio-activated samples. Furthermore, fraction 15 produced a significant mutagenic response in all sediment samples measured only in non bio-activated samples.<p> All raw extracts tested in the H295R assay caused an increase in estradiol production up to 4-fold from controls. Testosterone production increased slightly from controls in only two of the raw extract samples. Of the 18 fractions, fractions 7, 10 and 15 increased estradiol in at least three of the samples studied (Sigmaringen2006, Opfingen2006, Lauchert2006 and Lauchert2004). Furthermore, fraction 7 significantly decreased testosterone production compared to controls in three of the four sediment samples.<p> Taken as a whole, these results show the value of using multiple bioassays and fractionation to characterize sediments that covers a variety of different biological endpoints. This study also demonstrates the usefulness of the H295R assay when combined with a new fraction technique to assess endocrine disrupting chemicals in sediment samples.

Declines in some fish populations in the Upper Danube River, Germany, have been reported during the past decades despite extensive stocking efforts. Many theories exist for why such declines have occurred including habitat change, dams, invasive species, disease and pollution. One of the factors of concern in the Upper Danube River is pollution because a number of studies have shown that sediments collected from this area were acutely and/or chronically toxic to fish. Although it can be difficult to link bioassay results to direct effects on the population level, bioassays can give us insight into the potential of exposure of wildlife including fish to sediment. In combination with other researchers a large battery of sediment testing on the Upper Danube River is being performed. Testing includes sediment testing of estrogen receptor mediated processes, dioxin-like responses and genotoxic effects. In this study, four sediment extracts from the Upper Danube River in Germany were used with a novel fractionation technique to characterize the sediment extracts and fractions for their ability to disrupt steroidogenesis, for their mutagenic activities and their teratogenic effects. Fractionation of each of the four sediment samples was performed by separating compounds according to their polarity, planarity, and the size of the aromatic ring system in an on-line fractionation procedure on coupled high performance liquid chromatography columns.<p> Mutagenic activity was measured in the raw sediment extracts and all 18 fractions using the Ames fluctuation assay and the Danio rerio embryo assay was used to assess lethal endpoints. Furthermore, disruptions of steroidogenesis were assessed by first establishing methods and a proof of concept of the H295R assay by exposing H295R cells to 7 model chemicals and measuring changes from a control in estradiol, testosterone and aromatase activity. Once methods were established all sediments and their fractions were analyzed using the Assay.<p> Specifically, in the <i>Danio rerio</i> assay, two raw sediment extracts killed 100% of <i>Danio rerio</i> embryos at a concentration of 33.3 mg sediment equivalents (SEQ)/ml, but none of the 18 fractions of these samples produced any measured toxicity at a concentration of 100 mg SEQ/ml. In the Ames fluctuation assay, significant mutagenic activity was measured in raw sediment extracts and in the fractions. Fraction 10 produced a significant mutagenic response in all sediment samples measured only in S9 bio-activated samples. Furthermore, fraction 15 produced a significant mutagenic response in all sediment samples measured only in non bio-activated samples.<p> All raw extracts tested in the H295R assay caused an increase in estradiol production up to 4-fold from controls. Testosterone production increased slightly from controls in only two of the raw extract samples. Of the 18 fractions, fractions 7, 10 and 15 increased estradiol in at least three of the samples studied (Sigmaringen2006, Opfingen2006, Lauchert2006 and Lauchert2004). Furthermore, fraction 7 significantly decreased testosterone production compared to controls in three of the four sediment samples.<p> Taken as a whole, these results show the value of using multiple bioassays and fractionation to characterize sediments that covers a variety of different biological endpoints. This study also demonstrates the usefulness of the H295R assay when combined with a new fraction technique to assess endocrine disrupting chemicals in sediment samples.

The DDT metabolite 3-methylsulfonyl-DDE (3-MeSO2-DDE) is bioactivated by cytochrome P450 11B1 (CYP11B1) in the adrenal cortex of mice and forms irreversibly bound protein adducts, reduces glucocorticoid secretion, and induces cell death selectively in cortisol-producing adrenocortical cells. 3-MeSO2-DDE has therefore been proposed as a lead compound for an improved adrenocortical carcinoma (ACC) therapy. The aims of this thesis were to (1) develop in vitro test systems based on murine and human adrenocortical cell lines and to (2) investigate the mechanisms behind 3-MeSO2-DDE toxicity in adrenocortical cells. The cytotoxic and endocrine-modulating effects of 3-MeSO2-DDE were compared to those of o,p′-DDD (mitotane), the current ACC therapy, and to those of several structurally analogous compounds in both murine and human cell lines. 3-MeSO2-DDE bioactivation and cytotoxicity proceeded in a similar manner in the murine adrenocortical Y-1 cell line as in mice in vivo. The effects were highly structure-specific. Moreover, 3-MeSO2-DDE formed irreversibly bound protein adducts and caused cell death also in the human H295R cell line, and was slightly more cytotoxic than o,p′-DDD. However, 3-MeSO2-DDE toxicity in human cells was not affected by the CYP11B1 inhibitor etomidate, suggesting that bioactivation in human cells is performed by additional/other enzyme(s) than CYP11B1. 3-MeSO2-DDE generated biphasic responses in cortisol and aldosterone secretion and in expression levels of the steroidogenic genes CYP11B1, CYP11B2, and StAR. Such hormesis-like responses were not seen for o,p′-DDD or the precursor DDT metabolite p,p′-DDE. In addition, the two o,p′-DDD enantiomers (R)-(+)-o,p′-DDD and (S)-(-)-o,p′-DDD exhibited slight differences in cytotoxic and endocrine-modulating activity in H295R cells. In conclusion, this thesis  provides  extended  knowledge  on  the  mechanisms  of  action  of 3-MeSO2-DDE and points out important differences in effects between murine and human cells. Lead optimisation studies of 3-MeSO2-DDE using the herein presented in vitro test systems are ongoing.

Proopiomelanocortin (POMC) is a 241 amino acids precursor protein, which encodes various neuropeptides including corticotropin (ACTH), a-melanocyte-stimulating hormone (a-MSH), and b-endorphin (b-EP). POMC plays an important role in stress response, metabolism, energy homeostasis and anti-inflammation. Recent studies demonstrated that systemic POMC gene delivery potently suppresses the tumor growth and metastasis of B16-F10 melanoma in vitro and in vivo via inhibition of NF-£eB/COX2 pathway. However, systemic POMC expression also led to elevated urine excretion and water intake in mice. This was attributed to enhanced steroidgenesis as evidence by elevated plasma corticosteroids levels in animals and increased cortisol production in adrenal H295R cells after POMC gene delivery. Since corticosteroids are also potent anti-inflammatory agents, it remains unclear whether the ACTH-mediated cortisol synthesis also contributed to the POMC-induced tumor suppression. To address this issue, we generated a series of adenovirus vectors encoding POMC genes with mutation or deletion in ACTH domain including ACTH (K15A/R17A). Unlike the wild type POMC, gene delivery of ACTH (K15A/R17A) resulted in significantly lower cortisol production, CYP11B1 mRNA level, and glucocorticoid responsive element (GRE)-driven luciferase activities in H295R cells. ACTH (K15A/R17A) gene delivery did not affect the urination and water intake in mice. Above all, ACTH (K15A/R17A) gene delivery remained capable of inhibiting the colonies formation and invasiveness of B16-F10 melanoma cells. In summary, steroidgenesis is not essential to POMC-mediated melanoma suppression. In addition, ACTH (K15A/R17A) gene delivery may provide a better alternative for melanoma control.

Les lésions surrénaliennes surviennent dans la population générale à une fréquence d’environ 2-3%. Parmi les anomalies génétiques identifiées jusqu’à présent dans les tumeurs surrénaliennes, les mutations somatiques de β-caténine sont les plus prévalentes. Elles sont présentes dans environ 20% des adénomes et carcinomes cortico-surrénaliens. β-caténine est l’élément central de la voie canonique de WNT qui joue un rôle crucial dans le développement embryonnaire, l’homéostase et la tumourigenèse. Les mutations activatrices de β-caténine conduisent à l’accumulation nucléaire de β- caténine qui interagit avec les TCF/LEF-1 qui active la transcription des gènes cibles. Les gènes cibles de β-caténine, varient et dépendent du contexte cellulaire. Dans la glande surrénale, les gènes cibles de β-caténine sont inconnus. Nous avons effectué des études de microarray qui nous ont permis d’identifier 490 transcrits dérégulés dans les adénomes corticosurrénaliens porteurs de mutations ponctuelles de β-caténine. L’expression aberrante d’ISM1, RALBP1, PDE2A, CDH12, ENC1, PHYHIP et CITED2 dans les adénomes porteurs de mutations de β-caténine a été confirmée par PCR en temps réel. Le traitement des cellules humaines de carcinome cortico-surrénalien H295R (mutation de CTNNB1, Ser45Prol) avec les inhibiteurs de β-caténine/TCF (PKF115-584 et PNU74654) ont confirmé l'implication de β-caténine dans la régulation transcriptionelle d’ISM1, RALBP1, PDE2A, ENC1 et CITED2. En conclusion, nos travaux ont conduit à l’identification de nouveaux gènes cibles de β-catenin impliqués dans la tumourigenèse cortico-surrénalienne. / Adrenal lesions occur in the general population at a prevalence of about 2-3%. Several mutations have been identified in adrenocortical tumours. β-catenin mutations were recently found to be the most frequent genetic alteration in both sporadic adrenocortical adenomas and carcinomas (20-30%). β-catenin is the central player in canonical Wnt signaling which plays a key role in organ/ gland development, maintenance of homeostasis and tumourigenesis. Activation of Wnt signaling by altered regulation of β-catenin levels evokes -catenin accumulation in the nucleus, and interaction with the TCF/LEF-1 proteins that activates the transcription of target genes. These target genes are believed to be highly cell and context specific and are linked to developmental and cell cycling functions. β-catenin target genes in adrenocortical tumours are unknown. Using microarray technology, we found 490 transcripts that are deregulated in adrenocortical adenomas harbouring β-catenin activating mutations in comparison to non mutated adenomas and normal adrenal glands. These genes differ highly in function and many are poorly characterized genes. Differential expression of ISM1, RALBP1, PDE2A, CDH12, ENC1, PHYHIP and CITED2 in adenomas with activating β-catenin mutations was confirmed by real-time PCR. Treatment of human adrenocortical carcinoma cells, H295R (CTNNB1 Ser45Prol), with β-catenin/TCF inhibitors (PKF115-584 and PNU74654) further confirmed the implication of β-catenin on the transcriptional regulation of ISM1, RALBP1, PDE2A, ENC1 and CITED2. In conclusion, we have found new potential β-catenin target genes that may be involved in adrenocortical tumourigenesis.

Les lésions surrénaliennes surviennent dans la population générale à une fréquence d’environ 2-3%. Parmi les anomalies génétiques identifiées jusqu’à présent dans les tumeurs surrénaliennes, les mutations somatiques de β-caténine sont les plus prévalentes. Elles sont présentes dans environ 20% des adénomes et carcinomes cortico-surrénaliens. β-caténine est l’élément central de la voie canonique de WNT qui joue un rôle crucial dans le développement embryonnaire, l’homéostase et la tumourigenèse. Les mutations activatrices de β-caténine conduisent à l’accumulation nucléaire de β- caténine qui interagit avec les TCF/LEF-1 qui active la transcription des gènes cibles. Les gènes cibles de β-caténine, varient et dépendent du contexte cellulaire. Dans la glande surrénale, les gènes cibles de β-caténine sont inconnus. Nous avons effectué des études de microarray qui nous ont permis d’identifier 490 transcrits dérégulés dans les adénomes corticosurrénaliens porteurs de mutations ponctuelles de β-caténine. L’expression aberrante d’ISM1, RALBP1, PDE2A, CDH12, ENC1, PHYHIP et CITED2 dans les adénomes porteurs de mutations de β-caténine a été confirmée par PCR en temps réel. Le traitement des cellules humaines de carcinome cortico-surrénalien H295R (mutation de CTNNB1, Ser45Prol) avec les inhibiteurs de β-caténine/TCF (PKF115-584 et PNU74654) ont confirmé l'implication de β-caténine dans la régulation transcriptionelle d’ISM1, RALBP1, PDE2A, ENC1 et CITED2. En conclusion, nos travaux ont conduit à l’identification de nouveaux gènes cibles de β-catenin impliqués dans la tumourigenèse cortico-surrénalienne. / Adrenal lesions occur in the general population at a prevalence of about 2-3%. Several mutations have been identified in adrenocortical tumours. β-catenin mutations were recently found to be the most frequent genetic alteration in both sporadic adrenocortical adenomas and carcinomas (20-30%). β-catenin is the central player in canonical Wnt signaling which plays a key role in organ/ gland development, maintenance of homeostasis and tumourigenesis. Activation of Wnt signaling by altered regulation of β-catenin levels evokes -catenin accumulation in the nucleus, and interaction with the TCF/LEF-1 proteins that activates the transcription of target genes. These target genes are believed to be highly cell and context specific and are linked to developmental and cell cycling functions. β-catenin target genes in adrenocortical tumours are unknown. Using microarray technology, we found 490 transcripts that are deregulated in adrenocortical adenomas harbouring β-catenin activating mutations in comparison to non mutated adenomas and normal adrenal glands. These genes differ highly in function and many are poorly characterized genes. Differential expression of ISM1, RALBP1, PDE2A, CDH12, ENC1, PHYHIP and CITED2 in adenomas with activating β-catenin mutations was confirmed by real-time PCR. Treatment of human adrenocortical carcinoma cells, H295R (CTNNB1 Ser45Prol), with β-catenin/TCF inhibitors (PKF115-584 and PNU74654) further confirmed the implication of β-catenin on the transcriptional regulation of ISM1, RALBP1, PDE2A, ENC1 and CITED2. In conclusion, we have found new potential β-catenin target genes that may be involved in adrenocortical tumourigenesis.