Humans are exposed to both endogenous and exogenous sources of N-nitroso compounds, which can be metabolically activated to generate diazoacetate, a known active DNA carboxymethylation agent. Detection of DNA lesions remains one of the core tasks in toxicology and in evaluating human health risks associated with exposure to chemical carcinogens. In this study, we have developed a new highly sensitive and specific nanoflow LC-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS3) method for the simultaneous detection and accurate quantification of several carboxymethylated DNA lesions in XPA-deficient GM04429 human skin fibroblast cells upon treatment with the DNA carboxymethylation agent azaserine. Our results showed that, upon treatment with 150 �M of azaserine, N6-carboxymethyl-2'-deoxyadenosine (N6-CMdA) and O6-carboxymethyl-2'-deoxyguanosine (O6-CMdG) could be detected at the levels of 0.94�0.57 and 8.15�0.75 lesions per 107 nucleosides, respectively. An aliquot equivalent to 0.25 �g of DNA hydrolysate was used for analysis by this nanoLC-NSI/MS3 method. We are now in the process of assessing the dose-dependent formation of N6-CMdA, O6-CMdG, and other carboxymethylated DNA lesions and the involvement of O6-methylguanine-DNA methyltransferase (MGMT) in the repair of O6-CMdG.

Quantification of carboxymethylated DNA lesions in cells by nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry coupled with stable isotope-dilution method

248th National Meeting (2014)

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