Electron paramagnetic resonance studies of the reaction of aryl radicals with nucleic acids and their components

Abstract

Aryl radicals may be responsible for the DNA damage observed in both cellular systems and isolated DNA exposed to a number of systems (such as benzoyl peroxide or arenediazonium ion/metal ion couples) which are believed to be capable of generating such species, though it is unclear how this damage arises. EPR spectroscopy in conjunction with spin trapping [using 2-methyl-2-nitrosopropane (MNP)] has therefore been utilised to study the mechanism and sites of attack of aryl radicals (generated by treatment of the corresponding diazonium ions with Fe^{2 +}–EDTA or Ti³⁺) on DNA, RNA and their components. The results obtained suggest that, for the pyrimidine nucleobases, nucleosides and nucleotides, the major mode of reaction is addition to the alkenic C⁵–C⁶ double bond of the base moiety, though significant yields of other radicals, believed to arise from abstraction of hydrogen at the sugar moiety, are also observed with some of the nucleosides and nucleotides. Radicals arising from attack on adenosine 5′-triphosphate have also been detected. The increased yield of sugar-derived radicals in these reactions, when compared with those previously reported for (electrophilic) HO˙ and alkoxyl radicals, is in accord with the known nucleophilic nature of aryl radicals.

Studies with the polyU, polyA·polyU, polyC, RNA and DNA suggest that aryl radicals also damage these macromolecules, though the broad nature of the spectral lines and interference from the signal of the arylradical adduct to the spin trap prevent detailed identification of the site(s) of attack. For DNA and RNA the signals obtained are pH dependent. At pH 7.4 both slowly tumbling and rapidly tumbling spin adducts are observed with tRNA, which is consistent with the spin trapping of both large, substrate-derived, radicals and low-molecular-mass fragments, possibly from the sugar moieties. With DNA only spectra from rapidly tumbling species are seen at pH 7.4; these are again believed to be due to the presence of low-molecular-mass material. The formation of these small fragments suggests that aryl radicals are capable of generating strand breaks in nucleic acids, and therefore that such species may be responsible for the genetic damage observed in cells exposed to aryl-radical-generating systems.