Oxidatively-induced DNA damage was measured in the DNA of WBC from two groups of women: carriers of a BRCA mutation, but asymptomatic for disease, and healthy controls. Two oxidatively induced lesions were measured: a formamide remnant of pyrimidine base and the glycol modification of thymine. These lesions, employed previously in studies of the effects of smoking, antioxidant usage and ovarian cancer, are proving valuable indicators of oxidative stress. The BRCA carriers of mutations, with no overt sign of cancer, nevertheless had significantly higher levels of DNA damage than the controls. The level measured for the formamide lesion was 5.9 ± 1.0 (femtomoles/μg of DNA ± SEM) compared with 2.4 ± 0.3 in controls. The level of the glycol lesion was 2.9 ± 0.4 compared with 1.8 ± 0.2 in controls. The experimental design utilized DNA from WBC and employed LC-MS/MS to detect the lesions.

The goal of this research was to determine whether antioxidant usage could be correlated with changes in DNA damage levels. Liquid Chromatography‐tandem Mass Spectrometry (LC‐MS/MS) was used to simultaneously measure five different oxidatively‐induced base modifications in the DNA of WBC. Measurements of the five modifications were made before and after an 8‐week trial during which participants took the SU.VI.MAX supplement. Levels of the five DNA modifications were compared among different groupings: users versus non‐users of antioxidant supplements, before versus after the supplement intervention and men versus women. The statistical significance of differences between groups was most significant for pyrimidine base modifications and the observed trends reflect trends reported in epidemiological studies of antioxidant usage. A combination of modifications derived from pyrimidine bases is suggested as a superior indicator of oxidative stress.

Reactive oxygen species (ROS) generate a type of DNA damage called tandem lesions, two adjacent nucleotides both modified. A subcategory of tandem lesions consists of adjacent nucleotides linked by a covalent bond. Covalently linked tandem lesions generate highly characteristic liquid chromotography-tandem mass spectrometry (LC-MS/MS) elution profiles. We have used this property to comprehensively survey X-irradiated DNA for covalently linked tandem lesions. A total of 15 tandem lesions were detected in DNA irradiated in deoxygenated aqueous solution, five tandem lesions were detected in DNA that was irradiated in oxygenated solution.

BACKGROUND:Cigarette smoking is a known cause of cancer, and cancer may be in part due to effects of oxidative stress. However, whether smoking cessation reverses oxidatively induced DNA damage unclear. The current study sought to examine the extent to which three DNA lesions showed significant reductions after participants quit smoking.METHODS:Participants (n = 19) in this study were recruited from an ongoing 16-week smoking cessation clinical trial and provided blood samples from which leukocyte DNA was extracted and assessed for 3 DNA lesions (thymine glycol modification [d(TgpA)]; formamide breakdown of pyrimidine bases [d(TgpA)]; 8-oxo-7,8-dihydroguanine [d(Gh)]) via liquid chromatography tandem mass spectrometry (LC-MS/MS). Change in lesions over time was assessed using generalized estimating equations, controlling for gender, age, and treatment condition.RESULTS:Overall time effects for the d(TgpA) (χ2(3) = 8.068, p < 0.045), d(PfpA) (χ2(3) = 8.477, p < 0.037), and d(Gh) (χ2(3) = 37.599, p < 0.001) lesions were seen, indicating levels of each decreased significantly after CO-confirmed smoking cessation. The d(TgpA) and d(PfpA) lesions show relatively greater rebound at Week 16 compared to the d(Gh) lesion (88% of baseline for d(TgpA), 64% of baseline for d(PfpA), vs 46% of baseline for d(Gh)).CONCLUSIONS:Overall, results from this analysis suggest that cigarette smoking contributes to oxidatively induced DNA damage, and that smoking cessation appears to reduce levels of specific damage markers between 30-50 percent in the short term. Future research may shed light on the broader array of oxidative damage influenced by smoking and over longer durations of abstinence, to provide further insights into mechanisms underlying carcinogenesis.

The 1-carbamoyl-2-oxo-4,5-dihydroxyimidazolidine modification of cytosine is a known base modification produced in vitro by oxidative stress. However, the presence of this modification in vivo has not been established. In this study the introduction of this base modification into dinucleoside monophosphates was accomplished using the Fenton reaction. Subsequently, the modification was produced in short isotopically labeled oligomers. Labeled tetramers bearing the lesion were used as internal standards for LC-MS/MS determinations of the base modification in the DNA of white blood cells from healthy donors. The background level of the 1-carbamoyl-2-oxo-4,5-dihydroxyimidazolidine modification of cytosine was found to be larger than the levels of the formamide and thymine glycol base modifications.

A different approach to the measurement of DNA damage has been developed based on the fact that many lesions can be excised from DNA in the form of modified dinucleoside monophosphates. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used in conjunction with isotopically labeled internal standards to quantify the lesion. The method has several advantages, including high sensitivity for the detection of dinucleoside monophosphates. The method was applied to the measurement of the 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol) lesion in the DNA of mouse fibroblast cells exposed in culture to various treatments including ionizing radiation, UVC light and buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. The application of the method to the measurement of other DNA lesions is discussed. (c) 2006 by Radiation Research Society.

Abstract Greene, K. F., Budzinski, E. E., Iijima, H., Davidzik, J. B., DeFedericis, H-C., Patrzyc, H. B., Evans, M. S., Bailey, D. T., Freund, H. F. and Box, H. C. Assessment of DNA Damage at the Dimer Level: Measurement of the Formamide Lesion. Radiat. Res. 167, 146–151 (2007). UVC-radiation-induced DNA damage was measured in mouse fibroblast cells using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in conjunction with isotopically labeled internal standards. The thymine glycol and formamide lesions were assayed in the form of modified dinucleoside monophosphates. The 8-oxo-7,8-dihydroguanine lesion was measured as the modified nucleoside. DNA damage in cells treated with tirapazamine was also measured. Tirapazamine is a chemotherapeutic agent that acts via a free radical mechanism. The two agents, UVC radiation and tirapazamine, produce markedly different profiles of DNA damage, reflecting their respective mechanisms of action. Both agents produce significant amounts of thymine glycol and formamide damage, but only the former produced a measurable amount of the 8-oxo-7,8-dihydroguanine lesion. The merits of measuring DNA damage at the dimer level are discussed.

Singlet oxygen, hydrogen peroxide, hydroxyl radical and hydrogen peroxide are the reactive oxygen species (ROS) considered most responsible for producing oxidative stress in cells and organisms. Singlet oxygen interacts preferentially with guanine to produce 8-oxo-7,8-dihydroguanine and spiroiminodihydantoin. DNA damage due to the latter lesion has not been detected directly in the DNA of cells exposed to singlet oxygen. In this study, the singlet oxygen-induced lesion was isolated from a short synthetic oligomer after exposure to UVA radiation in the presence of methylene blue. The lesion could be enzymatically excised from the oligomer in the form of a modified dinucleoside monophosphate. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), the singlet oxygen lesion was detected in the form of modified dinucleoside monophosphates in double-stranded DNA and in the DNA of HeLa cells exposed to singlet oxygen. Pentamer containing the singlet oxygen-induced lesion and an isotopic label was synthesized as an internal standard for quantifying the lesion and served as well as for correcting for losses of product during sample preparation.

Purpose : To study the formation of the dihydrothymine lesion produced in DNA by ionizing radiation in an anaerobic environment.Materials and methods : The dihydrothymine lesion, along with other lesions, was isolated from an X-irradiated aqueous solution of the dinucleoside monophosphate d(TpA) and analysed by correlated two-dimensional nuclear magnetic resonance spectroscopy. The dihydrothymine lesion was obtained by enzymatic digestion of irradiated DNA in the form of modified dinucleoside monophosphates, d(T(d)A), where T-d stands for dihydrothymidine. Liquid chromatography-tandem mass spectrometry was used to detect the lesion in the DNA of X-irradiated mouse fibroblast cells.Results : The modified dinucleoside monophosphate, d(T(d)pA), fragments by two pathways so that altogether the lesion could be detected using two different sets of tandem mass spectrometry (precursor ion mass/daughter ion mass) values.Conclusion : The dihydrothymine lesion is a significant lesion in cells exposed to ionizing radiation in an anaerobic environment.

The two main cisplatin-induced DNA lesions, G∧G and A∧G, have been measured in cells exposed to the drug. (G∧G and A∧G denote the intrastrand bifunctional adducts formed between adjacent purine bases.) It has proven feasible, using liquid chromatography-tandem mass spectrometry (LC-MS/MS), observe the G∧G and A∧G lesions in mouse fibroblast cells exposed for 1 h to a 120μM concentration of cisplatin. After extraction of the DNA from the cells, the lesions were enzymatically isolated from the DNA in the form of modified dinucleoside monophosphates with the phosphodiester bond intact. MS/MS detection of the modified dinucleoside monophosphates in the negative ion mode manifests two transitions; from the negative ion to the loss of one NH3 group and from the ion less one NH3 group to the loss of both NH3 groups. The multiple reaction monitoring capability of LC-MS/MS was used to measure the three most abundant isotopes of the two main lesions for both transitions of each lesion (i.e., 12 MS/MS values in toto). Ion currents could be detected for all 12 pairs of MS/MS values in the DNA from exposed cells. Although this protocol results in some overlap of MS/MS values between the two lesions, a slight difference in elution times clearly distinguishes between them.

Oxidative DNA damage can result from environmental factors, such as radiation, as well as from the untoward consequences of normal metabolic processes. It is of interest to assay oxidative DNA damage in cells and tissues because this damage has been implicated in human disease, particularly cancer. Eleven indicators of oxidative DNA damage have been measured by Liquid Chromatography-Mass Spectrometry (LC-MS) in DNA extracted from cells exposed to oxidative stress. Mouse fibroblast cells were exposed to hydrogen peroxide and to UVC light and to the combined action of both agents. Significant increases of the 8-oxo-7,8-dihydropurine lesions over background were detected. Significant increases of the formamido lesions resulting from breakdown of pyrimidine bases were also observed. Of special interest was the observation of double lesions, tandem combinations of both aforementioned lesions, in cells exposed to oxidative stress.

DNA X-irradiated in oxygenated aqueous solution produces the formamido lesion from the breakdown of pyrimidine nucleosides. This pyrimidine breakdown product inhibits the hydrolysis by nuclease PI of the phosphoester bond 3' to the damaged nucleoside. Consequently, the lesion can be obtained from an enzymatic digest of the DNA as a modified dinucleoside monophosphate in which the 5' nucleoside contains the lesion. In this form, the formamido lesion can be detected with good sensitivity by liquid chromatography-mass spectrometry (LC-MS). Nucleosides that have lost the base moiety also inhibit nuclease P1. Together, the formamido and abasic lesions account for all of the substantial peaks in the LC-MS ion current profile.(C) 2002 by Radiation Research Society.

It was demonstrated previously that double lesions are produced in DNA by ionizing radiation. These double lesions consist of adjacent nucleotides each bearing a modified base. The goal of the present investigation was to determine whether Fenton chemistry can generate the same kind of lesions. DNA oligomers were exposed to metal-catalyzed H(2)O(2) reactions, and the products were characterized by chromatography and by mass spectrometry. Double lesions are produced by this treatment in which deoxyguanosine is oxidized to 8-oxo-7,8-dihydrodeoxyguanosine and an adjacent pyrimidine nucleoside is degraded to a formamido remnant.

Evidence is presented for the formation of products in irradiated dinucleoside monophosphates in which both bases are damaged. The dinucleoside monophosphates d(GpT), d(GpC), d(TpG) and d(CpG) were X-irradiated in oxygenated aqueous solution. Product identification was by NMR spectroscopy. In products containing double base lesions, guanine is converted to 8-hydroxyguanine and the pyrimidine base is degraded to a formamido remnant.

Evidence has been accumulating at the oligomer level that free radical-initiated DNA damage includes lesions in which two adjacent bases are both modified. Prominent examples are lesions in which a pyrimidine base is degraded to a formamido remnant and an adjacent guanine base is oxidized. An assay has been devised to detect double-base lesions based on the fact that the phosphoester bond 3′ to a nuclesoside bearing the formamido lesion is resistant to hydrolysis by nuclease P1. The residual modified dinucleoside monophosphates obtained from a nuclease P1 (plus acid phosphatase) digest of DNA can be 32P-postlabeled using T4 polynucleotide kinase. Using this assay the formamido single lesion and the formamido–8-oxoguanine double lesion were detected in calf thymus DNA after X-irradiation in oxygenated aqueous solution. The lesions were measured in the forms d(PFpG) and d(PFpGH), where PF stands for a pyrimidine nucleoside having the base degraded to a formamido remnant and GH stands for 8-oxo-deoxyguanosine. The yields in calf thymus DNA irradiated 60 Gy were 8.6 and 3.2 pmol/μg DNA, respectively.

Previously, double lesions in which two adjacent bases are modified were identified in DNA oligomers exposed in solution to ionizing radiation. However, the formation of such lesions in polymer DNA had not been demonstrated. Using reference oligomer containing a specific double lesion and employing liquid chromatography-mass spectrometry (LC-MS), it was possible to show directly that double lesions are formed in irradiated calf thymus DNA. The double lesion in which a pyrimidine base is degraded to a formamido remnant and an adjacent guanine base is oxidized to 8-oxoguanine was detected in DNA X-irradiated in oxygenated aqueous solution. The double lesion in which the methyl carbon atom of a thymine base is covalently linked to carbon at the 8-position of an adjacent guanine base was detected in DNA irradiated in a deoxygenated environment.

The products produced by X irradiation of an oxygenated aqueous solution containing d(CpApTpG) were analyzed by NMR spectroscopy and mass spectrometry. Thirteen different base modifications were detected, including a novel product formed by the addition of oxygen to guanine. Seven different strand break products were identified, including strands having 5'-phosphoryl groups, 3'-phosphoryl groups and groups having 3'-phosphoglycolates as termini. The products produced in largest yield contained base modifications: Pyrimidine bases degraded to a formamido moiety, the 8-oxo-7,8-dihydroguanine (8-oxoguanine) lesion, and double base lesions in which both the 8-oxo-7,8-dihydroguanine lesion and a formamido remnant are present.

Free radicals interact with DNA bases to produce secondary radicals. The secondary radicals are reactive species and tend to interact with neighboring bases, resulting in DNA lesions with two adjacent modified bases. In this study the DNA oligomers d(CpApTpG) and d(CpGpTpA) were exposed to free radicals generated in anoxic aqueous solution by X irradiation. Four new lesions were identified in which adjacent guanine and pyrimidine bases are covalently bonded. One of the tandem lesions formed in d(CpGpTpA) has the C5 carbon atom of cytosine covalently bonded to the C8 carbon atom of guanine. Interestingly, the same bond is formed between the terminal bases in d(CpApTpG), resulting in a cyclized molecule.

An improved method for separating the products of DNA oligomers irradiated in aqueous solution has been devised. Altogether 39 products were isolated from the tetramer d(CpGpTpA) X-irradiated in dilute anoxic solution. Of these, 16, including most of the major products, were identified through the use of proton nmr spectroscopy. The identified products fall into four categories: (1) base products, (2) strand scission products, (3) base modifications and (4) tandem lesion. A tandem lesion in which the methyl carbon atom of thymine is covalently linked to the guanine C8 carbon atom was produced in larger yield than any of the simple base modifications.