The small intestine and the liver regulate several physiological functions together including the absorption and bioavailability of drugs and bile and nitrogen homeostasis. It is important to study these two organs together to gain a holistic understanding of their communication with each other. However, there is a lack of culture models that investigate the use of primary cells/tissues from the liver and the intestine to study their interaction and importance in manifestation of drug toxicity. The studies described in this dissertation were conducted using inverted rat intestinal explants obtained from three regions of the jejunum, named as the proximal, medial and distal jejunum. Markers of enterocyte, goblet cell and Paneth cell function in the jejunum followed in vivo – like spatial trends reported for the entire small intestine. Jejunum explants were integrated with hepatocytes to model the intestine-liver axis. Integration of jejunum explants from the proximal region with hepatocytes had a beneficial effect on both hepatocyte urea secretion and jejunum mucin secretion, hinting at communication between these organs in culture. Integrated cultures of the rat jejunum and hepatocytes were used to investigate ethanol toxicity in vitro. Trends in activities of enzymes involved in ethanol metabolism and mucus secretion in integrated cultures with proximal jejunum explants corroborated with in vivo reports on ethanol toxicity. Various metabolites secreted and metabolized in vitro were also identified using mass spectrometry. Spatial trends in concentrations of several lipids including bile acids, lysophosphatidylcholines and fatty acids corroborated with in vivo reports of lipid metabolism. The integrated intestine-liver cultures can be used as a platform for future investigations of drug toxicity, lipid metabolism and inter-organ communication. / Doctor of Philosophy / The small intestine and the liver perform several functions together. The small intestine is responsible for the digestion of food, absorption of nutrients and metabolism of oral drugs. The liver is involved in the metabolism of glucose, protein, lipids and drugs. It is important to study these two organs together to gain a holistic understanding of their communication with each other. However, there is a lack of culture models that investigate the use of cells/tissues directly obtained from animal liver and intestine to study their interaction and importance in manifestation of drug toxicity. The studies described in this dissertation were conducted using tissues obtained from three regions of the jejunum segment of the rat small intestine. Functional markers of various cell types in the jejunum followed in vivo – like spatial trends reported for the entire small intestine. Jejunum tissues were integrated with liver cells to model the intestine-liver axis. Integration of jejunum tissues from the proximal region with liver cells had a beneficial effect on both liver and intestinal markers, hinting at communication between these organs in culture. Integrated cultures of the rat jejunum and liver cells were used to investigate alcohol toxicity in vitro. Trends in activities of enzymes involved in alcohol metabolism and mucus secretion in integrated cultures with jejunum tissues corroborated with in vivo reports on alcohol toxicity. Various metabolites secreted and metabolized in vitro were also identified using mass spectrometry. Spatial trends in concentrations of lipids including bile acids, lysophosphatidylcholines and fatty acids within the jejunum corroborated with in vivo reports of lipid metabolism. The integrated intestine-liver cultures can be used as a platform for future investigations of drug toxicity, lipid metabolism and inter-organ communication.

This thesis describes two experiments: one related to antihistamines, and the other related to antitubercular compounds.

This research was divided into four objectives for assessing the impacts of coal mining on ecosystem health. The first objective was to provide an ecotoxicological assessment in the upper Clinch River using standard bioassessment techniques. Analysis of sediments and interstitial water (porewater) indicate higher concentrations of trace metals in samples from sites located above both a power plant (CRP) and Dumps Creek mining influences. The furthest sampling site located near Pounding Mill, Virginia (CR-PM) had higher concentrations of aluminum (2,250.9 mg/kg), copper (5.9 mg/kg) and iron (12,322.6 mg/kg) compared to samples collected directly below the Dumps Creek confluence (site CR-2). Similar results were obtained from bioaccumulation in-situ tests with the Asian clam (Corbicula fluminea) in 2009. Aluminum (7.81 mg/kg), Fe (48.25 mg/kg) and Zn (7.69 mg/kg) were accumulated in higher concentrations at CR-PM site than CR-2. However, the site located below the CRP effluent discharges (CR-3L) on the left bank had substantially higher concentrations of Al (14.19 mg/kg), Cu (6.78 mg/kg), Fe (88.78 mg/kg) and Zn (7.75 mg/kg) than both CR-PM and samples collected directly opposite of this site at CR-3R. To further understand the potential impact active mining on the Clinch River, a more comprehensive ecotoxicological evaluation was conducting in the Dumps Creek subwatershed. Field bioassessments determined that biological impairment occurred directly below a deep mine discharge (CBP 001), which was characterized by a distinct hydrogen sulfide odor. Total abundance and richness of benthic macroinvertebrates decreased to 3.5-20 and 1.25-2.3, respectively at DC-1 Dn. The discharge also caused the proliferation of a sulfur-oxidizing bacterium, Thiothrix nivea. During continuous discharge of the effluent, the bacteria was observed coating all surfaces at DC-1 Dn and may also contribute to an Fe-encrusted biofilm observed on in-situ clams at downstream site, DC-2 Dn. Toxicity tests with mining effluents indicate some potential toxicity of the 001 discharge, but this was variable between test organisms. Selecting the most appropriate test species for sediment and water column assays has been a primary goal for ecotoxicologists. Standard test organisms and established test guidelines exist, but US EPA recommended species may not be the most sensitive organisms to anthropogenic inputs. Therefore, Chapter Three and Four addressed the use of mayflies in routine laboratory testing. Preliminary results of toxicity tests with the mayfly, Isonychia sp. (Ephemeroptera) suggested that Isonychia were moderately sensitive to NaCl after 96-hr with an average LC50 value of 3.10 g NaCl/L. When exposed to a coal-mine processed effluent, Isonychia generated LC50 values that ranged from 13 to 39% effluent and were more sensitive to the effluent than Ceriodaphnia dubia. Based on results of the feasibility study in presented in Chapter Four, field collected organisms appear to be too unpredictable in test responses and therefore, such tests would be unreliable as stand-alone indicators of effluent toxicity. / Ph. D.

Tall fescue is a widely grown, cool season grass prevalent in the eastern United States that is known for its resistance to abiotic and biotic stresses. A main reason for tall fescue's resistance to these stresses is attributed to the presence of a fungal endophyte. Unfortunately, this endophyte also adversely affects cattle production. Cows consuming the ergot alkaloids produced by these endophytes can exhibit decreased feed intake, growth performance, organ vasoconstriction, and increased rectal temperature. This work is interested in examining how endophyte toxin exposure impacts pregnancy in cattle. Reduced blood flow to the fetus and inadequate maternal nutrition contributes to intra uterine growth restriction (IUGR), and this work proposed that fescue endophyte toxicity affects the gestating cow and fetus. Three studies were completed. In experiment 1, gestating cows grazed high or low endophyte fescue pastures during late gestation to determine if exposure to ergot alkaloids in utero results in IUGR and if calves from these pregnancies have altered growth performance. Creep feeding was evaluated as a mitigation strategy for impaired calf growth due to fescue toxicity, and feedlot performance was evaluated to determine if consuming fescue during gestation and creep feeding would affect feedlot performance. Calf BW was different (P < 0.01) by treatment x time. Birth weights of calves were similar , prior to creep feeding calves exposed to high endophyte fescue were lower, and post-supplementation creep fed calves had increased BW. Days on feed and dressing percentage were decreased in the supplemented group, and marbling score was decreased for both the supplemented and unsupplemented groups following the completion of the feedlot phase (P < 0.05). The second study was setup similar to study one, however cows were exposed to fescue pastures from d 170 of gestation until calving. Calf birth weights did not differ, but weights were increased in the supplemented group post creep feeding (P < 0.05). Average daily gains (ADG) of supplemented calves were greater during the supplementation period (P < 0.01). In the third study, indwelling vaginal temperature probes were used to evaluate differences in body temperature of cows fed fescue seed with high or low levels of ergot alkaloids during early gestation, and in varying environmental conditions. In the winter trial, body temperature was measured hourly from days 0-14 of gestation. In the summer trial, body temperature was measured hourly from days 0-32 of gestation. Body temperatures were different (P < 0.01) between treatments during both trials. / Master of Science

A controlled study was performed to determine the potential toxic effects of the new nonsteroidal anti-inflammatory drug, eltenac (4-[(2,6-dichlorophenyl) amino]-3-thiopheneacetic acid), in horses. Four treatment groups were formed, each composed of 6 horses. The drug was injected intravenously, once daily, at a dose rate of 0.5 mg/kg, 1.5mg/kg or 2.5 mg/kg for 15 days. A control group was injected with sterile saline solution. Parameters assessed were hay and water consumption, daily clinical observations (evaluation of attitude, mentation, pulse and respiratory rate, fecal consistency, skin condition, and color and hydration of mucous membranes), physical examinations, complete hemogram, coagulation profiles, serum biochemical profiles, urinalysis, gastroscopic examinations and gross post-mortem and histopathological examinations on all organ systems. All examiners were blinded to group assignment and dosage levels until the completion of the study. A few glandular gastric ulcers, mild in severity, developed in seven animals during the treatment period. This occurred more often in horses treated with high doses of eltenac (P=.02). A dose dependent change of WBC count and neutrophil count were noted. Total protein, albumin and globulin levels had dose dependent decreases. One horse in the high dose group (2.5mg/kg) developed ventral edema as well as hypoproteinemia. N one of the horses in any of the dosage groups exhibited depression or anorexia. Gross post-mortem and histologic examination did not reveal any signs of drug related gastrointestinal, renal or hepatic abnormalities. Minimal toxic effects of eltenac given intravenously were greatest in horses treated with 2.5 mg/kg of the compound for 15 days. / Master of Science

Toxicity of copper to larval <I>Mercenaria mercenaria</I> was evaluated with static non-renewal and continuous renewal methods that permitted daily observation of mortality, activity, development, and metamorphosis without subsampling. Clam larvae, 100 - 150 microns, were held for up to two weeks in small, 30 mm, sealed petri plates during static assays with excellent survival of control organisms, low evaporative losses, and relatively low between replicate variability. An eight day LC50 of 12 micrograms / liter for six day old organisms was determined as well as EC50s (active swimming). EC50s at 24 hours were as much as much as seven times lower than LC50s after 24 hours of exposure. Flow - through assays were conducted with a modified petri dish design. Two sections from opposing sides of a 30 mm petri dish were removed and covered with 35 micron polyester screening. This dish (organism dish) was placed in an outer catch dish that captured the effluent toxin as it passed through the screening and routed it to a catch bottle for water quality analysis. The toxicant feed line entered through the catch dish cover and slowly dripped toxin into the organism dish. Water quality in the flow - through assay remained excellent. Survival of control organisms in the flow - through assay was lower than in static assays, but metamorphosis was not delayed as had been observed in static assays. Data variability was low enough that statistical distinctions were made between the effects of copper on metamorphosis. A non-standard "M" shaped survival response was observed in all assays. The responses generating the "M" shaped response in the static petri assay were statistically different from each other. Activity, as judged by swimming, in organisms was not observed to follow the "M" shaped pattern. Instead, it decreased exponentially with increasing copper concentrations. Static experiments with unfed clams, observations of activity, and data from experiments in copper accumulation by algae led to a theory relating the unusual dose response to food consumption and its relationship to the effective dose of copper to which the larval clams were exposed. / Master of Science

Currently, protocols for investigating the effects of chemical pollutants on periphyton communities under natural conditions are statistically flawed and/or potentially harmful to the ecosystems in which the studies are conducted. Toxicant-releasing substrates have been proposed to allow the delivery of different levels of chemical pollutants to replicate microbial communities in situ while minimizing the amount of toxicant released into the aquatic ecosystems under investigation. The purpose of this research was to compare the copper-induced responses of laboratory periphyton communities in artificial streams to those generated using standard, laboratory toxicity testing protocols during a summer and winter experiment. Chemical-releasing substrates were successfully used to deliver copper to periphyton communities in a predictable manner, over a broad range of doses, and at fairly constant rates during one week exposure periods. / Master of Science

Induced pluripotent stem cells (iPSCs) can be differentiated into multiple cell types in the body while maintaining proliferative capabilities. The generation of human iPSC-derived hepatocytes (iPSC-Heps) has resulted in a new source for hepatic cells. The current available options for human hepatocytes are primary human hepatocytes (PHHs) and cell lines. PHHs isolated from healthy human donors are difficult to obtain, while cell lines exhibit reduced hepatotoxic sensitivity. iPSC-Heps are being investigated as an alternative option as they are derived from a continuous, stable source and are able to maintain their original donor genotype, which opens the door for patient-specific studies. iPSC-Heps show promise for utilization in tissue engineering, hepatotoxicity studies as well as screening for patient-specific therapeutics. Various reports have concluded that iPSC-Heps exhibit reduced hepatocyte function in comparison to PHHs. Prior reports on iPSC-Heps have focused on improving their adult phenotype functions through variations in differentiation protocols or by altering their in vitro culturing environment. This thesis focuses on incorporating hepatic non-parenchymal cells to more closely mimic the tissue and cell architecture found in the liver tissue. We designed and assembled a 3D iPSC-Hep model that integrates liver sinusoidal endothelial cells, with the goal of achieving functional maturity. Hepatotoxicants were administered to our models and various hepatic markers were measured to analyze the toxic response. This work demonstrates the need for the inclusion of hepatic non-parenchymal cells in iPSC-derived liver tissues, specifically for hepatotoxicity applications. / Master of Science / Induced pluripotent stem cells (iPSCs) can be differentiated into multiple cell types in the body while maintaining proliferative capabilities. The generation of human iPSC-derived hepatocytes (iPSC-Heps) has resulted in a new source for hepatic cells. The current available options for human hepatocytes are primary human hepatocytes (PHHs) and cell lines. PHHs originating from healthy human donors are difficult to obtain, while cell lines may exhibit reduced hepatotoxic sensitivity to chemicals. iPSC-Heps are being investigated as an alternative option since they are derived from a continuous source and are able to maintain their original donor genetic make-up, allowing for patient-specific studies. iPSC-Heps can be used in tissue engineering, hepatotoxicity studies as well as screening for patient-specific therapeutics. Various reports have concluded that iPSC-Heps exhibit reduced function in comparison to PHHs. Prior reports on iPSC-Heps have focused on improving their function through variations in differentiation procedures or by changing their culture environment. This thesis focuses on incorporating other hepatic cells to more closely mimic the tissue and cell architecture found in the liver tissue. We designed and assembled a 3D iPSC-Hep model that integrates liver sinusoidal endothelial cells, with the goal of improving hepatocyte function. Chemicals were administered to our models and various hepatic markers were measured to analyze the toxic response. This work demonstrates the need for the inclusion of additional hepatic cell types in iPSC-derived liver tissues, specifically for hepatotoxicity applications.

In addition to their potential value for in situ bioremediation, the earthworm as a laboratory model may offer insight into mechanisms of xenobiotic toxicity. Using the filter paper contact toxicity test, the LD50s of a series of salicylates and phenolics were determined. The rank order in toxicity of these chemicals was compared with mammalian (rat, oral dosing) LD50s and found to be similar. To determine if protein secretion from chemical stress would be a more sensitive toxicity marker for the above xenobiotics, worms were exposed to either sodium salicylate or acetaminophen at a no effect level (NOAEL) and at the LD100 through filter paper contact. The ability of L. terrestris to metabolize drugs was investigated by using worm homogenate to treat various drugs in both encapsulated and free enzyme forms.

<p dir="ltr">Aquatic ecosystems and their inhabitants are no strangers to stressors. Natural stressors like disease, competition, and predation have a near constant presence in these environments and are often accompanied by human-induced stressors like climate change and chemical contaminants. Chemical contaminants like pesticides are often found in aquatic systems located near agriculture and can have detrimental effects on wildlife. Although natural stressors and pesticides often occur at the same time, their combined interactions still require further investigation to understand, as some pesticides, like fungicides, are frequently under researched. Additionally, fungicides also are lacking in research pertaining to combined chemical stressors. Studying the interactive effects of combined stressors, whether natural or human-induced, is crucial for applying laboratory findings to natural environments.</p><p dir="ltr">Here, I conducted an outdoor mesocosm experiment and multiple laboratory experiments to broadly assess the interactive effects of the fungicide chlorothalonil. More specifically, I explored 1) the interactive effects of the herbicide atrazine and chlorothalonil on an aquatic community, 2) the interaction between chlorothalonil and predator-induced stress of three tadpole species, and 3) the impacts of chlorothalonil on amphibian predator-prey dynamics using tadpoles and larval salamanders. First, to test my hypothesis that atrazine and chlorothalonil will have combined interactions that negatively impact an aquatic community, I conducted a mesocosm experiment where I exposed an aquatic community to atrazine, chlorothalonil, and the combination of the two pesticides. I found that the two pesticides do hold the potential to interact in certain cases, but their main effects alone are often just as damaging, especially for chlorothalonil where an environmentally relevant concentration caused near total morality for bullfrog tadpoles.</p><p dir="ltr">Next, I conducted a laboratory experiment with tadpoles and caged predators exposed to a sublethal concentration of chlorothalonil to test my hypothesis that the combination of predator-induced stress and chlorothalonil will decrease tadpole survival and alter tadpole behavior. I found that chlorothalonil alters tadpole behavior by significantly reducing activity levels in all three study species. However, no evidence was found for the interaction of the two stressors decreasing survival.</p><p dir="ltr">Lastly, to test my hypothesis that the toxicity of chlorothalonil will reduce tadpole behavioral responses and thereby increase their predation risk, I conducted a laboratory experiment with free-swimming tadpoles as prey and larval tiger salamanders as predators, exposed to different combinations of chlorothalonil exposure for the prey or predator. I found no meaningful differences in survival curves for the different combinations of prey and predator, but leopard frog tadpole final survival was lower in the higher chlorothalonil dose treatment due to predation.</p><p dir="ltr">Overall, these studies have assisted in filling research gaps on fungicides and their impact on predator-prey dynamics and aquatic communities. This work provided insights into the direct impacts of the herbicide atrazine and the fungicide chlorothalonil on aquatic species, and their potential to interact with natural stressors, emphasizing the need to protect natural ecosystems from chemical stressors.</p>