Thymine glycol and thymidine glycol in human and rat urine: a possible assay for oxidative DNA damage.

Abstract

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.Oxygen radicals and other reactive oxygen species are generated in vivo as a consequence of normal metabolism (1-4). The oxidation of certain cellular components by these oxygen species could contribute to aging and age-dependent diseases such as cancer. Oxygen radicals have been shown to produce base damage and strand breaks in DNA (1, 3), as well as to initiate the process of lipid peroxidation (1, 3). The latter results in the formation of lipid hydroperoxides, which, in the presence of cellular iron-containing compounds, can also break down to yield oxygen radicals (1).Numerous defense mechanisms within the cell have evolved to limit the levels of reactive oxygen species and the damage they induce (1-5). Among the cellular defenses are the enzymes superoxide dismutase, catalase, and glutathione peroxidase (1-4), as well as antioxidants such as (3-carotene (1-4), the tocopherols (6), and uric acid (7). However, low levels of reactive oxygen species can escape these cellular defenses (8) and produce damage to DNA, protein, and unsaturated fats. One product that is formed in DNA in vitro as a consequence of chemical oxidation or ionizing radiation (an oxidative mutagen) is thymine glycol (5,6-dihydroxydihydrothymine) (9,10). Mammalian DNA repair systems are known to excise this lesion from DNA in vitro (5,(11)(12)(13)(14)(15)(16)(17). We report here the identification, in both human and rat urine, of the cis isomer of thymine glycol (5,6-dihydroxydihydrothymine) and its deoxyribonucleoside thymidine glycol (5,6-dihydroxydihydrothymidine) and we propose that these bases are derived from repair of oxidized DNA.MATERIALS AND METHODS GBq), were oxidized to the cis-glycols by potassium permanganate (9). After removal of the unreacted permanganate with Na2S205, the supernatant was adjusted to 1 M NH4OAc (pH 8.8) and loaded onto a 0.5 x 2 cm boronate affinity column (Affi-Gel 601, Bio-Rad), equilibrated with the same buffer. The column was washed with 1 M NH4OAc (pH 8.8) (buffer A), the cis-glycol fraction was removed by elution with 0.1 M HOAc, and the eluate was concentrated by lyophilization.[3H]Thymine glycol and [3H]thymidine glycol ...

Identification of endogenous electrophiles by means of mass spectrometric determination of protein and DNA adducts.

“…One example, cisthymine glycol (TG) (Fig. 3), has been detected in urine by HPLC (33). We have now developed a GC-MS assay for TG, in which it is quantitated using a d3-labeled internal standard.…”

Role of chemically induced cell proliferation in carcinogenesis and its use in health risk assessment.

“…It has been suggested that hyperplasia per se can increase the probability of genetic change by increasing the likelihood of mutations arising from normal DNA replication processes or the conversion of DNA lesions produced by oxygen radicals formed endogenously to mutations (3,10). Various estimates have been made of the amounts of spontaneous DNA damage suffered by rats and humans (11,12), but to date, there have been no estimates of the frequency of mutations caused by such DNA damage. Thus, the significance of DNA damage resulting from endogenous reactants such as oxygen radicals remains speculative.…”

Do mitochondrial DNA fragments promote cancer and aging?

“…Oxygen-dependent DNA damage is also observed with aflatoxin B~ [40] and phorbol esters [41,42]. The formation of thymine glycol and thymidine glycol in vivo has been used as a possible assay for oxidative DNA damage [28,43]. Very recently, the formation of 8-hydroxydeoxyguanosine in DNA by oxygen radicals was reported [44][45][46][47][48][49].…”