It has been demonstrated repeatedly that ionizing radiation damages the DNA in cells, often leading to both chromosomal aberrations and mutations. The direct effects of ionizing radiation include breaks to single or both DNA strands, base modifications, and damage to the carbohydrate components associated with the DNA. The most important indirect effect of radiation is the production of free radicals from cellular water, which react with the DNA (reviewed in Reference 388). Tumors may develop when the cellular DNA repair mechanisms fail to detect and correct these changes or are themselves compromised.
Concomitant damage to both the base and sugar moieties of the same nucleotide subunit has been studied using sample preparation techniques that release the sugars and use acidic or enzymatic hydrolyses to free the bases. The sugars have been identified and quantified by GC/MS after reduction with NaBD4 to the corresponding polyalcohols, followed by silylation. The methodology has also been used to detect the 8,5'-cyclopurine nucleosides that are diagnostic of radiation damage. Products of damaged DNA could be quantified even at radiation doses as low 0.1 Gy (Gray, unit of absorbed dose of ionizing radiation, 1 Gy = 100 rad).389
Mass spectrometric methodologies have been developed for the study of oxidative DNA damage produced by free radicals, particularly by the highly reactive hydroxyl radical (• OH), as well as for the investigation of enzymatic repair of the DNA (reviewed in Reference 390). Enzymatic hydrolysis, with deoxyribonuclease and/or exonucleases, produced nucleosides from DNA. This was followed by acidic hydrolysis, often with formic acid, to separate the intact and modified bases by cleavage of the glycosidic bonds between the base and sugar moieties. The hydrolysis products were silylated and analyzed by GC/MS. Full scanning EI provided adequate structural details for compound identification, while SIM was used for quantification. The four intact DNA bases, ~20 modified DNA bases, ~12 modified DNA 2'-deoxynucleosides, and DNA base-amino acid cross-linked products could be identified and quantified in a single analysis. Analogs labeled with 13C and/or deuterium were used as i.s. in studies to assess the presence and position of carbonyl and deoxy groups in modified/damaged sugars. Using conventional GC/MS, LOD were of the order of 1 fmol/analyte or 1 to 3 modified residues in 106 bases.391 Another isotope dilution GC/MS technique has been used to identify and quantify nine modified DNA bases in children with acute lymphoblastic leukemia and in controls. More than threefold differences were found between the patients and controls in the cases of 5-hydroxy-5-methylhydantoin and 2,6-diamino-4-hydroxy-5-formamidoguanine.392
Base modifications arising from • OH radical-induced hydroxylation and cleavage reactions have been studied in DNA that was extracted from ductal carcinoma tissues excised from patients with invasive breast cancer. After hydrolyzing the DNA and forming TMS derivatives, GC/EIMS was used to quantify the modified bases. The concentrations of 8-hydroxyguanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyadenine were up to ninefold higher than those of DNA isolated from calf-thymus. There were no differences in the profiles of the unmodified bases from the two tissues. It has been suggested that the • OH attack on DNA may be associated with escalations in H2O2 concentrations that bring about "automutagenic" progressive alterations in DNA.393 Care should be taken in the interpretation of results from GC/MS techniques because these methods often produce derivatization-related artifacts that may be present in considerable quantities (reviewed in Reference 394).
Another application of the above methodologies has been the investigation of genomic base damage in the lymphocytes of lung cancer patients undergoing radiation therapy. Because the concentrations of 8-OH-guanine and some other modified bases have been shown to differ significantly among individuals,395 this research was based on lymphocyte chromatin samples from cancer patients using their own preradiation samples as controls. Observed DNA modifications that indicated damage by • OH radicals included several purine- and pyrimidine-derived modified bases. The quantitative differences were credited to the intensity of applied radiation (dictated by the nature of the neoplasm), individual radiation sensitivity, and the capability of the cells from different individuals to repair DNA damage. These modifications in the DNA, caused by the radiation, were in qualitative agreement with those resulting from the treatment of experimental animals with carcinogens.396 It was concluded that DNA damage by therapeutic radiation may contribute significantly to the development of secondary malignancies, such as leukemias.397
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