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Lawrence Berkeley National Laboratory Recent Work Title AGING: A THEORY BASED ON free radical
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UCRL-3078 Unclassified Health and Biology UNIVERSITY OF CALIFORNIA Radiation Laboratory Berkeley, CA
lifornia Contract No. W-7405-eng-48 AGING: A THEORY BASED ON FREE RADICAL AND RADIATION CHEMISTRY Denham Harman July 14, 1955 (\ Printed for the U, S. Atomic Energy Commission
AGING: A THEORY BASED ON FREE RADICAL AND RADIATION CHEMISTRY* De nharn Har rna n Donner Laboratory of Biophysics and Medical Physics University of California Berkeley
, California July 14, 1955
The phenomenon onsiderable specu
and death-aging.; cycle appears to
has· b.eert'the:'s.c::mr:te be a more or less
direct function of the metabolic rate
and this in turn largely depends on the
_species (animal or plant) on which are superimposed as second order effects
the factors of heredity and of the stresses and strain of life --- which alter
the metabolic activity.
The universality of this phenomenon suggests that the reactions which cause it are basically the same in all living things. Viewing this process, which in essence is cellular degeneration, in the light of present day free radical and radiation chemistry and of radiobiology it seems possible that one factor in aging may be related to "deleterious side attacks of free radicals
(which.are normally produced in the course of cellular metabolism) on cell constituents. 4 Irradiation of living things induces mutation, cancer, and aging . Inasmuch as these also arise spontaneously in nature it is natural to inquire if the processes might not be similar. It is believed that one mechanism of irradiation effect is through liberation of OH and H02 radicals 5. There is evidence, although indirect, that these two highly active free radicals are produced.normally in living systems
. In the first place free radicals are present in living cells; this was recently demonstrated in vivo by a paramagnetic resonance absorption rnethod6. Further, it was shown that the concentration of free radicals increased with increasing metabolic activity in conformity with the postulates set forth some years ago that free radicals were involved in biological oxidation - reduction reactions 7 · 8 · ·A.re some of the~e free radicals OH and/or H02, or radicals of a similar high order of reactivity, and where might they arise in the cell?
The most likely source of OH and H02 radicals, at least in the animal cell, would be the interaction of the respiratory enzymes involved in the direct utilization of molecular oxygen, particularly those containing iron, and by the action of catalase on hydrogen peroxide. This follows from the * This communication is written as the introductory article of a planned series of notes concerning the effect of free radicals on aging. Experiments have now been initiated in this laboratory to investigate and, if possible, prove the validity of the ideas presented.
fact' that it has been known for mand years tl~at iron salts catalyse the air oxidation of organic compounds
9· 1 ; OH radicals are believed to be involved in these reactions 4 . I'ron salts also catalyse the decomposition of hydrogen peroxide to water and oxygen - a reaction that involves the OH and H02 radicals ll Further, recent studies in this laboratory on t];le inactivation of rat liver
catalase suggest that the OH radical is involved. The catalase activity of the homogenates both in the presence and absence of hydrogen donors such as sodium bisulfite, sodium hypophosphite, pyrogallol and mercaptans remains relatively constant under an atmosphere of nitrogen. However, in the presence of air catalase activity rapidly decreases and the rate of decrease is accelerated in the presence of the hydrogen donors. In addition, methanol, ethanol, and sodium formate (compounds which are oxidized by hydrogen peroxide in the presence of catalase 12} stabilize the enzym'e in the presence of air. A free radical mechanism involving the OH radical has been formulated to account for these facts. The H02 radical . has been implicated in the analogous degradation of hemoglobin and myoglobin13
Thus, although the evidence is indirect, there are good reasons for assuming that the changes produced by irradiation and those which arise spontaneously in the living cell have a common source - the OH and H02 radicals. These al-ise on the one hand through the dissociation of water and on the other largely by the interaction of the oxidative enzymes with oxygen and hydrogen peroxide. (It is not unlikely that other metal containing enzymes, such as vitamin B 12 , which cont~ins cobalt, also contribute. }
The manner in which a highly reactive radical such as OH would exert
its effect on a cell is obscure. However, it would be expected to react for
the most part near the area where it was produced and to react with the more
easily oxidized substances such as DPNH2 or the reduced form
of the flavoproteins. They would also be expected to react to a certain extent with other
cellular constituents including the nucleoproteins and nucleic acids. The
organic radicals formed in this manner (by removal of a hydrogen atom)
could then undergo further reaction e. g. addition of oxygen leading to the
formation of peroxides and other oxygenated compounds, degradation into
smaller units, dimerization, etc., such as has been observed in simpler
free radical and polymer systems
14 In this manner the functional efficiency
and reproductive ability of the cell could eventually be impaired. In addition,
since genes would be expected to be attacked occasionally it would be anti-
cipated that mutations and cancer would result every now and then.
In a multicellular organism such as man the effects of cells on each other are superimposed on the above. Some cells are- more important than others in maintaining life. For example, as degenerative changes occur in the cells of the circulatory system
the flow of oxygen and metabolites to
other cells is interfered with thus leading to further degenerative changes in them. This theory is suggestive of chemical means of prolonging effective life. For example, maintenance of an increased cellular concentration of an easily reduced compound such as cysteine, which affords some Radiation Protection
, would be expected to slow down the aging process and thereby put off the appearance of the diseases associated with iL As a side effect radiation resistance would be enhanced. Further studies of the effect of hydroxy and other radicals, in .the presence and absence of oxygen and easily oxidized substances, on cellular constituents such as DNA and RNA may be quite productive. Some of the implications of this theory both as it pertains to aging and to cancer are now under study. Groups of mice of the AKR and C3H strains., which spontaneously develop lymphatic leukemia and mammary Cancer Res
pectively, are daily being given various radiation protection compounds in their dieL These animals will be followed to determine if the average age at which they develop leukemia or cancer is greater than in the controls. Mice in each group are also being sacrificed at periodic intervals for histologic study. A somewhat similar investigation has been started with Drosophila. Consideration of the biochemistry of cancer cells
and of the systemic effects observed in cancer from the standpoint of the theory presented in this paper led to the conclusion that hydrogen donors, such as cysteine for example, -might be of benefit in the fields of cancer chemotherapy and nutrition. Preliminary work with ascites tumor and LCS cancer in mice supports this conclusion. This research was done under the auspices of the Atomic Energy Commission.
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, (Saunders, Philadelphia, 1943). 4. Hempelmann, Z. H. and Hoffman, J. G., Ann. Rev. of Nuclear Science ~· 36 9 (19 53). 5. Stein, G. and Weiss, J. , Nature, 161, 6 50 (1948 ). 6. Commoner, B., Townsend, J. and Pake, S. E. Nature 174, 689 (1954). 7. Waters, W. A., "Chemistry of Free Radicals" (Oxford, 1948 ). 8. Michealis, L. in "The Enzymes", edited by J. B. Summer and K. Myrback. Vol. 2, Part 1, Chapter 44 (Academic Press, 1951). 9. Fenton, J., Chern. Soc., 65, 899 (1894); 75, 1 (1900). 10. Wieland, Annalin, 457, 1 (1927); 459, 1 (1930). 11. Uri, N., Chern. Rev. 50,375, 1952. 12. Chance, B. , in "The Enzymes", edited by J. B. Summer and K. Myrback, Vol. 2, Part 1, Chapter 56 (Academic Press, 1951). 13. George, P., in Advances in Catalysis, edited by Frankenburg, W. G., Komarewsky, V. I., and Rideal, E. K. Vol. 4, page 367. (Academic Press, 1952). 14. D 1Alelio, G. F., fundamental principles
of Polymerization (John Wiley and Sons
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