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O ácido 3-indol acético (IAA), um conhecido hormônio de planta, e seu metabólito indol-3-carboxialdeído (ICA) têm sido envolvidos em várias patologias humanas como fenilcetonúria e doenças renais. A formação de espécies reativas de oxigênio e de estados eletronicamente excitados, como o oxigênio singlete (1O2) e a carbonila triplete (R2-C=O*), durante a oxidação aeróbica do IAA catalisada por peroxidase de raiz forte (HRP) têm sido relacionada com efeitos citotóxicos do IAA. Nossos resultados indicam aumento na formação de 8-oxo-7,8-dihidro-2\'desoxiguanosina (8-oxodGuo) após o tratamento da base com o sistema IAA/HRP/O2 in vitro, medido por um detector eletroquímico acoplado a um sistema de cromatografia líquida de alta performance (HPLC-EC). O tratamento de células de mamíferos (CV1-P e neutrófilos) com este sistema induziu o aumento na formação de 8-oxodGuo no DNA, assim como o aumento no nível de lipoperoxidação das células CV1-P quando comparadas ao controle. Detectamos a presença do adulo 1,N2-etenodesoxiguanosina em DNA de neutrófilos submetidos ao IAA. Portanto, neste estudo, apresentamos evidências de danos em biomoléculas promovidos pelo sistema IAA/HRP/O2. / Indole-3-acetic acid (IAA), a2 plant hormone, and its metabolite indole-3-carboxialdehyde (ICA) has been involved in several human pathologies as phenylketonuria and renal diseases. Formation of reactive oxygen species and electronically excited states as singlet oxygen (1O2) and triplet carbonyl (R2-C=O*), during the aerobic oxidation catalyzed by horseradish peroxidase (HRP) has been reported to be involved in the IAA cytotoxic effects. Our results show an increase in the formation of 8-oxo-7,8-dihydro-2\'deoxiguanosine (8-oxoxdGuo) after IAA/HRP/O2 system treatment in vitro as inferred from high performance liquid chromatography-electrochemical detection (HPLC-EC) measurements. Treatment of mammalian cells (CV1-P and neutrophils) with the system induces 8-oxoxdGuo formation in DNA as well increased levels of lipid peroxidation CV1-P cells when compared to controls. The 1,N2-etenodeoxyguanosine was detected in neutrophils DNA. Therefore, in this study, we presented evidence of biomolecule damage by IAA/HRP/O2 system.

by Wang, Wei Vivian. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 237-266). / ACKNOWLEDGEMENTS --- p.viii / SUMMARY --- p.ix / PUBLICATION --- p.xiv / STATEMENT OF ORIGINALITY --- p.xv / LIST OF ABBREVIATIONS --- p.xvi / Chapter CHAPTER 1 --- INTRODUCTION --- p.3 / Chapter 1.1 --- Preamble --- p.3 / Chapter 1.2 --- Free oxygen radicals --- p.7 / Chapter 1.2.1 --- Free oxygen radicals and mechanism of radical damage / Chapter 1.2.1.1 --- What is a free radical? / Chapter 1.2.1.2 --- Mechanism of free radical damage / Chapter 1.2.2 --- Detection and characterisation of free radical species / Chapter 1.2.2.1 --- Direct methods / Chapter 1.2.2.1.1 --- Electron spin resonance (ESR) spectroscopy / Chapter 1.2.2.1.2 --- Chemiluminescence / Chapter 1.2.2.2 --- Indirect methods / Chapter 1.2.2.2.1 --- Lipid peroxidation / Chapter 1.2.2.2.2 --- Protein and DNA oxidation / Chapter 1.2.2.2.3 --- Purine and pyrimidine metabolites / Chapter 1.2.3 --- Free oxygen radicals and major disease / Chapter 1.2.4 --- Oxygen-derived free radicals and fetal hypoxia / Chapter 1.3 --- Acid-base status in cord blood --- p.41 / Chapter 1.3.1 --- Correlation between obstetric clinical events and cord blood acid-base / Chapter 1.3.2 --- Practical implications of cord blood acid-base studies / Chapter 1.4 --- Intrapartum cardiotocography (CTG) analysis --- p.58 / Chapter 1.4.1 --- Base line / Chapter 1.4.1.1 --- Baseline rate / Chapter 1.4.1.2 --- Baseline variability / Chapter 1.4.2 --- Accelerations and decelerations / Chapter 1.4.3 --- Fetal outcome of labour / Chapter 1.4.3.1 --- Fetal heart rate (FHR) changes during labour / Chapter 1.4.3.2 --- Acidaemia during labour / Chapter 1.4.4 --- Computerised analysis of cardiotocogram / Chapter 1.5 --- Intrapartum complications --- p.83 / Chapter 1.5.1 --- Meconium stained amniotic fluid / Chapter 1.5.2 --- Nuchal cord entanglement / Chapter 1.5.3 --- Prolonged 1st and 2nd stage of labour / Chapter 1.6 --- Objectives of project --- p.93 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.98 / Chapter 2.1 --- Materials --- p.98 / Chapter 2.1.1 --- Clinical materials / Chapter 2.1.2 --- Chemicals and reagents / Chapter 2.1.2.1 --- The measurement of malondialdehyde (MDA) / Chapter 2.1.2.2 --- The measurement of organic hydroperoxides (OHP) / Chapter 2.1.2.3 --- The measurement of purine and pyrimidine metabolites / Chapter 2.1.3 --- Equipment / Chapter 2.1.3.1 --- Fetal monitor / Chapter 2.1.3.2 --- Fetal heart rate analysis system / Chapter 2.1.3.3 --- Blood gas analyser / Chapter 2.1.3.4 --- UV-VIS Spectrophotometer / Chapter 2.1.3.5 --- Fluorescence Spectrophotometer / Chapter 2.1.3.6 --- High Performance Liquid Chromatography (HPLC) / Chapter 2.2 --- Investigation Methods --- p.105 / Chapter 2.2.1 --- Blood gas / Chapter 2.2.2 --- Lipid peroxidation in umbilical cord blood / Chapter 2.2.2.1 --- The measurement of MDA / Chapter 2.2.2.2 --- The measurement of OHP / Chapter 2.2.3 --- Purine and pyrimidine metabolites in umbilical cord blood / Chapter 2.2.4 --- Computer analysis of CTG / Chapter 2.2.4.1 --- Data and signal processing / Chapter 2.2.4.2 --- The algorithm / Chapter 2.3 --- Statistical analysis --- p.112 / Chapter CHAPTER 3 --- RESULTS --- p.116 / Chapter 3.1 --- Umbilical blood pH and gas measurements --- p.118 / Chapter 3.2 --- Lipid peroxidation in cord blood plasma --- p.121 / Chapter 3.2.1 --- Validation of assay / Chapter 3.2.1.1 --- Performance characteristics of the MDA assay / Chapter 3.2.1.2 --- Performance characteristics of the OHP assay / Chapter 3.2.2 --- "Inter-relationship among MDA, OHP and acid-base status" / Chapter 3.3 --- Nucleotide metabolites in cord blood plasma --- p.142 / Chapter 3.3.1 --- Calibration of assay / Chapter 3.3.2 --- Inter-relationship among nucleotides and acid-base status / Chapter 3.4 --- Analysis of FHR patterns --- p.150 / Chapter 3.4.1 --- Umbilical blood gas and CTG analysis / Chapter 3.4.2 --- Biochemical parameters and CTG analysis / Chapter 3.5 --- "Relations of umbilical arterial blood pH and gas, lipid peroxidation, purine or pyrimidine metabolites and FHR patterns with intrapartum complications" --- p.166 / Chapter 3.5.1 --- Meconium stained amniotic fluid / Chapter 3.5.1.1 --- Clinical features / Chapter 3.5.1.2 --- Relationship between meconium stained amniotic fluid and biochemical parameters / Chapter 3.5.1.3 --- Relationship between meconium stained amniotic fluid and FHR patterns / Chapter 3.5.2 --- Nuchal cord / Chapter 3.5.2.1 --- Clinical features / Chapter 3.5.2.2 --- Relationship between nuchal cord and biochemical parameters / Chapter 3.5.2.3 --- Relationship between nuchal cord and FHR patterns / Chapter 3.5.3 --- The length of second stage of labour / Chapter 3.5.3.1 --- Clinical features / Chapter 3.5.3.2 --- Relationship between the length of second stage and acidaemia or FHR patterns / Chapter 3.5.4 --- Apgar scores / Chapter 3.5.4.1 --- Clinical features / Chapter 3.5.4.2 --- Relationship between Apgar scores and biochemical parameters / Chapter 3.5.4.3 --- Relationship between Apgar scores and FHR patterns / Chapter 3.5.4.4 --- "Relationship between Apgar scores and nuchal cord, meconium or second stage of labour" / Chapter CHAPTER 4 --- DISCUSSION --- p.189 / Chapter 4.1 --- Blood pH and gas in fetal asphyxia --- p.189 / Chapter 4.2 --- Lipid peroxidation in cord blood at birth --- p.194 / Chapter 4.2.1 --- Method for measurement of the cord plasma MDA / Chapter 4.2.2 --- Method for measurement of the cord plasma OHP / Chapter 4.2.3 --- Relationship between the fetal asphyxia and lipid peroxidation in cord plasma / Chapter 4.3 --- Purine and pyrimidine metabolites in cord blood at birth --- p.203 / Chapter 4.3.1 --- Limitations imposed by the tcchniqucs used / Chapter 4.3.2 --- Relationship between the fetal asphyxia and purine and pyrimidine metabolites in cord plasma / Chapter 4.4 --- Computerised analysis of CTG --- p.210 / Chapter 4.4.1 --- CTG patterns and cord blood acid base balance / Chapter 4.4.2 --- CTG patterns and cord blood biochemical parameters / Chapter 4.5 --- "Intrapartum complications 2,9" / Chapter 4.5.1 --- Meconium stained amniotic fluid / Chapter 4.5.2 --- Nuchal cord / Chapter 4.5.3 --- The length of second stage / Chapter 4.5.4 --- Apgar scores / Chapter CHAPTER 5 --- CONCLUSION --- p.233 / REFERENCES --- p.237

Ngai Lei-Ka. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 113-125). / Abstracts in English and Chinese. / List of Abbreviations --- p.ix / List of Tables --- p.x / List of Figures --- p.xii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Flavonoids --- p.1 / Chapter 1.1.1 --- The six major classes of flavonoids --- p.1 / Chapter 1.1.1.1 --- Flavonones --- p.1 / Chapter 1.1.1.2 --- Flavones --- p.2 / Chapter 1.1.1.3 --- Flavonols --- p.2 / Chapter 1.1.1.4 --- Isoflavonoids --- p.2 / Chapter 1.1.1.5 --- Anthocyanidins --- p.3 / Chapter 1.1.1.6 --- Flavans --- p.3 / Chapter 1.1.2 --- Structural variation of flavonoids --- p.3 / Chapter 1.1.3 --- The roles of flavonoids --- p.6 / Chapter 1.2 --- "Free radicals, oxidative stress and antioxidants" --- p.7 / Chapter 1.2.1 --- Oxidants and free radicals --- p.7 / Chapter 1.2.2 --- Lipid peroxidation (LPO) --- p.8 / Chapter 1.2.3 --- Oxidative stress and human diseases --- p.9 / Chapter 1.2.4 --- Role of food antioxidants --- p.10 / Chapter 1.2.5 --- Synthetic and natural food antioxidants --- p.11 / Chapter 1.3 --- Antioxidant activity of flavonoids --- p.12 / Chapter 1.4 --- Determination of antioxidant activity --- p.13 / Chapter 1.4.1 --- Trolox equivalent antioxidant capacity (TEAC) assay --- p.13 / Chapter 1.4.2 --- DPPH radical scavenging assay --- p.14 / Chapter 1.4.3 --- β-carotene bleaching assay --- p.15 / Chapter 1.5 --- Single cell gel electrophoresis assay (Comet assay) --- p.16 / Chapter 1.6 --- Determination of the genotoxicity --- p.18 / Chapter 1.7 --- Research objectives --- p.19 / Chapter 2 --- Materials and Methods --- p.26 / Chapter 2.1 --- Standards and reagents --- p.26 / Chapter 2.2 --- Sample Preparation --- p.26 / Chapter 2.3 --- Trolox equivalent antioxidant capacity (TEAC) assay --- p.27 / Chapter 2.4 --- DPPH´Ø radical scavenging assay --- p.28 / Chapter 2.5 --- β-carotene bleaching assay --- p.29 / Chapter 2.6 --- The comet assay --- p.30 / Chapter 2.6.1 --- Preparation of reagents --- p.31 / Chapter 2.6.2 --- Blood sample --- p.32 / Chapter 2.6.3 --- Optimal condition of comet assay --- p.32 / Chapter 2.6.3.1 --- Induction of DNA damage --- p.32 / Chapter 2.6.3.2 --- Antioxidant pre-treatment --- p.32 / Chapter 2.6.4 --- Hydrogen peroxide (H2O2) treatment --- p.32 / Chapter 2.6.4.1 --- Pre-incubation system --- p.32 / Chapter 2.6.4.2 --- Co-incubation system --- p.33 / Chapter 2.6.5 --- Slide preparation --- p.33 / Chapter 2.6.6 --- Cell lysis --- p.33 / Chapter 2.6.7 --- Alkaline treatment and electrophoresis --- p.34 / Chapter 2.6.8 --- Neutralization --- p.34 / Chapter 2.6.9 --- Quantification of DNA damage --- p.34 / Chapter 2.6.10 --- Cell viability analysis --- p.35 / Chapter 2.6.11 --- Statistical Analysis --- p.35 / Chapter 2.7 --- Mutatox® test --- p.36 / Chapter 2.8 --- Statistical analysis --- p.36 / Chapter 3 --- Result --- p.45 / Chapter 3.1 --- Determination of antioxidant activity using Trolox equivalent antioxidant capacity (TEAC) assay --- p.45 / Chapter 3.1.1 --- Trolox standard reference --- p.45 / Chapter 3.1.2 --- Antioxidant activity: ABTS´Ø+ scavenging capacity --- p.45 / Chapter 3.2 --- Evaluation of antioxidant activity using free radical scavenging assay --- p.46 / Chapter 3.2.1 --- Free radical scavenging abilities at 5 minutes --- p.46 / Chapter 3.2.2 --- Antiradical efficiency --- p.47 / Chapter 3.3 --- Evaluation of antioxidant activity using β-carotene bleaching assay --- p.47 / Chapter 3.3.1 --- Optimal incubation time --- p.47 / Chapter 3.2.2 --- Antioxidant activities on β-carotene bleaching assay --- p.47 / Chapter 3.4 --- Structure and antioxidant activities relationship (SAR) of flavonoids --- p.48 / Chapter 3.4.1 --- Effect of hydroxylation on the antioxidant activities of flavonoids --- p.48 / Chapter 3.4.1.1 --- Hydroxylation positions --- p.48 / Chapter 3.4.1.2 --- Polyhydroxylation --- p.48 / Chapter 3.4.2 --- Importance of B ring structure --- p.49 / Chapter 3.4.2.1 --- Increase hydroxylation in B ring --- p.49 / Chapter 3.4.2.2 --- The othro-dihydroxyl arranagement in B ring --- p.49 / Chapter 3.4.3 --- Importance of C ring structure --- p.50 / Chapter 3.4.3.1 --- The presence of a hydroxyl group at C3 --- p.50 / Chapter 3.4.3.2 --- The blockage of hydroxylation at C3 --- p.50 / Chapter 3.4.3.3 --- The presence of a double bond between C2 to C3 --- p.50 / Chapter 3.4.3.4 --- The presence of a carbonyl group at C4 --- p.50 / Chapter 3.4.3.5 --- The conjugation of a carbonyl at with a double bond between C2 to C3 --- p.51 / Chapter 3.4.4 --- Effect of glycosylation --- p.51 / Chapter 3.5 --- Evaluation of protective effects on DNA damage using comet assay --- p.51 / Chapter 3.5.1 --- Optimization of conditions fro the determination of H2O2-induced DNA damage --- p.52 / Chapter 3.5.1.1 --- H2O2 concentration & treatment temperature --- p.52 / Chapter 3.5.1.2 --- H2O2 treatment time --- p.52 / Chapter 3.5.1.3 --- Sample volume --- p.52 / Chapter 3.5.2 --- Protective effect of vitamin C on DNA damage --- p.53 / Chapter 3.5.3 --- Protective effect of selected flavonoids on DNA damage --- p.53 / Chapter 3.5.4 --- Structure and antioxidant activities relationship (SAR) of flavonoids in comet assay --- p.54 / Chapter 3.5.4.1 --- The importance of B ring structures --- p.54 / Chapter 3.5.4.2 --- The importance of C ring structures --- p.54 / Chapter 3.6 --- Evaluation of genotoxicity of flavonoids using Mutatox test --- p.55 / Chapter 4 --- Discussion --- p.96 / Chapter 4.1 --- Comparison of antioxidant activities between hydrophilic and lipophilic assays --- p.97 / Chapter 4.2 --- Structure and antioxidant activity relationship of flavonoids --- p.98 / Chapter 4.3 --- The comet assay --- p.103 / Chapter 4.4 --- Comparison of protective effect on DNA damage between pre-incubation and co-incubation systems --- p.105 / Chapter 4.5 --- Structure and protective effect of flavonoids in the comet assay --- p.106 / Chapter 4.6 --- Genotoxicity of flavonoids --- p.108 / Chapter 4.7 --- Significance and future works --- p.108 / Chapter 5 --- Conclusion --- p.111 / References --- p.113

This thesis reports on the feasibility of the utilization of organotin hydrides as enaantioselective free radical reducing agents. The chiral organotin hydrides prepared contain the bulky chiral (1R,2S,5R)-menthyl substituent and in some cases also contain a stereogenic tin centre. Reaction of (1R,2S,5R)-menthylmagnesium chloride (MenMgC1) with triphenyltin chloride in THF proceeds with epimerization of the C-1 carbon of the menthyl group and results in a mixture of (1R,2S,5R)-menthyltriphenyltin and (1S,2S.5R)-menthyltriphenyltin. Addition of Lewis bases such as triphenylphosphine to the THF solution of triphenyltin chloride prior to the addition of the Grignard reagent suppresses epimerization and enables isolation of pure (1R,2S,5R)-menthyltriphenyltin. (1R,2S,5R)-Menthyltriphenyltin is the precursor for the synthesis of (1R,2S,5R)-menthyldiphenyltin hydride as well as (1R,2S,5R)-menthyl-containing organotin halide derivatives. A crystal structure of (1R,2S,5R)-menthylphenyltin dibromide and (1R,2S,5R)-menthylphenyltin dichloride confirmed the configuration of the menthyl substituent in these compounds. Reaction of MenMgC1 with diphenyltin dichloride in THF proceeds with no epimerization of the C-1 carbon of the menthyl group and bis((1R,2S,5R)-menthyl)diphenyltin is formed. A crystal structure of (1R,2S,5R)-menthyltriphenyltin confirmed the configuration of the menthyl substituent. Bis((1R,2S,5R)-menthyl)diphenyltin is used to form bis((1R,2S,5R)-menthyl)phenyltin hydride as well as other bis(1R,2S,5R)-menthyl derivatives. A series of chiral non-racemic triorganotin halides and triorganotin hydrides containing one or two (1R,2S,5R)-menthyl substituents as well as various potentially intramolecular coordination substituents were synthesized and characterized. The intramolecular substituents include the 8-(dimethylamino)naphthyl, 2-[(1S)-1-dimethylaminoethyl]phenyl, 2-(4,4-dimethyl-2-oxazoline)-5-methylphenyl and the 2-(4-(S)isopropyl-2-oxazoline)-5-methylphenyl substituents. Each compound containing a stereogenic tin centre was synthesized as diastereomeric mixtures. AM1 calculations of these compounds provide good qualitative predictability of the molecular geometries observed in the solid state as well as the diastereomeric ratios observed in solution. X-ray analysis of some of the organotin halides containing intramolecular coordination substituents revealed a tendency towards penta-coordination at the tin centre as a result of N-Sn interactions. The chiral organotin hydrides synthesized were found to be poor enantioselective free radical reducing agents. However, the addition of one molar equivalent of achiral or chiral Lewis acids to the free radical reduction reactions involving these organotin hydrides results in remarkable increases in enantioselectivity. There are numerous examples in which enantioselectivities exceed 80% and three examples of enantioselectivites which are equal and above 90% with one outstanding enantioselective outcome of ≥99%. These results appear to be the highest enantioselectivites for organotin hydride radical reductions reported to date. There is strong evidence to suggest that the chiral menthyl group of the organotin hydride directs the stereochemical outcome in the reduced product. The results also suggest that an increase in the number of menthyl substituents attached to tin or the introduction of intramolecular coordination substituents does not necessarily results in a greater increase in enantioselectivity. Preliminary studies into the synthesis of organotin hydrides containing Lewis acid functionalities are also reported. A zirconium chloride functionality was found to be incompatible with organotin hydride. However, an organotin hydride containing a trialkylboron Lewis acid functionality attached via an alkyl chain was successfully synthesized. Although this reagent was only stable in the preparative THF solution, it was still found to be effective at reducing benzaldehyde to benzyl alcohol.