The presence of biologically-active estrogenic endocrine disrupting compounds (EDCs) in treated effluents from biological wastewater treatment facilities has prompted wide-spread interest in the behavior of these contaminants during the activated sludge process. The yeast-estrogen screen (YES) was used to quantify the estrogenic activity of samples taken from different areas of three wastewater treatment facilities. An estrogenic mass-balance around these facilities revealed that the majority of influent estrogenic activity was removed in the activated sludge process, but the main route for EDC discharge to the natural environment was via the treated effluent. The estrogenic activity in the effluent from a membrane bioreactor (MBR) was lower compared to a fully aerobic activated sludge process using secondary clarification, suggesting that enhanced removal of particulate and colloidal material may improve EDC removal efficiency. Colloidal material was obtained from settled mixed liquor suspended solids (MLSS) collected from a pilot MBR and a full-scale activated sludge process that included anoxic and aerobic zones. The MLSS was sized fractionated by filtration, and used to quantify the sorption coefficients for pyrene, 17&#946;-estradiol (E2), and 17α-ethinylestradiol (EE2) by fluorescence quenching. The MLSS-derived colloidal organic carbon (COC) sorption coefficient (Kcoc) for pyrene ranged from (< 1 to 80) L/kgcoc, indicating a similar affinity for pyrene compared to natural organic matter. Kcoc coefficients for E2 ranged between (< 1 to 158) L/kgcoc for E2 and (< 1 to 228) L/kgcoc for EE2, and are the highest E2 and EE2 sorption coefficients reported in the literature to date. There was a strong correlation between the Kcoc coefficients and molar extinction coefficient at 280 nm (e280) for pyrene and E2, suggesting that the interaction of the π;-electrons is an important factor in determining overall sorption behavior. There was no such correlation for EE2. Based on the Kcoc coefficients and COC concentrations of the samples, between 1 and 50% of the aqueous E2 and EE2 concentrations were associated with colloidal material. In a novel application of the YES bioassay, the bioavailability of colloid-associated E2 was quantified by comparing the EC50 values of the dose-response curves generated in the presence and absence of size fractionated COC. An increase in EC50 values as a function of COC concentration was attributed to a reduction in bioavailability of E2, suggesting that MLSS-derived COC can reduce, but not eliminate, the biological impact of EDCs. However, there was a high degree of variability in the EC50 values, and estimates of the colloid-associated E2 fraction based on the Kcoc-e280 correlation were unsuccessful in accurately predicting increases in EC50 values. Nevertheless, the YES bioassay may represent a powerful tool in determining the bioavailability of EDCs in complex environmental samples. Results from this research effort suggest that the colloidal phase derived from activated sludge systems represents an important transport vehicle whereby EDCs and other trace organic compounds can enter into the natural environment. Consequently, wastewater treatment plants discharging to sensitive ecosystems or involved with direct water reuse programs should optimize the treatment process to remove colloidal material. / Ph. D.

Drinking water supplies contaminated with arsenic (As), a toxin and carcinogen, adversely impact the health of millions of people worldwide. Previous work has documented that different inorganic and organic As species vary with respect to their toxicities. It is, however, currently not well understood how As speciation affects bioavailability, defined as the capacity of a contaminant to cross an organism's cellular membrane, or how arsenic's form (dissolved vs. non-dissolved) can affect bioavailability. This dissertation addresses the effect of speciation and form on As bioavailability through a combination of field and laboratory studies. In the first project, a poultry litter application experiment was conducted to determine if trace elements (As, Cu, and Zn) are released from litter to underlying soil water, and if so, whether the trace elements are present in dissolved form or complexed to nanoparticles, colloids, or particles. Results showed that Cu and Zn released from the litter were dominantly complexed to organic matter or to iron oxides/clay particles, while As was dominantly dissolved or complexed to organic matter. In the second project, a luminescent E. coli bioreporter was created and exposed to different As species, including As(III), As(V), MSMA, and roxarsone. Results showed variable response, with As(III) producing the strongest response, followed by As(V) and MSMA; roxarsone showed no response. The bioreporter was exposed to As solutions with varying cation concentrations to examine the impact of sample geochemistry on performance. Increased monovalent (Na,K) concentrations enhanced luminescent response, while increased divalent (Ca) concentrations inhibited response. These altered responses reflect different As uptake pathways into the cell. The third study addressed bioavailability of As species to Corbicula fluminea, a clam commonly used for biomonitoring. Results demonstrate that As(III) is most bioavailable to Corbicula, followed by As(V), MSMA, and roxarsone. Corbicula also displayed the ability to change As speciation through internal processing and via their shell, demonstrating that Corbicula can affect As speciation in solution. Results of these studies enhance the scientific knowledge of how speciation and form affect As bioavailability, and can also inform regulators who use bioavailability to set remediation goals for As-contaminated systems. / Ph. D.

Synthetic substituted phenols can become incorporated into soil organic matter by processes similar to natural humification. Biological, (enzyme-catalyzed), and abiotic, (mineral surface catalyzed), reactions have been implicated in these processes which result in the nonextractable binding of phenolic contaminants to soil organic matter. Experiments conducted with phenol, 4-monochlorophenol (MCP), 2,4,6-trichlorophenol (TCP), and pentachlorophenol (PCP) indicated a statistically significant difference in the degree of nonextractable binding in oxic and anoxic environments. Soils were extracted with water and methylene chloride. The extracted soils were combusted at 950°C and the 14C02 evolved was quantified as a measure of the bound contaminant. The amount of 14C02 captured during combustion of the soils indicated that approximately two times greater contaminant binding occurred in the oxic atmosphere as compared to the anoxic atmosphere. Autoclaving the soil resulted in a reduction in contaminant binding. Addition of H₂O₂ increased MCP binding by 4.5 times. The nonextractable contaminant appeared to consist of (i) a biologically coupled; (ii) an abiotically coupled; and (iii) a desorbable, but diffusion-limited component. The initial aqueous concentration of the contaminant appeared to have the greatest effect on the degree of nonextractable binding. An empirical model was developed for predicting the extent of chlorophenol binding to soil as a function of the initial aqueous concentration of the contaminant. Nearly 50% of the nonextractable 4-MCP was bioavailable to inoculated microorganisms. / Ph. D.

Quercetin is a flavonol with potential health benefits including activities against cardiovascular disease, obesity, and oxidative stress. However, the benefits of quercetin are likely limited by poor bioavailability, primarily attributed to its poor aqueous solubility (due to its hydrophobicity and crystallinity) and extensive phase-II metabolism. Improving the apparent solubility of quercetin has the potential to improve its in vivo bioavailability. Strategies to increase solution concentrations in the small intestinal lumen have the potential to substantially increase quercetin bioavailability, and efficacy. We aimed to achieve this by incorporating quercetin into amorphous solid dispersions (ASDs) with cellulose derivatives, eliminating crystallinity, and selectively releasing amorphous quercetin under simulated intestinal conditions (pH 6.8, 37C). Amorphous quercetin was dispersed in cellulose esters including 6-carboxycellulose acetate butyrate (CCAB), hydroxypropylmethylcellulose acetate succinate (HPMCAS) and cellulose acetate suberate (CASub) to achieve stability and provide pH-triggered release. In addition, polyvinylpyrrolidone (PVP) containing CASub and CCAB blends were prepared to further promote enhanced dissolution. The ASD employing 10% quercetin in 20% PVP:70% CASub was most successful at enhancing the solution concentration of quercetin, providing an 18-fold increase in the area under the concentration/time curve (AUC) compared to quercetin alone. These results warrant in vivo assessment of quercetin-loaded ASDs formulated with CASub and its blend with PVP towards improving the bioavailability of quercetin. / Master of Science in Life Sciences

Creosote, containing many high molecular weight hydrophobic polycyclic aromatic hydrocarbons (PAH's), is present in the subsurface environment at the Oneida Tie-Yard in Oneida, Tennessee. Phytoremediation using hybrid poplar trees was chosen as the remedial technology on-site. Since monitoring began, the contaminant plume has been shrinking consistently and evidence has shown that remediation is taking place. However, remediation may be rate-limited by the desorption and dissolution kinetics of the PAH's on-site. The objectives of this research are to: (1) estimate the desorption and dissolution rates of 10 PAH's found in the subsurface and (2) estimate the amount of each PAH and total mass of contaminant that is irreversibly sorbed to the soil. Three laboratory desorption and dissolution experiments were performed using contaminated soil samples from the Oneida Tie-Yard site. The first experiment was a batch desorption equilibrium experiment, the second was a batch desorption kinetics experiment, and the third was a soil column dissolution kinetics experiment. The target compounds in this study were: naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, chrysene, and benzo(b)fluoranthene. The resulting data for the desorption equilibrium experiment revealed that rates of equilibrium were truly not instantaneous in the systems studied. However, because approximately 76% of PAH's desorbed by the first sampling event (3 days), an equilibrium isotherm was considered appropriate. Results showed that there is a sorbed reversible concentration that readily desorbs to the aqueous phase for each PAH. Additionally, it was determined that the percent removal of sorbed PAH's decreases with increasing molecular weight. Desorption curves based on experimental data were found to exhibit linear behavior over large variations in aqueous concentration, but showed exponential behavior as concentrations approached zero. Freundlich sorption equilibrium isotherms for the 10 monitored PAH's on-site were generally found to have N coefficient values over 1, especially over large variations in solution phase concentration, indicating a non-uniform sorbent. Dissolution of resistant PAH's under field-like conditions was determined to occur over long periods of time. Dissolution rates calculated from experimental data were shown to generally decrease with increasing molecular weight. Overall, desorption and dissolution kinetics of PAH's were shown to be rate-limiting factors to remediation at the Oneida Tie-Yard. / Master of Science

The efficacy of rifapentine, an oral antibiotic used in the treatment of tuberculosis, is reduced due to its degradation at gastric pH and low solubility at intestinal pH. We aimed to improve delivery properties in vitro by incorporating rifapentine into pH-responsive amorphous solid dispersions with cellulose derivatives including: hydroxypropylmethylcellulose acetate succinate (HPMCAS), cellulose acetate suberate (CASub), and 5-carboxypentyl hydroxypropyl cellulose (CHC). Most amorphous solid dispersions reduced rifapentine release at gastric pH, with the best performing polymer CASub showing >31-fold decrease in area under the curve compared to rifapentine alone. Lower solubility at gastric conditions was accompanied by a reduction in the acidic degradation product 3-formylrifamycin, as compared to rifapentine alone. Certain formulations also showed enhanced apparent solubility and stabilization of supersaturated solutions at intestinal pH, with the best performing polymer HPMCAS showing almost a 4-fold increase in total area under the curve compared to rifapentine alone. These in vitro results suggest that delivery of rifapentine via amorphous solid dispersion with cellulose polymers may improve bioavailability in vivo. / Master of Science in Life Sciences

No / Of particular importance in recent years has been the development of techniques for producing nanoparticles (NPs) of poorly-water soluble drugs with dimensions less than 1000 nm for which their high surface area can lead to improvements in bioavailability. Furthermore, the small size of these particles can also enable cellular uptake, particularly for positively charged systems. Therefore, an overall objective of this part of the project was to produce nanoparticles with different levels of positive surface charge using the bottom-up method.

6 Zusammenfassung Marianne Reinboth Bioverfügbarkeit des Flavonols Quercetin beim Hund Veterinär-Physiologisches Institut der Veterinärmedizinischen Fakultät der Universität Leipzig Eingereicht im Juni 2010 79 Seiten, 20 Abbildungen, 6 Tabellen, 211 Literaturangaben, 1 Anhang Schlüsselwörter: Quercetin, Bioverfügbarkeit, Hund, absolute Bioverfügbarkeit, Isoquercitrin, Rutin, Flavonole Für das pflanzliche Flavonol Quercetin werden vielfältige gesundheitsfördernde Wirkungen postuliert, so auch bei Hunden. Über die Bioverfügbarkeit des Flavonols bei dieser Spezies liegen bislang jedoch keinerlei Daten vor. Daher hatte diese Arbeit das Ziel, Bioverfügbarkeit und pharmakokinetische Parameter von Quercetin und wichtigen Quercetinglycosiden bei Hunden nach deren Verabreichung mit einer Testmahlzeit in einer praxisrelevanten Dosierung von 10 mg/kg Körpermasse zu untersuchen. Dazu erhielten 9 adulte Beagles beiderlei Geschlechts das zuckerfreie \"Aglycon\" Quercetin bzw. seine Glycoside Isoquercitrin (Quercetin-3-O-Glucosid) und Rutin (Quercetin-3-O-Glucorhamnosid) in jeweils äquimolarer Dosierung in einer Testmahlzeit verabreicht. Anschließend wurden Blutproben über einen Zeitraum von bis zu 72 Stunden entnommen und mittels HPLC die Konzentrations-Zeitverläufe der Metaboliten im Blutplasma, die Bioverfügbarkeit sowie weitere pharmakokinetische Parameter bestimmt. Weiterhin wurde die absolute Bioverfügbarkeit von Quercetin aus dem Vergleich einer oralen mit einer intravenösen Applikation bestimmt. Der weitaus größte Teil der Plasmametaboliten von Quercetin sowie seiner beiden Glycoside bestand aus glucuronidierten bzw. sulfatierten Quercetinkonjugaten. Nicht konjugiertes Quercetin-Aglycon kam nur in einem Anteil von etwa 20 % vor. Neben Quercetin machten seine Metaboliten Isorhamnetin und Kämpferol weniger als 10 % aller im Plasma zirkulierenden Flavonole aus. Die absolute Bioverfügbarkeit von Quercetin betrug nur etwa 4 %. Die relative Bioverfügbarkeit aus dem 3-O-Glucosid Isoquercitrin war mehr als doppelt so hoch wie aus dem Aglycon, die maximalen Plasmaspiegel lagen aber auch hier unter 1 µmol/l. Sowohl nach Aufnahme von Quercetin als auch nach Isoquercitrin kam es zu einer relativ schnellen Absorption aus dem Dünndarm mit einem ersten Plasmapeak ungefähr eine Stunde nach der Ingestion. Vier Stunden nach Aufnahme der beiden Flavonole trat ein zweiter Plasmapeak auf, der in der Regel höher als der erste ausfiel. Dies deutet auf einen enterohepatischen Kreislauf der über die Galle ausgeschiedenen Metaboliten hin. Nach Aufnahme von Rutin kam es zu einer verzögerten Absorption, da eine Deglycosylierung durch bakterielle Glycosidasen im Dickdarm Voraussetzung für die Absorption des Flavonols ist. Maximale Plasmakonzentrationen wurden im Mittel erst 11 Stunden nach Ingestion dieses Glycosids erreicht. Die maximalen Plasmakonzentra-tionen nach Rutin waren geringer als nach Quercetin oder Isoquercitrin, jedoch war die mittlere Verweildauer der Plasmametaboliten mit 18 Stunden auch wesentlich länger. Im Unterschied zu anderen Spezies war die relative Bioverfügbarkeit von Rutin gegenüber Quercetin nicht verringert. Obwohl Rutin eine relativ gute Quercetinquelle für Hunde zu sein scheint, muss bei der Einschätzung möglicher In-vivo-Wirkungen die relativ geringe Bioverfügbarkeit sowie die intensive Metabolisierung seines Aglycons Quercetin berücksichtigt werden. / 7 Summary Marianne Reinboth Bioavailability of the Flavonol Quercetin in Dogs Institute of Physiology of the Faculty of Veterinary Medicine, University of Leipzig Submitted in June 2010 79 pages, 20 figures, 6 tables, 211 references, 1 appendix Keywords: quercetin, bioavailability, dog, absolute bioavailability, isoquercitrin, rutin, flavonols The plant flavonol quercetin is supposed to exert multiple health-related effects in dogs. To date no information on its bioavailability in this particular species is avai-lable. This study intended to investigate bioavailability and pharmacokinetics of quercetin and certain quercetin glycosides in dogs after ingestion of a test meal sup-plemented with a quercetin dose equivalent to 10 mg/kg body weight. Nine adult beagle dogs of both sexes received the aglycon quercetin (sugarfree) or its glycosides isoquercitrin (quercetin-3-O-glucoside) and rutin (quercetin-3-O-glucorhamnoside) in equimolar amounts together with a test meal. Blood samples were taken over a period of up to 72 hours; bioavailability and pharmacokinetics were calculated from the HPLC-derived plasmaconcentration-time-curves. Absolute bioavailability was calculated by comparing an oral to an intravenous administration of quercetin. The majority of analysed plasma metabolites were glucuronidated and sulfated con-jugates of quercetin. Non-conjugated quercetin aglycon comprised only 20 %. Be-sides quercetin, its metabolites isorhamnetin and kaempferol made up less than 10 % of all circulating metabolites. The absolute bioavailability of quercetin was only 4 %. The relative bioavailability of quercetin from isoquercitrin was more than twice as high than from the aglycon, but even there maximal plasma concentrations were generally less than 1 μmol/l. Absorption from the small intestine was rather fast with a first plasma peak after 1 hour after ingestion of quercetin or isoquercitrin. A second, generally higher plasma peak occurred 4 hours after ingestion. This suggests an in-tensive enterohepatic recycling of biliary secreted metabolites. Absorption was significantly delayed after ingestion of rutin due to the necessity of bacterial deglycosilation in the large intestine. Plasma concentrations peaked only after 11 hours. Plasma concentrations after rutin were lower than after quercetin or isoquercitrin, but mean residence time of plasma metabolites was as long as 18 hours after rutin ingestion. Consequently, a once daily feeding of dogs with rutin might lead to relatively constant plasma metabolite concentrations. In contrast to other species, bioavailability from rutin was not smaller than that from quercetin. Although rutin seems to be a relative good quercetin source for dogs, estimations about potential in-vivo-effects of quercetin have to take into consideration its low bioavailabilty and intensive metabolism.

Field pea (Pisum sativum L.) seeds have high nutritional value but also contain phytate which can inhibit the absorption and utilization of nutrients. Phytate is the main storage form of phosphorus in the seeds but chelates Fe, Zn and some other micronutrients and is not well digested by monogastrics. Peas with pigmented seed coats contain polyphenols which also have anti-nutritional properties. To increase the nutritional value of field pea seeds, two low phytate lines (1-150-81 and 1-2347-144) containing higher inorganic phosphorus concentration (IN-P) and lower phytate-phosphorus concentration (PA-P) than the normal phytate varieties were developed from CDC Bronco in previous research. The objectives of this research were 1) to determine the effect of genotype and environment on iron bioavailability in a set of five pea varieties differing in phytate concentration and iron concentration using in vitro digestion/Caco-2 cell culture bioassay; 2) to determine the effect of seed coats on iron bioavailability by testing whole seeds compared to dehulled seeds in varieties differing in seed coat pigmentation using in vitro digestion/Caco-2 cell culture bioassay; 3) to determine the inheritance of iron bioavailability in field pea by evaluating recombinant inbred lines differing in phytate concentration using in vitro digestion/Caco-2 cell culture bioassay; 4) to determine the effects of pea with the low phytate trait on body weight and hemoglobin concentration of chickens. Iron concentration (FECON) did not differ significantly between normal and low phytate varieties. Iron bioavailability (FEBIO) of the two low-phytate lines was 1.4 to 1.9 times higher than that of the three normal phytate varieties, and growing environment also had a significant effect on FEBIO. Peas with pigmented seed coats contained 7 times lower FEBIO than peas with non-pigmented seed coat. The removal of the seed coat increased the FEBIO in peas with pigmented seed coat 5 to 6 times. From previous research on PR-15 recombinant inbred lines (RILs) which were developed from a cross between low phytate line 1-2347-144 and a normal phytate variety CDC Meadow, it was found that PA-P was controlled by a single gene. FEBIO, in this study, was also found to follow a bimodal frequency distribution, characteristic of single gene control, and it was highly correlated with PA-P in the PR-15 lines. In vivo studies were used to evaluate iron absorption of chickens fed with low and normal phytate pea diets. The diets containing the low-phytate pea lines had no significant effect on chicken body weight and hemoglobin level, compared with the diets containing normal phytate pea varieties. An unexpected high FECON was discovered in the diets that was traced to the ingredients of limestone and dicalcium-phosphate which likely affected the experimental results.

Heavy metal bioaccumulation in aquatic organisms may occur through direct or indirect uptake routes. Research indicates that the significance of uptake route varies with contaminant and organism exposed. The relative importance of different metal sources in aquatic systems was investigated by exposing freshwater snails to dietary or dissolved sources of cadmium. Snails were exposed to control, contaminated food only, contaminated water only, and contaminated food and water treatments. During the 15-day exposure, samples were taken to determine Cd concentration in snail soft tissue, snail shell, algal food, and overlying water. Analyses of snail soft tissue and shells indicate that exposure route significantly affects Cd concentrations in the tissues. In both cases, dissolved Cd is the primary contributor to metal body burden.