RESUMEN
BACKGROUND: A long-standing hypothesis is that oxidative stress is a risk factor for cancer. Support for this hypothesis comes from observations of higher levels of oxidative damage in the DNA of WBC of cancer patients compared with healthy controls. METHODS: Two generally overlooked types of DNA damage, the formamide modification and the thymine glycol modification, both derived from pyrimidine bases, were assayed as markers of oxidative stress. Damage levels were measured in the DNA of WBC of ovarian cancer patients and of healthy controls. RESULTS: The levels of both modifications were higher in ovarian cancer patients than in healthy controls although in the case of the formamide modification age could not be ruled out as a factor. CONCLUSION: Our results in combination with other published measurements of oxidative DNA damage support the hypothesis that oxidative damage, on average, is higher in WBC of cancer patients than in healthy controls.
Asunto(s)
Daño del ADN , Neoplasias Ováricas/metabolismo , Estrés Oxidativo , Pirimidinas/metabolismo , Adulto , Factores de Edad , Anciano , Femenino , Humanos , Leucocitos/metabolismo , Persona de Mediana Edad , Neoplasias Ováricas/genéticaRESUMEN
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.
Asunto(s)
Daño del ADN , ADN/química , ADN/efectos de la radiación , Fibroblastos/química , Fibroblastos/efectos de la radiación , Timina/química , Timina/efectos de la radiación , Animales , Células Cultivadas , ADN/metabolismo , Fosfatos de Dinucleósidos/química , Fosfatos de Dinucleósidos/efectos de la radiación , Espectroscopía de Resonancia Magnética , Ratones , Ratones Endogámicos C3H , Rayos XRESUMEN
A single free radical-initiating event can produce a pair of base lesions in DNA oligomers exposed to ionizing radiation. Whereas double base lesions have been observed previously, the present study shows that double lesions may sometimes consist of a base lesion and an associated strand break. The mechanism for the formation of double lesions is discussed. A redox process is postulated in which guanine is the source of the electron. It is suggested that double lesions may be formed in DNA either on adjacent nucleotides or, alternatively, on nucleotides separated by one, two or possibly more intervening nucleotides. It is hypothesized that intramolecular electron transfer facilitates the formation of double lesions on nonadjacent nucleotides.
Asunto(s)
Daño del ADN , ADN/efectos de la radiación , Cromatografía Líquida de Alta Presión , ADN/química , ADN/metabolismo , Radicales Libres/metabolismo , Radicales Libres/efectos de la radiación , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/efectos de la radiación , Radioquímica , Espectrofotometría UltravioletaRESUMEN
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.
Asunto(s)
Daño del ADN , Desoxiguanosina/análogos & derivados , Peróxido de Hidrógeno/toxicidad , Oligodesoxirribonucleótidos/efectos de la radiación , 8-Hidroxi-2'-Desoxicoguanosina , Catálisis , Cromatografía Líquida de Alta Presión , Cobre/farmacología , Desoxiguanosina/efectos de la radiación , Radicales Libres , Peróxido de Hidrógeno/farmacología , Hierro/farmacología , Espectrometría de Masas , Ácido Nitrilotriacético/farmacología , Oxidación-Reducción , Estrés Oxidativo , Pirimidinas/efectos de la radiación , Especies Reactivas de OxígenoRESUMEN
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.
Asunto(s)
Emparejamiento Base , Daño del ADN , ADN/efectos de la radiación , Oxígeno/química , Animales , Bovinos , Cromatografía Líquida de Alta Presión , ADN/química , Espectrometría de Masas/métodos , Rayos XRESUMEN
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 (32)P-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(P(F)pG) and d(P(F)pG(H)), where P(F) stands for a pyrimidine nucleoside having the base degraded to a formamido remnant and G(H) stands for 8-oxo-deoxyguanosine. The yields in calf thymus DNA irradiated 60 Gy were 8.6 and 3.2 pmol/microgram DNA, respectively.
Asunto(s)
ADN/química , ADN/efectos de la radiación , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Animales , Bovinos , Cromatografía Líquida de Alta Presión , ADN/metabolismo , Daño del ADN , Relación Dosis-Respuesta en la Radiación , Formamidas/química , Radicales Libres/química , Guanina/análogos & derivados , Guanina/química , Nucleósidos de Pirimidina/química , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Espectrometría de Masa Bombardeada por Átomos Veloces , Timina/análogos & derivados , Timina/química , Timo , Rayos XRESUMEN
A 32P-postlabeling assay has been developed for the simultaneous detection of the thymine glycol lesion and the formamido remnant of pyrimidine bases in DNA exposed to reactive oxygen species (ROS). The formamido lesion is a principal lesion produced in X-irradiated DNA oligomers when oxygen is available to mediate the damage process. Production of the well-known thymine glycol lesion is less dependent on the concentration of oxygen. These two lesions have the common property that they make the phosphoester bond 3' to the modified nucleoside resistant to hydrolysis by nuclease P1. Our assay uses 32P-postlabeling to measure these lesions in the form of modified dimers obtained from DNA by nuclease P1 digestion. Appropriate carriers and internal standards have been chemically synthesized to improve the reliability and accuracy of the assay. The measurements were accomplished on 1-microgram samples of DNA.
Asunto(s)
Daño del ADN , Nucleósidos de Pirimidina/química , Especies Reactivas de Oxígeno , Timina/química , Fosfatasa Ácida , Autorradiografía/métodos , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , ADN/efectos de los fármacos , ADN/efectos de la radiación , Radioisótopos de Fósforo , Polinucleótido 5'-Hidroxil-Quinasa , Estándares de Referencia , Endonucleasas Específicas del ADN y ARN con un Solo FilamentoRESUMEN
Two new modified uracil nucleosides, 5-carbamoylmethyuridine (ncm5U, I) and 5-carbamoylmethyl-2-thiouridine (ncm5s2U, II) were isolated from a 24 hr collection of a normal human urine. The structures were assigned on the basis of UV, NMR and mass spectral data and confirmed by comparison of the spectral data and HPLC mobilities with those of authentic samples. On the basis of experimental data it appears possible that 5-carbamoylmethyl-2-thio-uridine (ncm5s2U, II) may be a degradation product produced from a labile precursor by the chemical treatments during the isolation procedure. However, the other nucleoside (ncm5U,I) certainly appears to be of metabolic origin and was also found in the urines of one chronic myelogenous leukemia and one lung carcinoma patient. Abbreviations used are: tRNA-transfer ribonucleic acid, TMS-trimethylsilyl, RP-HPLC--reverse phase high performance liquid chromatography, EI--electron impact, cm5U-5-carboxymethyluridine, mcm5U-5-methoxycarbonylmethyluridine, cm5s2U-5-carboxymethyl-2-thiouridine, mcm5s2U-5-methoxycarbonylmethyl-2-thiouridine, t6A-9-beta-D-ribofuranosyl-[N(purin-6-yl)carbamoyl]-1-threonine, C-cytidine, acp3u-3-(3-amino-3-carboxypropyl)uridine, AICR-aminoimidazole carboxamide riboside, alpha-4-PCNR & beta-4-PCNR-9-alpha-D-(or beta-D)-ribofuranosyl-pyridin-4-one-3-carboxamide, H x 7R-7-beta-D-ribofuranosyl hypoxanthine, m3U-3-methyluridine, m1I-1-methylinosine, m1G-1-methylguanosine, DI-5'-deoxyinosine, dms5OA-5'-deoxy-5'-methylthioadenosine sulfoxide, m2(2)G-N2-dimethylguanosine, psi-psi-uridine, A-adenosine, I-inosine, CML-chronic myelogenous leukemia mam5s2U-5-methylaminomethyl-2-thiouridine, ncm5U-5-carbamoylmethyluridine, ncm5s2U-5-carbamoylmethyl-2-thiouridine, UV-ultraviolet, NMR-nuclear magnetic resonance, HPLC-high performance liquid chromatography, GC-MS-gas chromatography-mass spectrometry.
Asunto(s)
Tiouridina/análogos & derivados , Uridina/análogos & derivados , Cromatografía Líquida de Alta Presión , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/orina , Neoplasias Pulmonares/química , Neoplasias Pulmonares/orina , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas , Espectrofotometría Ultravioleta , Tiouridina/química , Tiouridina/aislamiento & purificación , Tiouridina/orina , Uridina/química , Uridina/aislamiento & purificación , Uridina/orinaRESUMEN
A DNA lesion which results from the breakdown of a pyrimidine base leaving a formamido remnant has been associated with oxidative stress. This lesion is shown to be produced in keratinocytes irradiated in culture with UVB light. The amount of formamido lesion produced is comparable to the amount of the 8-hydroxyguanine lesion. The two lesions were measured by 32P-postlabeling and electrochemical detection methods respectively.
Asunto(s)
Daño del ADN , ADN/metabolismo , ADN/efectos de la radiación , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Animales , Línea Celular , Guanina/análogos & derivados , Guanina/análisis , Guanina/metabolismo , Ratones , Rayos UltravioletaRESUMEN
A novel nucleoside, in the amount of 400 micrograms, was isolated from a 24-h collection of urine of a chronic myelogenous leukemia patient. On the basis of ultraviolet, nuclear magnetic resonance, and mass spectrometry and chromatography, its structure was established to be 7-beta-D-ribofuranosylhypoxanthine. The ultraviolet and mass spectral data and the thin layer chromatographic mobilities of the natural material were identical to those of a synthetic sample. High performance liquid chromatographic retention times and the coinjection high performance liquid chromatography of the natural material with the synthetic samples of the alpha and beta-anomers of 7-ribofuranosylhypoxanthines further confirmed the identity of the isolated material as 7-beta-D-ribofuranosylhypoxanthine.