http://hdl.handle.net/1957/18863 2013-06-17T00:41:17Z http://hdl.handle.net/1957/38762 Discovery and mechanism of action of anti-cancer compounds targeting the aryl hydrocarbon receptor O'Donnell, Edmond F. III The aryl hydrocarbon receptor (AhR) is a ligand activated transcription factor belonging the basic helix-loop-helix Per/Arnt/Sim (bHLH PAS) family of proteins. The AhR has long been studied for its role in mediating the effects of environmental toxicants, most notably 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). By studying the mechanism of action of TCDD, the AhR has been shown to be involved in numerous biological functions and processes, including metabolism of xenobiotics, the adaptive immune response, embryonic development, and cell cycle regulation. The AhR has also been shown to act as a tumor suppressor gene in several mouse models of cancer. In addition, the AhR is highly expressed in a number of different cancers. We hypothesized that structurally diverse ligands of the AhR have different effects on AhR function, and that it is possible to activate anti-cancer properties of the AhR without inducing dioxin-like toxicity. To this end, we screened a small molecule library of pharmacologically active compounds including FDA-approved drugs, with the specific intent of repurposing such drugs as AhR-based anti- cancer therapeutics. We used a parallel screening approach, with one assay utilizing a xenobiotic response element (XRE) based reporter assay to identify traditional ligands of the AhR, and a second system consisting of a novel heterologous AhR-dependent reporter assay designed to identify novel AhR ligands distinct from those that activate XRE-driven genes. Using these approaches, and evaluating the phenotypic effects of putative hits in target cancer cell types, four lead compounds were selected for further evaluation, two of which are currently in use in clinic: leflunomide, used to treatment rheumatoid arthritis, and raloxifene, a selective estrogen receptor (ER) modulator used to prevent osteoporosis and ER-positive breast cancer. The mechanisms of action of the lead compounds were characterized in terms of AhR activation and AhR- dependent anti-cancer effects. We showed that leflunomide inhibits growth of melanoma cells in an AhR-dependent manner through a mechanism distinct from its current use in the clinic. Two of the lead compounds were found to induce AhR-dependent apoptosis, with raloxifene exhibiting anti-cancer effects in hormone-independent breast cancer cells. The AhR ligands characterized in this work, including leflunomide and raloxifene, represent a novel small molecule probe set that can be used to better characterize the anti-cancer properties of the AhR. The AhR-dependent phenotypes of these compounds compared to AhR ligands such as TCDD will also be useful for establishing a molecular basis for how the AhR facilitates diverse biological outcomes in response to activation by distinct ligands. Development of these compounds, especially raloxifene, as AhR-targeted anti- cancer therapeutics will be useful for establishing the AhR as a viable anti-cancer target. Graduation date: 2013; Access restricted at author's request from May 22, 2013 - May 22, 2015 2013-05-01T00:00:00Z http://hdl.handle.net/1957/37979 From pesticide degradation products to legacy toxicants and emerging contaminants : novel analytical methods, approaches, and modeling Forsberg, Norman D. Environmental toxicologists and public health officials are responsible for assisting in the identification, management, and mitigation of public health hazards. As a result, there is a continued need for robust analytical tools that can aid in the rapid quantification and characterization of chemical exposure. In the first research phase, we demonstrated that a current tool for estimating human organophosphate pesticide exposure, measuring dialkyl phosphate (DAPs) metabolites in urine as chemical biomarkers of pesticide exposure, could represent exposure to DAPs themselves and not to pesticides. We showed that DAPs are metabolically stable, have high oral bioavailability, and are rapidly excreted in the urine following oral exposure. Results suggest that DAP measurements may lead to overestimates of human organophosphate pesticide exposure. In the second phase of research, a quick, easy, cheap, effective, rugged, and safe (QuEChERS) based analytical method was developed and validated for quantifying polycyclic aromatic hydrocarbons (PAHs) in biotic matrices with fat contents that ranged from 3 to 11%. Our method improved PAH recoveries 50 to 200% compared to traditional QuEChERS methods, performed as well or better than state of the art Soxhlet and accelerated solvent extraction methods, had sensitivity useful for chemical exposure assessments, and reduced sample preparation costs by 10 fold. The validated QuEChERS method was subsequently employed in a human exposure assessment. Little is known about how traditional Native American fish smoke-preserving methods impact PAH loads in smoked foods, Tribal PAH exposure, or health risks. Differences in smoked salmon PAH loads were not observed between Tribal smoking methods, where smoking methods were controlled for smoking structure and smoke source. PAH loads in Tribally smoked fish were up to 430 times greater than those measured in commercially available smoked fish. It is not likely that dietary exposure to non-carcinogenic PAHs at heritage ingestion rates of 300 grams per day poses an appreciable risk to human health. However, levels of PAHs in traditionally smoked fish may pose and elevated of risk of cancer if consumed at high rates over a life time. Accurately estimating PAH exposure in cases where aquatic foods become contaminated is often hindered by sample availability. To overcome this challenge, we developed a novel analytical approach to predict PAH loads in resident crustacean tissues based on passive sampling device (PSD) PAH measurements and partial least squares regression. PSDs and crayfish collected from 9 sites within, and outside of, the Portland Harbor Superfund site captured a wide range of PAH concentrations in a matrix specific manner. Partial least squares regression of crayfish PAH concentrations on freely dissolved PAH concentrations measured by PSDs lead to predictions that generally differed by less than 12 parts per billion from measured values. Additionally, most predictions (> 90%) were within 3-fold of measured values, while state of the art bioaccumulation factor approaches typically differ by 5 to 15-fold compared to measured values. In order to accurately characterize chemical exposure, new analytical approaches are needed that can simulate chemical changes in bioavailable PAH mixtures resulting from natural and/or remediation processes. An approach based on environmental passive sampling and in-laboratory UVB irradiation was developed to meet this need. Standard PAH mixtures prepared in-lab and passive sampling device extracts collected from PAH contaminated environments were used as model test solutions. UV irradiation of solutions reduced PAH levels 20 to 100% and lead to the formation of several toxic oxygenated-PAHs that have been previously measured in the environment. Site specific differences in oxygenated-PAH formation were also observed. The research presented in this dissertation can be used to advance chemical exposure estimation techniques, rapidly and cost-effectively quantify a suite of PAHs in biotic tissues, and simulate the effect of abiotic transformation processes on the bioavailable fraction of environmental contaminants. Graduation date: 2013; Access restricted to the OSU Community at author's request from April 3, 2013 - April 3, 2014 2013-03-20T00:00:00Z http://hdl.handle.net/1957/37899 Ligand selective regulation of cell growth by the Ah receptor through activation of TGFβ signaling; Ligand selective regulation of cell growth by the Ah receptor through activation of TGF-beta signaling Koch, Daniel C. The Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and member of the basic helix-loop-helix Per/ARNT/Sim (bHLH/PAS) family of chemosensors and developmental regulators. As a member of the PAS domain family of transcription factors responsive to exogenous signals, the AhR exerts influence on many processes relating to cellular fate. The activation of AhR is widely associated with toxic endpoints related to dioxin exposure. However, the AhR also activates endogenous gene programs related to development, cellular growth, and differentiation. The AhR is able to bind a variety of ligands, leading to a wide range of biological outcomes. Recent reports have shown that the AhR can mediate tumor suppressive effects. As a ligand-activated transcription factor, the AhR has the potential to actuate a variety of transcriptional programs that are dependent on the AhR ligand. Our central hypothesis is that AhR ligands can be identified that are capable of initiating tumor suppressive functions of the AhR. We utilized complementary cell-based and in silico virtual screening approaches to identify potential AhR ligands. We developed homology models of the AhR ligand-binding domain (LBD) for virtual ligand screening (VLS) of small molecule libraries. This led to the identification of new AhR ligands 5,7- dihydroxyflavanone!and 5-hydroxy-7-methoxyflavone. Additional small molecule libraries were screened in parallel that led to identification of flutamide as a putative AhR ligand. Flutamide is clinically approved for the treatment of prostate cancer due to its ability to antagonize androgen receptor mediated transcription. We investigated the biological effects of flutamide in AhR positive cancer cells that do not express the androgen receptor and found that flutamide inhibited the growth of HepG2 cells. Suppression of AhR expression reversed the anti-proliferative effects of flutamide. We tested 15 structural analogs of flutamide, including the flutamide metabolite 2-hydroxyflutamide for activation of AhR transcriptional activity. Flutamide is unique in its ability to activate the AhR, and suppresses hepatoma cell growth. These data suggests that flutamide-induced AhR transcriptional activity is required to initiate the tumor suppressive effects. We examined changes in cell cycle checkpoint proteins after flutamide treatment and discovered increased expression of cell cycle inhibitory proteins p27[superscript Kip1] and p15[superscript INK]. We also found that transforming Growth Factor β1 (TGFβ1), which regulates both p27[superscript Kip1] and p15[superscript INK], is upregulated by flutamide. We demonstrate that TGFβ1 is upregulated by flutamide in an AhR-dependent manner and is required for suppression of proliferation by flutamide. We identify specific and unique transcriptional signatures of the AhR upon activation by flutamide, that are distinct from the potent AhR agonist 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). In summary, we characterize flutamide as an AhR ligand and demonstrate its AhR-dependent tumor suppressive effects in hepatoma cells. We provide the first direct evidence that AhR regulates TGFβ signaling in a ligand dependent manner. We demonstrate that the AhR-induced downstream transcriptional signature and subsequent biological effects are specific to the AhR ligand. Our studies have broad impact for characterizing the AhR as a new therapeutic target in hepatocellular carcinoma. Graduation date: 2013; Access restricted to the OSU Community at author's request from March 28, 2013 - March 28, 2015 2013-02-08T00:00:00Z http://hdl.handle.net/1957/37428 Cardiac output and respiratory measurements in the rainbow trout and their application to the study of blood and water flow limitations on chemical flux at the gill Schmieder, Patricia K. (Patricia Kathleen) A method has been developed for the continuous automated monitoring of cardiac output in adult rainbow trout. Average cardiac output measured under control conditions and varied environmental conditions of hypoxia and post-hypoxia was significantly higher (P≤ 0.05) in male than female trout. The cardiac output of trout in spawning condition was significantly higher (P≤ 0.05) than that of trout not in spawning condition. Measurements of pulsatile cardiac output were made simultaneously with trout ventilation, and revealed ventilatory interactions with blood flow that varied depending on environmental oxygen condition. The method for monitoring gill blood flow was used with methods for automated measurement of gill water flow, oxygen uptake, and chemical flux in vivo. An experimental protocol was developed in which environmental oxygen was varied to obtain maximum increases in water flow over the gills without blood flow changes, and subsequent attainment of maximum increases in blood flow through the gills with decreasing water flow. The protocol was used as a probe to study variations in chemical flux with varied blood or water flow. The changes in gill flux of butanol (Log octanol/water partition coefficient (P) = 0.88) measured during control, hypoxia, and post-hypoxia correlated with observed changes in blood flow. A 70% increase in butanol flux was noted with a 50% increase in cardiac output, but there was no increase in butanol flux with a 100% increase in ventilation volume. Changes observed in the gill flux of decanol (Log P = 4.57) measured under varied environmental oxygen conditions correlated with observed changes in ventilation volume. A 100% increase in decanol flux was noted with a 160% increase in ventilation volume. The observed blood flow limitations to uptake of the low Log P butanol, and the water flow limitations to uptake of the high Log P decanol helped to verify assumptions made in recently proposed flow-limited models for prediction of chemical flux across fish gills. Graduation date: 1991 1990-07-19T00:00:00Z