Vol. 107, No.

4, 1982

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 1198-1205

August 31, 1982

FREE RADICALS AND TISSUE DAMAGE PRODUCED BY EXERCISE

Kelvin J.A. Davies*t

, Alexandre T. Quintanilhat,
and Lester Packert

George A. Brooks,

tThe Membrane Bioenergetics Group, Lawrence Berkeley Laboratory and the Dept.
of Physiology/Anatomy, University of California Berkeley, California 94720, USA
and
The Exercise Physiology Laboratory, Department of Physical Education,
University of California, Berkeley, California
94720, USA

Received July 14, 1982

Summary: We report a two- to three-fold increase in free radical (R') concentrations of muscle and liver
following exercise to exhaustion.
Exhaustive exercise
also resulted in decreased mitochondrial respiratory control, loss of sarcoplasmic
reticulum (SR) and endoplasmic reticulum (ER) integrity, and increased levels of
lipid peroxidation products.
Free radical concentrations, lipid peroxidation,
and SR, ER, and mitochondrial damage were similar in exercise exhausted control
animals and non-exercised vitamin E deficient animals, suggesting the possibility
of a common R" dependent damage process.
In agreement with previous work
showing that exercise endurance capacity is largely determined by the functional
mitochondrial content of muscle (1-4), vitamin E deficient animals endurance
was 40% lower than that of controls.
The results suggest that R" induced damage
may provide a stimulus to the mitochondrial biogenesis which results from
endurance training.

Daily exercise of low work intensity but prolonged

training)
cellular

results
stimulus

literature
exercise

muscle

conflicting

biogenesis

an increased

sensitivity

loss of lysosomal
(i0,ii).

levels of pentane

peroxidation,

is not known

although the

(1,2,5,6).

The

a single bout of exhaustive

damage

(7,8), but Gale

(9) has

to oxidizing agents and others have

latency and release of proteolytic

In addition,

Dillard et al.

enzymes in both

(12) have measured

(which may be formed as a lipid peroxidation

in the expired air of exercising humans.

the possibility

(endurance

content of muscle,

reports as to whether

can actually cause muscle subcellular

and liver

increased

for mitochondrial

contains

demonstrated
reported

in an increased mitochondrial

duration

that exhaustive

In this report we have investigated

exercise may produce

and loss of mitochondrial,

sarcoplasmic

free radicals
reticulum

Corresponding Author.
Current address:
Dept. of Physiology & Biophysics~Harvard Medical School
25 Shattuck St.~Bostont MA 02115
USA

0006- 29 IX/82/161198-08501.00/0
Copyright 1982 by Academic Press, Inc.
All rights o f reproduction in any form reserved.

product)

1198

(R-),

(SR), and

lipid

Vol. 107, No. 4, 1982

endoplasmic

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

reticulum

(ER) integrity,

damage caused by v i t a m i n
to exercise

and have c o m p a r e d

E deficiency.

induced mitochondrial

The p o s s i b l e

proliferation

the r e s u l t s w i t h c e l l u l a r

relevance

of our f i n d i n g s

is discussed.

M a t e r i a l s and Methods:
M a l e L o n g Evans rats w e r e o b t a i n e d 30 days a f t e r b i r t h
and fed either a control (21 IU v i t a m i n E/kg) or a v i t a m i n E d e f i c i e n t (<i IU
v i t a m i n E/kg) diet (BioServ Inc., Frenchtown, NJ).
A f t e r 100 days, rats fed
the d e f i c i e n t diet e x h i b i t e d 83% g r e a t e r e r y t h r o c y t e h e m o l y s i s in a s t a n d a r d
test (13) than rats fed the control diet.
A n i m a l s w e r e c o n t i n u e d on their
r e s p e c t i v e diets and all e x p e r i m e n t s w e r e c o n d u c t e d at 6 m o n t h s of age.
Two
types of e x e r c i s e tests on a m o t o r i z e d r o d e n t t r e a d m i l l w e r e u s e d as p r e v i o u s l y
d e s c r i b e d (1-4): a s u b m a x i m a l work i n t e n s i t y e n d u r a n c e test in w h i c h time to
e x h a u s t i o n was measured, and a test i n v o v i n g p r o g r e s s i v e l y i n c r e a s i n g w o r k
i n t e n s i t y to m e a s u r e m a x i m a l w o r k l o a d capacity.
E n d u r a n c e tests w e r e given two
days f o l l o w i n g p r o g r e s s i v e i n t e n s i t y tests and rats were then i m m e d i a t e l y killed.
M u s c l e (gastrocnemius, soleus, and p l a n t a r i s ) and liver h o m o g e n a t e s (10%
w/V) were c o n s t i t u t e d in 175 mM KCI, 15 M M Tris, pH 7.4.
Free r a d i c a l s were
m o n i t o r e d by E P R (Varian E 109E) in b o t h t i s s u e h o m o g e n a t e s and intact tissue at
23 C.
M i t o c h o n d r i a l r e s p i r a t o r y c o n t r o l indices (RCI) w e r e m e a s u r e d at 37
C in homogenates, w i t h a m e d i u m p r e v i o u s l y d e s c r i b e d (i-4), as rate of u n c o u p l e d
r e s p i r a t i o n / r a t e of basal respiration.
Basal r e s p i r a t i o n was m e a s u r e d (Rank 02
electrode) w i t h 1 ~M d i c y c l o h e x y l c a r b o d i i m i d e and e i t h e r I0 mM p y r u v a t e + 2.5
mM malate, 10 mM succinate + 4 ~M rotenone, or 20 mM glutamate; u n c o u p l e d
r e s p i r a t i o n was a c h i e v e d w i t h 1 ~M c a r b o n y l c y a n i d e ~ - t r i f l u o r o m e t h o x y p h e n y l hydrazone.
L i p i d p e r o x i d a t i o n was m e a s u r e d in h o m o g e n a t e s by the t h i o b a r b i t u r i c
acid m e t h o d (14).
S a r c o p l a s m i c r e t i c u l u m and E R m e m b r a n e i n t e g r i t y were a s s e s s e d
w i t h latency m e a s u r e m e n t s of a l k a l i n e p h o s p h a t a s e a c t i v i t y (15), u s i n g b o t h
initial and total (solubilized with T r i t o n i00) activities.

Results and Discussion:

homogenates
intensity
fold

of m u s c l e

and liver.

to exhaustion,

(representative

p e a k heights
E deficient

An R" signal

was o b s e r v e d by EPR in all

Following exercise

of s u b - m a x i m a l

the a m p l i t u d e of R" E P R signals

spectra

are shown in Fig.

is given in T a b l e I).
animals

(g ~ 2.004)

Signal

i n c r e a s e d two- to t h r e e -

1 and m o r e d e t a i l e d

amplitude

was greater than in h o m o g e n a t e s

workload

analysis

in h o m o g e n a t e s
from control

from vitamin

animals

To be sure that EPR signals were not the r e s u l t of h o m o g e n i z a t i o n

we also e x a m i n e d the spectra of washed,
seen from F i g u r e
exercise

2, d i f f e r e n c e s

exhausted

animals,

w e r e of the same m a g n i t u d e

whole

in signal p e a k h e i g h t s b e t w e e n

and b e t w e e n

control

in w h o l e - l i v e r

E P R spectral peaks were larger than those

buffer

dilution

constant,

in the latter case.

exercise

p e a k heights

induced or v i t a m i n

are r e p r e s e n t a t i v e

liver p r e p a r a t i o n s .

and v i t a m i n

of i n c r e a s e d R"

1199

procedures,

r e s t e d and

E deficient

animals

Whole-liver

due to the K C i - T r i s

Since all other c o n d i t i o n s

E deficiency

at rest.

As m a y be

as in liver h o m o g e n a t e s .
from h o m o g e n a t e s ,

of

were m a i n t a i n e d

induced increases

concentrations.

in E P R

Relatively

Vol. 107, No. 4, 1982

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

NON-EXERCISED

RATS

EXERCISE EXHAUSTED

Control Liver

Control Liver

E Deficient Liver

E Deficient Liver

Control Muscle

Control Muscle

E Deficient Muscle

E Deficient Muscle

RATS

50 gauss
Figure i:

Free radical signals (g ~ 2.004! observed by EPR in muscle and

liver homogenates, from control and vitamin E deficient rats,
at rest or following the endurance exercise test to exhaustion.
EPR settings: power 10 mW, modulation 5 gauss, frequency 9.51 GHz,
time constant 0.25 s, temperature 23 C.

s t a b l e R" h a v e p r e v i o u s l y b e e n shown to p e r s i s t in w e t t i s s u e s a m p l e s e v e n a f t e r
m e t a b o l i s m stops

(16).

Our d e m o n s t r a t i o n t h a t signal s t r e n g t h s w e r e c o n s i s -

t e n t l y g r e a t e r in t i s s u e s t h a t h a d b e e n v e r y active,

a g r e e s w i t h the e x p e c t a t i o n

t h a t h i g h l y r e a c t i v e R" are c o m m o n m e t a b o l i c i n t e r m e d i a t e s (17).

e x e r c i s e c o u l d lead to f a s t e r rates of u b i s e m i q u i n o n e t u r n o v e r
globin autooxidation
generation.

Increased

(18) and h e m o -

(19), r e s u l t i n g in h i g h e r l e v e l s of s u p e r o x i d e r a d i c a l

It has b e e n s u g g e s t e d t h a t R" o b s e r v e d in liver t i s s u e m a y a r i s e

from various mitochondrial components

(16,17), a n d the E P R signals we r e p o r t

1200

Vol. 107, No. 4, 1982

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

to
to

I-I
~ ,--I
~ ~1 " 0

"0
4.4

0.,~
m

O1 C ~

7.
Z

c.

o1 ~

o1
-I-1

~
0

-,-4 o

..~

cO

~C

~-~

o1

-,.-.I tO

"0

~,

r-,

'~

a2
to

~I
I
to,

.r.d

7, 7, 7, ~, 7,

A,

+I

+I

A,

~
v

-,'4
~

rd
~

k~ ..Q

+I

'0
tO

rO ,---I ~

g.~

O 0 0 r ~ + l

C; ~

to

O~

$A

to~4

a:l

O .~rO

ol

to ,.4-,.-I .,-.I
C

8 ~0 g
~

~
4J r.O -,-'1 m
r~
~
-,.4

.,-I

rO
--

1201

Vol. 107, No. 4, 1982

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EXERCISE EXHAUSTED RATS

NON-EXERCISED RATS
Control

Control

E Deficient

Figure 2:

50 gauss

Free radical signals (g ~ 2.004) in whole-liver tissue, from control

and vitamin E deficient rats, following rest or the endurance exercise
test to exhaustion. EPR conditions as in Fig. i.

m a y be r e l a t e d to u b i s e m i q u i n o n e or f l a v i n r a d i c a l s

(iron-sulfur protein signals

are u n l i k e l y u n d e r t h e s e c o n d i t i o n s a n d at 22 C).
L i v e r a n d m u s c l e h o m o g e n a t e s f r o m e x e r c i s e e x h a u s t e d rats e x h i b i t e d r e d u c e d
mitochondrial respiratory control compared with tissues from nonexercised rats
(Table I).

V i t a m i n E d e f i c i e n c y w a s a l s o f o u n d to d e c r e a s e r e s p i r a t o r y control.

T h e c a u s e of d e c r e a s e d m i t o c h o n d r i a l r e s p i r a t o r y c o n t r o l f o l l o w i n g e x e r c i s e or
vitamin E deficiency

w a s i n c r e a s e d r a t e s of b a s a l r e s p i r a t i o n r a t h e r than

d e c r e a s e d r a t e s of m a x i m a l r e s p i r a t i o n , s u g g e s t i n g an i n c r e a s e in i n n e r m e m b r a n e
" l e a k i n e s s " to p r o t o n s a n d d e c r e a s e d e n e r g y c o u p l i n g e f f i c i e n c y .

In o r d e r to

a s s e s s m i t o c h o n d r i a l f u n c t i o n as r a p i d l y as p o s s i b l e f o l l o w i n g exercise,

all

m e a s u r e m e n t s of r e s p i r a t o r y c o n t r o l w e r e p e r f o r m e d in w h o l e h o m o g e n a t e s r a t h e r
t h a n in i s o l a t e d m i t o c h o n d r i a .

B a s a l r a t e s of r e s p i r a t i o n w e r e o b t a i n e d w i t h

s u b s t r a t e + d i c y c l o h e x y l c a r b o d i i m i d e to b l o c k the A T P a s e F o c o m p l e x p r o t o n
leak.

This is a n e c e s s a r y p r e c a u t i o n in h o m o g e n a t e s due to e n d o g e n o u s ADP,

but has the a d d e d a d v a n t a g e of p e r m i t t i n g m e a s u r e m e n t of t r u e m e m b r a n e integrity.

The p a t t e r n of m e m b r a n e d a m a g e f o l l o w i n g e x h a u s t i v e e x e r c i s e or v i t a m i n E
d e f i c i e n c y m a y a l s o be d e d u c e d f r o m l a t e n c y m e a s u r e m e n t s of the SR and E R e n z y m e

1202

Vol. 107, No. 4, 1982

alkaline

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

phosphatase

and solubilized
to increase
activities
exercise

(Table I).

activities,

following

or v i t a m i n

ficiency

(Table I).

increased

Experiments

Vitamin

E is known

radical

chain b r e a k i n g

always

tionship

fashion

to a c c o m p a n y

is unknown,
in other

lipid p e r o x i d a t i o n

(21)]

damaging

effect of osmium
appear

that d a m a g e d

fugal

forces.

revealed

E de-

similarly

E deficiency.

E deficiency

Whether

in a
R"

were

or not the rela-

status

of tissues

tetroxide

(an oxidant)

mitochondria

induced by v i t a m i n

[a b y - p r o d u c t

increased

is altered

control

isolated

but it is well

cannot be s e d i m e n t e d

E deficiency,

may

and the

Mitochondria
(8,22),

of

by exercise

by exercise

(a reductant)

(9).

of

are also i n t e r e s t i n g

of p e n t a n e

air are s i g n i f i c a n t l y

at low centri-

for example,

resulted

yield.

the existence

of a temporal

and the onset of fatigue,

m i n at 26.8 m/min

and 15% grade

following

for muscle

and 1.06 for liver,

for muscle

and 3.2 for liver,

for liver,

and m a l o n d i a l d e h y d e

g for liver.

and v i t a m i n

that c o n c e n t r a t i o n s

or d i s r u p t e d

Immediately

and v i t a m i n

vitamin

(20). These results

of E P R R" signals

mals).

integrity.

and lipid h y d r o p e r o x i d e s

effect of g l u t e r a l d e h y d e

in a 20% lower m i t o c h o n d r i a l

To determine

exercise

to have high r e s p i r a t o r y

Damage

the total

SR a n d E R m e m b r a n e

following

radicals

found

that e x h a u s t i v e

by both exercise

dienes

were

but R" have been shown to act as initiators

The fact that the redox

both the p r o t e c t i v e

suggest

lipid peroxidation.

in expired

explain

whereas

isolated mitochondria

situations

in light of the d e m o n s t r a t i o n

known

with

initial

(14), a n d it should be noted that e l e v a t e d

with i n c r e a s e d

is causal

at e x h a u s t i o n

increased

both

activities

E deficiency,

in d e c r e a s e d

to react with o x y g e n

lipid p e r o x i d a t i o n

(12).

result

involved

the initial

Such results

of c o n j u g a t e d

observed

associated

unchanged.

was greatly

concentrations

concentrations

or v i t a m i n

E deficiency

Lipid peroxidation

calculations

but in all cases

exercise

were r e l a t i v e l y

Latency

relationship
a control

control

concentration

All of these results

1203

between

3.9

and 49.3%

a n d 42.2

values

ani-

1.21

was

for m u s c l e

was 36.3 for m u s c l e

are i n t e r m e d i a t e

were

(pyruvate-malate)

l a t e n c y was 47.4%

for 23

time for control

E P R R" signal p e a k heights

respiratory
lysosomal

the a p p e a r a n c e

rat was e x e r c i s e d

(half the m e a n endurance

exercise,

between

nmoles/

for r e s t e d

Vol. 107, No. 4, 1982

Table 2:

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Progressive work intensity and endurance capacity exercise tests.

Exercise Test

Animal Group

Progressive Intensity

Control

Vitamin E Deficient

VO2max (mlO2"kg-l'min-l)

62.9 + 4.6

61.9 + 1.8

VCO2max (mlCO2"kg-l'min -1 )

67.9 + 5.6

69.7 + 2.9

i.i + 0.01

Rmax (VCO2max/VO2max)

i.i + 0.02

50.9 + 2.1

Maximal Speed (m/min)

49.6 + 1.9

Endurance Capacity
Maximal Endurance (min)

46.3

+ 4.1

28.2 + 1.2

Whole-animal maximal CO 2 production (VCO2max) , respiratory exchange ratio

(Rmax) and maximal speed on a 15% grade were measured at the point of
whole animal maximal 02 consumption (V02max). The progressive intensity test
involved 6.7 m/min increases in treadmill speed every 2 min until VO2max
was attained. The endurance test was run at a constant speed of 26.8
m/min and 15% grade, and min to exhaustion are reported.
All values are
means + SEM for 6 rats per group.

control

rats and exercise

exhausted control

be c o n s i d e r e d preliminary,
exercise

Although

depending mainly

The r e s u l t s p r e s e n t e d

role for liver d u r i n g e n d u r a n c e

oxidative

to controls,
exchange
present

ratio,

also imply a rather m o r e a c t i v e

results

determined

relatively

was d e c r e a s e d by 40% c o m p a r e d

observations

capacity

low level of m e m b r a n e

ber of factors which cause

that e n d u r a n c e

c o n t e n t of muscle,

(Table II).

capacity

The

is l a r g e l y

and that V O 2 m a x

for o x y g e n c o n s u m p t i o n or A T P p r o d u c t i o n

imply t h a t the d e c r e a s e d

rats was p r e c i p i t a t e d

respiratory

at V O 2 m a x were u n a f f e c t e d

by the functional m i t o c h o n d r i a l

They further

deficient

workload

confirm previous

is not limited by m u s c u l a r
(1-4).

capacities)

(which h a d c o m p r o m i s e d

(whole-animal m a x i m a l 02 consumption),

and m a x i m a l

on

e x e r c i s e t h a n m a y be g e n e r a l l y appreciated.

and ATP p r o d u c t i o n

but V O 2 m a x

i n d u c e d by

(at c o n s t a n t workload)

T h e e n d u r a n c e c a p a c i t y of v i t a m i n E d e f i c i e n t rats
muscle

such data s h o u l d

it does a p p e a r that the low level damage

is gradual and cumulative,

the d u r a t i o n of work.

rats.

endurance

by p e r o x i d a t i v e
damage

c a p a c i t y of v i t a m i n E

damage to mitochondria.

The

i n d u c e d by e x e r c i s e m a y be one of a num-

fatigue d u r i n g p r o l o n g e d

sub-maximal

work in c o n t r o l

animals.
The m i t o c h o n d r i a l

c o n t e n t of muscle

can be increased by as m u c h as 100% by

1204

Vol. 107, No. 4, 1982

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

chronic endurance exercise training (1,2,5).

The magnitude of change, as well

as studies of cytochrome c turnover (6), suggest that rates of mitochondrial

biosynthesis are greatly increased.

In various other biological situations,

low-level damage has been shown to result in increased rates of biosynthesis,

often leading to supranormal steady-state concentrations.

Our results suggest

that the daily imposition of lengthy exercise bouts (endurance training) will
result in elevated rates of mitochondrial damage.

It is tempting to propose

that exercise induced free radicals may cause limited damage to mitochondrial
membranes which, in a chronic training situation, may be the initiating stimulus
to mitochondrial biogenesis.
Acknowledgements:
This research was supported by grants from the U.S. Department of Energy, The National Institutes of Health, and the Hoffmann La Roche
Company.
K.J.A.D. was the recipient of the Chancellors Award for Research,
University of California, Berkeley, CA 94720 USA.

References:
i.
2.
3.
4.
5.
6.
7.
8.
9.
10.
ii.
12.
13.
14.
15.
16.

17.
18.
19.
20.
21.
22.

Davies, K.J.A. (1981) Ph.D. Thesis.

University of California, Berkeley.
Davies, K.J.A., Packer, L., and Brooks, G.A. (1981) Arch. Biochem. Biophys.
209, 539-554.
Davies, K.J.A., Packer, L., and Brooks, G.A. (1982) Arch. Biochem. Biophys.
215, 260-265.
Davies, K.J.A., Maguire, J.J., Brooks, G.A., Dallman, P.R., and Packer, L.
(1982) Amer. J. Physiol.: Endocrinol. Metabol. 242, E418-E427.
Holloszy, J.O. (1967) J. Biol. Chem. 242, 2278-2282.
Booth, F.W., and Holloszy, J.O. (1977) J. Biol. Chem. 252,416-419.
Gollnick, P.D., and King, D.W. (1969) Amer. J. Physiol. 216, 1502-1509.
Terjung, R.L., Baldwin, K.M., Mol~, P.A., and Holloszy, J.O. (1972) 223,549-554.
Gale, J.B. (1974) Med. Sci. Sports 6, 182-187.
Dohm, G.L., Kasperek, G.J., Tapscott, E.B., and Beecher, G.R. (1980)
Biochem. J. 188, 255-262.
Kasperek, G.J., Dohm, G.L., Barakat, H.A., Strausbauch, P.H., Barnes,
D.W., and Snider, R.D. (1982) Hiochem. J. 202, 281-288.
Dillard, C.J., Litov, R.E., Savin, W.M., and Tappel, A.L. (1978) J. Appl.
Physiol.: Respirat. Environ. Exercise Physiol. 45, 927-932.
Draper, H.H., and Csallany, A.S. (1970) J. Nutr. 98, 390-394.
Buege, J.A., and Aust, S.D. (1978) in Methods in Enzymology (eds.,
Fleischer, S., and Packer, L.) Vol. LII, pp. 306-307, Academic Press, New York.
Forte, J.G., Forte, G.M., and Saltman, P. (1967) J. Cell Physiol. 69, 293-304.
Borg, D.C. (1972) in Biochemical Applications of Electron Spin Resonance
(eds.
Swartz, H.M., Bolton, J.R. and Borg, D.C.), pp. 265-284, Wiley
Interscience, New York.
Borg, D.C. (1976) in Free Radicals in Biology (ed. Pryor, W.A.) Vol. i,
pp. 69-145, Academic Press, New York.
Chance, B., Sies, H., and Boveris, A. (1979) Physiol. Rev. 59, 527-605.
Hochstein, P., and Jain, S.K. (1981) Fed. Proc. 40, 183-188.
Kellogg, E.W., III, and Fridovich, I. (1975) J. Biol. Chem. 250, 8812-8817.
Horvat, R.J., Lane, W.G., Ng, H., and Shepherd, A.D. (1964) Nature 203,523-524.
Brooks, G.A., Hittelman, K.J., Faulkner, J.A., and Beyer, R.E. (1971)
Am. J. Physiol. 220, 1053-1059.

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