Quantification of the 2-Deoxyribonolactone and Nucleoside 5′-Aldehyde Products of 2-Deoxyribose Oxidation in DNA and Cells by Isotope-Dilution Gas Chromatography Mass Spectrometry: Differential Effects of γ-Radiation and Fe²⁺−EDTA

Abstract

The oxidation of 2-deoxyribose in DNA has emerged as a critical determinant of the cellular toxicity of oxidative damage to DNA, with oxidation of each carbon producing a unique spectrum of electrophilic products. We have developed and validated an isotope-dilution gas chromatography-coupled mass spectrometry (GC-MS) method for the rigorous quantification of two major 2-deoxyribose oxidation products: the 2-deoxyribonolactone abasic site of 1’-oxidation and the nucleoside 5’-aldehyde of 5’-oxidation chemistry. The method entails elimination of these products as 5-methylene-2(5H)-furanone (5MF) and furfural, respectively, followed by derivatization with pentafluorophenylhydrazine (PFPH), addition of isotopically labeled PFPH derivatives as internal standards, extraction of the derivatives, and quantification by GC-MS analysis. The precision and accuracy of the method were validated with oligodeoxynucleotides containing the 2-deoxyribonolactone and nucleoside 5’-aldehyde lesions. Further, the well defined 2-deoxyribose oxidation chemistry of the enediyne antibiotics, neocarzinostatin and calicheamicin γ1I, was exploited in control studies, with neocarzinostatin producing 10 2-deoxyribonolactone and 300 nucleoside 5’-aldehyde per 106 nt per µM in accord with its established minor 1’- and major 5’-oxidation chemistry. Calicheamicin unexpectedly caused 1’-oxidation at a low level of 10 2-deoxyribonolactone per 106 nt per µM in addition to the expected predominance of 5’-oxidation at 560 nucleoside 5’-aldehyde per 106 nt per µM. The two hydroxyl radical-mediated DNA oxidants, γ-radiation and Fe2+-EDTA, produced nucleoside 5’-aldehyde at a frequency of 57 per 106 nt per Gy (G-value 74 nmol/J) and 3.5 per 106 nt per µM, respectively, which amounted to 40% and 35%, respectively, of total 2-deoxyribose oxidation as measured by a plasmid nicking assay. However, γ-radiation and Fe2+-EDTA produced different proportions of 2-deoxyribonolactone at 7% and 24% of total 2-deoxyribose oxidation, respectively, with frequencies of 10 lesions per 106 nt per Gy (G-value, 13 nmol/J) and 2.4 lesions per 106 nt per µM. Studies in TK6 human lymphoblastoid cells, in which the analytical data were corrected for losses sustained during DNA isolation, revealed background levels of 2-deoxyribonolactone and nucleoside 5’-aldehyde of 9.7 and 73 lesions per 106 nt, respectively. γ-Irradiation of the cells caused increases of 0.045 and 0.22 lesions per 106 nt per Gy, respectively, which represents a ~250-fold quenching effect of the cellular environment similar to that observed in previous studies. The proportions of the various 2-deoxyribose oxidation products generated by γ-radiation are similar for purified DNA and cells. These results are consistent with solvent exposure as a major determinant of hydroxyl radical reactivity with 2-deoxyribose in DNA, but the large differences between γ-radiation and Fe2+-EDTA suggest that factors other than hydroxyl radical reactivity govern DNA oxidation chemistry.

Vegetables′ juice influences polyol pathway by multiple mechanisms in favour of reducing development of oxidative stress and resultant diabetic complications

“…[2930] Therefore, evaluation of antioxidative property of antioxidants on both, genomic DNA as well as its ribose sugar may reveal crucial information on genetic toxicology of oxidative stress and preventive role offered by an antioxidant.…”

DNA damage induced by carbon ions (C3+) beam accessed by independent component analysis of infrared spectra

“…The peak at 1471 cm (5) 1 is related to C  C in phase stretching in a specific position, namely, if the double bond is conjugated to a carbonyl group (Rivero et al 2010), being the presence of this group is related with furfural. The presence of this product is claimed to be related with degradation products of nucleoside-5′-aldehyde which in turn is a product of oxidation of 2-deoxyribose in DNA (Chan et al 2010). The absorbance increase of the peak at 1055 cm (6) 1 can be also be associated with loss of water molecules as a result of DNA sample being submitted to vacuum during irradiation.…”

Methylarsenic compounds [MAK Value Documentation, 2014]

“…The hydroxyl radical is capable of producing single strand breaks as the result of hydrogen abstraction from 2-deoxyribose residues (Aydogan et al 2002;Chan et al 2010;Dedon 2008;Hartwig 2013;Pogozelski and Tullius 1998). Singlet oxygen reacts particularly with guanine with the formation of 8-oxo-7,8-dihydroguanine (8-oxoguanine), without thereby inducing the formation of further reaction products (Cadet et al 2008).…”