REACTIVE OXIDATIVE SPECIES (ROS)  

1. Laser spectroscopic studies of free radical reactions in DNA

2. Chemical identification of reaction products in DNA 

(This research is being carried out by Research Professor Vladimir Shafirovich in the Chemistry Department Laser Spectroscopy Facility)

The reactive oxidative species can contain both nitrogen and oxygen, or only oxygen atoms.  Some examples of ROS molecules, and their general importance are summarized in Figure 1. Our research is focussed on reactions of small ROS molecules and radicals with biological molecules, specifically on oxidative DNA damage initiated by reactive oxygen and nitrogen species. Many of these species are formed during normal metabolic activity.  The concentration levels are particularly elevated under conditions of oxidative stress associated with chronic inflammation, infections and other decease (Figure 1). The overproduction of reactive oxygen and nitrogen species gives rise to the oxidation of cellular macromolecules, including DNA. Many oxidative modifications of DNA (so-called DNA lesions) are mutagenic and their enhanced formation is linked to increased risk of cancer development. In DNA the attack of reactive oxidative species is focused on guanine, the most easily oxidizable nucleic acid base. Our central hypothesis is that neutral guanine radicals derived from the one-electron oxidation of guanine by strong oxidants are key intermediates for the generation of stable DNA lesions. To test this hypothesis, we have developed a very efficient technique for the site-selective one-electron oxidation of guanine in DNA using carbonate radicals generated by intense laser pulses. This technique allows us to monitor, in real time, the formation and fates of guanine radicals in DNA. We begin by a systematic mapping of the reaction pathways of guanine radicals and the reaction products of their transformation in DNA leading to the formation of stable DNA lesions. Recently, we have studied the reactions of the ROS NO₂·, which readily combines with guanine radicals to form the unstable 8-nitroguanine adduct (1).   In addition to 8-nitroguanine, another more unusual DNA lesion is formed: the 5-guanidino-4-nitroimidazole adduct.  This adduct is stable and can be synthesized photochemically in high yield (Figure 2).  The biological effects of these lesions are currently being investigated.

Another focus of our research is the aqueous chemistry of nitroxyl (HNO) and its anion generated by UV laser flash photolysis of Angeli’s salt. In collaboration with Dr. S. Lymar (Brookhaven National Laboratory) we showed that the ground state of nitroxyl anion is a triplet,  and that HNO in its singlet ground state has much lower acidity, pKa ~ 11.4, than previously believed (2). According to this novel concept, if nitrogen(+1) is produced in biological systems, it should be present in the form of singlet HNO. As a small neutral molecule, singlet HNO should be able to penetrate through biological membranes and its role in biology and medicine may be very important.

References

1.  Shafirovich, V., Mock, S., Kolbanovskiy, A., Geacintov, N. E. Photochemically catalyzed generation of site-specific 8-nitroguanine adducts in DNA by the reaction of long-lived neutral guanine radicals with nitrogen dioxide. Chem. Res. Tox. 15, 591-597 (2002).

2. Shafirovich, V., Lymar, S. Nitroxyl and its anion in aqueous solutions: Spin states, protic equilibria, and reactivities toward oxygen and nitric oxide. Proc. Natl. Acad. Sci. USA, 99, 7340-7345 (2002).

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