Mechanism of action studies on the FR-9000482 class of antitumor antibiotics

1997 Summer.Includes bibliographical references.The interactions of members of the FR-900482 class of antitumor antibiotic agents with DNA has been examined. Importantly, the first in vitro demonstration of nucleic acid interstrand cross-linking has been reported and the DNA base pair sequence specificity of the cross-linking event has been elucidated. These agents demonstrate a high degree of selectivity for 5'-CG-3' sequences of DNA. As such, bio-mechanistic analogy between these compounds and the clinically employed compound Mitomycin Chas been shown. Efforts have also examined extensively the ability of these agents to give rise to orientation isomers of each respective cross-link and their different properties. DNA-protein cross-linking by these agents has also been examined. A sequence-specific DNA-peptide binding motif has been identified which undergoes drug-mediated DNA-protein cross-linking. This is the first reported instance of a mitosene based-minor groove DNA-protein cross-link event. Significantly, the motif examined is characteristic of tissues which bear striking similarity to those of cancerous cell lines

Aktivität und relative Schutzwirkung zweier konkurrierender DNA-Reparaturwege für O6-Methylguanin in vivo

Aufgrund ihres hohen zytotoxischen, mutagenen und karzinogenen Potentials sind DNA-Alkylierungen an der O6-Position des Guanins äußerst riskant sowohl für Zellen als auch für Organismen. Deshalb verfügen Säugerzellen über effiziente Reparaturmechanismen, über die diese kritischen Läsionen aus dem Genom entfernt werden können. Neben der gut untersuchten direkten De-Methylierung durch das MGMT-Protein ist kürzlich ein alternativer Exzisions-Reparaturweg für O6-meG in humanen Zellen entdeckt worden. Im ersten Teil dieser Arbeit wurde nun geklärt, dass dieser Reparaturmechanismus auch in allen daraufhin untersuchten Primärzellen der Maus nachweisbar war und das die Proteine XPC und FancD2 dabei eine essenzielle Funktion hatten, während ein Verlust von XPA nur zu einer verlangsamten Exzisionsreparatur von O6-meG führte. Im Weiteren wurde analysiert, welche relative Schutzwirkung die beiden konkurrierenden Reparaturmechanismen unter in vivo-Bedingungen für das Risiko von biologischen Endpunkten wie Zelluntergang oder Tumorentstehung, haben. Dazu wurden Reparatur-profiziente und -defiziente Mäuse mit dem Alkylanz N-Methyl-N-Nitroseharnstoff (MNU) behandelt und die zytotoxische Auswirkung auf die Hämatopoese, dem Alkylierungs-sensibelsten Zellsystem der Maus, anhand der Myelosuppression im peripheren Blut registriert. Ferner wurde in Langzeituntersuchungen das Auftreten von malignen Veränderungen (Lymphomen) bei den MNU-exponierten Tieren analysiert. Der funktionelle Ausfall von XPC oder FancD2 führte, überaschenderweise unabhängig von der MGMT-Aktivität, zu einer signifikanten Verstärkung sowohl der Zytotoxizität bei hämatopoetischen Stamm- und Vorläuferzellen (Myelosuppression) als auch der Karzinogenese (T-Zell-Lymphome). Demgegenüber zeigte ein Funktionsverlust der MGMT-vermittelten Reparatur oder des XPA-Proteins deutlich geringe Auswirkungen auf Ausmaß beziehungsweise Häufigkeit der beiden Endpunkte. Waren die Mäuse hingegen sowohl für MGMT als auch für XPA defizient, zeigten sie die mit Abstand höchste Suszeptivität für Zelluntergang und maligne Transformation nach MNU-Exposition. Bei der Bestimmung der Mutationsrate in Knochenmarkzellen der verschiedenen Mauslinien anhand einer transgenen lacZ-Sequenz fand sich eine etwa zehnfache Erhöhung dieser Werte nach MNU-Behandlung, aber kein signifikanter Unterschied bei An- oder Abwesenheit der beiden Reparaturfunktionen. Nach Sequenzanalyse des Indikatorgens zeigten sich jedoch bei den Reparatur-defizienten Mäusen Hinweise auf das vermehrte Auftreten von Punktmutationen, die von persistierenden O6-meG-Addukten herleitbar waren. Der hier in seiner biologischen Funktion erstmals näher charakterisierte alternative Exzisions-Reparaturweg ist offenbar nicht nur auf die Entfernung von Guanin-O6-Alkylierungen aus der DNA beschränkt, sondern greift beispielweise auch auf Cisplatin-induzierte Läsionen zu und hat höchstwahrscheinlich einen großen Einfluss auf die therapeutische Wirksamkeit sowie auf das Nebenwirkungsspektrum von methylierenden Medikamenten in der OnkologieThe DNA alkylation damage O6-methylguanine (O6-meG) has a major biological impact on both individual cells and the whole organism due to its cytotoxic, mutagenic and carcinogenic potential. Mammalian cells posses very efficient repair mechanisms that can remove this critical lesion from the genome. In addition to the well-studied direct demethylation by the MGMT repair protein an alternative excision repair mechanism for O6-meG in human cells was identified recently. The first part of this thesis aimed to confirm that the alternative repair pathway is also active in various primary cell types of mice. The activity of the alternative repair pathway was shown to depend on the XPC (NER) and FancD2 (FA) proteins as essential factors for the excision repair of O6-meG-repair in vivo, whereas a loss of XPA only led to a slowed-down removal of O6-meG. Furthermore, this work addressed the relative contribution of both alternative repair mechanisms (MGMT and excision repair) to the protection from biological end points after MNU-exposure in vivo, such as cell death and tumor formation. Repair-proficient and –deficient mice were analyzed for the MNU-induced cytotoxicity in hematopoietic stem and progenitor cells by measuring the myelosuppression in the peripheral blood. In addition, the frequency of malignant alterations (occurrence of lymphoma) was monitored in MNU-exposed animals in long-term studies. In vivo analyses for MNU-induced cytotoxicity demonstrated the protective role of XPC and FancD2 for the hematopoietic system independent of MGMT-activity. Hematopoietic stem and progenitor cells of MNU-exposed XPC(-/-)- and FancD2(-/-)-mice exhibited more severe myelosuppression in comparison to wildtype or XPA(-/-)-mice. In parallel, the loss of functional XPC- or FancD2-protein led to reduced latency and increased frequency of lymphoma in MNU-exposed mice. However, the strongest hematotoxic and carcinogenic effects of DNA methylation were observed in mice with combined MGMT- and XPA-deficiency. The in vivo-mutation rates in lacZ-transgenic bone marrow cells of different mouse strains showed an up to 10-fold increase after exposure of MNU, with no significant difference between repair-deficient and wildtype mice and independent of pharmacological depletion of MGMT. However sequence analysis of the lacZ-gene in repair-deficient mice showed evidence of an increased occurrence of point mutations related to persisting O6-meG-adducts. Taken together, this work reveals some new biological functions of the alternative excision repair mechanism and gives insight into the molecular mechanism of this pathway. Preliminary adduct measurements of cisplatin-induced intrastrand crosslinks indicate that this system is not restricted to the repair of O6-G-alkylations, but is also essential for the removal of Pt (GpG)-lesions from DNA. The new findings may have an impact on the therapeutic effectiveness and on the biological side effects of methylating drugs in oncology

Bio-orthogonal conjugation for “wiring” redox-active proteins/enzymes to any conductive surface

This thesis first illustrates the utility and limitations of protein film electrochemistry of adsorbed species via the analysis of a fungal Lytic Polysaccharide Monooxygenase enzyme. While a useful enzyme assay of H2O2 reduction is developed, detailed analysis of the underpinning reversible enzyme electron transfer processes is limited by background redox contributions from the carbon nanotubes which are required to stabilise the enzyme in an electroactive configuration. The rest of this thesis describes the development of methodologies for achieving electroactive immobilisation of redox proteins/enzymes onto electrode surfaces. Electroactive immobilisation of redox proteins/enzymes via covalent bonds has applications in the fabrication of biosensors and the development of green technologies/biochemical synthetic approaches, yet most published methodologies for achieving covalent immobilisation of proteins have relied on performing ligations between amine-reactive motifs on electrode surfaces and protein lysine residues. This results in many redox proteins/enzymes becoming immobilised in non-electroactive orientations or in orientations that otherwise hinder the redox enzyme’s ability to perform catalysis. In an effort to address this, methodologies for the incorporation of bio-orthogonal aldehyde motifs into proteins were investigated, and a protein immobilisation approach was developed in which hydroxylamine-functionalised electrode surfaces undergo bio-orthogonal ligation to aldehyde-functionalised proteins. As characterising and controlling surface chemistries is notoriously difficult, a new method is also under development that aims to enable covalent orientation-selective electroactive redox protein/enzyme immobilisation via the direct electro-grafting of redox proteins/enzymes that have been site-specifically labelled with aryl diazonium cations. I report the first ever usage of triazabutadienes as photocaged sources of aryl diazonium cations for use as electrode derivatisation agents in aqueous solutions at near-neutral pH, and, to the best of my knowledge, the first site-specific installations of triazabutadienes/diazonium cations onto the surfaces of proteins. I hope that further development of this method will enable the orientation-selective electroactive immobilisation of redox proteins/enzymes

Sewage sludge heavy metal analysis and agricultural prospects for Fiji

Insoluble residues produced in Waste Water Treatment Plants (WWTP) as by products are known as sewage sludge (SS). Land application of SS, particularly in agricultural lands, is becoming an alternative disposal method in Fiji. However, currently there is no legislative framework governing its use. SS together with its high nutrient and organic matter contents, constitutes some undesired pollutants such as heavy metals, which may limit its extensive use. The focus of this study therefore was to determine the total concentrations of Pb, Zn, Cd, Cu, Cr, Ni and Mn in the SS produced at the Kinoya WWTP (Fiji) and in the non-fertile soil amended with the SS at 20, 40, 60, 80% application rates and in the control (100% Soil). The bioavailable heavy metals were also determined as it depicts the true extent of metal contamination. The treatment mixtures were then used to cultivate cabbage plants in which the total heavy metal uptake was investigated. Total Zn (695.6 mg/kg) was present in the highest amounts in the 100% SS (control), followed by Pb (370.9 mg/kg), Mn (35.0 mg/kg), Cu (65.5 mg/kg), Cr (20.5 mg/kg) and finally Cd (13.5 mg/kg) and hence a similar trend was seen in all treatment mixtures. The potential mobility of sludgeborne heavy metals can be classified as Ni > Cu > Cd > Zn > Mn > Cr > Pb. Total metal uptake in plant leaves and stems showed only the bioavailable metals Cu, Cd, Zn and Mn, with maximum uptake occurring in the leaves. Ni, despite being highly mobile was not detected, due to minute concentrations in the SS treatments. Optimum growth occurred in the 20 and 40% SS treatments. However maximum Cu and Mn uptake occurred in the 40% SS treatment thereby making the 20% treatment the most feasible. Furthermore the total and bioavailable metal concentrations observed were within the safe and permitted limits of the EEC and USEPA legislations

Advanced Electrochemical Analysis for Complex Electrode Applications

This thesis has investigated several complex situations that may be encountered in electrochemical studies. Three main situations have been examined, they include the formation of polymer films on electrode surfaces during measurements, a novel nanocatalyst modified electrode surfaces, and organised carbon nanotube (CNT) structures on electrode surfaces. These have been utilised for different electrochemical applications owing to their dissimilar properties. Voltammetric techniques of cyclic voltammetry (CV), square wave voltammetry (SWV) and Fourier transformed large amplitude ac voltammetry (FTACV) have been utilised to examine these reactions. Chapter 3 reports the investigation of catechol oxidation and subsequent polymerisation through crosslinking with D-glucosamine or chitosan. Hydrogel can be formed on the electrode surface during the process, which changes the viscosity of the solution and thus affects the diffusion of chemical species. This process has been examined by several voltammetric techniques. A further examination of the chemical system has also been conducted using FTACV for the first time. Chapter 4 describes the preparation of carbon microsphere supported molybdenum disulfide. The material has been utilised as electrocatalysts for hydrogen evolution reaction (HER) in acidic media, and the performance tested by traditional linear sweep voltammetry (LSV) and advanced FTACV techniques. The FTACV technique has been used for the first time for HER processes. In addition, the synthesised particles have also been used for thermal catalytic decomposition of hydrogen sulfide, which shows a significant improvement in the conversion rate over conventional examples. Chapter 5 demonstrates the direct growth of vertically aligned CNT forests on a gold electrode. The electrochemical response of the fabricated electrode has also been examined with ferrocyanide as the redox species. Furthermore, the immobilisation of anthraquinone onto CNT forest has been attempted. The fabricated electrode was utilised as a pH sensor via CV and SWV, and both indicates a well correlated pH-potential relationship in the pH range of 2 to 12. The sensor has also been assessed by the FTACV technique.Schlumberger Gould Research Campus for Research Excellence and Technological Enterprise (CREATE) programme in Singapor

Investigation of Volatile Organic Compounds (VOCs) released as a result of spoilage in whole broccoli, carrots, onions and potatoes with HS-SPME and GC-MS

Vegetable spoilage renders a product undesirable due to changes in sensory characteristics. The aim of this study was to investigate the change in the fingerprint of VOC composition that occur as a result of spoilage in broccoli, carrots, onions and potatoes. SPME and GC-MS techniques were used to identify and determine the relative abundance of VOC associated with both fresh and spoilt vegetables. Although a number of similar compounds were detected in varying quantities in the headspace of fresh and spoilt samples, certain compounds which were detected in the headspace of spoilt vegetables were however absent in fresh samples. Analysis of the headspace of fresh vegetables indicated the presence of a variety of alkanes, alkenes and terpenes. Among VOCs identified in the spoilt samples were dimethyl disulphide and dimethyl sulphide in broccoli; Ethyl propanoate and Butyl acetate in carrots; 1-Propanethioland 2-Hexyl-5-methyl-3(2H)-furanone in onions; and 2, 3-Butanediol in potatoes. The overall results of this study indicate the presence of VOCs that can serve as potential biomarkers for early detection of quality deterioration and in turn enhance operational and quality control decisions in the vegetable industry