An approach based on ultra-high pressure liquid chromatography-tandem mass spectrometry to quantify O6-methyl and O6-carboxymethylguanine DNA adducts in intestinal cell lines.

O⁶-methylguanine (O⁶-MeG) and O⁶-carboxymethylguanine (O⁶-CMG) are characteristic promutagenic and toxic DNA adducts formed by nitrosated glycine derivates and N-nitrosopeptides. Since endogenous nitrosation has been hypothesised as a plausible origin for the association between red and processed meat intake and colorectal cancer, a highly sensitive, fast and specific quantitative assay is needed to correlate the dose of individual DNA adducts with the effects of food consumption and individual digestive and metabolic processes. An ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for quantitation of O⁶-MeG and O⁶-CMG, using the deuterated analogues as internal standards (ISTD), was developed. Samples of calf thymus DNA containing O⁶-MeG and O⁶-CMG were purified by acid hydrolysis and solid phase extraction prior to quantification by UHPLC-MS/MS in the selected reaction monitoring mode. The method was successfully validated in terms of repeatability (RSD<10%), reproducibility (RSD<15%) and linearity (99.9%) by incubating 0.1mg calf thymus DNA with the known N-nitroso compound potassium diazoacetate (KDA). The limit of quantitation was 30 fmol mg⁻¹ DNA for O⁶-MeG or 1 adduct per 10⁸ nucleotides and 50 fmol mg⁻¹ DNA for O⁶-CMG or 1.7 adducts per 10⁸ nucleotides. Subsequently, the method was applied to human colon carcinoma cell lines, Caco-2 and HT-29, treated with KDA to illustrate its capability to quantify O⁶-MeG and O⁶-CMG DNA adducts using biological relevant models in vitro. This method will support further research to unravel the mechanistic basis of endogenous nitrosation processes upon consumption of red and processed meat products.
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