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RESEARCH PRODUCT

[33] Use of repair endonucleases to assess DNA damage by peroxynitrite

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Publisher Summary This chapter discusses the use of repair endonucleases to assess DNA damage by peroxynitrite. Repair endonucleases allow a convenient quantification of various types of oxidative modifications induced by peroxynitrite, both in cultured cells and in cell-free DNA. The high sensitivity of the assays allows highly ectotoxic exposure conditions to be avoided—as well as the generation of secondary DNA modifications—that often become a problem at high levels of damage because primary DNA oxidation products can be orders of magnitude more reactive than the original bases, as demonstrated for the reaction of 8-hydroxyguanine with singlet oxygen. The ratio of the various types of modification (damage profile) is a fingerprint of the reactive species directly responsible for the DNA damage. Thus, the damage profiles induced by hydroxyl radicals and peroxynitrite are clearly distinguishable from each other, but independent of the reactions used to generate the two species. The fingerprint character of the two damage profiles is further supported by the observation that scavengers and antioxidants influence the various types of modification similarly—that is, do not change the ratios.