thymidine-glycol and thymine-glycol

We have measured the levels of thymine glycol (TG) and thymidine glycol (dTG) in human urine, using an HPLC method. The results show that all 30 specimens examined (including 10 non-neoplastic and 20 neoplastic human urines) contained significant amounts of TG and dTG, average levels (mean +/- SEM) were 0.376 +/- 0.026 and 0.138 +/- 0.009 nmol/kg body weight/day respectively. The average levels of TG and dTG were 0.435 +/- 0.038 and 0.164 +/- 0.017 nmol/kg body weight/day in 10 healthy human urine specimens and 0.347 +/- 0.035 and 0.125 +/- 0.010 nmol/kg body weight/day in 20 neoplastic human urine specimens respectively. No significant differences were found between female and male as well as between the non-neoplastic and neoplastic human urine specimens. There were also wide interindividual variations, which were not age-dependent.

Topics: Adolescent; Adult; Aged; DNA Damage; DNA Repair; Female; Humans; Male; Middle Aged; Neoplasms; Oxidation-Reduction; Thymidine; Thymine

Oxidative damage to DNA is caused by reactive by-products of normal metabolism, as well as by radiation. Oxidized DNA bases excised by DNA repair enzymes and excreted in urine were measured in four different species to determine the relation between specific metabolic rate (ml of O2 consumed per gram of body weight per hr) and oxidative DNA damage. An average of 6.04 nmol of thymine glycol per kg/day and 2.58 nmol of thymidine glycol per kg/day were found in mouse urine and 1.12 nmol of thymine glycol per kg/day and 0.95 nmol of thymidine glycol per kg/day were found in monkey urine. On a body weight basis, mice excrete 18 times more thymine glycol plus thymidine glycol than do humans, and monkeys excrete 4 times more thymine glycol plus thymidine glycol than do humans. When results among mice, rats, monkeys, and humans are compared, specific metabolic rate correlates highly with oxidative DNA damage. These findings are consistent with the theory that free radical-induced DNA damage may play a central role in the aging process.

Topics: Aging; Animals; DNA Damage; Energy Metabolism; Glycols; Haplorhini; Mice; Oxidation-Reduction; Thymidine; Thymine

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Biomarkers; Deoxyguanosine; DNA Damage; Gas Chromatography-Mass Spectrometry; Guanine; Humans; Mice; Rats; Thymidine; Thymine

Thymine glycol is a DNA damage product of ionizing radiation and other oxidative mutagens. In an attempt to find a noninvasive assay for oxidative DNA damage in individuals, we have developed an HPLC assay for free thymine glycol and thymidine glycol in urine. Our results indicate that humans excrete about 32 nmol of the two glycols per day. Rats, which have a higher specific metabolic rate and a shorter life span, excrete about 15 times more thymine glycol plus thymidine glycol per kg of body weight than do humans. We present evidence that thymine glycol and thymidine glycol are likely to be derived from repair of oxidized DNA, rather than from alternative sources such as the diet or bacterial flora. This noninvasive assay of DNA oxidation products may allow the direct testing of current theories which relate oxidative metabolism to the processes of aging and cancer in man.

Topics: Animals; Chromatography, High Pressure Liquid; DNA Replication; Germ-Free Life; Glucose; Humans; Rats; Species Specificity; Spectrophotometry, Ultraviolet; Thymidine; Thymine; Tritium

5,6-Dihydroxy-5,6-dihydrothymine (thymine glycol) is formed in DNA by reaction with oxidizing agents and as a result of ionizing and near-ultraviolet radiation. We describe a rapid purification of cis-5,6-dihydroxy-5,6-dihydrothymine and cis-5,6-dihydroxy-5,6-dihydrothymidine (cis-thymidine glycol) and their use as markers in identifying the thymine glycol moiety in oxidized DNA. Both glycols were prepared by oxidation of [14C]thymine and -thymidine with KMnO4 followed by purification on Sephadex LH-20 (LH-20). [3H]DNA was oxidized with KMnO4 and the thymidine glycol in DNA identified by enzymatic digestion of the DNA followed by cochromatography of the digest with marker [14C]thymidine glycol on LH-20. The cis conformation of the glycol was confirmed by the change in the elution pattern when borate rather than water was used as eluent. Alkaline hydrolysis of a mixture of [14C]thymine glycol and oxidized [3H]DNA followed by trichloroacetic acid precipitation and LH-20 chromatographic analysis of the neutralized supernatant yielded a complex pattern of radioactive degradation products with coincidence of one 14C marker- and one [3H]-DNA-derived peak. All applied radioactivity was recovered. This methodology should be useful in determining thymine glycol content of irradiated DNA and in elucidating the mechanism by which these altered residues are removed from cellular DNA by repair enzymes.

Topics: Chemical Phenomena; Chemistry; DNA, Bacterial; Escherichia coli; Oxidation-Reduction; Potassium Permanganate; Thymidine; Thymine

5,6-Dihydroxy-5,6-dihydrothymine (thymine glycol) is formed in DNA by chemical oxidants and ionizing radiation. We describe the separation of thymine glycol, 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol), thymine, and thymidine by high-pressure liquid chromatography (HPLC). Enzymatic hydrolysates of chemically oxidized or gamma-irradiated single-stranded DNA were cochromatographed with 14C-containing marker compounds. In chemically oxidized DNA, thymidine glycol was the major derivative formed. In addition, there were four rapidly eluting thymine-derived components. In irradiated DNA, thymidine glycol constituted about 5% of the modified thymines, and the rapidly eluting fractions were proportionately increased. DNA isolated from gamma-irradiated and nonirradiated HeLa cells grown in the presence of [3H]thymidine was subjected to enzymatic hydrolysis and HPLC analysis. In control DNA, 0.3% of the thymines were modified. Thirty-six kilorads of gamma radiation caused a 30% increase in thymine damage. Thus, most of the base damage was due to internal beta radiation from incorporated [3H]thymidine. The chromatographic patterns of irradiated and nonirradiated samples were qualitatively the same, but the yields of some products increased 2-fold, while others remained unchanged. A comparison of the HPLC profiles of hydrolysates of in vitro oxidized and irradiated DNA with those of the cellular DNA revealed one fast eluting peak to be absent in cellular DNA, suggesting that it was formed only in single-stranded DNA. In cellular DNA, the major modified thymine was a more hydrophobic derivative not formed by in vitro radiation nor chemical oxidation. As in in vitro irradiated DNA, thymidine glycol constituted 5% of the modified thymines. The presence of cis-thymidine glycol in hydrolysates was confirmed by chromatography on Sephadex LH-20 using water and borate as eluants.

Topics: Chromatography, High Pressure Liquid; DNA; Gamma Rays; Oxidation-Reduction; Thymidine; Thymine