Pharmaceutical drugs are used extensively by humans and domestic animals. The detection of compounds such as non-steroidal anti-inflammatory drugs (NSAIDs) in effluents of sewage treatment plants and surface waters has raised concerns about whether these drugs have the potential to impact aquatic organisms. However, little is known about either the mechanism of action of NSAIDs or their impact on aquatic organisms. A key indicator of animal stress performance is the elevation in plasma cortisol levels, the primary circulating corticosteroid in teleosts, and the associated metabolic changes in response to stressor exposure. The secretion of cortisol is under the control of the hypothalamus-pituitary-interrenal (HPI) axis with the terminal step involving the activation of interrenal steroidogenesis by adrenocorticotropic hormone (ACTH) from the pituitary gland. Cortisol, predominantly via glucocorticoid receptor (GR) activation, is involved in a wide array of animal functions, including growth and metabolism, osmo- and iono-regulation, stress and immune function and reproduction, all of which play a role in regaining homeostasis after stressor insult. The overall objective of this thesis was to investigate the role of NSAIDs in impacting the evolutionarily-conserved adaptive stress response in a model teleost fish, the rainbow trout (Oncorhynchus mykiss). Specifically, the impact of NSAIDs on stress coping mechanisms was investigated by examining: i) interrenal steroid biosynthetic capacity and cortisol production, ii) target tissue GR function, iii) cellular heat shock protein response, iv) tissue-specific metabolic response to stressors, and iv) ionoregulatory performance in seawater. The experimental approach involved a series of whole animal in vivo and in vitro studies, using rainbow trout interrenal cell preparations, with two NSAIDs, salicylate and ibuprofen, commonly detected in our surface waters. Fish were subjected to stressors of varying intensity and duration, including handling disturbance, heat shock and salinity exposures, to identify targets impacted by NSAIDs in fish. NSAIDs did not affect resting plasma cortisol levels but disrupted the acute ACTH-stimulated corticosteroidogenesis in vitro and stressor-induced plasma cortisol response in vivo. The mode of action of NSAIDs in disrupting cortisol production involves inhibition of the key rate-limiting step, the steroidogenic acute regulatory protein (StAR), in steroidogenesis. Also, tissue (brain, liver and gill) GR protein content is a target for endocrine disruption by NSAIDs leading to abnormal negative feedback regulation of plasma cortisol levels and reduced target tissue responsiveness to cortisol after stressor exposure. The drugs also clearly affected the cellular stress response in rainbow trout by perturbing the expression of heat shock protein 70 (hsp70), a highly conserved stress coping mechanism. This impaired heat shock response with NSAIDs corresponded with an altered tissue metabolic capacity suggesting disturbances in biochemical adjustments to stressor. Specifically, the dynamics of glucose, the primary fuel to cope with the enhanced tissue metabolic demand, was disrupted in a drug-specific manner in rainbow trout. Exposure to NSAIDs also disrupted the ionoregulatory mechanisms critical for seawater acclimation in rainbow trout. The targets for ionoregulatory disturbance in seawater by NSAIDs include the major ion transporter gill Na+/K+-ATPase as well as gill GR, a key signaling protein for Na+/K+-ATPase upregulation in fish. Altogether, NSAIDs disrupt the adaptive endocrine and metabolic stress coping mechanisms in rainbow trout. The targets for endocrine disruption by NSAIDs include multiple sites along the HPI axis as well as target tissue response to cortisol action in fish. Specifically, the mode of action of NSAIDs involves disruption of StAR and GR, two key proteins critical for cortisol production and target tissue responsiveness to this steroid, respectively. While the work presented here identified the mechanism(s) of action of NSAIDs, the environmental relevance of this finding, specifically the impact of concentrations of NSAIDs present in our waterways on fish stress performance, remains to be explored.

Pharmaceutical drugs are used extensively by humans and domestic animals. The detection of compounds such as non-steroidal anti-inflammatory drugs (NSAIDs) in effluents of sewage treatment plants and surface waters has raised concerns about whether these drugs have the potential to impact aquatic organisms. However, little is known about either the mechanism of action of NSAIDs or their impact on aquatic organisms. A key indicator of animal stress performance is the elevation in plasma cortisol levels, the primary circulating corticosteroid in teleosts, and the associated metabolic changes in response to stressor exposure. The secretion of cortisol is under the control of the hypothalamus-pituitary-interrenal (HPI) axis with the terminal step involving the activation of interrenal steroidogenesis by adrenocorticotropic hormone (ACTH) from the pituitary gland. Cortisol, predominantly via glucocorticoid receptor (GR) activation, is involved in a wide array of animal functions, including growth and metabolism, osmo- and iono-regulation, stress and immune function and reproduction, all of which play a role in regaining homeostasis after stressor insult. The overall objective of this thesis was to investigate the role of NSAIDs in impacting the evolutionarily-conserved adaptive stress response in a model teleost fish, the rainbow trout (Oncorhynchus mykiss). Specifically, the impact of NSAIDs on stress coping mechanisms was investigated by examining: i) interrenal steroid biosynthetic capacity and cortisol production, ii) target tissue GR function, iii) cellular heat shock protein response, iv) tissue-specific metabolic response to stressors, and iv) ionoregulatory performance in seawater. The experimental approach involved a series of whole animal in vivo and in vitro studies, using rainbow trout interrenal cell preparations, with two NSAIDs, salicylate and ibuprofen, commonly detected in our surface waters. Fish were subjected to stressors of varying intensity and duration, including handling disturbance, heat shock and salinity exposures, to identify targets impacted by NSAIDs in fish. NSAIDs did not affect resting plasma cortisol levels but disrupted the acute ACTH-stimulated corticosteroidogenesis in vitro and stressor-induced plasma cortisol response in vivo. The mode of action of NSAIDs in disrupting cortisol production involves inhibition of the key rate-limiting step, the steroidogenic acute regulatory protein (StAR), in steroidogenesis. Also, tissue (brain, liver and gill) GR protein content is a target for endocrine disruption by NSAIDs leading to abnormal negative feedback regulation of plasma cortisol levels and reduced target tissue responsiveness to cortisol after stressor exposure. The drugs also clearly affected the cellular stress response in rainbow trout by perturbing the expression of heat shock protein 70 (hsp70), a highly conserved stress coping mechanism. This impaired heat shock response with NSAIDs corresponded with an altered tissue metabolic capacity suggesting disturbances in biochemical adjustments to stressor. Specifically, the dynamics of glucose, the primary fuel to cope with the enhanced tissue metabolic demand, was disrupted in a drug-specific manner in rainbow trout. Exposure to NSAIDs also disrupted the ionoregulatory mechanisms critical for seawater acclimation in rainbow trout. The targets for ionoregulatory disturbance in seawater by NSAIDs include the major ion transporter gill Na+/K+-ATPase as well as gill GR, a key signaling protein for Na+/K+-ATPase upregulation in fish. Altogether, NSAIDs disrupt the adaptive endocrine and metabolic stress coping mechanisms in rainbow trout. The targets for endocrine disruption by NSAIDs include multiple sites along the HPI axis as well as target tissue response to cortisol action in fish. Specifically, the mode of action of NSAIDs involves disruption of StAR and GR, two key proteins critical for cortisol production and target tissue responsiveness to this steroid, respectively. While the work presented here identified the mechanism(s) of action of NSAIDs, the environmental relevance of this finding, specifically the impact of concentrations of NSAIDs present in our waterways on fish stress performance, remains to be explored.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3245. Adviser: Robert Knox. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

A major feature of inflammation is the extravasation of cellular and macromolecular components of blood into the interstitium to attack and fend off an offending stimuli, thus initiating healing. When prolonged and/or uncontrolled, this process can begin to produce deleterious effects and inadvertently damage tissue. Such instances are observed in pathologies, such as atherosclerosis, organ transplant rejection and reperfusion injury. It is therefore vital to understand the various mechanisms that initiate and control the process of extravasation. Since the endothelium is the key component between blood and the interstitium, its regulation is largely responsible for control of extravasation. TNFα and IFNγ are cytokines that function in orchestrating signals between different cellular players involved in inflammation. In this dissertation, the effects of these cytokines on endothelial permeability were explored using in vivo and in vitro methods. I investigated two ways whereby the endothelial barrier could be controlled. Firstly, the molecular mechanisms at intercellular junctions could be regulated, thus influencing permeability via the paracellular pathway. One such mechanism involves cadherins which are transmembrane homotypic Ca²⁺-dependent cell-cell adhesion molecules implicated in the control of junctional organization and therefore, paracellular permeability. Secondly, manipulation of signaling pathways by cytokines that induce endothelial apoptotic cell death could create cell-sized gaps in the endothelial barrier. The results of this work demonstrated that combined cytokine treatments: (i) induced focal areas of cadherin-5 relocalization which corresponded to regions of macromolecular extravasation in a rat in vivo model and (ii) induced the upregulation and junctionalization of N-cadherin, concomitant with downregulation and decreased junctionalization of cadherin-5 in cultured endothelial cells. Thus, combined cytokine regulation of endothelial cadherin function was found central in both accommodating an initial microvascular permeability increase upon cytokine induction of inflammation and in limiting the extent of paracellular gap formation (via its effects on N-cadherin) under chronic inflammatory conditions. Furthermore, despite in vitro evidence of endothelial apoptosis, the combined cytokines induced most endothelial cells to express protective, anti-apoptotic molecules (e.g. A1) and therefore contributed to preserving endothelial barrier integrity. Through these experiments, specific mechanisms for regulating the endothelial barrier during inflammation have been identified and are better understood.

Women are born with a finite number of primordial follicles, the smallest follicles in the ovary. Once these follicles are destroyed, they cannot be replaced and after extensive loss, ovarian failure (menopause) can occur. The industrial chemical 4-vinylcyclohexene diepoxide (VCD) induces depletion of these follicles and causes premature ovarian failure in rats. VCD-induced ovotoxicity has been found to accelerate a natural process in the ovary, atresia, which occurs via apoptosis. The mechanism(s) by which VCD enhances follicular atresia are unknown; however, it has been shown to alter the expression of several genes and proteins associated with apoptosis. While downstream signaling events of VCD are becoming identified, the early signaling events of this pathway have not yet been determined, but may involve a receptor-mediated cascade. Therefore, these studies tested the hypothesis that VCD-induced ovotoxicity involves a nuclear receptor-mediated pathway that leads to increased atresia. Concurrent treatment of rats with VCD and estradiol selectively protected primary follicles from loss by an estrogen receptor-mediated mechanism via a reduction of caspase-3-induced apoptosis. VCD does not alter ER number, affinity, circulating estradiol levels, or directly bind ERbeta. Concurrent dosing of rats with VCD and an AhR antagonist prevented primordial and primary follicle loss via a reduction in caspase-3-induced apoptosis. Repeated dosing with VCD was shown to up-regulate expression of AhR mRNA; however, VCD did not alter expression of AhR-mediated genes glutathione-S-transferase Ya1 or Ya2 nor CYP 1A1 protein. AhR-deficient mice were still susceptible to VCD-induced follicle loss. Repeated dosing with VCD reduced Heat Shock Protein (HSP) 90 expression in small primary follicles. Analogs of the ER and AhR did not alter HSP90 protein, nor did a loss of HSP90 function induce follicle loss or potentiate VCD-induced follicle depletion. While the ER, AhR, and HSP90 are all co-localized in the oocyte nucleus of primordial and primary follicles, no evidence was seen to support that these proteins are interacting. Taken together, the ER is able to prevent VCD-induced ovotoxicity in primary follicles, the AhR is not required for VCD-induced follicle loss, and HSP90 does not appear to play a central role in follicle depletion caused by VCD.

Iron is essential for life and iron homeostasis is important for all species. Compared to the understanding of iron metabolisms in vertebrates, we know much less about insect intracellular iron homeostasis. The iron regulatory proteins (IRPs) play central roles in this process by interaction with iron responsive elements (IREs). Here, I report the cloning, sequencing, overexpression, purification and characterization of IRP1s from two insect species, Manduca sexta and Aedes aegypti. Electrophoretic mobility shift assays demonstrated that both IRP1s specifically bind IREs s not only from the same species, but also from human ferritin IRE. Another ferritin subunit also was cloned from Manduca sexta and an IRE was identified in the 5'-untranslated region of the mRNA, and the IRE reacted with Manduca IRP1 specifically. Transcription/translation assays demonstrated that both IRP1s repress ferritin synthesis in vitro, and the repression is IRE dependent. Iron administration to Manduca sexta increased hemolymph ferritin levels and decreased fat body IRP1/IRE binding activities without affecting either the IRP1 mRNA or protein levels. These data indicates that translational control of ferritin synthesis by IRP1/IRE interaction could occur in insects in a manner similar to that of mammals. To our knowledge this is the first report of the control of insect ferritin synthesis by IRP1/IRE interaction. The different responses to reducing agent of Manduca sexta and mammalian IRP1s could provide a potential future strategy for designing pesticides in insect control.

4-Vinylcyclohexene (VCH) is released during rubber manufacture as an environmental contaminant. It causes destruction of ovarian follicles and ovarian cancer in mice. To determine its long term effects on the female reproductive system, female B6C3F1 mice were administered sesame seed oil or VCH (800 mg/kg) ip daily for 30 days. Mice were killed when in diestrus at 30, 60, 120, 240, or 360 days after VCH administration. Ovaries and uteri were weighed and ovaries prepared for histology. Primary and secondary follicles were counted and FSH determined by RIA. The number of estrous cycles differed from controls in the 30 day group. Uterine weight differed in the 30, 60, and 360 day groups. Primary follicles, secondary follicles and ovarian weight decreased in the VCH groups. FSH increased in the 240 and 360 day VCH groups. These studies indicate that VCH causes a dramatic and permanent reduction in ovarian follicles with a reduction in ovarian weight the most obvious indicator of follicle destruction.

Mineral hydrocarbons (MHC) produce hepatic microgranulomas and granulomas following repeated administration to female Fischer-344 (F-344) rats. Female Sprague-Dawley (S-D) rats are less sensitive to these MHC-induced hepatic effects. Comparative studies were conducted to characterize the pharmacokinetics and disposition of a representative C26 MHC, [1-14C]1-eicosanylcyclohexane ([14C]EICO), in these two rat strains. Following a single oral dose of MHC, F-344 rats had a higher blood Cmax of [14C]EICO, a longer time to Cmax, and a greater blood AUC compared to S-D rats. Fecal excretion was the major route of elimination of parent [ 14C]EICO for both rat strains. Both rat strains eliminated 11% of the dose in the urine by 96 h. However, S-D rats eliminated the majority of [14C]EICO-metabolites in the urine by 16 h, while F-344 rats excreted [14C] equivalents in a time-dependent manner. At 96 h, 3% of the radioactive dose was recovered in livers of F-344 rats, but only 0.1% in S-D rats. Parent [14C]EICO was determined to be retained in livers of F-344 rats. Only metabolites of [14C]EICO were present in urine, the two major being 12-cyclohexyldodecanoic acid and 10-cyclohexyldecanoic acid. Inhibition of hepatic P450 activity using ABT prior to MHC administration resulted in a significant increase in [14C]EICO retention in livers of F-344 rats at 96 h. Pretreatment with clofibrate, a CYP4A inducer, decreased the amount of [14C]EICO retained in livers of F-344 rats at 96 h. Dietary exposure from MHC for 2 weeks resulted in an increased amount of MHC retained in livers of F-344 rats, whereas little was retained in treated S-D rats. The majority of MHC was retained in hepatocytes, whereas little was detected in Kupffer cells (KC). This was evidenced following transmission electron microscopy analysis of liver sections of F-344 rats. Multiple MHC droplets were observed in hepatocytes, while few droplets were detected in KC. Results from these studies suggest that inherent strain-differences in systemic exposure, rates of elimination, and hepatic retention of MHC exist following MHC exposure. The CYP4 enzyme family plays a role in the metabolism, clearance, and retention of [14C]EICO, while the phagocytic activity of KC is less significant after a single oral dose of MHC. In addition, repeated exposure of MHC alters the morphology of hepatocytes of F-344 rats but not S-D rats. These differences just described suggest that F-344 rats are inherently predisposed to the observed MHC-induced hepatic effects. (Abstract shortened by UMI.)

Nectarivorous birds are represented by three major radiations: hummingbirds, honeyeaters, and sunbirds. These lineages share a number of convergent features in ecology, morphology, physiology, and behavior, and have served as important models in the study of foraging strategies and energetics. Because their diet is rich in water and sugar but poor in nitrogen and electrolytes, nectarivores provide a striking opportunity for evaluation of physiological constraints. My research emphasizes a novel aspect of the water-energy interaction: water overingestion in nectar-eating birds. The dual purpose of my dissertation research was to investigate the physiological mechanisms that allow nectar-eating birds to cope with exceedingly high ingestion of water and to elucidate the consequences of ingesting and processing large quantities of water for energy intake and for the maintenance of balance of important metabolites such as glucose. In nectar-eating birds, water overabundance in food has the potential effect of constraining energy procurement by overwhelming osmoregulatory processes and limiting digestive function. My research has allowed the development of an integrated quantitative description of gut and kidney function under the broad range of water loads and hydration conditions that birds can experience in the wild. Understanding limits to water processing will provide general insights into how animals are designed, on how aspects of design constrain their ecological performance, and into how aspects of design in one physiological system can impose limits on other systems. The osmoregulatory processes of nectar-eating birds highlight the relevance of understanding the impact that events taking place in the gut can have for feeding behavior, and renal and metabolic function. Adopting a broadly comparative approach to understanding the interaction between feeding behavior, digestion, and osmoregulation is pertinent because is unclear whether the many extreme physiological characteristics of hummingbirds that have traditionally been assumed to be associated with a nectar-feeding habit are shared by other nectar-eating birds. In my dissertation research I have begun to examine the similarities, and have found some important differences, in the responses of two major radiations of nectar-eating birds to their sugary and watery diets.

Nectarivory has evolved independently eight times among birds. In the neotropics specialized nectarivory evolved in hummingbirds and flowerpiercers (genus Diglossa). Flowerpiercers are nectar-robbers of hummingbird-pollinated plants. Because flowerpiercers and hummingbirds are found in the same habitats feeding on the nectar of the same flowers, they provide us with a unique opportunity to understand the pressures that nectarivory imposes on animals. Flowerpiercers present beaks that have a long hook at the tip of their maxilla. The hook is used to hold tubular flowers in place while they stab them by projecting their lower mandible. Then, they insert their tongues in the puncture and extract nectar. I investigated the following questions: (1) What is the function of the flowerpiercer's hook, and which are the consequences of its evolution? (2) Are the digestive traits of flowerpiercers convergent with those of hummingbirds? (3) How do digestive traits affect sugar selection by nectar feeding birds? And (4) What are the effects of Diglossa baritula's physiology and behavior over its annual cycles? I found that nectarivorous flowerpiercers evolved from a frugivorous ancestor with a hookless beak. The evolution of a hooked bill allowed flowerpiercers to become efficient nectar-robers, but hindered their efficiency to feed on fruit. Using a phylogenetically informed approach, I found that the digestive traits of flowerpiercers and hummingbirds are not convergent. Unlike hummingbirds which have astounding intestinal sucrase activity levels, flowerpiercers had low sucrase activity. Nectar intake in D. baritula seems to be limited by its ability to digest sucrose. I also found that sugar preferences in nectar-feeding birds are concentration-dependent. At lower concentrations birds preferred hexoses, whereas at higher concentrations they preferred sucrose. Although nectar composition and concentration are often discussed as two different floral traits, they have a synergistic effect on the sugar preferences of nectar-feeding birds. D. baritula individuals exhibit biannual reproductive and molting cycles that are synchronized with flower and fruit abundance in the mountains of Mexico. The ability to rob nectar appears to have molded the evolution of the most important morphological, physiological and behavioral traits of D. baritula.