Recent studies indicate that endogenously ligand-activated Aryl Hydrocarbon Receptor (AHR) plays an important role in normal and pathological processes, including the induction and progression of breast cancer. As the known number of AHR-mediated processes grows, so too does the importance of identifying endogenous AHR ligands that influence breast cancer progression. The following studies focus on two tryptophan metabolism pathway branches, the kynurenine (KYN) branch and the indoxyl sulfate (IS) branch, to determine if ER-/PR-/HER2- breast cancer cells can produce, or are exposed to, AHR ligands from these branches, how these ligands affect cell migration and, if produced, how their production is controlled. It is hypothesized that: 1) malignant cells produce, or derive from their microenvironment, AHR ligands through the KYN and/or IS pathways, 2) these metabolites drive AHR-dependent breast cancer migration, 3) environmental AHR ligands mimic the effects of endogenous ligands, 4) rate-limiting kynurenine pathway enzymes are responsible for endogenous AHR ligand production and their downstream effects, and 5) the AHR controls expression of a rate-limiting kynurenine pathway enzyme(s) in a positive feedback loop. ER-/PR-/HER2- mammary epithelial cells were assayed for production of AHR-activating tryptophan metabolites and for the kynurenine pathway enzymes tryptophan 2,3-dioxygenase (TDO) and indolamine 2,3-dioxygenase (IDO). The relationship between kynurenine and IS pathways, AHR activity, cell migration, and aldehyde dehydrogenase 1 (ALDH1), a cancer stem cell marker associated with poor prognosis, was investigated using tryptophan metabolites, enzyme-specific gene knockdown or over-expression, qPCR, cell migration assays and ALDH1 activity assay. ER-/PR-/HER2- tumor cells produce KYN and its downstream metabolite xanthurenic acid (XA), at levels sufficient to activate the AHR. KYN, XA, and IS significantly accelerate migration in an AHR-dependent fashion, and at physiological doses, while environmental AHR ligands 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and benzo[a]pyrene (B[a]P) mimic this effect. IS induces ALDH1 activity. AHR knockdown or inhibition significantly reduces Tdo2 expression. Finally, gene expression dataset analyses reveal high Tdo2 levels in primary breast tumors, with the highest levels in ER-/PR-/Her2- and stage 4 tumors. These studies identify three tryptophan-derived AHR ligands that contribute to breast cancer progression and demonstrate a positive feedback loop, where AHR activity up-regulates Tdo2, which drives endogenous AHR ligand production.

No description available.

Serotonin has been implicated as a modulator of feeding behavior. This experiment was designed to alter brain serotonin levels through dietary means in hypothalamic ventromedial-lesioned and unlesioned rats. Daily food, water, and animal weights were measured. The purpose was to determine if VMH lesions altered the feeding pattern found in unlesioned rats. Although food intake for tryptophanenriched diets and tryptophan-deficient diets did not differ from their respective control groups, in some cases gross animal weights did differ significantly between experimental and control groups and between lesioned and unlesioned groups. A proposed model explains how a "low" energy signal and a "high" protein signal cycles amino acids through gluconeogenesis to comPensate for an energy deficit.

M. S.

The effects of intragastrically administered tryptophan and tyrosine, which are direct precursors of brain neurotransmitters affecting food intake, were investigated. Differences in sensitivities and/or responses in relatively fast-growing and slow-growing strains of chickens were also tested. The amino acids were intubated intragastrically in a series of four experiments. Food consumption following tryptophan intubation decreased in both the slow and fast-growing strains. Increased food intake was observed in the slow-growing birds as a result of tyrosine treatment suggesting that food consumption can be altered by manipulation of dietary amino acids. Tyrosine treatment did not stimulate food intake in the fast-growing strain, implying that chickens in this population were in a state of maximal or near maximal stimulation, and thus relatively incapable of increasing food intake. To determine whether the changes observed in feeding behavior were perhaps due to some general inhibition or stimulation of the central nervous system rather than the result of mediation of feeding mechanisms in the brain, the effects of intragastrically administered tyrosine and tryptophan on tonic immobility (TI) were also tested. Duration and susceptibility of TI were unaffected by tyrosine and tryptophan, suggesting that these amino acids acted upon mechanisms specifically involved in the regulation of food intake. / Master of Science

Three experiments, each with 24 pregnant female rats as subjects, were conducted. Four experimental diets contained either 7.5 mg tryptophan or 500 mg tryptophan/100 g and either 7.5 ppm or 50 ppm zinc. Dams on the low zinc diets throughout gestation consumed less food and gained less weight than those on the high zinc diets. Reproductive success was less on the low zinc diet. The incidences of maternal mortality, fetal mortality, and reabsorptions were higher in the dams fed a low zinc diet. There were no significant differences in water intake, urine volume, fetal weight, or fetal nitrogen content which were dependent upon dietary treatment. Dams on the low zinc diets had acute renal nephrosis which was most severe when dietary tryptophan levels were high. Fetal zinc content was higher in fetuses from dams fed the high zinc diets. Although the dams on the low zinc diet did exhibit signs of zinc deficiency, the deficiency was not severe enough to affect fetal weight. Diets containing between 6 and 9 ppm were not sufficient for normal parturition. / M. S.

La voie des kynurénines est l’une des trois voies de biosynthèse du NAD, qu’elle produit à partir de la dégradation du plus rare des acides aminés essentiels, le tryptophane. Cette voie catabolique est initiée par trois enzymes distinctes, dont l’indoléamine 2,3-dioxygénase 1 (IDO-1). L’IDO-1 est la seule des trois enzymes dont l’expression est induite par des stimuli pro-inflammatoires tels que l’IFN-γ, le TNF-α ainsi que des produits bactériens et viraux. De plus, son expression est induite de manière particulièrement importante au cours de la maturation des cellules dendritiques (DC) qui jouent un rôle clé, tant dans les réponses immunes innées qu’adaptatives. Par conséquent, outre la production d’un cofacteur essentiel du métabolisme cellulaire, le catabolisme du tryptophane participe également à la régulation de la réponse immune. <p><p>En effet, la fonction d’agent antimicrobien fut la première attribuée à l’IDO-1 parce qu’en réponse aux stimuli inflammatoires, celle-ci dégrade le tryptophane et limite la prolifération d’organismes pathogènes auxotrophes pour cet acide aminé. Ensuite, l’inhibition de l’activité catalytique de l’IDO-1 par une approche pharmacologique permit de mettre en évidence la contribution de cette enzyme au phénomène de tolérance maternelle pour les fœtus semi-allogéniques qu’elle porte. Enfin, sa participation à la tolérance périphérique fut proposée étant donné sa capacité à induire le développement de Treg ou à leur servir de mécanisme effecteur. Cependant, ces deux dernières fonctions ne sont pas très claires puisque les souris invalidées pour le gène de l’IDO-1 (IDO-1-/-) ne présentent ni défaut reproducteur, ni signe d’auto-immunité spontanée.<p><p>Comme deux publications récentes suggéraient que l’inhibition pharmacologique de l’IDO-1 affecte la maturation des moDC humaines, nous avons effectué une analyse détaillée du compartiment immunitaire des souris IDO-1-/-, avec une attention toute particulière pour les DC. Au début, nous avons également constaté un important défaut de la maturation des BMDC IDO-1-/- générées in vitro en présence de GM-CSF. Cependant, le défaut se révéla extrêmement dépendant des conditions de culture, puisqu’un changement de substrat de culture ou de facteur de croissance suffit à restaurer une maturation normale de ces cellules. De même, l’analyse de la maturation des DC spléniques démontra de manière claire que l’IDO-1 n’est certainement pas essentielle à la maturation des DC in vivo.<p> <p>Nous avons ensuite montré que l’expression fonctionnelle d’IDO-1 protège les cellules et potentiellement les souris qui l’expriment lorsqu’elles sont soumises à des stress oxydatifs, suggérant que l’IDO-1 puisse consommer les anions O2- afin d’assurer son activité catalytique. Contre toute attente, nous avons ensuite constaté que les souris IDO-1-/- survivent plus longtemps aux infections par la forme pléiomorphe du parasite Trypanosoma brucei brucei. Bien que la levée de l’inhibition de la lymphoprolifération chez les souris IDO-1-/- soit l’explication la plus évidente de l’augmentation de leur survie, nous suggérons plutôt que c’est la perte de la fonction antioxydante de l’IDO-1-/- qui leur confère cette résistance. <p><p>En conclusion, l’IDO-1 ne semble pas jouer un rôle important dans la différenciation et maturation des cellules dendritiques. Nos observations préliminiares indiquent cependant que cette enzyme pourrait jouer un rôle anti-oxydant, et protége donc les cellules dendritiques d’un stress oxydant potentiellement causé lors des réponses innées anti-microbiennes.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

No / Vitiligo is characterized by a mostly progressive loss of the inherited skin color. The cause of the disease is still unknown, despite accumulating in vivo and in vitro evidence of massive oxidative stress via hydrogen peroxide (H2O2) and peroxynitrite (ONOO−) in the skin of affected individuals. The most favored hypothesis is based on autoimmune mechanisms. Since depletion of the essential amino acid l-tryptophan (Trp) severely affects various immune responses, we here looked at Trp metabolism and signaling in these patients. Our in vivo and in vitro data revealed total absence of epidermal Trp hydroxylase activities and the presence of H2O2/ONOO− deactivated indoleamine 2,3-dioxygenase. Aryl hydrocarbon receptor signaling is severely impaired despite the ligand (Trp dimer) being formed, as shown by mass spectrometry. Loss of this signal is supported by the absence of downstream signals (COX-2 and CYP1A1) as well as regulatory T-lymphocytes and by computer modeling. In vivo Fourier transform Raman spectroscopy confirmed the presence of Trp metabolites together with H2O2 supporting deprivation of the epidermal Trp pool by Fenton chemistry. Taken together, our data support a long-expressed role for in loco redox balance and a distinct immune response. These insights could open novel treatment strategies for this disease.—Schallreuter, K. U., Salem, M. A. E. L., Gibbons, N. C. J., Maitland, D. J., Marsch, E., Elwary, S., Healey, A. R. Blunted epidermal l-tryptophan metabolism in vitiligo affects immune response and ROS scavenging by Fenton chemistry, part 2: epidermal H2O2/ONOO−-mediated stress in vitiligo hampers indoleamine 2,3-dioxygenase and aryl hydrocarbon receptor-mediated immune response signaling.

No / Vitiligo is characterized by a progressive loss of inherited skin color. The cause of the disease is still unknown. To date, there is accumulating in vivo and in vitro evidence for massive oxidative stress via hydrogen peroxide (H2O2) and peroxynitrite (ONOO−) in the skin of affected individuals. Autoimmune etiology is the favored theory. Since depletion of the essential amino acid l-tryptophan (Trp) affects immune response mechanisms, we here looked at epidermal Trp metabolism via tryptophan hydroxylase (TPH) with its downstream cascade, including serotonin and melatonin. Our in situ immunofluorescence and Western blot data reveal significantly lower TPH1 expression in patients with vitiligo. Expression is also low in melanocytes and keratinocytes under in vitro conditions. Although in vivo Fourier transform-Raman spectroscopy proves the presence of 5-hydroxytryptophan, epidermal TPH activity is completely absent. Regulation of TPH via microphthalmia-associated transcription factor and l-type calcium channels is severely affected. Moreover, dopa decarboxylase (DDC) expression is significantly lower, in association with decreased serotonin and melatonin levels. Computer simulation supports H2O2/ONOO−-mediated oxidation/nitration of TPH1 and DDC, affecting, in turn, enzyme functionality. Taken together, our data point to depletion of epidermal Trp by Fenton chemistry and exclude melatonin as a relevant contributor to epidermal redox balance and immune response in vitiligo.

Monitoring the impact of anthropogenic activities, particularly in industrial regions, requires ecological screening tools and frameworks that provide a comprehensive understanding of ecosystem responses to environmental changes. Biological indicators, organisms like algae, insects, fish, and sentinel mammals, are critical for assessing ecosystem health, particularly in areas of high industrial activity. The aim of this thesis was to identify a cross-species biomarker that can assess organismal health and environmental stress across various species, organs, and biological matrices. A range of biological systems and signaling pathways related to xenobiotic metabolism, energy homeostasis, immune responses, and stress adaptation were explored, leading to the identification of the Tryptophan-Kynurenine Pathway, which consumes 60-90% of tryptophan in vertebrates. Tryptophan and its metabolites play key roles in diverse physiological processes, including cell growth and maintenance, immunity, disease states, and the coordination of adaptive responses to environmental and dietary cues. This adaptive response suggests that kynurenine-tryptophan ratio (KTR) may serve as a marker for exposure to a variety of environmental stress conditions, including toxicants, nutrient scarcity, predatory stress, and habitat loss—stressors that are prevalent in areas of high industrial activity. In recent years, the KTR is increasingly recognized as a sensitive biomarker in human diseases induced or exacerbated by stress; however, its role in environmental exposure and wildlife health remains unexplored. This thesis explores the question of whether KTR can be utilized as a cross-species biomarker for environmental stress or environmental exposure to toxicants, particularly focusing on the Athabasca Oil Sands Region (AOSR). In vitro studies with mammalian hepatocytes exposed to polycyclic aromatic compounds (PACs): benzo[a]pyrene (BaP), and a Bitumen Water Accommodated Fraction (BitWAF) demonstrated that KTR increases were driven by elevated kynurenine levels, indicating disruption of tryptophan metabolism via the aryl hydrocarbon receptor (AhR). Further studies using acid extractable organics from Oil Sands Process-Affected Water (OSPW), Naphthenic Acid Fraction Components (NAFCs) showed metabolic reprogramming, including altered glucose and fatty acid uptake and mitochondrial dysfunction, mediated through PPARα activation and upregulation of Tdo2, the enzyme responsible for kynurenine production. In vivo studies of longnose and white suckers from the AOSR were conducted to assess the relationship between KTR and CYP1 enzyme activity (EROD). These studies revealed species-specific responses, with an inverse correlation between KTR and EROD in longnose suckers and a direct correlation in white suckers. These findings validate KTR as a biomarker for environmental exposure in wildlife, with significant implications for monitoring ecosystem health. Collectively, this work demonstrates the potential of KTR as a novel biomarker for environmental toxicology, offering a valuable tool for assessing organismal stress across species in response to environmental contaminants. / Thesis / Doctor of Philosophy (PhD) / Human activities, especially industrial operations, can significantly impact the environment. To monitor these effects, scientists use various tools and organisms to assess ecosystem health. This research introduces a new approach to measuring environmental stress in wildlife by focusing on two key molecules: tryptophan and kynurenine. These molecules are part of a conserved biological pathway that helps all organisms manage stress, repair cells, adapt to their environment, and maintain overall health. Tryptophan, an essential amino acid, is broken down into kynurenine, and the balance between them— known as the kynurenine-tryptophan ratio (KTR)—can indicate the level of stress an organism is experiencing. This thesis investigates whether KTR can detect environmental stress caused by industrial activity, particularly from petroleum-derived chemicals in the Athabasca Oil Sands Region (AOSR). In laboratory experiments, mammalian liver cells were exposed to oil sands compounds and complex mixtures from oil sands wastewater. These compounds changed KTR, showing that the liver’s stress response was activated, and tryptophan metabolism was disrupted. The study also found that these chemicals affected cellular energy use and the way cells process fats and sugars. Furthermore, we examined fish species in the AOSR: longnose and white suckers. Results showed that KTR varied depending on the species and the location of exposure. In white suckers, KTR increased in response to stress, while in longnose suckers, it decreased, indicating species-specific responses to environmental changes. Overall, our findings suggest that KTR could serve as a useful tool for measuring environmental stress in different species and ecosystems, especially in areas affected by anthropogenic or industrial activity. Understanding how KTR changes in response to pollution can help scientists better monitor and protect wildlife and ecosystem health.