AGING: A THEORY BASED ON FREE RADICALAND RADIATION CHEMISTRY

DENHAM HARMAN, M.D., Ph.D.(From the Donner Laboratory of Biophysics and Medical Physics,

University of California, Berkeley)

The phenomenon of growth, decline anddeathaginghas been the source of consider-able speculation (1, 8, 10). This cycle seemsto be a more or less direct function of the meta-bolic rate and this in turn depends on thespecies (animal or plant) on which are super-imposed the factors of heredity and the effectsof the stresses and strains of lifewhich alterthe metabolic activity.

The universality of this phenomenon sug-gests that the reactions which cause it are basic-ally the same in all living things. Viewing thisprocess in the light of present day free radicaland radiation chemistry and of radiobiology, itseems possible that one factor in aging may berelated to deleterious side attacks of free radicals(which are normally produced in the course ofcellular metabolism) on cell constituents.*

Irradiation of living things induces mutation,cancer, and aging (9). Inasmuch as these alsoarise spontaneously in nature, it is natural toinquire if the processes might not be similar.It is believed that one mechanism of irradiationeffect is through liberation of OH and HO2radicals (12). There is evidence, although in-direct, that these two highly active free radicalsare produced normally in living systems. In thefirst place, free radicals are present in livingcells; this was recently demonstrated in vivo bya paramagnetic resonance absorption method(3). Further, it was shown that the concentra-tion of free radicals increased with increasingmetabolic activity in conformity with the postu-lates set forth some years ago that free radicalswere involved in biologic oxidation-reductionreactions (11, 13). Are some of these free radi-cals OH and/or HO2, or radicals of a similarhigh order of reactivity, and where might theyarise in the cell?

The most likely source of OH and HO2 radi-cals, at least in the animal cell, would be theinteraction of the respiratory enzymes involved

Submitted for publication March 23, 1956.* This research was performed under the auspices of the Atomic

Energy Commission.Published on a grant from the Forest Park Foundation to the

Journal of Gerontology.

in the direct utilization of molecular oxygen,particularly those containing iron, and by theaction of catalase on hydrogen peroxide. Thisfollows from the fact that it has been knownfor many years that iron salts catalyze the airoxidation of organic compounds (5, 6, 14, 15);OH radicals are believed to be involved in thesereactions (13). Iron salts also catalyze the de-composition of hydrogen peroxide to water andoxygena reaction that involves OH and HO2radicals (16). Further, recent studies in thislaboratory on the inactivation of rat liver cat-alase suggest that the OH radical is involved.The catalase activity of the homogenates bothin the presence and absence of hydrogen donorssuch as sodium bisulfite, sodium hypophosphite,pyrogallol, and mercaptans remains relativelyconstant under an atmosphere of nitrogen.However, in the presence of air, catalase activityrapidly decreases and the rate of decrease isaccelerated in the presence of the hydrogendonors. In addition, methanol, ethanol, andsodium formate (compounds (2) which areoxidized by hydrogen peroxide in the presenceof catalase) stabilize the enzyme in the presenceof air. A free radical mechanism involving theOH radical has been implicated in the analo-gous degradation of hemoglobin and myoglobin(7).

Thus, although the evidence is indirect, thereare good reasons for assuming that the changesproduced by irradiation and those which arisespontaneously in the living cell have a commonsourcethe OH and HO2 radicals. These ariseon the one hand through the dissociation ofwater and on the other largely by the interac-tion of the oxidative enzymes with oxygen andhydrogen peroxide. (It is not unlikely thatother metal-containing enzymes, such as vita-min B12, which contains cobalt, also contrib-ute.)

The manner in which a highly reactive radi-cal such as OH would exert its effect on a cellis obscure. However, it would be expected toreact for the most part near the area where it

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was produced and to react with the more easilyoxidized substances such as DPNH2 or the re-duced form of the flavoproteins. They wouldalso be expected to react to a certain extent withother cellular constituents including the nucleo-proteins and nucleic acids. The organic radicalsformed in this manner (by removal of a hydro-gen atom) could then undergo further reac-tions, e.g., addition of oxygen leading to theformation of peroxides and other oxygenatedcompounds, degradation into smaller units,dimerization, etc., such as has been observed insimpler free radical and polymer systems (4).In this manner the functional efficiency and re-productive ability of the cell could eventuallybe impaired. In addition, since genes would beexpected to be attacked occasionally it would beanticipated that mutations and cancer wouldresult every now and then.

In a multicellular organism such as man theeffects of cells on each other are superimposedon the above. Some cells are more importantthan others in maintaining life. For example,as degenerative changes occur in the cells ofthe circulatory system, the flow of oxygen andmetabolites to other cells is interferred with,thus leading to further degenerative changes inthem.

In addition to the reactions within the cellsthemselves, one would expect that there wouldbe also a slow oxidation of the connective tissueby molecular oxygen catalyzed by metals suchas iron, cobalt, and manganese.

This theory is suggestive of chemical meansof prolonging effective life. For example, main-tenance of an increased cellular concentrationof an easily reduced compound such as cysteine,which affords some radiation protection, wouldbe expected to slow down the aging process andthereby put off the appearance of the diseasesassociated with it. As a side effect radiationresistance would be enhanced. Further studiesof the effect of hydroxy and other radicals, inthe presence and absence of oxygen and easilyoxidized substances, on cellular constituentssuch as DNA and RNA may be quite produc-tive. Some of the implications of this theoryboth as it pertains to aging and to cancer arenow under study. Groups of mice of the AKRand C3H strains, which spontaneously developlymphatic leukemia and mammary cancer, re-spectively, are daily being given various radia-tion protection compounds in their diet. Theseanimals will be followed to determine if the

average age at which they develop leukemia orcancer is greater than in the controls. Mice ineach group are also being sacrificed at periodicintervals for histologic study.

Consideration of the biochemistry of cancelcells and of the systematic effects in cancer Fromthe standpoint of the theory presented in thispaper led to the conclusion that hydrogondonors, such as cysteine for example, might beof benefit in the fields of cancer chemotherapyand nutrition. Preliminary work with ascitestumor and LCS cancer in mice supports thisconclusion.

SUMMARYAging and the degenerative diseases associ-

ated with it are attributed basically to the dele-terious side attacks of free radicals on cell con-stituents and on the connective tissues. The freeradicals probably arise largely through reactionsinvolving molecular oxygen catalyzed in the cellby the oxidative enzymes and in the connectivetissues by traces of metals such as iron, cobalt,and manganese.

REFERENCES1. Brody, S.: Bioenergetics and Growth. Reinhold

Publishing Corp., New York, 1945.2. Chance, B.: The Iron-Containing Enzymes. The

Enzyme-Substrate Compounds and Mechanism ofAction of the Hydroperoxidases, Chapter 56 C. InThe Enzymes, edited by Sumner, J. B. and Myr-back, K., Vol. 2, Part 1. Academic Press, Inc., NewYork, 1951.

3. Commoner, B., Townsend, J., and Pake, G. E.:Free Radicals in Biological Materials. Nature, 174:689-691, 1954.

4. D'Alelio, G. F.: Fundamental Principles of Poly-merization. John Wiley and Sons, Inc., New York,1952.

5. Fenton, H. J. H.: Oxidation of Tartaric Acid inPresence of Iron. /. Chem. Soc. 65: 899-910, 1894.

6. Fenton, H. J. H., and Jackson, H.: The Oxidationof Polyhydric Alcohols in Presence of Iron. /.Chem. Soc, 75: 1-11, 1899.

7. George, P.: The Specific Reactions of Iron in SomeHemoproteins. In Advances in Catalysis, edited byFrankenburg, W. G., Komarewsky, V. I., andRideal, E. K., Vol. 4, 367-428. Academic Press, Inc.,New York, 1952.

8. Heilbrunn, Z. V.: Outline of General Physiology,2nd Edition. W. B. Saunders, Philadelphia, 1943.

9. Hempelmann, L. H., and Hoffman, J. G.: PracticalAspects of Radiation Injury. Ann. Rev. NuclearScL, 3: 369-389, 1953.

10. Lansing, A. I., editor: Cowdry's Problems of Age-ing, 3rd Edition, Williams and Wilkins Co., Balti-more, 1952.

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11. Michealis, L.: Theory of Oxidation-Reduction,Chapter 44 in The Enzymes, edited by Sumner,J. B. and Myrback, K., Vol. 2, Part 1. AcademicPress, Inc., New York, 1951.

12. Stein, G., and Weiss, J.: Chemical Effects of Ioniz-ing Radiations. Nature, 161: 650, 1948.

13. Waters, W. A.: The Chemistry of Free Radicals.Oxford University Press, London, 1946.

14. Wieland, H., and Frage, K.: Ober den Mechanis-mus der Oxidationsvorgange. XX. Bernsteinsaure-Dehydrase. Ann., 477: 1-32, 1930.

15. Wieland, H., and Franke, K.: tJber den Mechanis-mus der Oxydationsvorgange. XII. Die Aktivierungdes Hydroperoxyds durch Eisen. Ann., 457: 1-70,1927.

16. Uri, N.: Inorganic Free Radicals in Solution,Chem. Rev., 40: 375-454, 1952.

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