The Dedon Lab

Chronic inflammation is strongly associated with many different forms of cancer. The link between cancer and inflammation may involve the secretion of reactive chemical species from phagocytes (Fig. 1) that contribute to the elimination of infectious agents. Of these inflammatory chemical mediators, nitric oxide (NO·) is one of the most studied and important biological molecules and is produced at relatively high rates by activated macrophages. Nitric oxide can diffuse into the surrounding host tissue and react with oxygen to generate nitrous anhydride (N2O3), a powerful nitrosating agent most likely responsible for DNA and RNA nucleobase deamination in vivo. Although, evidence for the emergence of deaminated nucleobases has been observed after in vitro exposure of deoxynucleosides, oligonucleotides and DNA plasmids to NO· and O2, studies revealed limited reactivity of cellular DNA toward deamination during NO·-related stress in vivo.

Figure 1: Reactive oxygen and nitrogen species produced during inflammation [Dedon et al. (2004) Arch. Biochem. Biophys. 423: 12-22].

Anticipating that the helical structure of DNA most probably shields nucleobases from reaction with N2O3, we have begun to refocus biomarker development from DNA bases to the more solvent-exposed free nucleotides and explore the cellular nucleotide pool as a potential target for nitrosative deamination among other products arising from exposure to chemical mediators of inflammation (Fig. 2). The first step is to develop a sensitive analytical liquid chromatography/mass spectrometry (LC/MS) method to quantify the deaminated nucleotides either in solution or in cell extracts.

The analytical method will then be applied to characterize the kinetics of in vitro deamination of pure nucleotides exposed to biologically relevant concentrations of N2O3 in a novel NO· delivery system (Fig. 3) recently developed by the Deen Group in MIT. The NO· delivery system and analytical method will also be employed to monitor the kinetics of nitrosative deamination of nucleotides in E. coli cells and human TK6 B-lymphoblastoid cells exposed to the same N2O3 concentrations.

Figure 3: Schematic of Silastic tubing NO˙ delivery system [Wang et al. (2003) Ann. Biomed. Eng. 31: 65-79].

The previous in vitro and in vivo experimental measurements will be combined to provide an estimate for the actual N2O3 concentration that nucleotides are actually exposed to in the cell cytosol under conditions similar to those at sites of inflammation in humans.