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The DNA damaging activity of RSU 1069 and seven of its analogues (RSU 1131, RSU 1164, RSU 1150, RB 7040, RSU 1172, RSU 1137 and RSU 1170) plus misonidazole and CB 1954 were investigated using the SOS-Chromotest The SOS-Chromotest is a genotoxicity assay that monitors the induction of the SOS response, which is induced in response to DNA damage. The strains used were PQ37, which possesses a uvrA mutation and is deficient in UvrA excinuclease activity, and PQ35, which is uvr and UvrABC excinuclease conpetent. These strains were exposed to the compounds being investigated under both oxic and hypoxic conditions. The results showed that RSU 1069 and some of its analogues were more active than misonidazole under both oxic and hypoxic conditions. This increase was due to their aziridine side-chains. With the exception of RSU 1137 and RSU 1170 all of the compounds showed altered SOS induction activities between oxic and hypoxic conditions. This alteration was shown to correlate with increased reduction of their nitro-groups under hypoxia. There was a difference in the hypoxic activities of RSU 1069 and some of its analogues between the uvrA-strain and the uvr -strain. With the uvrA-strain RSU 1069 showed decrease activity under hypoxia compared to oxia, whereas, the converse applied with the uvr -strain. This was interpreted to mean that RSU 1069 caused some damage that required an active UvrABC excinuclease to produce an SOS response. It has been proposed that this damage takes the form of DNA crosslinks. RSU 1137 showed insignificant SOS induction and this was demonstrated to be due to its nitro-group not being reduced. It was suggested that the ring opened aziridine side chain of RSU 1137 in some way inhibited its bioreduction. The order of activity of the drugs for SOS induction activities did not correlate with that for their toxicities. This indicated that DNA lesions other than, or in addition to, those responsible for cytotoxicity induced the SOS response. The DNA damaging activity and mutagenicity of RSU 1069 was also investigated using Ml3 phage rfDNA. Radiation reduced RSU 1069 was shown to produce some relatively long lived product that was more active towards DNA than unreduced RSU 1069, as judged by phage survival. Unreduced RSU 1069 was shown to be non-mutagenic, producing mutation rates under 1.5 times background level. The effect of strict hypoxic conditions upon the SOS response was investigated using the SOS-Chromotest with the uvrA tester strain. The results showed that the SOS response was induced under strictly anaerobic conditions in E. coli but that the response was altered compared to that obtained aerobically. The nature of the alteration was not determined as six different compounds, with five different modes of action, were used as SOS-inducers and all showed different types of response under hypoxic conditions.

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Methotrexate is a folic acid analogue widely used as a chemotherapeutic agent. It is known to be a potent inhibitor of the enzyme dihydrofolate reductase, therefore, perturbing intracellular pools of purine and pyrimidine bases for DNA synthesis, as well as pools of reduced folates used in a variety of metabolic reactions. It has been postulated, and subsequently widely accepted, that methotrexate kills cells by perturbing the intracellular ratio of dUTP:dTTP thereby leading to dUMP misincorporation into DNA. This would initiate an excision repair pathway designed to rid cellular DNA of this aberrant base. However, because of the imbalance of nucleotide pools, dUMP may well be re-incorporated during repair thus initiating a futile cycle of dUMP misincorporation and repair eventually leading to single-strand breaks in the DNA. From the results presented in this thesis, no evidence for dUMP misincorporation could be found in the two human cell lines studied (HeLa and CCRF-HSB2), despite the drug exhibiting dose-dependent cytotoxicity to both cell lines. This was true after a variety of methotrexate treatment times and at two different drug concentrations. Subsequent analysis of the drug treated cells, using the nucleoid sedimentation technique, for evidence of single-strand breaks in DNA yielded some anomalous results. Single-strand breaks, in the form of slower sedimenting nucleoids, were easily detectable after exposure of cells to low doses of methotrexate. However, treatment with higher doses resulted in the creation of faster sedimenting nucleoids. Subsequent analysis using other techniques showed that this faster sedimentation was occurring in the presence of DNA single-strand breaks. Collaborative work involving electron microscopy revealed methotrexate induced gross morphological changes in chromatin structure. Analogies with other unrelated anti-tumour agents interacting with topoisomerase enzymes are discussed.

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