David S. Bergtold

Characterization of free radical-induced base damage in DNA at biologically relevant levels

DNA damage induced by oxygen radicals, e.g., hydroxyl radicals generated in living cells either by cellular metabolism or external agents such as ionizing radiations, appears to play an important role in mutagenesis, carcinogenesis, and aging. Elucidation of the chemical nature of such DNA lesions at biologically significant quantities is required for the assessment of their biological consequences and repair. For this purpose, a sensitive method using gas chromatography-mass spectrometry with the selected-ion-monitoring technique (GC-MS/SIM) was developed in the present work. DNA was exposed to hydroxyl radicals and hydrogen atoms produced by ionizing radiation in N2O-saturated aqueous solution. DNA samples were subsequently hydrolyzed with formic acid, trimethylsilylated, and analyzed by GC-MS/SIM. Characteristic ions from previously known mass spectra of DNA base products as their trimethylsilyl derivatives were recorded and the area counts of each ion were integrated. From these acquired data, a partial mass spectrum of each product was generated and then compared with those of authentic materials. This technique permitted the detection and characterization of a large number of free radical-induced based products of DNA, i.e., 5,6-dihydrothymine, 5-hydroxy-5,6-dihydrothymine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-hydroxycytosine, thymine glycol, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine, simultaneously in a single sample after radiation doses from 0.1 to 10 Gy. Detectable amounts of the base products were found to be as low as approximately 10 fmol per injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation of oxy radicals in biosystems.

Many recent lines of evidence indicate that endogenous free radicals contribute to spontaneous mutagenesis through the direct induction of DNA damage. However, the mechanisms underlying this process are not yet fully understood. A brief overview of the knowledge that is currently available is provided here, with emphasis on the generation of oxy radicals in biosystems, the reactions of those radicals with biomolecules, and the induction of oxidative DNA base damage that might lead to mutation.

Dietary modulation of DNA damage in human

Manipulation of human diet can modulate urinary biomarkers of oxidative DNA base damage (UBODBD), reflecting changes in levels of DNA damage. When dietary composition is maintained but caloric intake is decreased (caloric restriction), UBODBD excretion is suppressed. At isocaloric dietary intake the level of damage depends on diet composition. For diets consisting of foods containing carbohydrates, proteins, and fats but lacking fruits and vegetables, the level of damage is higher than for diets including fruits and vegetables, which are rich in natural antioxidants. Assay of urinary biomarkers is suggested as a potential test for quantitative assessment of the carcinogenic or anticarcinogenic properties of foods, food components, and diets and for individual responses to nutritional regimens.

The 8-hydroxyguanine content of isolated mitochondria increases with lipid peroxidation☆

When lipid peroxidation was induced in isolated mitochondria there was a marked increase in the 8-hydroxyguanine content of the nucleic acids extracted from these mitochondria. The elevation of 8-hydroxyguanine levels was associated with an extensive alteration of normal electrophoretic mobility of mitochondrial DNA. However, suppression of lipid peroxidation with alpha-tocopherol proportionally attenuated 8-hydroxyguanine production and limited the electrophoretic mobility change of mitochondrial DNA.

Biomarkers of oh Radical Damage In Vivo

Mechanisms of formation of o-tyrosine (o-Tyr) and thymine glycol (TG), the two possible markers of OH radical generation in biosystems and in vivo are described. The o-Tyr measurements require invasive approaches, while TG detection may be accomplished by noninvasive analysis in the urine.

URINE BIOMARKERS FOR OXIDATIVE DNA DAMAGE

The maintenance of macromolecular integrity and function, particularly in DNA, appears to be a major determinant of the longevity and functional capacity of biological systems.1 DNA damage induced by active oxygen species therefore may be of primary importance in cancer and aging2–4. This suggests the need for specific biomarkers (a) to elucidate the biochemical nature of DNA lesions and their repair and (b) to monitor noninvasively the generation and removal of DNA damage in biological systems. Several types of damage are known to result from the interactions of free radicals with chromatin, including single- and double-strand breaks, base and sugar alterations, and DNA-protein crosslinks.5 Each of these types of damage seems to be amenable to chemical analysis by the techniques of high performance liquid chromatography (HPLC), gas chromatography/mass spectrometry (GC/MS), alkaline and neutral elution, and other chromatographic and filter techniques. Of particular interest have been the measurements of specific products of oxidative damage to DNA and proteins, such as thymine glycol, thymidine glycol, base-amino acid crosslinks, and altered amino acids. Recently, the measurement of thymine glycol (TG), thymidine glycol (dR-TG), and 5-hydroxymethyluracil (HMU) in urine has been suggested to be a suitable approach for the in situ assessment of oxidative DNA damage caused by every day metabolic processes.References

Urinary Biomarkers in Radiation Therapy of Cancer

The ability to monitor noninvasively the biological effect of a radiation dose in humans is potentially beneficial for screening cancer patients during the course of radiotherapy. Yet no universal approach exists for this purpose other than routine examination of patients and visible tumors, although in limited situations specific parameters are measured after treatment is completed (e.g., determinations of antibody levels after breast cancer therapy). In all cases, no appreciable indicators of the effects of the irradiation are apparent during the early part of the therapy regimen.

Oxygen Radicals, Lipid Peroxidation and DNA Damage in Mitochondria

Oxygen activation in eukaryotic cells occurs mainly within mitochondria, where the respiratory electron transport chain metabolizes approximately 90% of cellular oxygen. While mitochondrial oxygen metabolism supplies the cell with most of its ATP, it also results in the production of hazardous oxygen radicals.1,2 Oxygen radical production has been estimated to account for approximately 1 to 2% of mitochondrial oxygen consumption.3 Although the mitochondrion is protected by an elaborate system of antioxidants and scavengers,2,4 free radicals may escape their surveillance and cause damage to mitochondrial components. The close spatial relationship between the site of mitochondrial oxygen activation, the peroxidizable lipids of the Inner membrane, and mitochondrial DNA (mtDNA) suggests that oxygen radicals and lipid peroxidation may cause mutation of the mitochondrial genome. The condition of mitochondrial DNA structure is of fundamental importance because this genome encodes key enzymes of the respiratory chain.5 In view of growing evidence indicating that abnormalities of mtDNA may contribute to the etiology of aging6 and a number of diseases, including cancer,7,8 the relationship of mitochondrial oxygen radical production and mtDNA integrity merits further consideration.

Standard reference materials (SRM's) for measuring genetic damage

SummaryStandard reference materials were designed for the measurement of radiolytic products resulting from OH radical reaction with DNA in patients treated by radiation therapy. Deuterated thymine glycol and thymidine glycol are proposed as such SRMs; the synthesis of the former is described in detail. They might be of importance for optimising the therapy.