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Thesis (M.Sc.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Environmental Health Sciences, School of Public Health. Title from pdf file main screen (viewed on April 27, 2010). Includes bibliographical references.

Two adsorption/microcoulometric methods have been investigated for total organic halogen (TOX) in water. TOX, a proposed water-quality parameter, is a rapid, surrogate method to detect halides microcoulometrically and does not require compound identification before water quality can be judged. An XAD resin is used to concentrate organic halides that are eluted by a two-step, two-solvent procedure, followed by analysis using :chromatography or pyrolysis to convert organic halides to halide. In the granular activated carbon (GAC) method, the entire GAC-organic halide sample is pyrolyzed. TOX measurements of model compounds are comparable by both methods, but GAC was found to be superior to XAD for adsorption of chlorinated humics in drinking water and chlorinated lake water.

This thesis examines the biology, ecology and anthropogenic threats of Indo-Pacific bottlenose dolphins (Tursiops aduncus) off Zanzibar, Tanzania, based on research conducted and samples collected between 2000 and 2008. Distribution and occurrence are described based on incidental catches (bycatch) in gillnet fisheries. Biology and ecology are examined by ageing and studying the reproductive biology and stomach contents of collected specimens. The composition of organohalogen compounds is determined in blubber samples, and assessment and mitigation of bycatch are conducted using observers onboard fishing vessels. Fisheries bycatch data showed that Indo-Pacific bottlenose dolphins occur year round in all areas around Zanzibar. Sexual maturity was attained between 7 and 8 years and body length 190-200 cm in females and at 16 years and body length 213 cm in males. The gestation period was estimated to be 12.3 months, with calving occurring throughout the year, peaking November-March and with an interval of 2.7 years. The estimated pregnancy rate was between 0.10 and 0.58 depending on methods used. Stomach contents revealed a relatively large number of prey species, but that only a few small- and medium-sized neritic fish and cephalopods contribute substantially to the diet. Estimates of total annual bycatch were >9% which is not considered sustainable. An experiment showed that pingers can be a short term mitigation measure to reduce bycatch of dolphins in both drift- and bottom set gillnets. Methoxylated polybrominated diphenyl ethers (Meo-BDEs) were found at higher concentrations than anthropogenic organic pesticides (OCPs), with only traces of polychlorinated biphenyls (PCBs) detected. This study reveals the magnitude and apparent susceptibility of Indo-Pacific bottlenose dolphins off Zanzibar to anthropogenic threats, especially fisheries bycatch, and it is clear that immediate conservation and management measures are needed to reduce bycatch. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript. Paper 5: Submitted.

A study has been conducted for the qualitative and Quantitative analysis of chlorinated organic compounds in water. The study included the adaptation of Amberlite XAD macroreticular resin techniques for the concentration of municipal wastewater samples, followed by GC/MS analysis. A new analytical method was developed for the determination of volatile halogenated organics using liquid-liquid extraction and electron capture gas chromatography. And, a computer program was written which searches raw GC/MS computer files for halogen-containing organic compounds.

Various animal or vegetable origin bio-indicators are used for the assessment of the environmental contamination. The wild animals were chosen for monitoring of xenobiotic based organohalogen compounds. The district health facility staff collected the samples of wild boars in the territory of Central Bohemia. The controlled substances were isolated from the matrix by an extraction. The extraction was made by a petrolether and then it was purified by a column chromatography. A final cleansing of the extract was made by an acid hydrolysis. The determination of the selected analytes was finished by the method of gas chromatography with an electron capture detector. The results gave us basic information about the wild boar contamination of organohalogen pollutants.

Thyroid hormones, which are secreted by the thyroid gland, are one of the most important halogenated compounds in the body. Thyroid hormones control almost every processes in the body including growth, body temperature, protein synthesis, carbohydrate and fat metabolism, heart rate, and cardiovascular, renal and brain function. Thyroid gland secretes L-thyroxine or 3,3',5,5'-tetraiodothyronine (T4) as a prohormone. While the biologically active hormone 3,3',5-triiodothyronine (T3) is produced by selective phenolic ring deiodination of T4, selective tyrosyl ring deiodination of T4 produces a biologically less active metabolite 3,3',5'-triiodothyronine (rT3). Tyrosyl and phenolic ring deiodination of T3 and rT3, respectively, also produces a biologically inactive metabolite 3,3'-diiodothyronine (3,3'-T2). Regioselective deiodinations of thyroid hormones are catalysed by three isoforms of a selenoenzyme iodothyronine deiodinase (DIO1, DIO2, DIO3). DIO1 can remove iodine from both the tyrosyl and phenolic rings of thyroid hormones, whereas DIO2 and DIO3 are selective towards phenolic and tyrosyl ring, respectively. Although the Figure 1. (A) Deiodination of thyroid hormones by iodothyronine deiodinases (DIOs) (A) and naphthyl-based selenium and/or sulphur compounds (B). mystery behind the origin of regioselectivity of deiodination by DIOs remains unsolved, formation of halogen bonding between selenium in the active site of DIOs and iodine of thyroid hormones has been widely accepted as the mechanism of deiodination. Halogen bonding, a noncovalent interaction between halogen and an electron donor such as nitrogen, oxygen, sulphur, selenium etc., elongates the C-I bond and impart a carbanionic character on the carbon atom that gets protonated after the removal of iodide. Apart from the deiodination, thyroid hormones also undergo decarboxylation, oxidative deamination, sulphate-conjugation to form iodothyronamines, iodothyroaetic acids and sulphated thyroid hormones, respectively. Figure 2. (A) Proposed mechanism of deiodination of thyroid hormones by deiodinase mimics. (B) Halogenation of uracil- and cytosine-containing nucleosides by hypohalous acid (HOX). Recently, naphthyl-based selenium/sulphur-containing compounds, such as compound 1 (Figure 1B), have been reported to mediate the selective tyrosyl ring deiodination of T4 and T3 to form rT3 and 3,3'-T2, respectively. Interestingly, replacement of the selenol moiety in compound 1 with a thiol decreases the activity, whereas replacement of the thiol moiety with another selenol dramatically increases the deiodination activity. Based on the detailed experimental and theoretical investigations, a mechanism involving the Se···I halogen bonding was proposed (Figure 2A). In addition to the halogen bonding between selenium and iodine atom, chalcogen bonding between two nearby chalcogen atoms was also shown to be important for the deiodination activity. Another important class of halogenated compounds in the body are the halogenated nucleosides. Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes, which can convert halide ions (X¯) into a toxic reactive halogen species hypohalous acid (HOX) in presence of hydrogen peroxide (H2O2). Uracil- and cytosine-containing nucleosides are known to undergo halogenation at the 5-position of the nucleobase to form the halogenated nucleosides (Figure 2B). Interestingly, halogenated nucleosides such as 5-halo-2'-deoxyuridine are known to be incorporated in the DNA of dividing cells essentially substituting for thymidine. Incorporation of halogenated nucleosides into the DNA leads to mutagenesis, carcinogenesis and loss of genome integrity. Thymidylate synthase (TSase), the key enzyme involved in the biosynthesis of 2'-deoxythmidine-5'-monophosphate (dTMP) from 2'-deoxyuridine-5'-monophosphate (dUMP), can catalyse the dehalogenation of halogenated nucleotides in presence of external thiols. This thesis consists of five chapters. The first chapter provides a general introduction to halogen bonding, thyroid hormones and halogenated nucleosides. This chapter also briefly describes the halogen bond-mediated biochemical and biomimetic deiodinations of thyroid hormones by iodothyronine deiodinases and naphthyl-based organoselenium compounds. Dehalogenation of halogenated nucleotides by thymidylate synthase and thiol-based small molecules has also been discussed in this chapter. The second chapter of this thesis contains the regioselective deiodination of iodothyronamines (TAMs) by deiodinases mimics. TAMs are the endogenous metabolites produced by the decarboxylation of β-alanine side chain of thyroid hormones (THs). 3,3',5-triiodothyronamine (T3AM) and 3,5-diiodothyronamine (3,5-T2AM) undergoes selective tyrosyl ring deiodination by deiodinase mimics to form 3,3'-diiodothyronamine (3,3'-T2AM) and 3-iodothyronamine (3-T1AM), respectively. Interestingly, when the initial rates of deiodinations of T3 and T3AM were compared, deiodination of T3 was found to be several fold faster than that of T3AM under identical reaction conditions. To understand the ability of the iodine atoms to form Figure 3. (A) HPLC chromatogram of deiodination of T3. (B) Proposed mode of interaction of dimeric T3 and monomeric T3AM with organoselenium compounds. halogen bonding, a model selenolate (MeSe¯) was optimized with the T3 and T3AM. Although both T3 and T3AM forms the expected Se···I halogen bonding with MeSe¯, the strength of halogen bonding was found to be less for T3AM than T3. Furthermore, detailed kinetic and spectroscopic studies indicate that T3 and T3AM exist as dimeric and monomeric species in solution. The dimerization of T3 in solution was shown to have remarkable impact on the activation energy and pre-exponential factor of the deiodination reactions. Single crystal X-Ray crystallography and theoretical calculations indicated that in addition to Se···I halogen bonding, I···I halogen bonding may play an important role in the deodination of thyroid hormones by deiodinase mimics. Furthermore, the presence of heteroatoms such as nitrogen, oxygen and sulphur in the close proximity of one of the selenium atoms of deiodinase mimics was shown to have significant effect on the rate of deiodination reactions. The third chapter of the thesis focusses on the conformational polymorphism and conformation-dependent halogen bonding of L-thyroxine. Synthetic version of L-thyroxine (T4) is a life-saver for millions of people who are suffering from hypothyroidism, a thyroidal disorder recognised by low levels of T4 and elevated levels of TSH in blood plasma. Synthetic version of L-thyroxine is available in the Figure 4. Ball and stick model of the single crystal X-Ray structure of the conformational polymorphs of L-thyroxine. Form I and Form II was exclusively crystallized from methanol and acetonitrile, respectively. Water molecules are omitted for clarity. market with various brand names. However, adverse effects have been observed in the patients when they switch their brand of thyroxine. Based on these observations, the American Thyroid Association (ATA), the Endocrine Society (TES), and the American Association of Clinical Endocrinologists (AACE) declared that the different brands of T4 are not bioequivalent, thus leading to differences in the bioavailability of the drug. We have shown that the commercially available thyroxine exists in at least two stable forms (Form I and Form II) with different three-dimensional structures (Figure 4). These two forms exhibit different intermolecular interactions in crystal packing, spectral behaviours, thermal stabilities, optical activity and very interestingly, different solubility in acidic and basic pH. At pH 4, solubility of Form I is about 42% and 45% greater than that of Form II and bulk T4, respectively, whereas at pH 9, the solubility of Form II is about 38% and 42% higher than that of Form I and bulk T4, respectively. As T4 is a narrow therapeutic index drug, these differences in solubility may have remarkable impact on the bioavailability of the drug. In addition to this, we have shown that the ability of the iodine atoms in the C-I bonds to form halogen bond with donor atoms can be altered by changing the relative orientation of tyrosyl and phenolic rings in T4. In the fourth chapter, the three-dimensional structures and conformations of thyroid hormones (THs) and iodothyronamines (TAMs) are discussed. TAMs, the endogenous decarboxylated metabolites of THs, exhibit different binding affinities to the transport proteins and iodothyronine deiodinases (DIOs) compared to the THs. Figure 5. Change in the structure and conformations of thyroid hormones and iodothyronamines with the decarboxylation of amino acid side chain and deiodination of phenolic and tyrosyl ring. Furthermore, the substrate specificities of DIOs have been found to be dependent on the position of iodine atoms on the phenolic and tyrosyl ring of TAMs and THs. Single crystal X-ray structures of TAMs indicate that decarboxylation of amino acid side chain of THs induces significant changes in the structure and conformation. Furthermore, the positional isomers of THs and TAMs exhibit remarkably different conformations, which may have significant effect on the binding of these metabolites to the active site of DIOs. In addition to the structure and conformations, different categories of the intermolecular halogen···halogen (X···X) interactions in the crystal packing of THs and TAMs have also been discussed. Natural bond orbital (NBO) analysis have been done on the halogen-bonded geometries to understand the electronic nature of these interactions. In the fifth chapter, the dehalogenation of halogenated nucleosides and nucleobases by naphthyl-based sulphur/selenium compounds is discussed. Purine and pyrimidine nucleosides are halogenated at various positions of the aromatic ring by different peroxidases such as myeloperoxidase and eosinophil peroxidase present in the white blood cells. Incorporation of the halogenated nucleosides into the DNA of replicating cells leads to DNA-strand breaks, mutagenesis, carcinogenesis and loss of Figure 6. (A) Dehalogenation of halogenated nucleosides. Effect of base-pairing wih adenine and guanine on the deiodination of IU (B) and debromination of BrU (C) by compound 2. genome integrity. We have shown that the naphthalene-based organoselenium compounds such as compound 2 can mediate the dehalogenation of 5-iodo-2'-deoxyuridine (5-IdUd) and 5-bromo-2'-deoxyuridine (5-BrdUd) to produce 2'-deoxyuridine (dUd) (Figure 6A). The deiodination of 5-IdUd was found to be faster than the debromination of 5-BrdUd by compound 2. The mechanism of dehalogenation of halogenated nucleosides by compound 2 was found to be dependent on the nature of halogen. While the deiodination of 5-IdUd by compound 2 follow halogen bond-mediated pathway like thyroid hormones, debromination of 5-BrdUd follow a Michael addition-elimination pathway. Similar results were obtained when 5-iodo-2'-deoxycytidine (5-IdCd) or 5-bromo-2'-deoxycytidine (5-BrdCd) was used as substrate for dehalogenation reaction. Base-pairing of 5-iodouracil (IU) and 5-bromouracil (5-BrU) with adenine and guanine has a significant effect on the rate of dehalogenations of IU and BrU by compound 2 (Figure 6B and 6C).