Thesis (M.Sc.)--Memorial University of Newfoundland, 2001. / Bibliography: leaves 80-94.

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Thesis (PhD)--University of South Australia, 1995

Thesis (PhD)--University of South Australia, 1995

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 223-234).

Thesis (Ph. D.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on June 8, 2009) Includes bibliographical references.

Toxicities of sulfur-based drugs have been attributed to formation of highly reactive sulfur oxo-acids and depletion of glutathione by the formation of reactive metabolites. Metabolic activation of these sulfur centers to conceivably toxic reactive metabolites (RMs) that can covalently modify proteins is considered the initial step in drug-induced toxicity. Despite considerable effort and research, detection and characterization of these RMs during drug development and therapy remains a challenge. Methimazole (MMI) and 6-propyl-2-thiouracil (PTU) are two commonly used antithyroid, sulfur-based drugs. Though effective, these drugs are associated with idiosyncratic toxicity. PTU has acquired a black box warning and physicians are calling for its withdrawal. RMs resulting from bioactivation of these drugs have been implicated in the aforementioned adverse reactions. Unfortunately, isolating and detecting RMs using traditional analytical techniques has not been successful due to their high reactivity and short life span, typically less than a minute. Current approaches in drug metabolism studies use microsomal incubations to generate RMs, which are then trapped using nucleophiles. Antithyroid drugs, however, are known to deactivate enzymes involved in their oxidation. Moreover, due to the complex nature of biological matrices and low abundance of possible toxic conjugates, this technique results in poor selectivity and sensitivity. This study developed and optimized an analytical method based on coupling electrochemical redox reactions and mass spectrometry to generate, detect and identify RMs from antithyroid drugs. The metabolites were also compared to those that were generated using chemical oxidants and biological microsomes. Mimicry of enzymatic oxidation of the antithyroid drugs was carried out by electrochemically oxidizing them using a coulometric cell coupled on-line to electrospray ionization mass spectrometry (EC/ESI-MS). Oxidation of MMI and subsequent trapping with nucleophile resulted in formation of adducts with N-acetylcysteine, revealing reactive metabolites. The most-postulated metabolite, sulfenic acid, had never been isolated or detected until now, using electrochemistry on-line with electrospray ionization. The results showed that bioactivation of MMI proceeds predominantly through the S-oxide and not through formation of thiyl radicals. These same trapping experiments were also conducted with PTU, but no conjugates were detected. The lack of conjugates from PTU does not preclude formation of RMs, but asserts radical pathway might be dominant in EC oxidation. A double mixing stopped flow was used to investigate the kinetics and mechanism of reaction of the MMI and the biologically relevant hypochlorous acid (HOCl), a product of oxidation of chloride (Cl-) ions by myeloperoxidase. The products from the chemical oxidations were compared to the electrochemically generated metabolites, some differences were apparent. Human liver microsomes (HLM) were also used, to investigate oxidation of PTU. Oxidation of PTU, resulted in the supposedly toxic S-oxide, but this has never been isolated, save for speculation. A comparison of metabolites that were found with HLM to those generated electrochemically showed some degree of similarity. These results show that in vitro techniques such as chemical oxidations and electrochemistry coupled to mass spectrometry can be used to mimic oxidative metabolism and subsequent high throughput screening of reactive metabolites.

The cytochrome P-450 dependent monooxygenase system plays a key role in the bioactivation and detoxication of xenobiotics. Isozyme-specific inhibitors of cytochrome P-450 may be useful in elucidating the role of particular isozymes in xenobiotic metabolism or in suppressing the bioactivation of xenobiotics and enhancing detoxication. The antibiotic chloramphenicol is a selective mechanism-based inactivator of rat liver cytochromes P-450, inactivating 6 of the 12 isozymes monitored, including the major phenobarbital-inducible isozyme PB-B. Analogs of chloramphenicol have been tested to determine the importance of various functional groups in regulating the effectiveness and isozyme selectivity of chloramphenicol as a mechanism-based inactivator of cytochromes P-450. This information will aid in the design of more effective and isozyme specific mechanism-based inactivators. The dihalomethyl group and the propanediol moiety were found to be important in determining the efficacy of inactivation and the ability to inactivate the enzyme by virtue of the modification of the protein as opposed to the modification of the heme moiety. The propanediol side chain also plays a role in the isozyme selectivity. Unlike chloramphenicol, N (2-p-nitrophenethyl)dichloroacetamide (pNO₂DCA), which contains an ethyl group in place of the propanediol side chain of chloramphenicol, is an effective inactivator of BNF-B, the major beta-naphthoflavone-inducible isozyme, as well as PB-B, in vitro and in vivo. Alkaline hydrolysis and enzymatic digestion of the covalently modified isozymes has shown that chloramphenicol and pNO₂DCA are both metabolized by cytochromes P-450 to oxamyl chlorides which bind to lysine and other amino acid residues of the enzyme. However, the mechanism by which pNO₂DCA inactivates BNF-B differs significantly from that by which chloramphenicol inactivates PB-B, although both involve an impairment of the transfer of electrons from NADPH-cytochrome P-450 reductase, suggesting that there are differences in the active sites of these two isozymes.

Due to the high childhood obesity rates within the United States, it is necessary to develop efficacious strategies to combat childhood obesity. To explore whether early intervention can produce lasting metabolic improvements, we used a mouse model of genetically-induced hypothalamic leptin resistance (LeprNkx2.1knockout, hereby known as KO) that exhibits early-onset hyperphagia and obesity. We found that KO mice exhibit reduced capacity of the brown adipose tissue (as seen by disorganized mitochondrial structure). Brown adipose tissue capacity can be restored by paired-feeding in the peri-weaning period, leading to persistent improvements in later adiposity even after restriction ends. These studies lead us to investigate the maturation process of brown adipose tissue in the peri-weaning period. We found that brown adipose tissue expansion between 2 to 3 weeks of age is accompanied by a reduced thermogenic capacity in control mice, as determined by protein levels of uncoupling protein 1 and disorganization of the mitochondrial cristae. Thermogenic function was restored by 5 weeks of age, as demonstrated by a peak of uncoupling protein 1, in control mice but not KO mice. Paired-feeding of KO mice in the peri-weaning period rescued this peak at 5 weeks of age. These studies elucidate a critical period when brown adipose tissue expansion is followed by activation. The magnitude of brown adipose tissue activation at this time might be predictive of future obesity and metabolic rate, highlighting a potential therapeutic time window in which to intervene in pediatric obesity.

The aerobic transformation of TCE and cis-DCE by a tetrabutoxysilane-grown microorganism (Vancheeswaran et al., 1999) led to the investigation of novel substrates, including benzyl alcohol, for promoting cometabolism. The culture grew on carboxylic compounds and alcohols, but did not grow on formate, methanol, methane, propane, butane, ethylene, benzene, toluene, or p-xylene. Cis-DCE transformation was observed when the culture grew on butyrate, glucose, 1-propanol, 1-butanol, ethanol, benzyl alcohol, and phenol, and effectively transformed TCE, cis-DCE, and vinyl chloride when grown on phenol or benzyl alcohol. Several cycles of growth on benzyl alcohol led to increases in TCE transformation rates and transformation capacities. Products of benzyl alcohol degradation shifted from benzaldehyde to 2-hydroxy benzyl alcohol (2HBA) during the several cycles of growth. In resting cells studies, 2HBA production rates were highly correlated with TCE transformation rates. TCE transformation and 2HBA production rates doubled when the culture was grown on phenol and rates of TCE transformation were correlated with 2HBA production rates. Benzyl alcohol- and phenol-grown cells oxidized toluene to o-cresol, which indicated the similarity between benzyl alcohol ortho-monooxygenase, phenol hydroxylase, and toluene ortho-monooxygenase. 2-Butyne and 1-hexyne (but not acetylene) inhibited benzyl alcohol- and phenol-grown cells similarly, indicating the same ortho-monooxygenase was responsible for TCE cometabolism. Resting cell kinetic studies were performed with cells grown on phenol or benzyl alcohol. Benzyl alcohol degradation followed a Monod kinetics while phenol degradation followed a Haldane kinetics. The maximum transformation rates (k[subscript max]) of TCE, cis-DCE, and VC achieved by phenol-grown cells were about a factor of two higher than achieved with benzyl alcohol-grown cells, while the half-saturation constants (K[subscript s]) were in a similar range. Transformation capacities (Tc) for TCE, cis-DCE, and VC were about a factor of two to four higher with phenol-grown cells. The modeling of TCE, cis-DCE, and VC transformation using independently measured k[subscript max] and K[subscript s] values matched well with observed data from batch tests. Benzyl alcohol was shown to be an effective novel substrate for the aerobic cometabolism of TCE, cis-DCE, and vinyl chloride. Being a non-regulated compound, it might have applications for in-situ bioremediation. / Graduation date: 2004