One-electron oxidation of DNA and inflammation processesJean Cadet, Thierry Douki & Jean-Luc Ravanat

Nitrosoperoxycarbonate anion, a reactive species generated in inflammation processes, is able to specifically oxidize guanine bases with a sequence selectivity that is almost opposite from that usually observed for one-electron oxidants.

Evidence is accumulating for the involvement of oxidative DNA damage in various pathologies, including cancer, and in physiological processes such as aging and inflammation. For example,8-oxo-7,8-dihydroguanine (8-oxoGua), a ubiq-uitous oxidation product of DNA, has been shown to induce deleterious G→T transversions if the lesion is not repaired1. Substantial atten-tion paid to this area during the last decade has led to the elucidation of the DNA degradation pathways2,3 mediated by reactive oxygen (•[OH], [O2]•– and 1O2) and nitrogen species ([NO]•, ONOO–) that may be released during the respi-ratory burst or produced upon acute exposure to oxidative stress agents such as ionizing radia-tion4 and the UVA component of solar light5. Another major DNA oxidation process involves electron abstraction from nucleotide bases (and eventually the 2-deoxyribose moiety) initiated by highly energetic photons of gamma and X-ray radiation. However, one-electron oxidation of nucleobases, and particularly of guanine, which has the lowest ionization potential among DNA constituents, could be achieved in a much milder way by reactive species that may be bio-logically relevant2,4,6. Whereas high-intensity laser UV pulses typically oxidize nucleobases by two-photon ionization processes, photosensitiz-ers such as riboflavin, once excited, can abstract

one electron from DNA6. In these cases guanine is either the primary target or the final sink of a hole-transfer process that is dependent on DNA sequence context and occurs over a few to several dozens of base pairs from a remote one electron–oxidized base or sugar moiety7–9.

Experimental and theoretical studies have sug-gested that tracts of adjacent guanines (and particularly the 5′ base in such tracts) are more susceptible to one-electron oxidation, most likely as a result of charge migration to the sites of lowest ionization potential10.

Jean Cadet, Thierry Douki and Jean-Luc Ravanat are at the Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique-Unité Mixte de Recherche-E no. 3 (Commissariat à l’Energie Atomique/Université Joseph Fourier), Département de Recherche Fondamentale sur la Matière Condensée, CEA/Grenoble, F-38054 Grenoble Cedex 9, France.e-mail: jean.cadet@cea.fr

Figure 1 Main expected degradation pathways of the guanine moiety of DNA, which are mediated by the inflammation processes involving the release of ONOOCO2

– or its decomposition radical species, including [CO3]•– and [NO2]•. The one-electron oxidation pathway that may be specifically induced by photoexcited riboflavin is in green, whereas the nitration pathway is in red. The 8-nitroguanine lesion is likely to be converted into an abasic site, another alkali-sensitive site, owing to the lability of theN-glycosidic bond. It should be noted that 8-oxoGua and FapyGua have been shown to be generated2,4 in cellular DNA upon exposure to oxidative stress agents including ionizing radiation via the generation of •OH radicals. iNOS, inducible nitric oxide.

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What accounts for this altered sequence selectivity in nitrosoperoxycarbonate-medi-ated oxidation of DNA? At present, the authors hypothesize that either DNA secondary struc-ture or the negative charge of the oxidant may account for the observed paradoxical sequence selectivity. Obviously, these hypotheses must be tested by further experimental work. Future studies may determine whether other related lesions generated by one-electron oxidation can be detected in cellular DNA. For example, 8-nitroguanine, the product of [NO2]• reaction with DNA14 (Fig. 1), may serve as a confirma-tory marker for ONOOCO2

– oxidation. A large body of information on the reactions of the gua-nine radical cation has been derived from model studies2,6. It has been shown that nucleophilic addition at C8 of either a water molecule or the free amino group of a lysine residue gives rise to various adducts: 8-oxoGua and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) are the main final products of attack by water2,4,6, whereas a protein cross-link is formed by reaction of a lysine amino group at C8 (ref. 15). However, competitive deprotonation of the guanine radical cation generates the highly alkali-labile 2,2,4-triaminooxazolone (oxazo-lone)2. Methods for the sensitive detection and quantification of various oxidative DNA lesions, including HPLC-ESI MS-MS2,4,6, have greatly facilitated our understanding of DNA damage and repair. Future efforts will be required to design a sensitive and specific method for map-ping, at the sequence level, the base damage gen-erated in cellular DNA by ONOOCO2

– or other

In a recent comprehensive study, Dedon and co-workers11 observed a different specificity in the distribution of oxidized-guanine bases in duplex DNA upon exposure to nitrosoper-oxycarbonate anion (ONOOCO2

–). This one-electron biochemical oxidant is the product of the reaction of carbonate with peroxyni-trite (ONOO–), which is formed by addi-tion of superoxide radical to nitric oxide12. Nitrosoperoxycarbonate is produced in macro-phages during inflammation processes, in which all of the reactive-intermediate precursors are generated in relatively high yields (Fig. 1). Model studies have shown that guanine is the exclusive target of ONOOCO2

–, or more likely of [CO3]•–, a highly reactive inorganic radical anion that is released (along with the less reactive [NO2]• radical) upon homolysis of the O-O bond of nitrosoperoxycarbonate12. Interestingly, hot spots for base modifications, which the authors revealed as either piperidine-labile sites or formamidopyrimidine-sensitive lesions, were found to be mostly located at poorly oxidizable G-C sites11. These results stand in direct contrast to those of previous studies showing that gua-nine bases with the lowest ionization potential (usually the 5′ guanine in a doublet or triplet of guanines) are highly susceptible to one-electron abstraction from photoexcited riboflavin10. The unusual sequence reactivity of ONOOCO2

– in duplex DNA is also supported by the observa-tion of mutagenic hot spots in similar sequence contexts13 and by the authors’ observation that oxidation of single-stranded oligonucleotides was not sequence dependent11.

reactive species that are produced during inflam-mation. It is also possible that proteins that are bound to DNA modulate the effects of charge transport along the oligonucleotide chain7,8 and therefore modify the hot-spot pattern of guanine oxidation. The present findings11 are likely to stimulate both further intensive work on one-electron oxidation reactions within cel-lular DNA and also assessment of their biologi-cal impact by taking advantage of the possible induction of the ONOOCO2

– mediator during inflammation.

1. Bjelland, S. & Seeberg, E. Mutat. Res. 531, 37–80 (2003).

2. Cadet, J., Douki, T., Gasparutto, D. & Ravanat, J.-L. Mutat. Res. 531, 5–23 (2003).

3. Neeley, W.L. & Essigmann, J.M. Chem. Res. Toxicol. 19, 491–505 (2006).

4. Pouget, J.-P. et al. Radiat. Res. 157, 589–595 (2002).

5. Cadet, J., Sage, E. & Douki, T. Mutat. Res. 571, 3–17 (2005).

6. Douki et al. Top. Curr. Chem., 236, 1–25 (2004).7. Giese, B. Curr. Opin. Chem. Biol. 6, 612–618

(2002).8. Delaney, S. & Barton, J.K. J. Org. Chem. 68, 6475–

6483 (2003).9. Schuster, G.B. & Landman, U. Top. Curr. Chem. 236,

139–161 (2004).10. Saito, I. et al. J. Am. Chem. Soc. 120, 12686–12687

(1998).11. Margolin, Y. et al. Nat. Chem. Biol. 2, 365–366

(2006).12. Dedon, P.C. & Tannenbaum, S.R. Arch. Biochem.

Biophys. 423, 12–22 (2004).13. Kim, M.Y., Dong, M., Dedon, P.C. & Wogan G.N. Chem.

Res. Toxicol. 18, 76–86 (2005).14. Misiaszek, R., Crean, C., Geacintov, N.E. & Shafirovich,

V. J. Am. Chem. Soc. 127, 2191–2200 (2005). 15. Perrier, S. et al. J. Am. Chem. Soc. 128, 5703–5710

(2006).

NO place to hideNathaniel Finney

Nitric oxide (NO) regulates a broad range of biological processes, yet many intracellular details of NO-mediated processes remain hidden. A new fluorescein derivative capable of direct detection provides a key advance in determining NO function in vivo.

Nathaniel Finney is in the Organic Chemistry Institute, University of Zurich, Winterthurerstrasse 190, Zurich CH-8057, Switzerland.e-mail: finney@oci.unizh.ch

NO is an important small molecule in the cell, serving as both a direct-response ele-ment and an initiator of secondary activi-ties. Numerous proteins that produce or bind NO have been identified, as have pro-teins and small molecules (cyclic guanosine

monophosphate, or cGMP, in particular) whose production is regulated by NO. The impact of these discoveries has been tremen-dous, and the 1999 Nobel Prize in Physiology or Medicine recognized pioneering work in the field1–3. Despite the significance of NO as a cellular player, our knowledge of its behavior is limited. Of particular importance are the following questions: where and when is NO produced? Where is it localized? How long does it persist? The difficulty in answering these questions arises from the limited tools available for determining exactly where and

when NO is present. Here, Lippard and co-workers demonstrate the utility of a new turn-on fluorophore for immediate and selective NO detection4.

As NO is a colorless diatomic species, there have been no viable methods for its direct intracellular detection and thus all work on this molecule so far has necessarily relied on indirect detection or inference. In the early stages of the field, researchers made obser-vations based on indirect responses by, for example, controlling expression of NO syn-thases (NOS), following the NO-dependent

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