Xenobiotics are plant derived compounds metabolized by phase I and II liver enzymes. Phase I enzymes increase, and phase II enzymes decrease, xenobiotic toxicity. Xenobiotics considered were ergotamine, associated with fescue toxicosis, and sulforaphane, a phase II inducer. Hypothesized responses in liver gene expression and enzyme activity due to exposure to these xenobiotics were tested. Polymorphic mice were gavaged with sulforaphane, ergotamine or control over four daily dosing periods (2, 5, 8 and 11 d), with at least 5 mice per treatment. Mice were killed and livers collected 24 h after last dosing. With ergotamine, expression of phase II genes catecholâ Oâ amine methyltransferase 1 (P = 0.009) on d 8, and glutathioneâ Sâ transferase (Gst) mu1 (Gstm1; P = 0.049) on d 11 was increased, and sulfotransferase 5a1 on d 11 decreased (P = 0.02). Sulforaphane increased expression of cytochrome P450 1a2 on d 5 (P = 0.02) and flavin containing monooxygenases 1 on d 11 (P = 0.002), both phase I genes. It also increased expression of a phase II gene transcription factor (P = 0.03) and quinone reductase 02 (P = 0.007) on d 5, and Gstm1 on d 8 (P = 0.04) and d 11 (P = 0.01). Moreover, sulforaphane treated mice had higher (P < 0.05) Gstm1 expression across days. Among enzymes, only sufloraphane treated mice had higher (P < 0.05) Gst activity. The increase in both Gstm1 expression and Gst activity indicate a consistent benefit of sufloraphane on phase II enzyme activity. / Master of Science

Alkaloid toxicities negatively impact livestock health and production and are of serious economic concern to animal industries. To date, few strategies have been developed to evaluate alkaloid levels in feed or to counteract alkaloid toxicities. The present research evaluated the applicability of imprinting technology to synthesize polymers that have potential to interact with ergot alkaloids and therefore reduce their bioavailability in the GIT. The studies also evaluated applicability of synthesized polymers for use in the ruminal environment using an in vitro ruminal fermentation model, and for the ability to ameliorate vasoconstriction using ex vivo myographic evaluations. In the first experiment, styrene-based molecularly imprinted polymer (MIP) was synthesized using ergotamine as the imprinting template and evaluated for specificity of adsorption to various ergot alkaloids. Cross reactivity with related alkaloids exists due to similarities in structure and functional groups. Both polymers (MIP and NIP) showed strong adsorption intensity and no difference was observed for estimated maximum adsorption capacity between MIP and NIP. Morphologically, MIP was highly porous with greater surface area than NIP. Solid phase extraction indicated stronger adsorption of MIP than NIP to ergot alkaloids suggesting the potential for MIP as a sorbent material for solid phase extraction (SPE) columns used for sample clean-up prior to HPLC or LC-MS/MS analysis of complex samples. In Experiment 2, methacrylic acid-based polymers were synthesized with ergotamine as a template. Among the 4 alkaloids evaluated for selectivity, adsorption difference between MIP and NIP interacted with alkaloid concentration, although differences were generally consistent across concentrations. Imprinting did not affect lysergol and bromocriptine adsorption, but resulted in higher adsorption to methylergonovine. However, there was no difference between MIP and NIP for adsorption of ergotamine. Hydrophobic interactions and H-bonding were the primary interactive forces between polymers and alkaloid adsorbents. Morphologically, MIP had greater surface area and porosity implying a larger surface for adsorption. In addition to its application as SPE sorbent, this MIP was a suitable candidate for application as a feed adsorbent to reduce the bioavailability of certain alkaloid in the gut. In experiment 3, methacrylic acid-based polymers were evaluated for their effect on in vitro ruminal fermentation. There were no interactions between polymer type and inclusion level, and no differences between polymer types for cumulative gas production or rate of gas production. Total gas production and rate of gas production were unaffected by inclusion level. Polymers did not affect total or individual VFA concentrations, ammonia-N or methane concentration at any inclusion level. However, a logarithmic increase in polymer dose level decreased the pH linearly with maximum depression of 0.24 units. This study indicated that, within the range of expected use levels, polymers were essentially inert and would not be expected to affect ruminal fermentation. In experiment 4, ex vivo myographic bioassays were used to determine the impact of polymers on ergotamine bioavailability. Responses measured in the ex vivo myographic studies had similar trend as the responses generated from in vitro isothermal adsorption studies. Results of that study also showed that ex vivo myographic responses could be predicted from in vitro isothermal adsorption studies with more than 80% accuracy. These studies indicate that synthetic polymers are potentially effective adsorbents to mitigate ergot toxicity with little evidence of substantial differences between MIP and NIP.

Ergotalkaloide sind sekundäre Stoffwechselprodukte des parasitären Schlauchpilzes Claviceps purpurea der auf Getreide Mutterkörner (Sklerotien) bildet. In Sklerotien sind toxische Ergotalkaloide enthalten. Durch C. purpurea werden vorrangig sechs verschiedene Ergotalkaloid-Epimerenpaare gebildet, die toxischen C8-(R)-Epimere und die biologisch nicht relevanten C8-(S)-Epimere, die ineinander umgewandelt werden können. Das Ziel der Arbeit war es, die Epimerisierung der Ergotalkaloide während der Probenvorbereitung im Vergleich zu bisher bekannten Probenaufarbeitungsverfahren zu minimieren. Dieses gelang durch den Verzicht des Zusatzes starker Säuren oder Basen. Die aufgereinigten Extrakte können bei Raumtemperatur über 96 Stunden epimerisierungsfrei in einer tensidischen Acetonitril-Wasser-Lösung gelagert werden. Die Probenaufarbeitung mit anschließender Auftrennung über die Hochleistungsflüssigchromatographie und fluorimetrischer Detektion (HPLC-FLD) wurde für Roggenmehl und Speiseöl validiert und auf diese Martices angewendet. So konnten erstmals die Ergotalkaloidgehalte auch in Weizenkeimöl quantifiziert werden. Im zweiten Teil der Arbeit wurde das Epimerisierungsverhalten von Ergotalkaloiden bei Backversuchen und in vitro Verdauexperimenten untersucht. Das Backen resultierte in eine Verschiebung des Epimerengleichgewichtes auf die Seite der (S)-Epimere. Das angewendete in vitro Verdaumodell führte für die Ergotalkaloidepimerenpaare Ergotamin und Ergosin zu einer Verschiebung des Epimerengleichgewichtes auf die Seite der toxischen (R)-Epimere. Dagegen zeigten die Ergotalkaloide der Ergotoxingruppe eine Verschiebung des Epimerengleichgewichtes auf die Seite der (S)-Epimere. Der dritte Teil der Arbeit beschäftigt sich mit Ergotalkaloid-Konjugaten, die unter dem Einfluss von UV-Licht entstehen. Es wurden sechs Ergotalkaloid-Fettsäure-Konjugate synthetisiert und in Sklerotien über die HPLC in Verbindung mit massenspektrometrischer Detektion nachgewiesen. / Ergot alkaloids are secondary metabolites of the parasitic fungus Claviceps purpurea that forms sclerotia on cereals. These sclerotia contain toxic ergot alkaloids. C. purpurea forms six epimeric pairs of ergot alkaloids predominantly the toxic C8-(R)-epimers and the biologically inactive C8-(S)-epimers. In view of the fact that both epimeric forms can be transformed into one another, the objective of this work was to develop a novel sample preparation method that minimizes the epimerization rate compared to previously published methods. The presented sample preparation procedure minimizes epimerization of ergot alkaloids, as it operates without the addition of strong acidic or alkaline modifiers for matrix removal. After sample preparation, an ergot alkaloid containing extract in a sodium hexanesulfonate solution is obtained in which no epimerization after 96 hours was observed. Thus, the sample preparation allows extract storage at ambient temperature for prolonged HPLC analysis. This novel sample preparation followed by HPLC-flourescence analysis was validated for the matrices rye flour and wheat germ oil and was applied for food samples. This is the first time that the ergot alkaloid content in wheat germ oil was quantified. The second part of this work was the study of the epimerization behaviour of ergot alkaloids during baking and in vitro digestion. Baking of cookies resulted in a shift of the epimeric ratio towards the (S)-epimers. The in vitro digestion showed an ergot alkaloid specific shift of the epimeric ratio. The initial percentage of the (R)-epimer increased for ergotamine und ergosine. In contrast, ergot alkaloids of the ergotoxine type showed an epimeric shift towards their (S)-epimers. The third part of this work was the study of ergot alkaloid derivatives that are formed in combination with UV-light. Six different ergot alkaloid fatty acid derivates were synthesized and detected in sclerotia using a HPLC-MS/MS method.