Nucleobase deamination as a biomarker of inflammatory processes

Abstract

(cont.) kinetic studies. Moderate increases ([approximately]30%) in nucleobase deamination products were observed in the SJL mice bearing the RcsX tumor, but the biological meaning of these increases awaits studies of DNA repair kinetics. dO was not detected in any study at levels above 5 per 10⁸ nt, leading to the prediction that it will not be present at significant levels in inflamed tissues in humans. As a complement to the LC-MS method for the quantification of nucleobase deamination products, enzymatic probes were also developed for oxidative and nitrosative DNA lesions. These probes would not only allow differential quantification of the two types of DNA damage, but would also allow the lesions to be mapped in any DNA sequence by coupling their activity with the technique of ligation-mediated PCR. As an extension of the biomarker study, the effects of ONOO⁻ dose and dose-rate on the DNA damage and mutations induced in the supF gene were investigated. The observations suggest that both the dose and dose-rate at which a genetic target is exposed to ONOO⁻ substantially influence the damage and mutational response and these parameters will need to be considered in assessing the potential effects of ONOO⁻ in vivo. Finally, an extended study using the analytical method developed in this thesis yielded results in E. coli consistent with a new paradigm: perturbations of nucleobase metabolism may lead to incorporation of the purine precursors hypoxanthine (I) and xanthine (X) into DNA. This can be regarded as another endogenous process causing DNA damage that may lead to human diseases such as cancer ...

The objective of this thesis project was to develop nucleobase deamination products as biomarkers of inflammation and to study the role of these DNA lesions in the pathophysiology of inflammation-induced carcinogenesis. The basis of this research is the epidemiological evidence that chronic inflammation is associated with an increased risk of cancer, yet the link between the inflammatory process and the development of cancer has eluded definition. Biomarker development began with the establishment of a sensitive liquid chromatography-mass spectrometry (LC-MS) method to quantify four of the nucleobase deamination products: 2'-deoxyxanthosine (dX) and 2'-deoxyoxanosine (dO) from the deamination of dG; 2'-deoxyuridine (dU) from dC; and 2'-deoxyinosine (dl) from dA. The analytical method was then validated and tested with both in vitro and in vivo studies involving quantification of nucleobase deamination products in isolated plasmid DNA and human lymphoblastoid cells exposed to nitric oxide (NO)̇ at controlled physiological concentrations. Finally, the formation of nucleobase deamination products was analyzed in SJL/RcsX mice, an established mouse model of NO ̇over production. This set of studies revealed several important features of the nitrosative chemistry of NO ̇derivatives. The in vitro formation of dX, dl and dU was found to occur at nearly identical rates (k = 1.2 x 10⁵ M⁻¹s⁻¹). Low levels of nucleobase deamination products were formed in cells exposed to NO,̇ which suggests that cellular factors significantly influence the nitrosative chemistry. However, cellular glutathione (GSH) was found to play a smaller role than expected, considering the effects of GSH on steady-state concentrations of N₂0₃ in in vitro