title: Assessment of drug induced genotoxicity in mammalian cells and the contribution of topoisomerase II inhibition
Creators: name: Fellows, Michael
Publisher: name: University of Liverpool Repository
Year: 2015

description: There is a common belief that mammalian cell gene mutation assays are prone to false positives, thus questioning the relevance of these tests in regulatory screening paradigms and the mechanisms responsible for these uninterruptable results. False positives can lead to unnecessary animal testing and delays in the development of efficacious new medicines. The initial aim of this thesis was to put into perspective the rate of positives and firstly to consider the extent off target aneugenicity (chromosome loss or gain) may contribute to this rate. Secondly the contribution of topoisomerase II poisoning and its relationship to genotoxicity was considered. Topoisomerase II maintains DNA topology by inducing transient breaks in one strand so a second strand can pass. Chemicals that interact with the enzyme (topoisomerase II poisons; e.g. the antibiotic gemifloxacin and the chemotherapeutin etoposide), yield topoisomerase II bound DNA cleavage complexes, making breaks permanent, leading to mutation or cell death. Structurally, topoisomerase II poisons are diverse, so their genotoxicity is difficult to predict. To estimate the incidence of positives seen in pharmaceutical research, a retrospective review of data from 10 years of mouse lymphoma assays (MLA) conducted at AstraZeneca was undertaken. This showed that the rate of unexplainable positives was only 5%, vindicating the use of the test in screening paradigms. Consideration was then made of what mechanisms might contribute to this 5%. Aneugenicity was considered but it was shown that the MLA was a poor screen, failing to identify 7 known anuegens. To gain a better understanding of the relationship between topoisomerase II and genotoxicity, assays to assess enzyme poisoning were examined, including the ability of the cell free decatenation assay to predict the results of the in vitro micronucleus test. However, even when combined with an estimate of cellular uptake the predictivity was low. Assays to investigate topoisomerase II poison / DNA cleavage complexes in vivo were then investigated. The TARDIS and ICE assays both use antibodies to target topoisomerase II bound in the complex. TARDIS was found to be insensitive, failing to identify cleavage complex formation with gemifloxacin. Using the ICE assay, cleavage complex formation was seen for etoposide (0.1 μmol/L; FITC intensity 3.53 ± 0.79) and gemifloxacin (100 μmol/L; FITC intensity 3.37 ± 0.86), but not at equivalent concentrations to those inducing micronuclei (MN) (0.03 μmol/L etoposide; MN/1000 6 ± 2.6 and 10 μmol/L gemifloxacin; MN/1000 5 ± 3.4), thus questioning assay sensitivity, or suggesting a role for other mechanisms of genotoxicity e.g. reactive oxygen species (ROS). The hOGG Comet assay showed that neither etoposide nor gemifloxacin induced ROS related genotoxicity. Improvements to the sensitivity of the cleavage complex assays were made by preparation of mouse specific antibodies, but TARDIS was still unable to identify gemifloxacin. This work also suggested that when developing antibodies for DNA bound topoisomerase II, the n-terminus of the enzyme should be targeted. Over the last 4 years, emerging data linked the topoisomerase IIβ isoform to genotoxicity. As research within this thesis had investigated the activity of topoisomerase IIα, this may have explained the difficulty encountered equating topoisomerase II poisoning to genotoxicity. Following siRNA knockdown of topoisomerase IIα, the genotoxicity of etoposide and gemifloxacin was investigated. It was shown that for 0.3 μmol/L etoposide, topoisomerase IIα knockdown of 42% (± 2%) was associated with a reduction in micronuclei of 49% (± 9.7%). For 30 μmol/L gemifloxacin, topoisomerase IIα knockdown of 37% (± 9.5%) was associated with a reduction in micronuclei of 48% (± 0.2%). This was the first time such direct relationships had been demonstrated between the alpha isoform and genotoxicity. In conclusion, the predictivity of the MLA was confirmed but it was clear the assay is not a suitable screen for aneugenicity. The relative sensitivity of assays to measure topoisomerase II poisoning was shown and linked to genotoxicity. Whilst it was not possible to demonstrate cleavage complex formation at concentrations below which genotoxicity was seen, this was likely due to the insensitivity of the assays used rather than topoisomerase II poisons having other genotoxic mechanisms. For the first time the link between topoisomerase IIα and genotoxicity was confirmed and use of knockdown cells holds real promise as a tool for investigating off target topoisomerase II poisoning.