What are the Potential Sites of DNA Attack by N-Acetyl-p-benzoquinone Imine (NAPQI)?

Metabolic bioactivation of small molecules can produce electrophilic metabolites that can covalently modify proteins and DNA. Paracetamol (APAP) is a commonly used over-the-counter analgesic, and its hepatotoxic side effects have been postulated to be due to the formation of the electrophilic metabolite N-acetylbenzoquinone imine (NAPQI). It has been established that NAPQI reacts to form covalent bonds to the side-chain functional groups of cysteine, methionine, tyrosine, and tryptophan residues. While there have been scattered reports that APAP can form adducts with DNA the nature of these adducts have not yet been fully characterised. Here the four deoxynucleosides, deoxyguanosine (dG), deoxyadenosine (dA), deoxycytidine (dC), and deoxythymidine (dT) were reacted with NAPQI and the formation of adducts was profiled using liquid chromatography–mass spectrometry with positive-ion mode electrospray ionisation and collision-induced dissociation. Covalent adducts were detected for dG, dA, and dC and tandem mass spectrometry (MS/MS) spectra revealed common neutral losses of deoxyribose (116 amu) arising from cleavage of the glyosidic bond with formation of the modified nucleobase. Of the four deoxynucleosides, dC proved to be the most reactive, followed by dG and dA. A pH dependence was found, with greater reactivity being observed at pH 5.5. The results of density functional theory calculations aimed at understanding the relative reactivities of the four deoxynucleosides towards NAPQI are described.