Development of an in vitro genotoxicity screening assay: combining different genotoxic endpoints

Genotoxic agents are a major threat to the integritiy of chromosomes and viability of cells, specially if the damage is not repaired, because it can lead to chromosome instability, cell cycle arrest, cell dysfunction, induction of apoptosis or carcinogenesis. For genotoxicity, two main endpoints are gene mutations and chromosome aberrations; the latter can either be structural (clastogenic) or numerical (aneugenic). There are several in vitro and in vivo genotoxicity tests available. However, none of these tests are capable to detect both endpoints of genotoxicity simultaneously. The pUR288 mouse model, carrying the lacZ transgene on a plasmid vector, used for this thesis, is in theory capable of detecting large deletions (>500 base pairs) in addition to small deletions and point mutations. Since it was not previously demonstrated that the pUR288 mouse model is indeed sensitive enough to detect clastogens, a second mouse model was used, the Rad54/Rad54B deficient mice. This model has a defect in the repair of chromosomal aberrations, caused by clastogens. Determination of the lacZ mutant frequency (lacZ MF) as well as hybridization studies showed that the Rad54/Rad54B deficient mice are only moderately more sensitive towards clastogens as compared to pUR288 mice. It was concluded that, although still a promising test, both in vivo models were not sensitive enough to detect the effect of exposure to clastogens. In contrast to the in vivo studies, the in vitro studies using mouse embryonic fibroblasts (MEFs) derived from pUR288 and Rad54/Rad54B mice, have shown a dose-dependent induction in the lacZ MF and the micronuclei frequency (for the MNT) after exposure to both mutagens as well as clastogens in both MEFs models. Moreover, MMC-treatment resulted in a higher lacZ MF in Rad54/Rad54B MEFs compared to pUR288 MEFs. This genotype effect after MMC-exposure, also shown in microarray studies, was due to an impaired DNA damage response in MMC-treated Rad54/Rad54B MEFs. This will lead to a weaker DNA repair response and therefore a larger percentage of the cells will carry a lacZ mutation, hence a higher lacZ MF in the Rad54/Rad54B MEFs compared to pUR288 MEFs. Since MMC induces predominantly DNA crosslinks, the obtained results suggests that the Rad54 and/or Rad54B genes are involved in DNA crosslink damage recognition and repair. As a conclusion, the lacZ MF assay using pUR288 MEFs is able to detect both mutagens as well as clastogens simultaneously. Using this in vitro test can be of great advantage, since this can lead to a reduction in the number of tests needed. The reduction in time and costs and the increase in the sensitivity make this in vitro lacZ assay a good alternative for existing tests for screening of genotoxic compounds. However, before this test can be used as a high-throughput screenings test, it needs to be validated with a wider range of genotoxic compounds, non-genotoxic compounds, non-carcinogens and most importantly also low potency genotoxic compounds. Then it can be assessed whether this assay is reproducible and whether the results obtained can replace existing genotoxicity assays