Time-and dose-effect relationships of DNA adducts formation induced by nitrogen mustards in Neuro-2a cells

To identify the biomarkers of effect in DNA damage induced by nitrogen mustard treatment.

Neuro-2a cells were subjected to in vitro treatment with nitrogen mustards (HN1, HN2 or HN3). The types and levels of HN-DNA adducts were quantitatively analyzed using the multiple reaction monitoring (MRM) mode coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).

Good linearity was observed in the nine HN-DNA adduct standards within the range of 0.02 to 100 ng/mL, with coefficients of determination above 0.99. The limits of detection (LOD) and limits of quantitation (LOQ) ranged from 0.01 to 0.02 ng/mL and from 0.02 to 0.05 ng/mL, respectively. The relative standard deviations (RSD) for intra-assay and inter-assay precision were less than 15%. Such adducts as HN-N3-adenine (AN3), HN-N7-guanine (GN7), and HN-Bis-GN7 were detected in cells treated with the three nitrogen mustards. Adduct levels increased immediately after treatment and decreased 8 to 12 h later, showing a positive correlation with the dose of treatment.

By dynamically monitoring the temporal and quantitative changes of DNA adducts in Neuro-2a cells, this study confirms their potential as effect biomarkers in nitrogen mustard treatment while providing data for investigating the repair processes and mechanisms of DNA damage induced by nitrogen mustards.