2/21/02 Research Interests. Tuning-up human metabolism, which varies with genetic constitution and changes with age, is likely to be a major way to minimize DNA damage, improve health and prolong healthy lifespan. The decay of mitochondria with age due to oxidation of RNA/DNA, proteins, and lipids is a major interest; we are making progress in reversing some of this decay in old rats by feeding them normal mitochondrial metabolites at high levels and are extending the work to humans. Another major interest is determining optimum micronutrient intakes for minimizing human DNA damage, as an aid in the prevention of cancer, and other degenerative diseases associated with aging [1]. High levels of B vitamins raise the corresponding coenzyme levels in humans and will be beneficial to that considerable fraction of the population who have a variant enzyme with a suboptimal binding affinity for its coenzyme [2].
Delaying the Mitochondrial Decay of Aging. Mitochondria decay with age due to oxidation of RNA/DNA, proteins, and lipids. Oxidative mitochondrial decay is a major contributor to aging [3-7]. We are making progress in reversing some of this decay in old rats by feeding them normal mitochondrial metabolites (acetyl carnitine and lipoic acid) at high levels and are extending the work to humans. The principle behind this effect appears to be that with age increased oxidative damage to protein causes a deformation of structure of key enzymes, with a consequent lessening of affinity (Km) for the enzyme substrate [8]. The effect of age on the enzyme binding affinity can be mimicked by reacting it with malondialdehyde (a lipid peroxidation product. Feeding the substrate (acetyl carnitine) with lipoic acid, a mitochondrial antioxidant, restores the velocity of the reaction, Km for acyl carnitine transferase, and mitochondrial function [8]. In old rats (vs. young rats) mitochondrial membrane potential, cardiolipin level, respiratory control ratio, and cellular O2 uptake are lower; oxidants/02, neuron RNA oxidation, and mutagenic aldehydes from lipid peroxidation are higher [5, 8-10]. Ambulatory activity and cognition declines with age [9, 10]. Feeding old rats acetyl carnitine and lipoic acid for a few weeks restores mitochondrial function; lowers oxidants, neuron RNA oxidation, and mutagenic aldehydes; and increases rat ambulatory activity and cognition (as assayed with the Skinner box and Morris water maze) [8-15].
DNA Damage Increases with Age. Apurinic/apyrimidinic (AP) sites are common DNA lesions that arise from spontaneous depurination or by base excision repair (BER) of modified bases. A biotin-containing aldehyde-reactive probe (ARP) is used to measure AP sites in living cells [16]. The assay was applied to living cells and nuclei. The number of AP sites in old human fibroblasts (IMR90 cells) was about two to three times higher than that in young cells, and the number in human leukocytes from old donors was about seven times that in young donors. The repair of AP sites was slower in senescent compared with young IMR90 cells. An age-dependent decline is shown in the activity of the glycosylase that removes methylated bases in IMR90 cells and in human leukocytes. The decline in excision of methylated bases from DNA suggests an age-dependent decline in 3-methyladenine DNA glycosylase, a BER enzyme responsible for removing alkylated bases.
Micronutrients and DNA Damage. Approximately 40 micronutrients are required in the human diet. Deficiency of vitamins B12, folic acid, B6, niacin, C, or E, or iron, or zinc, appears to mimic radiation in damaging DNA by causing single- and double-strand breaks, oxidative lesions, or both [1]. The percentage of the U.S. population that has a low intake (<50% of the RDA) for each of these eight micronutrients ranges from 2% to 20+%; half of the population may be deficient in at least one of these micronutrients [1]. We have shown [17] that folate deficiency breaks chromosomes due to massive incorporation of uracil in human DNA (4 million/cell) with subsequent single strand breaks in DNA formed during base excision repair: two nearby single strand breaks on opposite strands cause the chromosome to fall apart. The level of folate where we see high uracil and breaks was present in 10% of the U.S. population and close to half of poor urban minorities due to poor diets. Vitamin B12 (14% elderly) and B6 (10% of U.S.) deficiencies also cause high uracil in human DNA and chromosome breaks as indicated by our new evidence and as expected from mechanistic considerations. We are currently attempting to determine the level of these three vitamins that minimizes both nuclear and mitochondrial DNA damage in humans. We have evidence that inadequate iron (19% of women of menstruating age in the U.S.) causes oxidative damage to mtDNA in rats [18, 19] and that inadequate zinc intake causes DNA damage.
Micronutrient deficiency may explain, in good part, why the quarter of the population that eats the fewest fruits and vegetables (5 portions a day is advised) has about double the cancer rate for most types of cancer when compared to the quarter with the highest intake [1]. A number of other degenerative diseases of aging are also associated with low fruit and vegetable intake. 80% of American children and adolescents and 68% of adults do not eat 5 portions a day [1]. Common micronutrient deficiencies are likely to damage DNA by the same mechanism as radiation and many chemicals, appear to be orders of magnitude more important, and should be compared for perspective [1, 20].
Micronutrients and Sperm. We are investigating the effect of inadequate micronutrient intake on genetic damage to sperm [1]. We have shown that folic acid deficiency decreases the sperm count in the rat by 90%, and that uracil is found in the sperm DNA of men on low fruit and vegetable diets. Our recent work in humans [21] shows an inverse association between the level of the non-methyl THF pool, but not the methyl-THF pool, with both sperm count and quality, consistent with a uracil misincorporation mechanism. We had previously shown that men with low vitamin C intake had more oxidative damage to their sperm DNA and that male smokers (smoking depletes the vitamin C level markedly) had more oxidative damage to their sperm [1]. Recent epidemiology supports the notion that smoking males have more offspring with childhood cancer.
1. Ames, B.N., DNA Damage from micronutrient deficiencies is likely to be a major cause of cancer. Mutat. Res., 2001. 475: 7-20.
2. Ames, B.N., I. Elson-Schwab, and E. Silver, High-dose vitamin therapy stimulates variant enzymes with decreased coenzyme-binding affinity (increased Km): relevance to genetic disease and polymorphisms. Am J Clin Nutr, 2002. 75: April.
3. Shigenaga, M.K., T.M. Hagen, and B.N. Ames, Oxidative damage and mitochondrial decay in aging. Proc. Natl. Acad. Sci. USA, 1994. 91: 10771-10778.
4. Beckman, K.B. and B.N. Ames, The free radical theory of aging matures. Physiol. Rev., 1998. 78: 547-581.
5. Hagen, T.M., D.L. Yowe, J.C. Bartholomew, C.M. Wehr, K.L. Do, J.-Y. Park, and B.N. Ames, Mitochondrial decay in hepatocytes from old rats: Membrane potential declines, heterogeneity and oxidants increase. Proc. Natl. Acad. Sci. USA, 1997. 94: 3064-3069.
6. Helbock, H.J., K.B. Beckman, M.K. Shigenaga, P. Walter, A.A. Woodall, H.C. Yeo, and B.N. Ames, DNA oxidation matters: The HPLC-EC assay of 8-oxo-deoxyguanosine and 8-oxo-guanine. Proc. Natl. Acad. Sci. USA, 1998. 95: 288-293.
7. Beckman, K.B. and B.N. Ames, Mitochondrial aging: open questions. Ann. N. Y. Acad. Sci., 1998. 854: 118-27.
8. Liu, J., D. Killilea, and B.N. Ames, Age-associated mitochondrial oxidative decay: Improvement carnitine acetyltransferase substrate binding affinity and activity in brain by feeding old rats acetyl-L-carnitine and/or R-a-lipoic acid. Proc Natl Acad Sci U S A, 2002. 99: 1876-1881.
9. Liu, J., E. Head, A.M. Gharib, W. Yuan, R.T. Ingersoll, T.M. Hagen, C.W. Cotman, and B.N. Ames, Memory loss in old rats is associated with brain mitochondrial decay and RNA/DNA oxidation: Partial reversal by feeding acetyl-L-carnitine and/or R-a-lipoic acid. Proc Natl Acad Sci U S A, 2002. 99: 2356-2361.
10. Hagen, T.M., J. Liu, J. Lykkesfeldt, C.M. Wehr, R.T. Ingersoll, V. Vinarsky, J.C. Bartholomew, and B.N. Ames, Feeding acetyl-L-carnitine and lipoic acid to old rats significantly improves metabolic function while decreasing oxidative stress. Proc Natl Acad Sci U S A, 2002. 99: 1870-1875.
11. Hagen, T.M., R.T. Ingersoll, C.M. Wehr, J. Lykkesfeldt, V. Vinarsky, J.C. Bartholomew, M.-H. Song, and B.N. Ames, Acetyl-L-carnitine fed to old rats partially restores mitochondrial function and ambulatory activity. Proc. Natl. Acad. Sci. USA, 1998. 95: 9562-9566.
12. Lykkesfeldt, J., T.M. Hagen, V. Vinarsky, and B.N. Ames, Age-associated decline in ascorbic acid concentration, recycling and biosynthesis in rat hepatocytes ‹ reversal with (R)-a-Lipoic acid supplementation. FASEB J., 1998. 12: 1183-1189.
13. Hagen, T.M., R.T. Ingersoll, J. Liu, J. Lykkesfeldt, C.M. Wehr, V. Vinarsky, J.C. Bartholomew, and B.N. Ames, (R)-a-Lipoic acid-supplemented old rats have improved mitochondrial function, decreased oxidative damage, and increased metabolic rate. FASEB J., 1998. 13: 411-418.
14. Hagen, T.M., C.M. Wehr, and B.N. Ames, Mitochondrial decay in aging. Reversal through dietary supplementation of acetyl-L-carnitine and N-tert-butyl-a-phenylnitrone. Ann. N. Y. Acad. Sci., 1998. 854: 214-223.
15. Hagen, T.M., V. Vinarsky, C.M. Wehr, and B.N. Ames, (R)-a-lipoic acid reverses the age-associated increase in susceptibility of hepatocytes to tert-butylhydroperoxide both in vitro and in vivo. Antiox. Redox Signal., 2000. 2: 473-483.
16. Atamna, H., I. Cheung, and B.N. Ames, A method for detecting abasic sites in living cells: age-dependent changes in base excision repair. Proc Natl Acad Sci U S A, 2000. 97(2): 686-91.
17. Blount, B.C., M.M. Mack, C. Wehr, J. MacGregor, R. Hiatt, G. Wang, S.N. Wickramasinghe, R.B. Everson, and B.N. Ames, Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: Implications for cancer and neuronal damage. Proc. Natl. Acad. Sci. USA, 1997. 94: 3290-3295.
18. Atamna, H., P.W. Walter, and B.N. Ames, The role of heme and iron-sulfur clusters in mitochondrial biogenesis, maintenance, and decay with age. Arch. Biochem. Biophys., 2002. 397: 345-353.
19. Walter, P.W., M.D. Knutson, A. Paler-Martinez, S. Lee, Y. Xu, F.E. Viteri, and B.N. Ames, Iron deficiency and iron excess damage mitochondria and mitochondrial DNA in rats. Proc Natl Acad Sci U S A, 2002. 99: 2264-2269.
20. Ames, B.N. and L.S. Gold, Paracelsus to parascience: the environmental cancer distraction. Mutat Res, 2000. 447(1): 3-13.
21. Wallock, L.M., T. Tamura, C.A. Mayr, K.E. Johnston, B.N. Ames, and R.A. Jacob, Low seminal plasma folate concentrations are associated with low sperm density and count in male smokers and nonsmokers. Fertil. & Steril., 2001. 75: 252-259.
Dr. Ames is a Professor of Biochemistry and Molecular Biology, University of California, Berkeley. He is a member of the National Academy of Sciences and he was on their Commission on Life Sciences. He was a member of the board of directors of the National Cancer Institute, the National Cancer Advisory Board, from 1976 to 1982. He was the recipient of the General Motors Cancer Research Foundation Prize (1983), the Tyler Environmental Prize (1985), the Gold Medal Award of the American Institute of Chemists (1991), the Glenn Foundation Award of the Gerontological Society of America (1992), the Lovelace Institutes Award for Excellence in Environmental Health Research (1995), the Honda Prize of the Honda Foundation, Japan (1996), the Japan Prize, (1997), the Kehoe Award, American College of Occup. and Environ. Med. (1997), the Medal of the City of Paris (1998), the U.S. National Medal of Science (1998), The Linus Pauling Institute Prize for Health Research (2001), and the American Society for Microbiology Lifetime Achievement Award (2001). His over 450 publications have resulted in his being among the few hundred most-cited scientists (in all fields):23rd most-cited (1973-1984). http://socrates.berkeley.edu/mutagen