Nitric oxide (NO) is a free radical ubiquitous in mammalian organisms with a key role in innate immunity. Upon activation, macrophages synthesize NO that reacts with oxygen and superoxide to form the potent nitrosating agent, nitrous anhydride (N2O3), and the strong oxidants peroxynitrite and nitrosoperoxycarbonate, respectively. N2O3 causes nitrosative deamination of DNA and RNA nucleobases, yielding xanthine (X) and oxanine (O) from guanine, hypoxanthine (I) from adenine and uracil (U) from cytosine, with the damage playing a role in the pathology of inflammation in cells and tissues. We have undertaken studies to assess the potential of deaminated RNA bases as quantitative biomarkers for modeling the chemistry of NO-induced nitrosation in vivo.  To this end, we first exposed purified total RNA from human lymphoblastoid TK6 cells to NO and O2 at constant steady-state levels of 1.7 �M and 210 �M, respectively, in a novel delivery system.  Deaminated ribonucleosides were then quantified using a sensitive HPLC-coupled mass spectrometry method. Analysis of the kinetics of nitrosative deamination of the RNA revealed that adenine was significantly less reactive than guanine, which was also apparent in DNA subjected to the same conditions.  Further, we observed concurrent formation of inosine, oxanine and xanthosine with rate constants of 5 x 104, 9 x 105 and 10 x 105 M-1s-1, respectively. These results were compared to similar studies performed by exposing cultures of TK6 cells to NO and O2 in the reactor.  We observed smaller increases in the levels of RNA deamination by at least an order of magnitude compared to purified RNA, which points to protection against nitrosative deamination in the cellular milieu.  The results of these studies provide new insights into the chemistry of inflammation-induced nitrosative deamination and allow us to develop a quantitative model for N2O3-mediated chemistry in vivo.

Kinetic analysis of nitrosative deamination of RNA in cells

244th National Meeting (2012)

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