title: Optimization and utilization of the comet assay for differentiation of various types of DNA damage
Creators: name: Hong Wu, Jian
Publisher: name: University of Liverpool Repository
Year: 2023

description: Mutagenic chemicals exert their effects through various mechanisms resulting in different types of DNA damage. For example, 7,l2-dimethylbenz[a]anthracene (DMBA), a potent skin carcinogen, is metabolically activated to reactive intermediates that bind to DNA to form both stable adducts and apurinic (AP) sites. Whether stable DNA adducts or AP sites are more responsible for tUmour initiation by DMBA is controversial. 8-methoxy-psoralen (8-MOP) plus UVA (pUVA), commonly used for the treatment of hyper-proliferative skin disorders, has been found to be associated with an increased risk of squamous cell cancer. Interstrand crosslink (ICL) formation by PUVA treatment is considered the major factor contributing to the carcinogenesis. However, it is recently suggested that PUVAinduced oxidative damage may playa role. Therefore, it is of importance to detect and subsequently characterize different types ofDNA lesions induced by mutagenic chem.icals to. obtain a better understanding of their potential contributions to carcmogeneSIS. This project aimed to: i) establish a technique which permits rapid and accurate measurement of various types of DNA lesions, such as AP sites, stable bulky adducts, ICLs, and oxidative DNA damage; ii) apply this technique to investigate the proportion of stable adducts and AP sites generated by DMBA in human keratinocyte cell line (HaCaT) and in human primary keratinocytes; iii) utilize the technique to study PUVA-induced ICLs and oxidative DNA damage in HaCaT cells and in reconstructed human epidermis; and iv) employ the method to assess PUVAinduced ICL formation and repair in Fanconi anaemia (FA)-like Chinese hamster cell mutants. The results indicated that AP sites were induced in both HaCaT cells and in primary human keratinocytes treated with DMBA, at a dose range of 1O~M to 1OO~M, in a dose-response manner, with HaCaT cells showing approximately 2-fold higher AP site formation than the primary human keratinocytes. Attempts to utilise the alkaline comet assay to measure stable adducts with the lesion-specific enzyme UvrABC were not successful. PUVA-induced ICLs were measured using the alkaline comet assay with a post-lysis y-irradiation. A clear dose-dependent response ofHaCaT cells to PUVA exposure is observed with a combination of a fixed UVA dose at O.05J/cm2 and a dose of 8MOP ranging from lO~M to lOO~M. Results also indicated that the ICL repair was concentration-dependent. All attempts to establish the comet assay in the reconstructed human epidermis failed due to the inability to obtain single cell suspensions from the tissues. It was also found that the comet assay is insensitive in detecting oxidative damage without lesion specific enzymes. The analysis of induction and repair of PUVA-induced ICLs in the putative FaneL defective Chinese hamster mutant DEB1 demonstrated that it exhibits slow repair of these lesions compared to its wild-type parent V79. Repair was also examined in DEB1 cells transfected with the wild-type and mutant (point mutation in ubiquitin ligase motif) versions of human FANCL cDNA. The former showed restoration of the rate ofICL repair, whilst the mutant cDNA only conferred partial correction.