AGING: A THEORY BASED ON FREE AND RADIATION DENHAM HARMAN, MD ., Ph .D. (From the Donner Laboratory of Biophysics and Medical Physics, Uniuersity of Califomia, Berkeley)

The phenomenon of growth, decline and in the direct utilization of molecular oxygen, death-aging-has been the source of consider- particularly those containing iron, and by the able speculation (1, 8, 10) . This cycle seems action of catalane on hydrogen peroxide. This to be a more or less direct function of the meta- follows from the fact that it has been known bolic rate and this in turn depends on the for many years that iron salts catalyze the air species (animal or plant) on which are super- oxidation of organic compounds (5, 6, 14, 15); imposed the factors of heredity and the effects OH radicals are believed to be involved in these of the stresses and strains of life-which alter reactions (13) . Iron salts also catalyze the de- the metabolic activity. composition of hydrogen peroxide to water and The universality of this phenomenon sug- oxygen-a reaction that involves OH and HO, gests that the reactions which cause it are basic- radicals (16) . Further, recent studies in this ally the same in all living things. Viewing this laboratory on the inactivation of rat liver cat- process in the light of present day free radical alase suggest that the OH radical is involved. and radiation chemistry and of radiobiology, it The catalane activity of the homogenates both seems possible that one factor in aging may be in the presence and absence of hydrogen donors related to deleterious side attacks of free radicals such as sodium bisulfite, sodium hypophosphite, (which are normally produced in the course of pyrogallol, and mercaptans remains relatively cellular metabolism) on cell constituents.' constant under an atmosphere of nitrogen. Irradiation of living things induces mutation, However, in the presence of air, catalane activity cancer, and aging (9). Inasmuch as these also rapidly decreases and the rate of decrease is arise spontaneously in nature, it is natural to accelerated in the presence of the hydrogen inquire if the processes might not be similar. donors. In addition, , , and It is believed that one mechanism of irradiation sodium formate (compounds (2) which are effect is through liberation of OH and HO, oxidized by hydrogen peroxide in the presence radicals (12) . There is evidence, although in- of catalane) stabilize the enzyme in the presence direct, that these two highly active free radicals of air. A free radical mechanism involving the are produced normally in living systems. In the OH radical has" been implicated in the analo- first place, free radicals are present in living gous degradation of hemoglobin and myoglobin cells; this was recently demonstrated in vivo by (7) . a paramagnetic resonance absorption method Thus, although the evidence is indirect, there (3) . Further, it was shown that the concentra- are good reasons for assuming that the changes tion of free radicals increased with increasing produced by irradiation and those which arise metabolic activity in conformity with the postu- spontaneously in the living cell have a common lates set forth some years ago that free radicals source-the OH and HO, radicals. These arise were involved in biologic oxidation-reduction on the one hand through the dissociation of reactions (11, 13) . Are some of these free radi- water and on the other largely by the interac- cals OH and/or HO or radicals of a similar tion of the oxidative enzymes with oxygen and high order of reactivity, and where might they hydrogen peroxide. (It is not unlikely that arise in the cell? other metal-containing enzymes, such as vita- The most likely source of OH and HO, radi- min Bl which contains cobalt, also contrib- cals, at least in the animal cell, would be the ute.) interaction of the respiratory enzymes involved The manner in which a highly reactive radi- Submitted. forpobiintion Much 23, 1956. would exert its effect on a cell 7ûie research was performed under the auspices of the Atomic cal such as OH Fmeru Commtnlon. is obscure. However, it would be expected to Published on a gttnt from the Forest Park Foundation to the journal of Gerontology. react for the most part near the area where it

AGING: FREE RADICAL THEORY 299

was produced and to react with the more easily average age at which they develop leukemia or oxidized substances such as DPNH, or the re- cancer is greater than in the controls. Mice in duced form of the flavoproteins. They would each group are also being sacrificed at periodic also be expected to react to a certain extent with intervals for histologic study. other cellular constituents including the nucleo- Consideration of the biochemistry of cancer proteins and nucleic acids. The organic radicals cells and of the systematic effects in cancer from formed in this manner (by removal of a hydro- the standpoint of the theory presented in this gen atom) could then undergo further reac- paper led to the conclusion that hydrogen tions, e.g., addition of oxygen leading to the donors, such as cysteine for example, might be formation of peroxides and other oxygenated of benefit in the fields of cancer chemotherapy compounds, degradation into smaller units, and nutrition. Preliminary work with ascites dimerization, etc., such as has been observed in tumor and LCS cancer in mice supports this simpler free radical and polymer systems (4) . conclusion. In this manner the functional efficiency and re- productive ability of the cell could eventually SUMMARY be impaired. In addition, since genes would be Aging find the degenerative . diseases associ- expected to be attacked occasionally it would be ated with it are attributed basically to the dele- anticipated that mutations and cancer would terious side attacks of free radicals on cell con- result every now and then. stituents and on the connective tissues. The free In a multicellular organism such as man the radicals probably arise largely through reactions effects of cells on each other are superimposed involving molecular oxygen catalyzed in the cell on the above. Some cells are more important by the oxidative enzymes and in the connective than others in maintaining life. For example, tissues by traces of metals such as iron, cobalt, as degenerative changes occur in the cells of and manganese. the circulatory system, the flow of oxygen and metabolites to other cells is interferred with, REFERENCES thus leading to further degenerative changes in 1. Brody, S.: Bioenergetics and Growth. Reinhold them. Publishing Corp., New York, 19$5. within the cells 2 Chance, B. : The Iron-Containing Enzymes. The In addition to the reactions Enzyme-Substrate Compounds and Mechanism of themselves, one would expect that there would Action of the Hydroperoxidases. Chapter 56 C. In be also a slow oxidation of the connective tissue The Enzymes, edited by Sumner, 1. B. and Myr- by molecular oxygen catalyzed by metals such Nick, K, Vol. 2, Part 1. Academic Press, Ina, New as iron, cobalt, and manganese. York, 1951. 3. Commoner, B., Townsend, J., and Pake, G. E: This theory is suggestive of chemical means Free Radicals in Biological Materials. Nature, 174: of prolonging effective life. For example, main- 689-691, 1954. tenance of an increased cellular concentration 4. D'Alelio, G. F. : Fundamental Principles of Poly- of an easily reduced compound such as cysteine, merization. John Wiley and Sons, Ina, New York, which affords some radiation protection, would 1952 be expected to slow down the aging process and 5. Fenton, H. J. H.: Oxidation of Tartaric Acid in . thereby put off the appearance of the diseases Presence of Iron. J. Chem. Soc. 65: 899-910, 1894 it. As a side effect radiation 6. Fenton, H. J. H., and Jackson, H. : The Oxidation associated with of be enhanced. Further studies of. Polyhydric Alcohols in Presence Iron. ]. resistance would Chem. Soc., 75: 1-11, 1899. of the effect of hydroxy and other radicals, in 7. George, P. : The Specific Reactions of Iron in Some the presence and absence of oxygen and easily Hemoproteins . In Advances in Catalysis, edited by oxidized substances, on cellular constituents Frankenburg, W. G., Komarewsky, V. L, and such as DNA and RNA may be quite produc- Rideal, E K, Vol. 4,367-428. Academic Press, Ina, tive. Some of the implications of this theory New York, 1952 both as it pertains to aging and to cancer are 8. Heilbrunn, Z. V.: Outline of General Physiology, W. 1943. now under study. Groups of mice of the AKR 2nd Edition. B. Saunders, Philadelphia, which spontaneously develop 9. Hempelmann, L. H., and Hoffman, J. G.: Practical and C3H strains, Aspects of Radiation Injury. Ann. Rev. Nuclear lymphatic leukemia and mammary cancer, re- Sci., 3: 369-389,1953. spectively, are daily being given various radia- 10. Lansing, A. L, editor: Cowdry's Problems of Age- tion protection compounds in their diet. These ing, 3rd Edition, Williams and Wilkins Co., Balti- animals will be followed to determine if the more, 1952

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11. Michealis, L: Theory of Oxidation-Reduction, 14. Wieland, H, and Frage, K: über den Mechanis- Chapter 44 in The Enzymes, edited by Sumner, mus der Oxidationsvorgänge. XX. Bersteins ure- J. B. and Myrbiick, K, Vol. 2, Part 1. Academic Dehydrase. Ann., 477: 1-32, 1930. Press, Inc., New York, 1951. 15. Wieland, FL, and Franke, K: über den Mechanis- 12. Stein, G., and Weiss, J.: Chemical Effects of Ioniz- mus der Oxydationsvorgänge. X1I Die Aktivierung ing Radiations. Nature, 161: 650, 1948. des Hydroperoxyds durch Eisen. Ann., 457: 1-70, 1927. 13. Waters, W. A: The Chemistry of Free Radicals 16. Uri, N.: Inorganic Free Radicals in Solution, Oxford University Press, London, 1946. Chem. Rev., 40: 375-154, 1952.