The use of DNA repair inhibitors and the comet assay-an overview

The standard comet assay detects DNA strand breaks and alkali-labile sites, but these lesions are nonspecific. They may result directly from genotoxic agents or arise as intermediates during the repair of other DNA damage, such as oxidized bases or bulky DNA adducts. Different approaches have been developed to generate or trap these repair intermediates, making them detectable with the comet assay. Recently, the combination of the comet assay with DNA repair inhibitors like hydroxyurea and cytosine arabinoside has been proposed to detect bulky DNA adducts. These lesions are mainly repaired through nucleotide excision repair, a process that transiently produces strand breaks when damaged oligonucleotides are excised. Normally, these intermediates are rapidly repaired by DNA resynthesis and ligation. However, by inhibiting this repair step, strand breaks persist and can be detected by the comet assay. This strategy has been applied in various fields, including genotoxicity testing, environmental toxicology, human biomonitoring, and studies on DNA repair kinetics. This review focuses specifically on the use of hydroxyurea, cytosine arabinoside, and aphidicolin in in vitro experiments to evaluate the utility and specificity of this method for detecting different types of DNA lesions. Notably, in ~70% of studies reviewed, the inclusion of DNA repair inhibitors led to a significant increase in DNA damage, highlighting the added value of this approach. However, although the method enhances sensitivity to bulky adducts, it also responds to other types of damage, such as those induced by alkylating or oxidative agents.