Abstract
Abstract
The standard alkaline comet assay is a straightforward and cost-effective genotoxicity test commonly used in genetic toxicology to detect strand breaks and alkali-labile sites in DNA. Furthermore, with certain modifications, other altered bases such as oxidised and alkylated bases and cross-links can also be detected. However, it cannot detect bulky DNA adducts, which are a significant DNA lesion formed by the attachment of a chemical to DNA. Given that bulky adducts are mainly repaired by nucleotide excision repair (NER), the modification of the comet assay by adding blockers of the repair synthesis stage of NER, lead to the accumulation of incision intermediates, i.e. DNA breaks. Although this modification has been proposed as a method for detecting bulky adducts, the literature suggests that it may also detect other DNA lesions and that it has not yet been validated. To study the DNA lesion specificity of the in vitro comet assay in combination with DNA repair inhibitors, we tested a set of seven genotoxic compounds with different mechanisms of action and two non-genotoxic compounds, in TK6 cells. The assays were performed in the absence or presence of the inhibitors, specifically the combination of hydroxyurea (HU) and cytosine arabinoside (Ara-C), or aphidicolin (APC). The panel of genotoxic compounds included a bulky DNA adducts inducer (BPDE), two alkylating agents (methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS)), two oxidising agents (KBrO₃ and H₂O₂), and two cross-linking agents (cisplatin (CisPt) and mitomycin C (MitC)). The MTS and comet assays were performed to determine cytotoxicity and DNA damage, respectively. Results showed that this approach increases the sensitivity of the comet assay for detecting DNA damage, but not specifically bulky DNA adducts.