Free Radicals-A Primary Cause of Aging

Free Radicals---A Primary Cause of Aging


Abstracts from the International Symposium, Biomedical Center and the Institute for

Gerontology, University of Uppsala, Uppsala, Sweden

Dr. Richard D. Lippman, editor.


1. Biological and Biochemical Aspects of Aging: An Introduction.


Ambjorn Agren, M.D, Medical Cell Biology, Biomedicum, Uppsala


    Molecular nechanisms behind the aging processes have attained great interest.

However, little has been known about the fundamental causes of aging until

recently. The most general assumption is that they may be due to accumulated

insults such as random "hits" from background radiation of free radicals

produced in other ways. This "error catastrophe" theory of aging predicts a

build-up of debris with a cell, for example, age pigmentation (lipofuscin).

In recent years the importance of the presence of free radicals in cells has

been considered, and their role in the aging processes of different organisms

has been tested. This "free radical theory" states that in the body, free radicals

initiate random and irreversible reactions which produce a cascade of deleterious

reactions, e.g. with DNA, MA, protein synthesis and lipid peroxidation.

    When a polyunsaturated fat or protein is damaged by such reactions, age

pigments such as lipofuscin, ceroid and amyloid are formed in cells and tissues.

Lipofuscin progressively accumulates in the cell during its lifespan. It may occupy

up to 35% of the total cell volume and hinder metabolite transport and cell

function. Several antioxidants and cell-protective substances have been found

to decrease this type of accumulation and hindrance, i.e. kavain, orotate,

magnesium thiazolidin and tocopherol p-chlorophenoxyacetate. Piammalian

experiments show that these substances can increase both maximum and

average lifespan. Whether this finding is valid for man remains to be firmly

established in more extensive, long-term clinical studies.


2. Involvement of Oxygen Free Radicals in Tissue Injury


Howard H. Thaw, Rolando F. Del Maestro and Karl-E. Arfors:

Department of Experimental Medicine, Pharmacia AB, Uppsala,

Sweden and University Hospital, London, Canada.


Oxidative damage that is associated with the pathogenesis of inflammation,

radiation and ischemia has been suggested to be mediated through active-oxygen

species. During oxidative metabolism and normal intracellular enzyme function,

superoxide anion radicals (O2-.), hydrogen peroxide (H2O2) and hydroxyl radicals

(*OH) are formed at rates which are directly proportional to oxygen tension. A

number of intrinsic enzymatic and scavenging mechanisms have emerged

under evolutionary pressures in our oxygen rich environment enabling us to cope with

these inevitable oxygen byproducts. The cytochrome oxidase complex

in the mitochondria adds four electrons t o oxygen, reducing it directly to

water and thereby circumventing the formation of any intermediates.




  Most oxygen is reduced to water quadravalently (by the addition of

four electrons), however, univalent or one-electron step reduction does occur

resulting in the formation of intermediates. Intrinsic enzymatic and scavenging

mechanisms are responsible for controlling these intermediates.

The superoxide dismutases catalyze the dismutation of Oz while other enzymes

such as catalase (CAT) and glutathione peroxidase remove H202. Furthermore,

antioxidants such as ascorbic acid, glutathione and vitamin E protect against

the effects of many different active-oxygen species.

xanthine oxidase systems (figure B ) , on human glial cells in culture, hyaluronic

acid and microcirculation of the hamster cheek pouch.

We have investigated the influences of these species generated by substrates.




H202 + 02 .-> .OH + OH- + lo2 (31


Figure B. The enzyme model for producing active oxygen species involves both a

univalent ( 1 ) and divalent (2) pathway. The simultaneous presence of H2o and

05 suggests further interaction and the formation of a strong oxidizing species.


  Progressive and irreversible morphological changes culminated in death for

the glial cells, and hyaluronic acid broke down under the influence of the free

radical generating system. In the microcirculation of the hamster cheek pouch,

increased microvascular permeability and pronounced alterations were observed

in polymorphonuclear leucocyte-endothelial interaction and adhesion. These results

suggest that active-oxygen species mediate macromolecular, cellular and

microcirculatory changes in our model systems. These processes should be

considered in an attempt to find new approaches to our understanding of these

complex phenomena. The mechanisms underlying free radical generation in

normal, disease and aging.


3 The Importance of Free Radicals in Aging


Mathias Uhlen, professor, Microbiology, Biomedicum, Uppsala and

The Institute for Gerontology , Stockholm, Sweden


   The free-radical theory of aging is based on the fact that free radicals,

directly or indirectly, lead to irreversible membrane damage or cross-linking

of biomolecules. The initiation step for the production of free radicals in

biological systems is often the production of the superoxide anion radical (02-)

by the addition of a single electron to molecular oxygen. There are many sources

for this electron, i.e. pollutants, drugs and radiation, but the primary

source is the electron transport system of mitochondria. Many enzymatic and

nonenzymatic systems have been developed by aerobic organisms to reduce

the flux of its byproducts, i.e. hydrogen peroxide (H202) and hydroxyl radicals (.OH).

Two enzyme types, the superoxide dismutases and glutathione peroxidase,

together with a wide variety of cell protectors such as vitamin E are important

defense systems in eukaryotic organisms. Many cell protectors incorporated into

special food supplements may slow the processes of aging by rendering

oxy-radicals harmless. In this regard, numerous investigators have described positive

effects upon lifespan when suitable free-radical inhibitors have been added to

the diet. Another approach is to measure the concentration of oxy-radicals in

vivo with the aid of synthesized chemiluminescent probes. Results from cultivated

normal human diploid glial cells confirmed that different antioxidant mixtures strongly

inhibit damages from superoxide and transition metals such as

copper and iron. An optimized antioxidant mixture, ACF 228, inhibited to less

than 40% of control values in these human brain cells.


  1. Age Pigments, Free Radicals and Lipid Peroxidation.


V.P. Collins, M.D.,  Department of Tumour Pathology, I n s t i t u t e of

Pathology, Karolinska I n s t i t u t e , Stockholm, Sweden


    To our knowledge the first description in the literature of intracellular

pigment, later to be known as age pigment, was that of Hanover, 1842, (1) who

described the occurrence of this pigment in the neurones of a number of species.

Koneff, 1886, (2) was to show that the accumulation of this pigment occurs with

aging. The natural fluorescence of age pigment was demonstrated by Stubel, 1911,

( 3 ) and since then the histochemical characteristics of this material have been

well defined. Age pigment has characteristics which clearly show it to be produced

by the peroxidation of lipids and proteins. Peroxidation of isolated subcellular

organelles in vitro gives rise to material with characteristics similar

to that of age pigment, Tappel, 1975, ( 4 ) . Age pigments have been found to

be localized to the lysosomal system intracellularly and to have a variable

fine structure.

     In v i t r o , cultivated human glial cells have been shown to contain age pigment

material who's histochemical fluorescent and ultrastructure is identical

with that found in vivo, Collins and Brunk, (5). The rate of accumulation

of age pigments in density-growth-inhibited human glial cells under routine culture

conditions has been documented, Collins and Brunk, (6). Thus normal

glial cells in v i t r o offer a well defined system for studies of the mechanism

of development of age pigments and the effects of their aggregation on cell

function as well as how various changes in the culture conditions effect the

rate of accumulation. Pseudoauto phagocytosis, induced by the exposure of glial

cells to non-homologous fractions of mitochondria (which they phagocytose) gives

rise to a significant increase in the cytoplasmic volume density of age pigment

material as compared to controls, Collins, et. al, (7). We conclude that

cell autophagocytosis of organelles such as mitochondria may represent

the introduction into the lysosomal system of material already damaged by free radicals

or provide the raw material which in the presence of such reactions will

produce age pigments. The effects of the accumulation of age pigments on cell

function are today unclear, but preliminary studies show no decrease in cell

culture life span even after the accumulation of cytoplasm volume densities of

30% or more.



1. u - Mover, A. : Mikioskcpiske ser af nervesystemet. Bianco Lunos

2. D c e f f , H.: Reit:-age zur Kepntniss der Yervenzellen in den peripheren Gang- I.

Xogrrvkkr ri, K j ijbenhavn, IS42

lien. Suchdriickerei Paul €Isl;er, Bern, 1886, 321

3. Stubel, H.: Archiv Gesamte Physiologie. 142:1, 1911

4. Tappel , A.L.: in Pathobiology of cell membranes (eds. B. Trump and A.U. Arstila)

p. 145, Academic Press, N.Y., 1975

5. Collins, V.P. and Brunk, U.T.: Mech. Aging Dev. 5:193, 1976

6. Collins, V.P. and Brunk, U.T.: Mech. Aging Dev. 6:139, 1978

7. Collins, V.P., Arborgh, B., Brunk, U.T., and Schellens, J.P.M.: Lab. Invest.

42:209, 1980


5. Measurement of and Protection from Free Radicals in Rat and Man

Dr. Richard D. Lippman:, Medical Cell Biology, Biomedicum, Uppsala

and The Institute for Gerontology, Stockholm, Sweden


  The superoxide anion radical (02-) was measured in the inner matrix of both rat and human liver-mitochondria by the use of a chemiluminescent, site-specific

probe. Mitochondrial, inner-membranial respiratory-chain leakage of was determined to be 6+2% of O2 uptake in intact rat-mitochondria, Lippman (1) ACF228.

Probed mitochondria were further incubated with a wide variety of radical-scavenger mixtures and geroprotectors (RSM&G). These RSM&G optimized a mixture which resulted in 100% scavengering of them in metabolically-active glial cells, HeLa cells. human liver-mitochondria, oxidase and vitamin E by a constant, homeostatic redox of their relatively stable radical analogues and oxidized products. In the ultrastructure of the mitochondria inner membrane, vitamin E storage is limited to one molecule per fifty molecules of lipid (2) despite an approximate femtomole per cell per minute flux of inner membranal generated 05 in state 3 respiration 3). This 1:50 ratio limits vitamin E's molecular protection of inner-membranal unsaturated fats, Primarily arachidonic acid which as a polyolefin has four unsaturated double bonds that are easily damaged.

   Therefore, despite megadosage of RSM&G and continual homeostatic RSM&G recycling, these bonds may remain somewhat labile to active-oxygen attack and nucleopholic addition reactions. Another reaction is the quantitative epoxidation of olefins (4) which indicates a clear link between in V ~ V O generation of carcinogenic epoxides, i.e. benzo(a)-pyran epoxide, and inhibition by RSM&G. In regard to senescence, these reactions result in organelle dysfunction

and lysis on the cellular level, cross-linking of elastin and collagen on the tissue level and general "aging" on the level of the whole organism--a marginal decrease in the rate of senescence (review, ref. 1).

   The limiting factors are possibly this 1:50 ratio and RSM&G bioavailability. In broader perspective, therapeutic scavengering of oxygens; and their eventual peroxidation byproducts promote greater protection A) from sunlight-induced tissue irradiation, B) from P-scission of unsaturated fatty acids in membranes, of thiol active-sites of enzymes and D) from irreversible, oxidized crosslinking of Schiff bases vital to the elasticity of collagen and elastin. In V ~ VO, R SM&G recycle such geroprotectors as membrane-bound glutathione per- RSM&G therapy in numerous mammalian studies has unquestionally demonstrated inactivation.

See ACF228 for more information regarding active oxygen scavengering.



1. Lippman, R.D.: Exp. Geront. 15(5):339, 1980

2. Molenaar, I., Vos 3. and Homes, F.A.: Vit. Horn. 30:45, 1972

3. Lippman, R.D. and Agren, A.: unpublished manuscript

4. Stanley, J.P.: J. Org. Chem. 45:1413, 1980