During primate evolution, a major factor in lengthening life-span and decreasing age-specific cancer rates may have been improved protective mechanisms against oxygen radicals. We propose that one ofthese protective systems is plasma uric acid, the level of which increased markedly during primate evolution as a consequence of a series of mutations. Uric acid is a powerful antioxidant and is a scavenger of singlet oxygen and radicals. We show that, at physiological concentrations, urate reduces the oxo-heme oxidant formed by peroxide reaction with hemoglobin, protects erythrocyte ghosts against lipid peroxidation, and protects erythrocytes from peroxidative damage leading to lysis. Urate is about as effective an antioxidant as ascorbate in these experiments. Urate is much more easily oxidized than deoxynucleosides by singlet oxygen and is destroyed by hydroxyl radicals at a comparable rate. The plasma urate level in humans (about 300 ILM) is considerably higher than the ascorbate level, making it one of the major antioxidants in humans. Previous work on urate reported in the literature supports our experiments and interpretations, although the findings were not discussed in a physiological context.Toxicity by oxygen radicals has been suggested as a major cause of cancer, heart disease, and aging (1-13). Oxygen radicals and other oxidants appear to be toxic in large part because they initiate the chain reaction of lipid peroxidation (rancidity). Lipid peroxidation generates various reactive species-such as radicals, hydroperoxides, aldehydes, and epoxides-with the capability of causing damage to DNA, RNA, proteins, cellular membranes, and cellular organization. Aerobic organisms have an array of protective mechanisms both for preventing the formation of oxidants and lipid peroxidation and for repairing oxidative damage. The protective systems include enzymes, such as superoxide dismutase (12) and the selenium-containing glutathione peroxidase (9, 10), and antioxidants and radical scavengers, such as a-tocopherol (vitamin E) and a-carotene in the lipid portion ofthe cell and glutathione and ascorbic acid in the aqueous phase (9, 10). These protective mechanisms are now being recognized as anticarcinogenic and, in some cases, even as life-span extending (5-7).A marked increase in life-span has occurred in human evolution during the descent from prosimians over the past 60 million years (4). At the same time an enormous decrease in the age-specific cancer rate has occurred in humans compared to short-lived mammals (14, 15). It seems likely that a major factor in lengthening life-span and decreasing age-specific cancer rates may have been the evolution ofeffective protective mechanisms against oxygen radicals (2-7, 10). We propose that one such mechanism is high plasma uric acid. MATERIALS AND METHODS'y-Ray Irradiation. Solutions of substrate (0.3 mM) in potassium phosphate buffer (20 mM, pH 7.4) were purged with 2, N2, or N20, sealed, and irradiated at room temperature with a 60Co y-ray source [12.7 krads/mi...

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A new tester strain, TA102, is described as an addition to the set of strains for the Salmonella/microsome mutagenicity test. This strain contains A-T base pairs at the site of the mutation (determined by DNA sequence analysis) in contrast to the other Salmonella tester strains that detect mutagens damaging G-C base pairs. This strain differs from previous tester strains in that the mutation has been introduced into a multicopy plasmid, so that -30 copies of the mutant gene are available for back mutation. The new strain detects a variety of oxidative mutagens, including x-rays, bleomycin, hydrogen peroxide and other hydroperoxides, streptonigrin and other quinones, and phenylhydrazine; a variety of aldehydes, including formaldehyde, glyoxal, kethoxal, glutaraldehyde, and malondialdehyde; a number of psoralens (in the presence of near-UV light), mitomycin C, neocarzinostatin, and UVlight. Some ofthese mutagens have been previously shown to damage thymine in DNA. Several auxiliary tester strains also are described, including TA96, a frameshift tester strain with a hot spot for mutation at a run of five A-T base pairs with a specificity similar to that of TA102. The importance of oxidative mutagens is discussed.Damage to DNA is likely to be a major cause ofcancer and other diseases (1, 2). The Salmonella mutagenicity test (3), along with other short-term assays (4), is being extensively used to survey a variety of substances in our environment for mutagenic activity. The test measures back-mutation in several specially constructed mutants of Salmonella. A homogenate of rat liver (or other mammalian tissue) is added to the bacterial suspension as an approximation ofmammalian metabolism (3). By using this system, over 80% of the organic carcinogens tested have been detected as mutagens (5-7).All of the histidine-requiring mutants in the standard set of Salmonella tester strains have G-C base pairs at the critical site for reversion: -C-C-C-in the base-pair substitution strain TA100,* -C-C-C-C-C-C-in the frameshift strain TA97 (8), and -C-G-C-G-C-G-C-G-in the frameshift tester strain TA98 (9). The present study describes tester strains that have A-T base pairs at the critical site for reversion. These strains detect a variety of oxidants and other agents as mutagens which were not detected in the standard tester strains. Cloning and Sequence Analysis of hisG428. The ochre mutation, hisG428 (11), was cloned by in vivo recombination into a derivative of phage M13Hol76 carrying the hisD6610 mutation (8). M13Hol76 contains the histidine operator, G, D, and part of the C genes from Salmonella. The male derivative of hisG428, TA2894, was infected with M13Hol76hisD6610 and recombinant phage were selected by their ability to complement the hisA(D)2121 deletion host, TA2891, and the inability to complement the hisA(G)8476 deletion host, TA2892. Singlestranded DNA from this HisD+ HisG-recombinant phage was isolated (12) and subjected to sequence analysis (13). MATERIALS AND METHODSPlasmid pAQl. M13Hol76hisG428 DNA was...

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 ...

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