Mark K. Shigenaga

Metabolism, like other aspects of life, involves tradeoffs. Oxidant by-products of normal metabolism cause extensive damage to DNA, protein, and lipid. We argue that this damage (the same as that produced by radiation) is a major contributor to aging and to degenerative diseases of aging such as cancer, cardiovascular disease, immune-system decline, brain dysfunction, and cataracts. Antioxidant defenses against this damage include ascorbate, tocopherol, and carotenoids. Dietary fruits and vegetables are the principal source of ascorbate and carotenoids and are one source of tocopherol. Low dietary intake of fruits and vegetables doubles the risk of most types of cancer as compared to high intake and also markedly increases the risk of heart disease and cataracts. Since only 9% of Americans eat the recommended five servings of fruits and vegetables per day, the opportunity for improving health by improving diet is great.

We argue for the critical role of oxidative damage in causing the mitochondrial dysfunction of aging. Oxidants generated by mitochondria appear to be the major source of the oxidative lesions that accumulate with age. Several mitochondrial functions decline with age. The contributing factors include the intrinsic rate of proton leakage across the inner mitochondrial membrane (a correlate of oxidant formation), decreased membrane fluidity, and decreased levels and function of cardiolipin, which supports the function of many of the proteins of the inner mitochondrial membrane. Acetyl-L-carnitine, a high-energy mitochondrial substrate, appears to reverse many age-associated deficits in cellular function, in part by increasing cellular ATP production. Such evidence supports the suggestion that age-associated accumulation of mitochondrial deficits due to oxidative damage is likely to be a major contributor to cellular, tissue, and organismal aging.

Oxidative damage to DNA is shown to be extensive and could be a major cause of the physiological changes associated with aging and the degenerative diseases related to aging such as cancer. The oxidized nucleoside, 8-hydroxy-2'-deoxyguanosine (oh8dG), one of the approximately 20 known oxidative DNA damage products, has been measured in DNA isolated from various organs of Fischer 344 rats of different ages. oh8dG was present in the DNA isolated from all the organs studied: liver, brain, kidney, intestine, and testes. Steady-state levels of oh8dG ranged from 8 to 73 residues per 10(6) deoxyguanosine residues or 0.2-2.0 x 10(5) residues per cell. Levels of oh8dG in DNA increased with age in liver, kidney, and intestine but remained unchanged in brain and testes. The urinary excretion of oh8dG, which presumably reflects its repair from DNA by nuclease activity, decreased with age from 481 to 165 pmol per kg of body weight per day for urine obtained from 2-month- and 25-month-old rats, respectively. 8-Hydroxyguanine, the proposed repair product of a glycosylase activity, was also assayed in the urine. We estimate approximately 9 x 10(4) oxidative hits to DNA per cell per day in the rat. The results suggest that the age-dependent accumulation of oh8dG residues observed in DNA from liver, kidney, and intestine is principally due to the slow loss of DNA nuclease activity; however, an increase in the rate of oxidative DNA damage cannot be ruled out.

Damage to the DNA of germ cells can lead to mutation, which may result in birth defects, genetic diseases, and cancer. The very high endogenous rate of oxidative DNA damage and the importance of dietary ascorbic acid (AA) in preventing this damage has prompted an examination of these factors in human sperm DNA. The oxidized nucleoside 8-hydroxy-2'-deoxyguanosine (8-oxo-7,8-dihydro-2'-deoxyguanosine; oxo8dG), 1 of approximately 20 major products of oxidative damage to DNA, was measured in DNA isolated from human sperm provided by healthy subjects and compared to the seminal fluid AA levels. This relationship was studied in two groups. In a group of 24 free-living individuals 20-50 years old high levels of oxo8dG were correlated with low seminal plasma AA. The endogenous level of oxo8dG in this group was 13 fmol per microgram of DNA or approximately 25,000 adducts per sperm cell. The second group of individuals was maintained on a controlled diet that varied only in AA content. When dietary AA was decreased from 250 to 5 mg/day, the seminal fluid AA decreased by half and the level of oxo8dG in sperm DNA increased 91%. Repletion of dietary AA for 28 days (from 5 mg/day to 250 or 60 mg/day) caused a doubling in seminal fluid AA and reduced oxo8dG by 36%. These results indicate that dietary AA protects human sperm from endogenous oxidative DNA damage that could affect sperm quality and increase risk of genetic defects, particularly in populations with low AA such as smokers.