This thesis, which is divided into four chapters, represents an account on the isolation, identification and the assessment of bioactivity of cytotoxic compounds from the genus Centaurea (Family: Asteraceae alto Compositae), a large genus of about 500 species. The first three chapters deal with an introduction of natural products and Centaurea species, followed by the isolation and characterisation of compounds from twelve Centaurea species. The last chapter describes the bioactivities of extracts and isolated compounds from these species. A total of 45 compounds were isolated from twelve Centaurea species, and only C. americana, C. cyanus, C. dealbata and C. macrocephala had previously been studied. Four of these are novel compounds. Four lignans arctiin, matairesinoside, matairesinol and lappaol A were isolated fromthe methanol extract of C. macrocephala seeds. Arctiin and matairesinoside were also isolated from the methanol extract of C. americana, C. bornmuel/eri, C. dealbata, C. huber-morathii, C. mucronifera, C. pamphylica, C. schischkinii and C. urvillei. The methanol extract of C. americana also afforded 20-hydroxyecdysone, 24-hydroxyecdysone, lappaol A, arctigenin and a novel compound, 3"-O-caffeoyl(9"'→3")-arctiin. The methanol extract of C. cyanus produced lariciresinol 4-0-B-D-glucoside, berchemol, moschamine and cis-moschamine. Arctigenin, astragalin, afzelin, matairesinol and a novel indole alkaloids, schischkiniin, were isolated from the methanol extract of C. schischkinii. Extract from C. bornmuelleri afforded arctigenin, astragalin, afzelin and matairesinol. The methanol extract of C. mon/ana yielded berchemol, berchemol 4'-O-B-D-glucoside, p-coumaroylquinic acid, cis-pcoumaroylquinic acid, pinoresinol, pinoresinol mono methyl ether, pinoresinol dimethyl ether, pinoresinol 4-0-B-D-glucoside, pinoresinol 4,4'di-0-B-D-glucoside, pinoresinol 4-0-apiose-(1→2)-B-D-glucoside, centcyamine, cis-centcyamine, N-(4-hydroxycinnamoyl)-5-hydroxytryptamine, cis-N-(4-hydroxycinnamoyl)-5-hydroxytryptamine, moschamine, cis-moschamine, tryptamine and two novel compounds, flavanone-apiose-glucuronic acid and montamine. C. gigantea afforded arctiopicrin, 8-0-(4-hydroxy-3-methylbutanoyl)-salonitenolide, chlorogenic acid, cirsiliol, isoquercetrin, orientin, isoorientin and 4"-hydroxybenzoyl-isoorientin. General toxicity, cytotoxicity and antioxidant activity of the extracts and isolated compounds were evaluated, respectively, by the brine shrimp lethality assay, MTT assay on human colon cancer cell line (CaCo-2) and DPPH assay. Among all the species, the methanol extract of C. bornmuelleri, C. gigantea, C. huber-morathii and C. montana were the most toxic extracts in brine shrimp lethality and MTT assay. Arctigenin (IC50=7.0 mM), matairesinol, montamine (IC50=43.9 mM) and lappol A, schischkiniin, arctiopicrin (IC50=8.5 mM) and 8-0-(4-hydroxy-3-methylbutanoyl)-salonitenolide (IC50=26.4 mM) showed higher cytotoxicity against MTT assay. Matairesinoside (IC50=2.2 x 10-3 mg/mL), matairesinol (IC50=2.0 x 10-3 mg/mL) and schischkiniin (lC50=3.8 x 10-3 mg/mL) exhibited significant free radical scavenging activities towards DPPH assay.

Statins are the mainstay of cholesterol-lowering therapy and are taken by millions of people worldwide. These drugs are generally well-tolerated but can cause myopathy ranging from mild muscle pain to fatal rhabdomyolysis. The mechanism of statin-induced myopathy (SIM) is not fully understood and there is currently no convenient and reliable marker of SIM, but mitochondrial dysfunction has been implicated. We sought to investigate the effect of statins on mitochondrial DNA (mtDNA) levels in order to gain information on the mechanism of SIM and to explore the possibility of utilizing changing mtDNA levels as a marker of SIM. Several approaches were used. First, mtDNA levels were quantified in skeletal muscle biopsies collected from a previously published 8-week clinical trial of high-dose simvastatin or atorvastatin versus placebo. Forty-eight hypercholesterolemic subjects were randomly assigned to receive placebo (N=16), high dose atorvastatin 40mg/day (N=16), or high dose simvastatin 80mg/day (N=16) for 8 weeks. Muscle mtDNA content was assessed by real-time PCR atbaseline and after 8-weeks on statin treatment and found to be significantly reduced in the groupreceiving simvastatin (P=0.005) but not the other two. In addition, a significant positive correlation was observed between mtDNA and muscle ubiquinone in all groups (R=0.63, P<0.01), with the strongest association found in the simvastatin-treated subjects (R=0.75, P=0.002). Next, in an attempt to determine whether statin-induced muscle pain may be associated with muscle mtDNA depletion, archived muscle biopsies collected from statin users with muscle complaints were sought through a review of a muscle biopsy database and possible study samples were identified; however, this was put on hold as too much information was missing from the pathology reports. Third, a series of cell culture experiments were carried out in which human skeletal muscle myotubes were exposed to various concentrations of simvastatin or atorvastatin, in order to determine an appropriate dose range for subsequent mitochondrial toxicity experiments. Lastly, mtDNA content and expression was quantified in skeletal muscle biopsies collected from 10 patients with statin-induced rhabdomyolysis (SIR) and compared to 8 healthy controls to investigate whether muscle mtDNA is altered in rhabdomyolysis. No differences in mtDNA content or expression were observed between the two study groups, but this may have been be due to the SIR subjects' marked heterogeneity. Statin therapy can be associated with considerable alterations in mtDNA content, which may play a role in the aetiology of SIM. MtDNA levels alterations with statin exposure should be investigated further to explore the involvement of mitochondrial alterations in the mechanism of SIM, and determine whether these may represent a useful clinical tool for assessing statin-induced muscle toxicity. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Background: A third of HIV-infected patients are co-infected with HCV in the developed world, and more of co-infected patients than ever before are dying because of liver related diseases today. Drug-related hepatotoxicity is a growing concern among human immunodeficiency virus (HIV) / hepatitis C virus (HCV) co-infected population. Nucleotide analogues containing HIV antiretroviral therapy, namely highly active anti-retroviral therapy (HAART), can induce mitochondrial toxicity. However, little is known about the effect of nucleotide analogues on the liver at the cellular and molecular level, and how it may affect treatment. Objective: To investigate whether liver tissue from HIV/HCV co-infected individuals will show greater liver mitochondrial toxicity if currently receiving antiviral HIV medication, compared to those who are not taking it. Methods: Liver biopsies were collected from 23 HIV/HCV co-infected males. Fourteen patients were on stable HAART (ON-HAART) and 9 were OFF-HAART, including 4 who stopped HAART >6 months prior and 5 who were HAART-nave. Liver mitochondrial toxicity was assessed by transmission electron microscopy-based quantitative stereological analyses of hepatocyte and mitochondrial morphometry, as well as by mitochondrial DNA (mtDNA) and mtRNA (COX1/(ß-actin) real-time-PCR quantification. Results: Hepatocytes tended to be larger in the ON-HAART group than in the OFF-HAART group (p=0.05), but they both showed similar mitochondrial volume fraction of the cell and mitochondrial crista density. Liver mtDNA and mtRNA levels were not significantly differentbetween ON-HAART and OFF-HAART. Hepatocyte lipid accumulation was significantly higher in HCV genotype 3 compared to genotype 1 infection ()=0.002), but was not associated with HAART status. Conclusions: We found no evidence or trend of increased mitochondrial toxicity in HIV/HCV co-infected individuals currently on HAART compared to those who are not. This finding could be relevant to the decision-making process with respect to initiating HCV therapy in this population. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Toxic metals are major pollutants of the aquatic environment and are able to cause survival impairment of the early life stage of the aquatic organisms. They can affect the osmoregulatory system and electrolyte balance in fishes as well as the expression of genes which are essential in the formation and development of the organs at the early embryonic stages of development. There are a lack of studies concerning the toxic effects of waterborne copper and silver on the osmoregulation, electrolytes balance and expression the genes which are responsible for the formation and development of heart and metal binding proteins in the early life stage of zebrafish. The current study aimed to assess the toxic effects of waterborne concentrations of copper as an essential trace element, and silver as a non-essential trace element, on biochemical processes and the molecular biology of the early life stages (ELS) of zebrafish. The first experiment of the current study (Chapter 3) aimed: 1. to determine the time of nkx2.5 gene expression, a gene involved in cardiac development, relative to the time of embryonic development. 2. To assess the toxic concentration of the copper and most vulnerable and sensitive stage of the embryos < 1 hour post fertilization (hpf) exposed to the copper via water route. The result of the experiment showed that the expression of the nkx2.5 gene reached a maximum at 16 hpf. The first 10 hpf of the embryonic development was the most vulnerable and critical stage of the developing embryos, and characterized by increased mortality as copper concentration increased, and delayed and decreased hatching success. Exposure of embryos for 72 hpf to a concentration of 500 µg L-1 Cu increased heart rate, whereas the exposure of the embryos at the blastula stage only, showed decreases in heart rate. The third part of the experiment evaluated the protective effect of calcium as a major cation of water hardness on Cu toxicity. Embryos age < 2 hpf were exposed to copper (0, 100, 250, and 500 µg L-1), with or without added calcium (40 mg L-1). An increase in embryonic Cu accumulation was observed in live and dead embryos exposed to Cu, with and without added calcium. Calcium concentration increased with embryonic copper tissue concentration in dead embryos. Na+ and K+ concentrations were higher in live embryos compared to dead embryos, and a 4 fold decrease in Na+K+-ATPase activity was seen in live embryos exposed to copper compared to controls. There was no effect of copper on total glutathione. Expression of nkx2.5 as one of the essential genes for the formation and development of the heart increased significantly; approximately 10 fold in the presence of Cu+Ca in comparison to the unexposed control or Cu exposure alone. Whereas expression of mt2 increased significantly 6 fold compared to the control during Cu exposure without added Ca2+. The second experiment (Chapter 4) aimed to investigate the effect of the dissolved Ag+ as AgNO3 on the survival of the early life stage of zebrafish. Embryos < 2 hpf were exposed to silver 0 (no added Ag), 2.5, 5, 7.5, 10 and 15 µg L-1 Ag as AgNO3 for up to 72 h. Although, the survival was not affected by increasing concentrations of total silver, a decrease in hatching and increase in heart beat was observed. A significant increase in embryonic silver accumulation in both live embryos (at 24 and 72 hpf) and dead embryos (at 24 hpf) was observed. The accumulation of silver in 24 hpf live embryos was more significant than in dead embryos. Dead and live embryos at 72 hpf exposed to Ag had lower Na+ and K+ concentrations. Live embryos also showed a transient increase in Ca2+ concentration at 24 h. Four fold increases in Na+K+-ATPase activity, Mt2, and total glutathione concentration were seen in embryos after 72 h of exposure to AgNO3 compared to controls. In contrast, nkx2.5 gene expression was significantly decreased by 3 fold in 24 h aged embryos exposed to silver compared to controls. Due to the lack of studies that investigate the effect of silver on protein expression profiles during the early stages of development of zebrafish, the third experiment (Chapter 5) aimed to investigate the effect of silver on the changes of the expressed proteins of zebrafish embryos at the segmentation stage (24 hpf). The proteomics analysis successfully identified total of 810 proteins in the embryonic homogenate and quantified changes in their abundance in response to silver exposure. MS analysis showed the induction of new proteins which were absent in control embryonic homogenates. Also the analysis revealed there were increased expression of proteins such as zona pellucida glycoprotein, ATP synthase subunit α and β, stressed proteins such as metal chaperones and heat shock proteins, antioxidant proteins such as catalase (CAT), superoxide dismutase (Cu-Zn), glutathione S-transferase M, and glutathione S-transferase and proteins related to muscular development such as myosin heavy polypeptide 2, actin alpha 1 skeletal muscle, slow myosin heavy chain 1, actin cytoplasmic 1, and tropomyosin proteins. Overall, the thesis confirmed that the early life stages of zebrafish are sensitive to metals and that there are critical windows of toxicity during development. Metal exposure at early stages of the development initiate several disturbances in biochemical processes as well as changes in molecular biology that affect fish survival.

This study looked at effects of organic solvents dimethylsulfoxide, dimethylformamide and acetone at 0.01%, 0.05% and 0.1% concentration on germination and seedling development wetland plants. Even at 0.01% level, all solvents affected some aspect of seed germination or seedling growth. Acetone at 0.01% was least toxic. Root morphological characteristics were most sensitive compared to shoot morphological characteristics. This study also looked at bioconcentration patterns of antimicrobial compounds triclosan, triclocarban and methyl-triclosan in wetland plants exposed to Denton Municipal Waste Water Treatment Plant effluent. Bioconcentration patterns of antimicrobial compounds varied among species within groups as well as within organs of species. The highest triclocarban, triclosan and methyltriclosan concentration were in shoot of N. guadalupensis, root of N. lutea and in shoots of P. nodous respectively.

A dissertation submitted to the Faculty of Science, in partial fulfilment of the requirements for the award of Master of Science degree, University of the Witwatersrand, Johannesburg, 2018 / Three mixed-ligand metal-metal bonded complexes containing one unsymmetrical anionic bridging ligand were successfully synthesized and characterized as to their electrochemical and spectroscopic properties. The investigated mono-substituted diruthenium complexes have the general formula, Ru2(OAc)3(L)Cl, where OAc = acetate anion and L = anilinopyridinate bridging ligand (ap, 2-Meap, 2-Fap). UV/Visible spectroscopy studies reveal that the investigated diruthenium complexes exist in the forms Ru2(OAc)3(L)Cl and [Ru2(OAc)3(L)]+ in solution. The two forms are observed as a split band in the 500 – 700 nm visible region. A collapse of one band is seen upon reaction of the complexes with excess halide (Cl-, Br-) indicating an equilibrium shift towards the neutral species in solution, whereas a reaction with AgBF4 precipitates the chloride as the AgCl salt, leaving only the cationic species in solution. Electrochemical characterization of the mixed-ligand diruthenium complexes conclusively reveals a stable Ru25+ oxidation state in all three complexes. Upon an applied potential in a non-coordinating solvent, each complex undergoes a reversible one-electron oxidation and reduction process accessing the Ru26+, and Ru24+ oxidation states respectively. The treatment of human breast adenocarcinoma MCF-7 cells with these water-soluble complexes results in a less than 50 % cell survival. This demonstrates significance of solubility in the development of metallodrugs for cancer treatment. / XL2018

The structures of the new pyrethroids were elucidated utilizing nuclear magnetic spectroscopy and elemental analysis. The biological activity of the pyrethroids has been evaluated for houseflies and yellow fever mosquitoes. A correlation of increase in rapid knockdown and toxicity with decrease in size of halogen has been made. The most effective compound, the difluorovinyl ester, has a toxicity of 123 times greater than pyrethrins. This degree of toxicity has not previously been reported for a 5-benzyl-3-furanmethyl ester. The relative lipophilicities of the dihalovinyl esters, Rm, have been determined by reverse phase partition chromatography. The R values for the dihalo esters correlate with their m effectiveness as rapid knockdown agents. The least lipophilic compound, the difluorovinyl ester, possessed the highest knockdown in this dihalovinyl ester series. The difluorovinyl ester has incorporated a high level of toxicity while maintaining excellent effectiveness as a knockdown agent against houseflies. This combination of high knockdown and high toxicity in a single compound has not been previously reported. Several other pyrethroids were synthesized and evaluated for knockdown and toxicity, in order to make specific comparisons with pyrethroids of known effectiveness. The replacement of the isobutenyl group of the chrysanthemates by the dihalovinyl group has been effective against houseflies and yellow fever mosquitoes in enhancing or maintaining toxicity and knockdown in every pyrethroid series investigated. In the allethronyl series, this dihalovinyl replacement has resulted in a 12-fold increase in rapid knockdown activity against mosquitoes.

Yes / Two approaches for the prediction of which of two vehicles will result in lower toxicity for anticancer agents are presented. Machine-learning models are developed using decision tree, random forest and partial least squares methodologies and statistical evidence is presented to demonstrate that they represent valid models. Separately, a clustering method is presented that allows the ordering of vehicles by the toxicity they show for chemically-related compounds.

The objective of this study was to evaluate the clinical, cerebrospinal fluid (CSF), and histopathologic effects of an epidurally administered NSAID (ketorolac) in dogs. This was performed as a blinded, placebo controlled study using twenty-two adult mixed breed dogs with 16 treatment and 6 control dogs. Dogs were anesthetized and epidural catheters were placed at the lumbosacral space. Catheter placement was evaluated fluoroscopically. Ketorolac (0.4 mg/kg) or placebo (5% ethanol) was administered epidurally over a 52 hour period, with 5 injections given at 12 hour intervals. At 1, 2, 4, or 8 hours after the first and last injection of ketorolac, dogs were anesthetized and CSF was obtained. Control dogs had CSF sampled 1 hour after the first and last ethanol injection. Neurologic function and pain response was evaluated before and during the study. Selected dogs were then euthanized and necropsies performed. None of the dogs exhibited any clinical or neurological abnormalities during the study. No statistical difference was noted in pain response or CSF analysis between treatment and control dogs. Gross necropsy revealed gastrointestinal ulceration of varying degrees in all treatment dogs. Histopathologic analysis of the spinal cord and meninges revealed minimal focal leptomeningeal phlebitis in 2 of 8 treatment dogs and minor subdural inflammation in one control dog. No changes to the neural structures were noted in any dogs. Epidural administration of ketorolac did not cause clinical signs, alteration in CSF values, or pathologic changes to the spinal cord when used for short duration. Gastrointestinal ulceration was common when ketorolac was administered epidurally at 0.4 mg/kg every 12 hours for 5 treatments. This study documented the safety of epidurally administered ketorolac in dogs before an efficacy trial can be performed. Gastrointestinal ulceration may limit use to short duration or single injection. / Master of Science

Epidemiological studies have linked exposure to elevated levels of airborne particulate matter with increased incidences of several types of respiratory disease, hospital admissions and morbidity. Millions of tons of airborne particulate matter are generated and released into the atmosphere each year. However, particulate matter resulting from the combustion of fuel oil and coal are of particular concern, because they are generally composed of small particles that can easily penetrate deep into the lungs, and can contain significant concentrations of toxic transition metals, such as zinc, iron and vanadium. Pulmonary toxicity (i.e. damage caused to lung tissues) of particulate matter is currently evaluated via time-consuming in-vivo testing, or via in-vitro testing. Compared to in-vivo testing, in-vitro testing offers significant advantages in terms of time savings and sample throughput. Unfortunately, the number of in-vitro testing methods are currently very limited, and do not allow a thorough investigation of the mechanisms of particulate matter toxicity. In light of these issues, the goals of the study described here were three-fold: *To adapt several in-vitro toxicity assays currently used in other applications to use in measuring particulate matter toxicity on lung cell layers; *To use these adapted assays to quantify the toxicity of numerous types of oil and coal ashes with varying particle sizes and transition metal concentrations, and; *To use the same assays to quantify the toxicities of several transition metals found in coal and oil ashes to better understand their relative contributions to overall particulate matter toxicity. Three colorimetric in-vitro assays were chosen for adaptation, and proved effective in measuring adverse cellular response to particulate matter exposure. Particle size was shown to have a large effect on the overall cytotoxicity of particulate matter; fine (less than 2.5 μm aerodynamic diameter) particles proved substantially more toxic than coarse (larger than 2.5 μm aerodynamic diameter) particles. Dose-response experiments measuring the toxic effects of the transition metals zinc, vanadium and iron revealed that zinc was the most toxic; a concentration of 0.6 mM caused a 50% drop in cellular metabolism, compared to 3 mM and 4 mM for vanadium and iron respectively.