Investigation of the abasic sites induced by hydrogen peroxide and methyl methanesulfonate in calf thymus DNA and BEAS-2B cells

The primary goals of this study were to investigate the formation of abasic sites (AP sites) induced by methyl methanesulfonate (MMS) and hydrogen peroxide (H₂O₂), and to characterize specific types of these pro-mutagenic DNA lesions in calf thymus DNA (CT-DNA), and BEAS-2B human lung normal cell line. Furthermore, these profiles were compared with those observed in leukocytes derived from healthy controls (HC), breast cancer patients (BCP) before treatment, and 5-year survivors. Results indicated that both H₂O₂ and MMS induced the concentration- and time-dependent formation of AP sites in CT-DNA. To characterize the specific types of AP sites induced by H₂O₂ or MMS, we performed AP site cleavage assay using putrescine, T7 exonuclease (T7 Exo), and exonuclease III (Exo III). Results showed that the AP sites induced by H₂O₂ in CT-DNA were predominantly 5’-and 3’-nicked AP sites and no intact AP sites were detected. By contrast, the majority of AP sites generated by MMS in CT-DNA are not excisable and are classified as residual and intact AP sites. Similar approaches were performed in human BEAS-2B cells and comparable observations were confirmed in the cell-based model. Further investigation indicated that the profile of the AP sites observed in Taiwanese HC is identical to that of BEAS-2B cells treated with H₂O₂ whereas the pattern of AP sites detected in BCP is similar to that of CT-DNA exposed to H₂O₂, suggesting that these AP sites were produced primarily through reactive oxygen species (ROS) generation. More than 70 % of the AP sites in leukocytes derived from BCP were 5’-nicked and residual AP sites. Furthermore, the characteristics of the AP sites detected in 5-year survivors are comparable with the ones in HC by using putrescine cleavage assay. Overall, we speculate that deficiency in the DNA repair cascade may play a role in mediating the formation of specific types of AP sites detected in BCP.