Dioxins are environmental contaminants that raise concern because they are potent and persistent. The most potent dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), causes a wide variety of biochemical and toxic effects in laboratory animals and in humans. Major toxicities of TCDD are initiated by their binding to the AH receptor (AHR), a ligand-activated transcription factor that regulates expression of numerous genes. However, the specific genes whose dysregulation leads to major toxicities such as wasting, hepatotoxicity, and lethality are unknown. The objective of this thesis research was to identify the molecular mechanisms by which dioxins cause lethality. To this end, a powerful genetic rat model was utilized – the Han/Wistar (Kuopio) rat which is highly resistant to dioxin toxicity due to a major deletion in the AHR’s transactivation domain (TAD) leading to 3 potential AHR variant transcripts. We found that insertion-variant transcripts (IVs) are the dominant forms of AHR expressed in H/W rats, constitutively and after TCDD treatment. Gene expression array analysis revealed that the total number of TCDD-responsive genes in liver was significantly lower in H/W rats (that carry the TAD deletion) than in dioxin-sensitive rats (that carry wildtype AHR). Genes that are well-known to be AHR-regulated and dioxin-inducible  such as CYP1 transcripts  remained responsive to TCDD in H/W rats; thus the TAD deletion selectively interferes with expression of a subset of hepatic genes rather than abolishing global AHR-mediated responses. Genes that differed in response to TCDD between dioxin-sensitive rats and dioxin-resistant rats are integral parts of pathways known to be disrupted by dioxin treatment such as protein synthesis/degradation, fatty acid transport/metabolism, and apoptosis. These genes are worthy candidates for further mechanistic studies to test their role in major dioxin toxicities. Numerous differentially-regulated genes were downregulated; however, microRNAs, which downregulate mRNA levels in other systems, likely play no role in downregulation of mRNAs by dioxins in adult liver and are unlikely to be involved in hepatotoxicity. Findings in this research support the hypothesis that H/W rats are resistant to TCDD lethality because the TAD deletion prevents the AHR from dysregulating specific mRNA transcripts but not hepatic miRNAs.

Persistent organic pollutants (POPs), including polychlorinated biphenyls (PCBs) are widespread in aquatic systems. These toxicants bioaccumulate in the tissues of aquatic organisms, especially fish as they occupy a position near the top of the aquatic food web. Teleost fish respond to stressors, including toxicants, by activating a co-ordinated network of adaptive responses, collectively termed the integrated stress response, which allows animals to regain homeostasis. Depending on the nature of the stressor, this stress response may be a generalised endocrine response that occurs at the organismal level and/or a cellular response involving protein synthesis. The cellular response to PCB insult involves aryl hydrocarbon receptor (AhR) activation and the induction of biotransformation enzymes, including cytochrome P4501A (Cyp1A). However, little is known about the mode of action of PCBs in affecting the adaptive stress response in animals. The objective of this thesis was to investigate the role played by AhR in mediating PCB impact on the highly conserved physiological responses to secondary stressors in fish. The experimental approach involved whole animal exposure studies with PCBs both in a laboratory setting as well as using feral fish. Also, in vitro mechanistic studies with pharmacological agents [AhR agonist (β-naphthoflavone) and antagonist (resveratrol), Hsp90 inhibitor (geldanamycin), proteasomal inhibitor (MG-132) and transcription (Actinomycin D) and translational inhibitors (cycloheximide D)] were carried out to understand AhR regulation in primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes. Also, a targeted trout cDNA microarray was developed as a tool to identify stress-responsive genes and signaling networks in fish. Short-term (3 day) exposure to PCBs, while inducing liver AhR and Cyp1A expression, did not modify the adaptive plasma cortisol response to an acute handling disturbance in rainbow trout. However, PCBs exposure did modify the metabolic response that is critical for recovery from an acute stressor in rainbow trout. To assess the impact of chronic PCB exposure on cellular stress response, two feral populations of Arctic char (Salvelinus alpinus) from Bjørnøya Island, Norway, were utilized. This is because the average PCB load in char liver from Lake Ellasjøen was approximately 25-fold higher than in individuals from Lake Øyangen, providing a natural setting to compare long-term toxicant impact on stress proteins. Liver Cyp1A expression was elevated in the high PCB fish suggesting AhR activation. Changes in mRNA abundance and/or protein expression of glucocorticoid receptor (GR), heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) in fish from the high PCB lake leads to the proposal that chronic exposures to PCBs is proteotoxic to the fish. In vitro mechanistic studies with trout hepatocytes revealed for the first time that AhR is autoregulated in response to ligand activation in rainbow trout. Furthermore this AhR regulation as well as AhR signaling involves both the molecular chaperone Hsp90 and the proteasome in hepatocytes. AhR signaling appears to play a role in the cellular response to heat shock in trout hepatocytes. Specifically, AhR signaling appears to be involved in the heat shock-induced Hsp70 and Hsp90 protein expression in trout hepatocytes. This modulation of Hsps by AhR may involve the proteasome. Overall, the results point to a cross-talk between the AhR and Hsps signaling pathways, while the precise mechanism(s) remains to be elucidated. A targeted rainbow trout cDNA microarray was constructed as a tool to identify stress-responsive genes in trout. This custom cDNA array consisted of 147 rainbow trout genes designed from conserved regions of fish sequences available in GenBank. The targeted genes had established roles in physiological processes, including stress and immune function, growth and metabolism, ion and osmoregulation and reproduction. This targeted array revealed changes in gene expression suggesting a rapid liver molecular reprogramming as critical for the metabolic adjustments to an acute stressor in fish. Also, transcripts not previously implicated in the stress response process in fish, including genes involved in immune function and protein degradative pathways, were found to be stress-responsive. Many of these transiently elevated stress-responsive transcripts were also shown previously to be glucocorticoid-responsive in fish implicating a key role for genomic cortisol signaling in stress adaptation. Overall, this thesis demonstrates that PCBs impact the organismal and cellular stress response in fish. AhR autoregulation may be a key aspect of PCBs impact on the cellular stress response pathways. Hsp90 and the proteasome may be involved in AhR regulation and PCB-mediated signaling in fish. The results suggest a cross-talk between AhR and Hsp signaling pathways in fish. Finally, the targeted cDNA microarray will be a useful tool to further expand our knowledge on PCBs impact on the cellular stress signaling pathways in fish.

Persistent organic pollutants (POPs), including polychlorinated biphenyls (PCBs) are widespread in aquatic systems. These toxicants bioaccumulate in the tissues of aquatic organisms, especially fish as they occupy a position near the top of the aquatic food web. Teleost fish respond to stressors, including toxicants, by activating a co-ordinated network of adaptive responses, collectively termed the integrated stress response, which allows animals to regain homeostasis. Depending on the nature of the stressor, this stress response may be a generalised endocrine response that occurs at the organismal level and/or a cellular response involving protein synthesis. The cellular response to PCB insult involves aryl hydrocarbon receptor (AhR) activation and the induction of biotransformation enzymes, including cytochrome P4501A (Cyp1A). However, little is known about the mode of action of PCBs in affecting the adaptive stress response in animals. The objective of this thesis was to investigate the role played by AhR in mediating PCB impact on the highly conserved physiological responses to secondary stressors in fish. The experimental approach involved whole animal exposure studies with PCBs both in a laboratory setting as well as using feral fish. Also, in vitro mechanistic studies with pharmacological agents [AhR agonist (β-naphthoflavone) and antagonist (resveratrol), Hsp90 inhibitor (geldanamycin), proteasomal inhibitor (MG-132) and transcription (Actinomycin D) and translational inhibitors (cycloheximide D)] were carried out to understand AhR regulation in primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes. Also, a targeted trout cDNA microarray was developed as a tool to identify stress-responsive genes and signaling networks in fish. Short-term (3 day) exposure to PCBs, while inducing liver AhR and Cyp1A expression, did not modify the adaptive plasma cortisol response to an acute handling disturbance in rainbow trout. However, PCBs exposure did modify the metabolic response that is critical for recovery from an acute stressor in rainbow trout. To assess the impact of chronic PCB exposure on cellular stress response, two feral populations of Arctic char (Salvelinus alpinus) from Bjørnøya Island, Norway, were utilized. This is because the average PCB load in char liver from Lake Ellasjøen was approximately 25-fold higher than in individuals from Lake Øyangen, providing a natural setting to compare long-term toxicant impact on stress proteins. Liver Cyp1A expression was elevated in the high PCB fish suggesting AhR activation. Changes in mRNA abundance and/or protein expression of glucocorticoid receptor (GR), heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) in fish from the high PCB lake leads to the proposal that chronic exposures to PCBs is proteotoxic to the fish. In vitro mechanistic studies with trout hepatocytes revealed for the first time that AhR is autoregulated in response to ligand activation in rainbow trout. Furthermore this AhR regulation as well as AhR signaling involves both the molecular chaperone Hsp90 and the proteasome in hepatocytes. AhR signaling appears to play a role in the cellular response to heat shock in trout hepatocytes. Specifically, AhR signaling appears to be involved in the heat shock-induced Hsp70 and Hsp90 protein expression in trout hepatocytes. This modulation of Hsps by AhR may involve the proteasome. Overall, the results point to a cross-talk between the AhR and Hsps signaling pathways, while the precise mechanism(s) remains to be elucidated. A targeted rainbow trout cDNA microarray was constructed as a tool to identify stress-responsive genes in trout. This custom cDNA array consisted of 147 rainbow trout genes designed from conserved regions of fish sequences available in GenBank. The targeted genes had established roles in physiological processes, including stress and immune function, growth and metabolism, ion and osmoregulation and reproduction. This targeted array revealed changes in gene expression suggesting a rapid liver molecular reprogramming as critical for the metabolic adjustments to an acute stressor in fish. Also, transcripts not previously implicated in the stress response process in fish, including genes involved in immune function and protein degradative pathways, were found to be stress-responsive. Many of these transiently elevated stress-responsive transcripts were also shown previously to be glucocorticoid-responsive in fish implicating a key role for genomic cortisol signaling in stress adaptation. Overall, this thesis demonstrates that PCBs impact the organismal and cellular stress response in fish. AhR autoregulation may be a key aspect of PCBs impact on the cellular stress response pathways. Hsp90 and the proteasome may be involved in AhR regulation and PCB-mediated signaling in fish. The results suggest a cross-talk between AhR and Hsp signaling pathways in fish. Finally, the targeted cDNA microarray will be a useful tool to further expand our knowledge on PCBs impact on the cellular stress signaling pathways in fish.

Many physiological responses to dioxin-like compounds (DLCs), including polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are mediated by the aryl-hydrocarbon receptor (AhR). In birds, activation of the AhR stimulates the transcription of cytochrome P4501A (CYP1A) genes, including CYP1A4 and CYP1A5, and ultimately leads to expression of biotransformation enzymes, including ethoxyresorufin-O-deethylase (EROD). It is well established that potencies of different DLCs range over several orders of magnitude. There is also a wide variation among birds in their responsiveness to DLCs both in efficacy and threshold for effects. A molecular basis for this differential sensitivity has been suggested. Specifically, a comparison of the AhR ligand-binding domain (LBD) indicated that key amino acid residues are predictive of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) sensitivity. Based on sequencing of the AhR LBD from numerous avian species a sensitive classification scheme has been proposed (in order of decreasing sensitivity, chicken (type I; sensitive) > Common pheasant (type II; moderately sensitive) > Japanese quail (type III; insensitive)). A series of egg injection studies with White-leghorn chicken (<i>Gallus gallus domesticus</i>), Common pheasant (<i>Phasianus colchicus</i>) and Japanese quail (<i>Coturnix japonica</i>) were performed to determine whether molecular and biochemical markers of exposure to DLCs are predictive of the proposed classification scheme. In addition, I was interested in determining whether this classification scheme applies to other DLCs, specifically dibenzofurans. Determining which species are "chicken- like", "pheasant-like" and "quail-like" in their responses to DLCs should allow more refined risk assessments to be conducted as there would be less uncertainty about the potential effects of DLCs in those species for which population-level studies do not exist.<p> Several concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), or 2,3,7,8-tetrachlorodibenzofuran (TCDF) (triolein vehicle) were injected into the air cells of Japanese quail, Common pheasant and chicken eggs. Liver from 14 d post-hatch chicks was harvested for analysis of CYP1A4 and CYP1A5 mRNA abundance by quantitative real-time PCR (Q-PCR), and EROD activity. Lowest observed effective concentration (LOEC) and relative potency (ReP) values for CYP1A mRNA abundance and EROD activity were determined and used to make comparisons of sensitivity between each species and DLC potency within each species.<p> The TCDD is widely considered to be the most potent DLC and this is supported by the rank order of LOEC values for CYP1A5 mRNA abundance in White-leghorn chicken (TCDD > PeCDF > TCDF). CYP1A4 mRNA abundance and EROD activity in White-leghorn chicken were significantly increased in the lowest dose exposure groups of each of the three DLCs, so the potency of these compounds based on these endpoints was not established. Interestingly, TCDD was not the most potent DLC in Common pheasant and Japanese quail. In Common pheasant, PeCDF is the most potent as a CYP1A4 mRNA inducer, followed by TCDD and TCDF. However, TCDF was the most potent EROD activity inducer for Common pheasant, followed by PeCDF, and then TCDD. No significant increases were found in CYP1A5 mRNA abundance in pheasant within the tested dose ranges for all the three DLCs. No significant increases in either CYP1A5 mRNA abundance or EROD activity were found in Japanese quail. In addition, PeCDF and TCDF, but not TCDD, significantly increased CYP1A4 mRNA abundance.<p> According to the predicted relative sensitivity by comparing the AhR LBD amino acid sequences, the White-leghorn chicken is more responsive to DLCs than the Common pheasant which is more responsive than the Japanese quail. By comparing the relative sensitivity calculated based on the LOEC values from my study, the sensitivity order to TCDD and TCDF support the proposed molecular based species sensitivity classification scheme (chicken > pheasant > quail), while pheasant is almost as sensitive as chicken to PeCDF ( pheasant ¡Ý chicken > quail).<p> Taken together, the data suggest that TCDD is the most potent DLC in White-leghorn chicken, but not in Common pheasant, or or Japanese quail. The data suggest that in type II avian species PeCDF may be more potent than TCDD. In addition, I found in my study that different biomarkers have different responses, which depends on species and chemicals as well. These data provide further insight into avian sensitivities to DLCs.</p>

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 113-122). Also available on the Internet.

This thesis presents the study of organic layers covalently assembled onto carbon surfaces. As a result of their attachment, the properties of carbon surfaces were controllably adjusted so that these surfaces could be used for desired applications. In order that a wide range of properties were imparted onto the carbon surface, many different modifiers were attached and thoroughly characterised. Three applications that the modified carbon surfaces were used for were the subsequent coupling of molecular species, adsorption of protein and assembly of aldehyde/sulfate-functionalised polystyrene (PS) and citrate-capped gold nanoparticles (NPs). Finally, patterning of different organic layers at pre-determined spatially defined locations on the one carbon surface was also investigated. The carbon surfaces used in this work were glassy carbon (GC) and pyrolysed photoresist film (PPF) surfaces. For PPF, methods for the reproducible fabrication of electrochemically suitable surfaces were investigated. The properties of GC and PPF surfaces are very similar apart from the surface roughness. PPF has near atomic smoothness and has RMS roughness values that are approximately four times smaller than GC. The first series of modifier layers attached to the carbon surfaces was via the oxidation of seven different primary amines. The different layers allowed the modulation of the wettability of the surface. Both n-tridecylamine (TDA, monoamine) and 1,12-diaminododecane (DAD, diamine) are able to form multilayers. The stability of TDA and DAD layers were tested by scanning, soaking and sonicating the layers in different media. Changes in the layer were monitored by the probe response of ferrocene monocarboxylic acid (FCA). However, atomic force microscope (AFM) depth profiling experiments showed that changes in the probe response did not indicate cleavage of the covalently attached layer and mechanisms are proposed to account for the changes in the response of the probe. Surface concentrations of the amine modifiers were estimated by the coupling of an electrochemically active species, FCA and nitrobenzoyl chloride (NBC). The electrochemical reduction of the 4-nitrophenylethylamine (NPEA) layer in acid caused the layer to 'shrink'. Surface concentration estimates of NPEA from acid reduction of layers with different thicknesses suggested that only a limited fraction of the p-nitrophenyl groups were reduced in acid. However, in ACN (acetonitrile)/0.1 M [Bu4N]BF4 (tetrabutyl ammonium fluoroborate) the relationship between the concentration of electroactive surface groups and layer thickness was linear. The other series of modifiers that was attached to alter the surface properties was performed by the reduction of aryl diazonium salts. Subsequent coupling reactions of tetraethylene glycol diamine (TGD) to para methylene carboxylic acid phenyl (MCA) and NBC to electrochemically reduced para nitro phenyl (NPh) layers were carried out. Surface concentrations of NPh as estimated from reduction scans was higher when reduction was performed in ethanol/water compared to acid. Four peaks at N1s binding energies were observed in x-ray photoelectron spectroscopy (XPS) spectra for both acid and ethanol/water reduced layers. The ability of attached amine and aryl layers to modulate the adsorption of protein was investigated using fluorescently labelled protein, bovine serum albumin-fluorescein isothiocynate (BSA-FITC) and fluorescence microscopy. TGD, para methyl phenyl (MP), para hexyl phenyl (HP) and para polyethylene glycol phenyl (PEG)-modified GC surfaces promoted protein adsorption relative to as-prepared GC, whereas n-hexylamine (HA) and polyethylene glycol diamine (PGD) layers reduced protein adsorption. The assembly of two types of NPs, aldehyde/sulfate-functionalised PS and citrate-capped gold NPs, onto amine-containing modifiers layers was examined. Citrate-capped gold NPs were synthesised and characterised. The surface coverage of the gold NPs was controlled by using different modifiers of different chemical compositions, tuning the modification conditions and adjusting the immersion time, concentration and pH of gold NP solution. Approaches to creating patterns of modifiers in pre-determined spatially defined locations on GC and PPF surfaces using poly(dimethyl)siloxane (PDMS), poly(vinyl)alcohol (PVA) and thin metal films were investigated. With the "fill-in" approach using PDMS, the smallest pattern of modifiers was the parallel lines with a line width of 20 µm and straight edges and was created by performing electrochemistry in PDMS microchannels which has not been previously investigated. Visualisation techniques, based on optical and scanning electron microscopy, were demonstrated for the molecular patterns.

Obesity, metabolic syndrome, and diabetes represent a major source of morbidity and mortality in the United States and worldwide. Chronic misalignment of an organism’s internal circadian clock with diurnal, cyclic changes in the external environment, prevalent in professions that require shift work, contributes significantly to Type 2 Diabetes development. Experimentally, only short-term models of circadian disruption have been explored. Therefore, the goal of this study was to establish an animal model of chronic circadian disruption, which would more closely mimic the harmful misalignment associated with metabolic syndrome in clinical studies. Moreover, since high fat diet consumption alters circadian behavior and rhythmic gene expression, contributing to the diet-induced phenotype, I hypothesized that chronic circadian disruption interacts with a high fat diet to worsen metabolic syndrome. To investigate circadian misalignment and diet-induced metabolic syndrome, I examined the contribution of the Aryl Hydrocarbon Receptor (AhR). AhR has similar PAS domain containing motifs as circadian clock proteins allowing for protein/protein interactions and crosstalk between AhR signaling and circadian rhythms. Furthermore, AhR activation is implicated in Type 2 Diabetes risk. To examine chronic circadian disruption, male wild-type (WT; C57Bl/6J) and AhR +/- mice were entrained to 12/12-hour light/dark cycles where lights were on from 10pm-10am and off from 10am-10pm. Misalignment was initiated by delaying the time of lights on by 8 hours on Monday. Mice were exposed to the misalignment schedule Monday-Friday then returned to the entrainment schedule Saturday and Sunday to mimic readjustment to society during the weekend. Circadian misaligned mice were exposed to the altered light schedule for 15 weeks and control animals remained on the12/12-hour light/dark cycle. Mice were fed a normal chow diet (10% fat) or a high fat diet (60% fat). Animals were sacrificed and samples were collected at 4-hour intervals on day 2 of the weekend. Exposure to chronic circadian misalignment by light disruption or high fat diet altered circadian rhythms of behavior, metabolic outputs, and expression of circadian clock, clock-controlled nuclear receptor, and lipid metabolism genes. A combination of light misalignment and high fat diet exacerbated the effects of either treatment alone further disrupting behavior, enhancing % body fat and fasting glucose, and dampening circadian clock gene expression. AhR +/- mice also were protected from the metabolic consequences of chronic misalignment and a high fat diet by resistance to altered behavioral and molecular circadian rhythms and disruption of metabolic outputs. With metabolic syndrome and Type 2 Diabetes occurrence on the rise, it is important to understand all contributing factors, including circadian disruption. Differences between chronic circadian misalignment and high fat diet-induced obesity in WT and AhR +/- mice furthers our understanding of the complex mechanisms that underlie Type 2 Diabetes development and advocates the discovery of potential therapeutic targets for the development of novel treatment options.

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No description available.

Supporting Information attached / <p> The development of a convenient route for the preparation of fluorous-tagged compounds for use with the fluorous labeling strategy (FLS) is described in this thesis. The FLS is a new and convenient method for the preparation of radiotracers and therapeutics in high effective specific activity (HESA) without the use of preparative HPLC. The objective of this thesis was to expand the general utility of the FLS by enabling the introduction of fluorous tags into molecules using a palladium-catalyzed cross-coupling reaction. To this end, a fluorous distannane, hexa(1H,1H,2H,2Hperfluorooctyl) distannane, was prepared from the corresponding fluorous tin hydride and used to produce trialkylarylstannanes from aryl and vinyl halides. Using the developed methodology, fluorous precursors for two radiopharmaceuticals, fialuridine (FIAU) and idoxuridine (IUdR), were prepared. The fluorous-tagged products were radiolabeled with iodine-125 to afford the desired compounds in high effective specific activity and in good radiochemical yield. </p> <p> Hexa(1H,1H,2H,2H perfluorooctyl)distannane was prepared from the corresponding tin hydride in nearly quantitative yield in the presence of Pd(PPh3)4. The distannane was combined with a series of seventeen aryl bromides and iodides and the appropriate palladium catalyst to afford trialkylarylstannanes in 15-59% isolated yield. </p> <p> The use of a phosphaadamantane ligand reported by Capretta et al. in the cross-coupling was also investigated, and the yields for the model compounds ranged from 13-67% Although no substantial change in yields was observed for aryl halides compared to the traditional catalyst Pd(PPh3)4, the phosphaadamantane ligands were more effective for the synthesis of precursors to [125I]fialuridine (FIAU) and [125I]idoxuridine (IUdR). Using this ligand system, the FIAU precursor was prepared in 38% overall yield from a dibenzoyl-protected vinyl bromide, and the IUdR precursor was prepared in 21% yield from a vinyl iodide. </p> <p> Following preparation of the FIAU and IUdR precursors, direct iodinolysis using a sub-stoichiometric amount of iodine was performed and the products isolated in excellent yield and purity using fluorous solid-phase extraction (FSPE). Following these experiments, the precursors were radiolabeled with [125I]NaI (50 – 500 μCi, 1.9 – 19 MBq) in the presence of Iodo-Gen® as an oxidant. Average radiochemical yields for three trials were 88% for FIAU and 94% for IUdR. The precursor was not observed in the FSPE-purified reaction mixture by UV-HPLC within the instrument’s detection limit. </p> <p> The fluorous labeling strategy allows molecular imaging and associated therapy agents to be produced in high effective specific activity in a rapid and convenient manner. With the development of the fluorous distannane and the associated coupling reactions reported here, the general utility of the fluorous labeling strategy has been greatly expanded. </p> / Thesis / Master of Science (MSc)