RADIATION-INDUCED CWGES I N TKE PROFILES OF
CERTAIN KEY ENZYMES I N RAT MYOCARDIUM AND SERUM:
EFFECTS OF EXERCISE AND DIETARY SUPPLEMENTATION WITH VITAMIN E
Lyle Dean MacWilliam
B. Sc
. , ( ~ i o c h e m i s t r ~Simon
)
Fraser
U n i v e r s i t y , 1971
A THESIS SUBMITTED I N PARTIAL FULFILLMENT OF
m l ~ nn n n T r r n m t m T r n c -
m n n m r v nwr+Dw;1T;r nw
11lI ~ & ~ U L ~ & I " ~
L VYL \I U
lllU
UUUI\UU
V I
MASTER OF SCIENCE (KINESIOLOGY)
i n t h e Department
of
Kinesiology
@
LYLE DEAN MACWILLIAM 1974
SIMON FRASER UNIVERSITY
A p r i l 1974
A l l r i g h t s r e s e r v e d . This t h e s i s may not be reproduced i n whole
o r i n p a r t , by photocopy o r o t h e r means, w i t h o u t permission of
t h e author.
APPROVAL
NAME :
Lyle Dean MacWilliam
DEGREE :
Master of Science (~inesiolo~~)
7
TITLE OF THESIS : Radiation-induced Changes in the Profiles of
Certain Key Enzymes in Rat Myocardium and
Serum: Effects of Exercise and Dietary
Supplementation with Vitamin E.
EXAMINING COMMITTEE:
Chairman: Dr. T.W. Calvert
Dr. N.M.G. Bhakthan
Senior Supervisor
Dr. A.J. Davison
Dr. K.K. Nair
Dr. E.W. Banister
External Examiner
Professor , Chairman
Department of Kinesiology
Simon Fraser University
Date Approved:
iii
ABSTRACT
Levels of l a c t a t e dehydrogenase, c r e a t i n e k i n a s e and glutamate
o x a l o a c e t a t e transaminase i n serum show q n i t i a l e l e v a t i o n s w i t h i n twelve
hours of exposure t o 2,000 r a d s of gamma r a d i a t i o n t o t h e t h o r a c i c region
of r a t s .
S i g n i f i c a n t decreases of t h e s e enzymes i n homogenates of h e a r t
muscle c o i n c i d e w i t h i n i t i a l e l e v a t i o n s i n t h e serum and may suggest t h a t
enhanced leakage of enzymes i s a consequence of r a d i a t i o n i n j u r y t o h e a r t
muscle.
However, i n s i g n i f i c a n t a l t e r a t i o n s i n mitochondria1 l e v e l s of
glutamate o x a l o a c e t a t e transaminase following exposure i n d i c a t e t h a t in
y i y ~inSi_l_ryf g
-
f he mff g ~ h ~ n d r ff argm t h p y a p p 1 1 t . f ~ l e v e l s nf
i s questionable.
pmmn radiat.-inn
The r e s u l t s suggest t h a t i o n i z i n g r a d i a t i o n causes a l t e r -
a t i o n s i n t h e dynamic p e r m e a b i l i t y of membranes, allowing leakage of biol o g i c a l l y a c t i v e m a t e r i a l out of t h e i n j u r e d c e l l .
1
Levels of l a c t a t e dehydrogenase, c r e a t i n e k i n a s e and glutamate
o x a l o a c e t a t e transaminase i n t h e serum of r a t s e x e r c i s e d immediately
following t h o r a c i c exposure t o 2,000 r a d s of gamma r a d i a t i o n become e l e v a t e d
w i t h i n twelve hours of t r e a t m e n t .
Although LDH l e v e l s i n serum of i r r a d i a t e d ,
e x e r c i s e d animals were s i g n i f i c a n t l y g r e a t e r t h a n s i m i l a r l y t r e a t e d sedentary
s u b j e c t s , serum l e v e l s of CK and GOT d i d not d i f f e r s i g n i f i c a n t l y between
t h e two groups.
.
These o b s e r v a t i o n s a r e i n t e r p r e t e d i n terms of changes i n
t h e p e r m e a b i l i t y of membranes.
Temporary r e d u c t i o n s i n l e v e l s of LDH i n
homogenates of h e a r t corresponded t o i n i t i a l e l e v a t i o n s of LDH i n serum and
suggest t h a t enhanced leakage of t h e enzyme i s a consequence of r a d i a t i o n
injury t o the heart.
I n c o n t r a s t t o demonstrated l o s s e s of CK and GOT from
homogenates of h e a r t i n i r r a d i a t e d sedentary animals, t h e l e s s e r amount
of leakage i n e x e r c i s e d s u b j e c t s suggests t h a t e x e r c i s e i n i r r a d i a t e d ,
1
t r a i n e d s u b j e c t s may p r o t e c t a g a i n s t l o s s of b i o l o g i c a l m a t e r i a l from
t h e damaged system.
Levels of GOT i n i s o l a t e d r a t h e a r t mitochondria
were temporarily decreased twenty-four hours a f t e r exposure.
However,
enzyme l e v e l s a f t e r i r r a d i a t i o n d i d not d i f f e r s i g n i f i c a n t l y between
e x e r c i s e d and sedentary animals, i n d i c a t i n g t h a t e x e r c i s e , a s an added
metabolic s t r e s s i n i r r a d i a t e d r a t s , d i d not cause any remarkable a l t e r a t i o n s i n t h e i n t e g r i t y of mitochondria1 membranes.
Comparisons of percentage e l e v a t i o n s i n serum 1;DH l e v e l s between
i r r a d i a t e d r a t s previously maintained on t o c o p h e r o l - f o r t i f i e d and standard
l a b chow d i e t s d i d not r e v e a l any s i g n i f i c a n t r a d i o p r o t e c t i v e a c t i o n of
vitamin E .
However, a marked decrease i n LDH a c t i v i t y i n t h e serum of
both sham-irradiated, d i e t a r y groups suggests t h a t a-tocopherol may induce
a f u r t h e r s t a b i l i z a t i o n of normal membrane permeability.
Although no
evidence of formation of l i p i d peroxides induced by i r r a d i a t i o n could
be d e t e c t e d , t h e r e was a s i g n i f i c a n t l y decreased l e v e l of TBA r e a c t a n t s
/
i n h e a r t homogenates of a l l t o c o p h e r o l - f o r t i f i e d
animals.
Be very c a r e f u l when you a r e looking f o r a t h i n g o r you w i l l be s u r e
t o f i n d it.
Louis Pas t e u r
ACKNOWLEDGMENTS
The completion of t h i s t h e s i s would have been d i f f i c u l t without
t h e h e l p of many people.
The author i s indebted t o D r . N.M.G.
f o r h i s valued guidance and continued s u p p o r t , t o D r . A . J .
D r . K.K.
Bhahthan
Davison and
Nair f o r t h e i r h e l p f u l c r i t i c i s m s and s u g g e s t i o n s , t o Tony
Legault f o r h i s a s s i s t a n c e w i t h computer programs and s t a t i s t i c a l analy s i s , and D r . R . Cornelson f o r h i s t e c h n i c a l a s s i s t a n c e i n i r r a d i a t i o n
procedures.
F i n a l l y , I would l i k e t o thank my w i f e , Arlene, f o r h e r
hours of work i n t h e t y p i n g of t h i s manuscript.
This work was f i n a n c i a l l y supported by t h e National Research
Council of Canada.
viii
TABLE OF CONTENTS
PAGE
...................................................... x i
OF FIGURES ..................................................... x i v
LIST OF TABLES
LIST
CHAPTER
I
I1
................................................. 1
Importance of t h e Study .................................... 4
O b j e c t i v e s of t h e Study .................................... 8
LITERATURE REVIEW ........................................ 1 0
INTRODUCTION
Enzymological C o n s i d e r a t i o n s
............................ 10
............... 1 4
L i p i d P e r o x i d a t i o n and Damage t o Membranes .............. 1 7
A/ General Considerations ............................ 1 7
B/ R a d i o l o g i c a l Considerations ....................... 21
V i t a n i n E; Antioxidant o r Metabolic I n t e r m e d i a t e ? ...... 24
RADIATION-INDUCED ENZYME: EFFLUX FROM RAT HEART: I . SEDENTARY
ANIMALS ...................................................... 28
I n t r o d u c t i o n ..................................... ........ 28
M a t e r i a l and Methods ....................................... 29
I r r a d i a t i o n Procedure ................................... 29
Sampling Procedure ...................................... 30
Heart Muscle P r e p a r a t i o n ................................ 31
Enzyme Assays ........................................... 32
P r o t e i n Determination ................................... 34
Data A n a l y s i s ........................................... 35
Evidence of R a d i a t i o n I n j u r y t o Membranes
ix
CHAPTER
PAGE
...................................................
Serum Assays ...........................................
Homogenate A s s a y s ......................................
Mitochondria1 Assays ...................................
Discussion ...............................................
RADIATION-INDUCED ENZYME EFFLUX FROM RAT HEART : I1 . EXERCISETRAINED RATS ................................................
I n t r o d u c t i o n ..............................................
M a t e r i a l and Methods ......................................
T r a i n i n g Procedure .....................................
I r r a d i a t i o n Procedure ..................................
R e s u l t s ...................................................
Results
IV
E f f e c t s of y-Radiation of E x e r c i s e d Rats
...............
........................................
Homogenate Assays ...................................
Mitochondria1 Assays ................................
Serum Assays
35
35
43
50
60
64
64
65
65
66
66
66
66
74
87
The E f f e c t of E x e r c i s e T r a i n i n g on Sham-Irradiated
Control Rats
87
Comparison of Radiation-Induced A l t e r a t i o n s i n Enzyme
Concentrations i n Serum. Heart Homogenate and Heart
Mitochondria i n E x e r c i s e d and Sedentary Rats
91
...........................................
...........
Serum Enzyme E l e v a t i o n s .............................
H e a r t Homogenate Enzyme Levels ......................
Heart Mitochondria1 Enzyme Levels ...................
Discussion
91
94
97
............................................... 97
X
PAGE
CHAPTER
.
................................................... 103
I n t r o d u c t i o n ........................................ 103
M a t e r i a l and Methods ....................................... 104
I r r a d i a t i o n Procedure ................................... 105
Heart Muscle P r e p a r a t i o n f o r T h i o b a r b i t u r a t e Analysis ... 105
T h i o b a r b i t u r a t e Analysis f o r L i p i d Peroxides ............ 105
R e s u l t s .................................................... 106
Serum Enzyme Analysis ................................... 106
L i p i d Peroxide Determination ............................ 114
Discussion ................................................. 1 2 1
VI
GENERAL CONCLUSIONS .......................................... 126
I r r a d i a t i o n E f f e c t s i n Sedentary Animals ................... 126
I r r a d i a t i o n E f f e c t s i n Exercised Animals ................... 127
I r r a d i a t i o n E f f e c t s i n Vitamin E - F o r t i f i e d Animals ......... 128
BIBLIOGRAPHY ....................................................... 130
APPENDIXES .......................................................... 144
V
RADIATION-INDUCED ENZYME EFFLUX FROM RAT HEART: 111 EFFECT
OF DIETARY SUPPLEMENTATION WITH VITAMIN E ON LIPID PEROXIDE
FORMATION
LIST OF TABI;ES
TABLE
1
PAGE
Serum l e v e l s of l a c t a t e dehydrogenase a t v a r i o u s i n t e r v a l s
Significant
a f t e r exposure t o 2,000 r a d s . o f y - r a d i a t i o n .
d i f f e r e n c e s between c o n t r o l and t e s t means were determined
by t - t e s t , u s i n g a pooled e s t i m a t e of v a r i a n c e
37
Serum l e v e l s of c r e a t i n e k i n a s e a t v a r i o u s i n t e r v a l s a f t e r
Significant differexposure t o 2,000 r a d s of y - r a d i a t i o n .
ences between c o n t r o l and t e s t means were determined by tt e s t , u s i n g a pooled e s t i m a t e of v a r i a n c e
40
Serum l e v e l s of glutamate o x a l o a c e t a t e transaminase a t
v a r i o u s i n t e r v a l s a f t e r exposure t o 2,000 r a d s of yr a d i a t i o n . S i g n i f i c a n t d i f f e r e n c e s between c o n t r o l and
t e s t means were determined by t - t e s t , u s i n g a pooled
e s t i m a t e of v a r i a n c e
44
..............
2
...................
3
........................................
4
5
6
Eelrt hnmngenate levels ~f lactate dehydrngenase Fct. v ~ r i n l l s
i n t e r v a l s a f t e r exposure t o 2,000 r a d s of y - r a d i a t i o n .
S i g n i f i c a n t d i f f e r e n c e s between c o n t r o l and t e s t means were
determined by t - t e s t , u s i n g a pooled e s t i m a t e of v a r i a n c e
...
47
Heart homogenate l e v e l s of c r e a t i n e k i n a s e a t v a r i o u s
i n t e r v a l s a f t e r exposure t o 2,000 r a d s of y - r a d i a t i o n .
S i g n i f i c a n t d i f f e r e n c e s between c o n t r o l and t e s t means
were determined by t - t e s t , u s i n g a pooled e s t i m a t e of
variance
....................................................
51
Heart homogenate l e v e l s of glutamate o x a l o a c e t a t e t r a n s aminase a t v a r i o u s i n t e r v a l s a f t e r exposure t o 2,000 r a d s
of y - r a d i a t i o n .
S i g n i f i c a n t d i f f e r e n c e s between c o n t r o l
and t e s t means were determined by t - t e s t , u s i n g a pooled
e s t i m a t e of v a r i a n c e
54
........................................
7
57
Serum l e v e l s of l a c t a t e dehydrogenase i n e x e r c i s e - t r a i n e d
r a t s a t v a r i o u s i n t e r v a l s a f t e r exposure t o 2,000 r a d s of
y-radiation.
S i g n i f i c a n t d i f f e r e n c e s between c o n t r o l and
t e s t means were determined by t - t e s t , u s i n g a pooled
estimate of variance
68
........................................
1.A
(r
Mitochondria1 l e v e l s of glutamate o x a l o a c e t a t e transaminase
at v a r i o u s i n t e r v a l s a f t e r exposure t o 2,000 r a d s of yr a d i a t i o n . S i g n i f i c a n t d i f f e r e n c e s between c o n t r o l and
t e s t means were determined by t - t e s t , u s i n g a pooled
e s t i m a t e of v a r i a n c e
...............................L.. ....
PAGE
Serum l e v e l s of c r e a t i n e k i n a s e i n e x e r c i s e - t r a i n e d rats
a t v a r i o u s i n t e r v a l s a f t e r exposure t o 2,000 r a d s of yr a d i a t i o n . S i g n i f i c a n t d i f f e r e n c e s between c o n t r o l and
t e s t means were determined by t - t e s t , u s i n g a pooled
e s t i m a t e of v a r i a n c e
71
Serum l e v e l s of glutamate o x a l o a c e t a t e transaminase i n
e x e r c i s e - t r a i n e d r a y s at v a r i o u s i n t e r v a l s a f t e r exposure
t o 2,000 r a d s of y - r a d i a t i o n .
Significant differences
between c o n t r o l and t e s t means were determined by tt e s t , u s i n g a pooled e s t i m a t e of v a r i a n c e
75
Heart homogenate l e v e l s of l a c t a t e dehydrogenase i n
e x e r c i s e - t r a i n e d r a t s a t v a r i o u s i n t e r v a l s a f t e r exposure
t o 2,000 r a d s of y-radiation.
Significant differences
between c o n i r o i auci test m e z x v c r e d z t = l m h e r l h - r tt e s t , u s i n g a pooieli estimzte of \-artace
78
Heart homogenate l e v e l s of c r e a t i n e k i n a s e i n exerciset r a i n e d r a t s a t v a r i o u s i n t e r v a l s a f t e r exposure t o 2,000
r a d s of y - r a d i a t i o n .
S i g n i f i c a n t d i f f e r e n c e s between
c o n t r o l and t e s t means were determined by t - t e s t , u s i n g
a pooled e s t i m a t e of variance
81
Heart homogenate l e v e l s of glutamate o x a l o a c e t a t e t r a n s aminase i n e x e r c i s e - t r a i n e d r a t s a t v a r i o u s i n t e r v a l s
a f t e r exposure t o 2,000 r a d s of y - r a d i a t i o n .
Significant
d i f f e r e n c e s between c o n t r o l and t e s t means were determined
by t - t e s t , u s i n g a pooled e s t i m a t e of v a r i a n c e
84
Mitochondria1 l e v e l s of glutamate o x a l o a c e t a t e transaminase
i n e x e r c i s e - t r a i n e d r a t s a t v a r i o u s i n t e r v a l s a f t e r exposure
t o 2,000 r a d s of y - r a d i a t i o n .
Significant differences
between c o n t r o l and t e s t means were determined by t - t e s t ,
using a pooled e s t i m a t e of v a r i a n c e
88
Enzyme l e v e l s of sham-irradiated, e x e r c i s e d and sedentary
c o n t r o l s . A comparison of exercise-induced changes i n
enzyme a c t i v i t i e s of serum ( s ) , h e a r t homogenate ( H ) , and
h e a r t mitochondrial ( M ) f r a c t i o n s
92
Comparison of a l t e r a t i o n s i n serum enzyme l e v e l s of posti r r a d i a t e d s e d e n t a r y and e x e r c i s e d r a t s
93
........................................
...................
-.I
.................-.
...............................
..............
.........................
...........................
.....................
xiii
TABLE
10A
PAGE
Comparison of a l t e r a t i o n s i n homogenate enzyme l e v e l s of
p o s t - i r r a d i a t e d s e d e n t a r y and e x e r c i s e d r a t s
95
Comparison of a l t e r a t i o n s i n mitochondria1 GOT l e v e l s of
p o s t - i r r a d i a t e d s e d e n t a r y and e x e r c i s e d r a t s
96
................
................
P o s t - i r r a d i a t i o n serum l a c t a t e dehydrogenase l e v e l s i n r a t s
maintained on s t a n d a r d P u r i n a l a b chow. S i g n i f i c a n t d i f f e r ences between c o n t r o l and t e s t means were determined by tt e s t , u s i n g a pooled e s t i m a t e of v a r i a n c e
................... 107
P o s t - i r r a d i a t i o n serum l a c t a t e dehydrogenase l e v e l s i n rats
maintained on vitamin E - f o r t i f i e d l a b chow f o r two weeks
p r i o r t o i r r a d i a t i o n . S i g n i f i c a n t d i f f e r e n c e s between
c o n t r o l and t e s t means were determined by t - t e s t , u s i n g
a -,soled estlmate of v a r i z r , c e
............................... 110
Comparison of a l t e r a t i o n s i n serum l a c t a t e dehydrogenase
l e v e l s between r a t s f e d s t a n d a r d l a b chow and tocopherolfortified diets
............................................. 113
Comparison of l a c t a t e dehydrogenase serum a c t i v i t y between
sham-irradiated c o n t r o l r a t s f e d s t a n d a r d l a b chow and
tocopherol-fortified d i e t s
.................................. 115
Concentrations of TBA r e a c t a n t s , expressed a s nmolar v a l u e s ,
i n t h e f i n a l s u p e r n a t a n t of h e a r t homogenates from r a t s f e d
s t a n d a r d P u r i n a l a b chow b e f o r e i r r a d i a t i o n . S i g n i f i c a n t
d i f f e r e n c e s between c o n t r o l and t e s t means were determined
by t - t e s t , u s i n g a pooled e s t i m a t e of v a r i a n c e
.............. 116
Concentration of TBA r e a c t a n t s , expressed a s nmolar v a l u e s ,
i n t h e f i n a l s u p e r n a t a n t of h e a r t homogenates from r a t s f e d
t o c o p h e r o l - f o r t i f i e d d i e t . S i g n i f i c a n t d i f f e r e n c e s between
c o n t r o l and t e s t means were determined by t - t e s t , u s i n g a
pooled e s t i m a t e of v a r i a n c e
................................. 119
Comparison of malonaldehyde c o n c e n t r a t i o n s i n t h e f i n a l
s u p e r n a t a n t of h e a r t homogenates from sham-irradiated c o n t r o l
r a t s f e d normal d i e t and vitamin E - f o r t i f i e d d i e t . . . . . . . . . . . .
120
xiv
LIST OF FIGURES
FIGURE
PAGE
1
Serum l e v e l s of l a c t a t e dehydrogenase a t various i n t e r v a l s
a f t e r exposure t o 2,000 r a d s of y - r a d i a t i o n .
2
Serum l e v e l s of c r e a t i n e k i n a s e a t v a r i o u s i n t e r v a l s a f t e r
exposure t 3 2,000 r a d s of y-radiation
3
Serum l e v e l s of glutamate o x a l o a c e t a t e transaminase at
v a r i o u s i n t e r v a l s a f t e r exposure t o 2,000 r a d s of yradiation
L..
.................. 38
........................ 41
...............................
................ 45
4
Heart homogenate l e v e l s of l a c t a t e dehydrogenase a t various
i n t e r v a l s a f t e r exposure t o 2,000 r a d s of y-radiation
6
Heart homogenate l e v e l s of glutamate oxaloacetate t r a n s aminase at v a r i o u s i n t e r v a l s a f t e r exposure t o 2,000 rads
of y-radiation
........ 48
............................................... 55
7
Mitochondria1 l e v e l s of glutamate oxaloacetate transaminase
at various i n t e r v a l s a f t e r exposure t o 2,000 r a d s of yradiation
.................................................... 58
1A
Serum l e v e l s of l a c t a t e dehydrogenase i n e x e r c i s e - t r a i n e d
r a t s a t v a r i o u s i n t e r v a l s a f t e r exposure t o 2,000 rads of
y-radiation
.................................................. 09
2A
Serum l e v e l s of c r e a t i n e k i n a s e i n e x e r c i s e - t r a i n e d r a t s
at various i n t e r v a l s aftel: exposure t o 2,000 rads of yradiation
.................................................... 72
3A
Serum l e v e l s of glutamate o x a l o a c e t a t e transaminase i n
e x e r c i s e - t r a i n e d r a t s a t v a r i o u s i n t e r v a l s a f t e r exposure
t o 2,000 r a d s of y-radiation
................................. 76
4~
Heart homogenate l e v e l s of l a c t a t e dehydrogenase i n exerciset r a i n e d r a t s a t various i n t e r v a l s a f t e r exposure t o 2,000 rads
of y - r a d i a t i o n
79
..............................................
5A
Heart homogenate l e v e l s of c r e a t i n e kinase i n e x e r c i s e - t r a i n e d
r a t s a t v a r i o u s i n t e r v a l s a f t e r exposure t o 2,000 r a a s of yradiation
82
....................................................
xv
PAGE
Heart homogenate l e v e l s of glutamate o x a l o a c e t a t e t r a n s aminase i n e x e r c i s e - t r a i n e d r a t s a t v a r i o u s i n t e r v a l s
a f t e r exposure t o 2,000 r a d s of y-radiation
85
Mitochondria1 l e v e l s of glutamate o x a l o a c e t a t e transaminase
i n e x e r c i s e - t r a i n e d r a t s a t v a r i o u s i n t e r v a l s a f t e r exposure
t o 2,000 r a d s of y - r a d i a t i o n
89
.................
................................
P o s t - i r r a d i a t i o n serum l a c t a t e dehydrogenase l e v e l s i n rats
maintained on s t a n d a r d P u r i n a l a b chow
...................... ,108
P o s t - i r r a d i a t i o n serum l a c t a t e dehydrogenase l e v e l s i n rats
maintained on vitamin E - f o r t i f i e d l a b chow f o r two weeks
prior t o irradiation
........................................ 111
Concentration of TBA r e a c t a n t s , expressed a s nmolar malon- 1 a - h . ~Y ~ I n t h e f i . 2 ~ 1
.~--penlat.nnt, of h e a r t , homogenates
fr~;;;r a t s fed ~ t ~ y d a rIf&i chew zyd t e c @ e r o l - f o r t i f i p d
d i e t s before irradiation
-&--*A
J.
--
.................................... 117
CHAPTER I
INTRODUCTION
Radiation i s not a new phenomenon, b u t i s an o l d hazard t h a t has
s i l e n t l y e x i s t e d through t h e ages.
'
Upon t h e i n i t i a l discovery of X-rays
by Roentgen i n 1895 and radium by Curie i n 1897, new v i s t a s f o r science
and technology were opened and, a s a r e s u l t , new and unknown dangers were
exposed.
I o n i z i n g r a d i a t i o n , i n c l u d i n g a l p h a and b e t a p a r t i c l e s , gamma r a y s ,
X-rays,
neutrons and p r o t o n s , soon became n o t o r i o u s f o r f a r reaching harm-
f u l e f f e c t s t o b i o l o g i c a l systems.
Although t h e s e forms comprise only a
p o r t i o n of t h e t o t a l r a d i a t i o n spectrum, t h e i r unique hazard l i e s i n t h e i r
,
i n n a t e a b i l i t y t o e n e r g i z e m a t t e r by i o n i z a t i o n . I n b i o l o g i c a l systems
t h i s can produce numerous p h y s i c a l and chemical e f f e c t s which can u l t i m a t e l y
l e a d t o massive biochemical breakdown and r e s u l t a n t death of t h e t a r g e t
tissue.
The r e p o r t s of Marcuse i n 1896 a r e t h e f i r s t documented d e s c r i p t i o n s
of t h e i n j u r i o u s e f f e c t s of X - i r r a d i a t i o n on t h e s k i n .
Lesions c o n s i s t i n g
of e p i l a t i o n , erythema and r a d i o d e r m a t i t i s were slow t o h e a l , e x c r u c i a t i n g l y
p a i n f u l , and were prone t o develop s k i n tumors.
P i e r r e Curie i n 1900 r e p o r t e d r a d i o d e r m a t i t i s and r e s u l t a n t deep
t i s s u e d e s t r u c t i o n a f t e r applying radium t o a small a r e a of h i s forearm,
and i n handling a c t i v e products, Mme. Curie developed erythema, d e r m a t i t i s ,
and severe b l i s t e r i n g of t h e f i n g e r s .
S i m i l a r l y , through t h e y e a r s numer-
ous massive i n j u r i e s and a c c i d e n t a l deaths have occured from major mishaps
w i t h i n l a b o r a t o r i e s and i n d u s t r i a l o r g a n i z a t i o n s .
The i n t e r a c t i o n s of i o n i z i n g r a d i a t i o n w i t h m a t t e r occurs by d i r e c t
o r i n d i r e c t action.
D i r e c t a c t i o n i s t h e i n t e r a c t i o n of r a d i a t i o n with
t a r g e t molecules, r e s u l t i n g i n an energy t r a n s f e r t o t h e molecule.
This
absorbed energy can r a i s e o r b i t a l e l e c t r o n s t o higher quantum l e v e l s ,
r e s u l t i n g i n highly e x c i t e d and u n s t a b l e molecules, o r can cause complete
e j e c t i o n of an e l e c t r o n from an o r b i t a l s h e l l .
Processes of s t a b i l i z a t i o n may r e s u l t i n d i s s o c i a t i o n of t h e
molecule i n t o f r e e r a d i c a l s , i n i o n i z a t i o n , o r i n lower l e v e l s of e x c i t a t i o n , t h e u l t i m a t e r e s u l t being t h e formation of s t a b l e products which
may d i f f e r from t h e o r i g i n a l molecule.
I n d i r e c t a c t i o n i s t h e r e a c t i o n between s o l u t e molecules and t h e
r e a c t i v e s p e c i e s formed by d i r e c t a c t i o n of r a d i a t i o n .
I n the living
c e l l , which i s an extremely complex s t r u c t u r e , water i s t h e u n i v e r s a l
solvent.
Therefore, when c e l l s a r e i r r a d i a t e d , most of t h e d i r e c t energy
t r a n s f e r occurs with water molecules simply because water p r e s e n t s t h e
g r e a t e s t number of t a r g e t s .
When i r r a d i a t e d , water l i k e any o t h e r m a t e r i a l
i s i o n i z e d , r e s u l t i n g i n t h e formation of u n s t a b l e intermediates which
quickly d i s s o c i a t e i n t o various r a d i o l y t i c products, such a s t h e extremely
r e a c t i v e hydrogen aqd hydroxyl f r e e r a d i c a l s .
The r e a c t i o n sequence can b e expressed a s
Free r a d i c a l s , once formed, d i f f u s e through t h e i r r a d i a t e d system r e a c t i n g
randomly with neighbouring molecules and thereby i n f l i c t damage t o t h e
i n t a c t components of t h e t a r g e t c e l l .
The b i o l o g i c e f f e c t s of i o n i z i n g r a a i a t i o n r e p r e s e n t t h e
e f f o r t s of l i v i n g t h i n g s t o d e a l w i t h energy l e f t i n them
a f t e r an i n t e r a c t i o n of one of t h e i r atoms w i t h an i o n i z i n g
r a y o r p a r t i c l e . For any l i v i n g system, t h i s energy w i l l
b e i n excess of t h e system's requirements f o r normal f u n c t i o n ;
it w i l l be a d e v i a t i o n from t h e proper energy r e l a t i o n s h i p s
w i t h i n t h a t system. 1
D e t e c t i b l e i n j u r y t o l i v i n g systems as a consequence of such
i n t e r a c t i o n s i s t h e r e s u l t of a long and complex sequence of events
i n i t i a t e d by r a d i o l o g i c a l damage t o b i o l o g i c a l l y p e r t i n e n t molecules,
and culminating i n t h e m a n i f e s t a t i o n of major p h y s i o l o g i c a l and biochemical a l t e r a t i o n s .
According t o L a t a r j e t and Grey (1954) t h e
development of observable i n j u r y i n an i r r a d i a t e d l i v i n g c e l l occurs i n
four steps.
The f i r s t s t e p involves t h e i n t e r a c t i o n between t h e i o n i z i n g
r a d i a t i o n and t h e component molecules of t h e c e l l ; t h e second c o n s i s t s of
r e a c t i o n s between s h o r t - l i v e d r a d i c a l s and molecules of b i o l o g i c a l
importance.
I n t h e t h i r d s t e p chemical chain r e a c t i o n s amplify t h e
primary damage r e s u l t i n g from t h e previous s t a g e s t o t h e p o i n t where
i n t h e f i n a l s t e p "observable" i n j u r y i s a p p a r e n t , involving c y t o l o g i c a l
l e s i o n s , g e n e t i c damage, and major biochemical changes.
It i s g e n e r a l l y
f e l t t h a t t h i s development of expression of t h e i n i t i a l i n j u r y occurs
through t h e i n c o r p o r a t i o n of t h e i n j u r y i n t o t h e ongoing metabolism of
t h e c e l l , and may s i m i l a r l y b e compared t o t h e e v e n t u a l breakdown of a
machine which i s o p e r a t e d i n a s l i g h t l y damaged c o n d i t i o n .
ID.
J. P i z z a r e l l o and R.L. Witcofski.
Lea and Febiger, P h i l a d e l p h i a , 1967, p. 3.
Basic Radiation Biology,
IMPORTANCE OF THE STUDY
Although t h e h e a r t has o f t e n been regarded a s one of t h e most
r a d i o r e s i s t a n t organs of t h e body ( ~ l l i n g e r1 9 5 7 ) , t h e o n s e t of r a d i a t i o n induced h e a r t d i s e a s e has been recognized a s an important m a n i f e s t a t i o n
of t h o r a c i c exposure ( s t e w a r t and Fajardo 1 9 7 1 ) .
There i s evidence t h a t
t h e h e a r t may, i n f a c t , be s e r i o u s l y damaged by i o n i z i n g r a d i a t i o n
( s t e w a r t 1967)
.
The r a d i o l o g i c a l re-treatment of Hodgkins
'
disease i n
t h e t h o r a x was found by Stewart and Fajardo (1971) t o c a r r y an exceedingly
h i g h r i s k of subsequent i n j u r y t o t h e h e a r t .
Although it has been shown
t h a t t h e myocardium i s most f r e q u e n t l y i n j u r e d ( F e r r i and Dosi 1 9 6 5 ) , a l l
components of t h e h e a r t show apparent p o s t - i r r a d i a t i o n damage.
Radiation-induced e l e c t r o c a r d i o g r a p h i c a l t e r a t i o n s have been
observed i n humans f ~ o n e sand Wedgwood
and r o d e n t s ( F u l t o n and Sudak 1954).
1 9 6 0 ) ~dogs ( ~ h i l l i -~
e ts a l . 1964)
S a l e t (1971) r e p o r t e d evidence of
i n c r e a s e d p u l s a t i o n f r e q u e n c i e s of r a t c a r d i a c c e l l s i n t i s s u e c u l t u r e
w i t h i n minutes of exposure t o m i c r o - i r r a d i a t i o n by l a s e r .
I n addition,
congestive h e a r t f a i l u r e induced by l a r g e r a d i o l o g i c a l doses i n both
r a b b i t s ( ~ f s k i n d1954) and dogs ( ~ i s h -oe t~ a l . 1965) i n d i c a t e s t h a t t h e
h e a r t i s indeed s u c c e p t i b l e t o severe r a d i a t i o n i n j u r y .
The c l i n i c a l i n d i c a t i o n s t h a t t h e s u r v i v a l of p a t i e n t s undergoing
r a d i a t i o n t r e a t m e n t f o r carcinoma of t h e l e f t lung was poorer t h a n t h a t
of p a t i e n t s t r e a t e d f o r r i g h t lung cancer, prompted a study by Takaoka
(1969) involving t h e i n v e s t i g a t i o n of c a r d i a c damage by t h e r a p e u t i c
radiation.
The r e s u l t s showed t h a t t h e degree of myocardial damage was
r e l a t e d t o t h e s i z e of t h e i r r a d i a t e d a r e a and t o t h e r a d i o l o g i c a l dose
given.
S i m i l a r l y , t h e prognosis of p a t i e n t s r e c e i v i n g i n t r a t h o r a c i c
r a d i o t h e r a p y may be i n f l u e n c e d by t h e i n c i d e n t a l l y induced myocardial
damage.
Cardiac s t r u c t u r a l a l t e r a t i o n s i n p a t i e n t s r e c e i v i n g t h e r a p e u t i c
r a d i a t i o n t o t h e mediastinum ( ~ a j a r d o-e t a l . 1968) showed p r o l i f e r a t i o n
of connective t i s s u e w i t h p e r i c a r d i t i s and e x t e n s i v e f i b r o s i s of t h e
myocardium.
I n a l a t e r study of t h e mechanism of radiation-induced
myocardial f i b r o s i s i n r a b b i t s , Fajardo and Stewart (1973) determined
t h a t immediate i n jury causes an a c u t e , t r a n s i e n t inflammatory exudate
i n a l l t i s s u e s of t h e h e a r t .
I n a d d i t i o n , t h e experiments c l e a r l y
i n d i c a t e d t h e c a p i l l a r y e n d o t h e l i a l c e l l a s an important t a r g e t of
radiation.
An e x t e n s i v e study on t h e m a n i f e s t a t i o n of chronic c a r d i a c i n j u r y
induced by r a d i a t i o n h a s been r e p o r t e d by Burch -e t a l . (1968).
The
p a t i e n t , a 37 y e a r o l d male p h y s i c i a n , underwent post-operative r a d i a t i o n
t r e a t m e n t f o r t h y r o i d f o l l i c u l a r carcinoma, r e c e i v i n g a dose of 4,800
r a d s t o t h e t h y r o i d bed w i t h an a d d i t i o n a l dose of 5,200 r a d s t o t h e
mediastinum through t h e c h e s t w a l l .
Although t h e r e was no r e c u r r e n c e
of t h e carcinoma, t h e p a t i e n t d i e d seven y e a r s l a t e r of congestive h e a r t
failure.
Microscopic o b s e r v a t i o n r e v e a l e d d i s r u p t i o n of t h e m y o f i b r i l s
and c e l l o r g a n e l l e s w i t h p o s s i b l e cytoplasmic l e a c h i n g .
Mitochondria1
damage was c h a r a c t e r i z e d by s w e l l i n g and r e d u c t i o n of i n t e r n a l c r i s t a e
concomitant w i t h a l o s s of o r d e r l y arrangement.
Although i t - w a s not
p o s s i b l e t o d e f i n e a s p e c i f i c s u b c e l l u l a r l e s i o n , it appears t h a t t h e
changes seen were r e l a t e d t o t h e i n i t i a l r a d i a t i o n damage.
Evidently
l a r g e t h e r a p e u t i c r a d i a t i o n doses can cause e x t e n s i v e damage t o t h e h e a r t
t h a t may not manifest i t s e l f u n t i l y e a r s a f t e r t r e a t m e n t .
The wealth of c l i n i c a l and p a t h o l o g i c evidence i n d i c a t i n g t h a t
i o n i z i n g r a d i a t i o n can produce myocardial damage shows t h e importance
of t h e e a r l y d e t e c t i o n of such i n j u r y ( ~ u g g i a-e t a l . 1970).
Since t h e
u l t i m a t e m a n i f e s t a t i o n of r a d i a t i o n i n j u r y t o l i v i n g systems i s r e l a t e d
t o t h e degree of i n i t i a l damage, t h e q u a n t i t a t i v e e s t i m a t e s of such
damage may provide a u s e f u l c l i n i c a l p r e d i c t i o n of t h e long term e f f e c t s .
Q u a n t i t a t i v e understanding of t h e biochemical and morphologic e f f e c t s
of c a r d i a c i r r a d i a t i o n i s e s s e n t i a l f o r t h e development of any preventa t i v e schemas.
Determination of enzyme leakage from t h e t a r g e t organ
may t h e r e f o r e provide e f f e c t i v e means of e v a l u a t i n g t h e i n i t i a l damqe
and t h u s s e r v e a s a u s e f u l r a d i o b i o l o g i c a l i n d i c a t o r .
A t p r e s e n t , t h e r e i s s u r p r i s i n g l y l i t t l e information r e l a t i n g
r a d i a t i o n damage of t h e h e a r t t o e l e v a t i o n of serum enzymes.
However,
Kubota (1969) has r e p o r t e d e l e v a t i o n s of l a c t a t e dehydrogenase, g l u t a mate o x a l o a c e t a t e transaminase and g l u t m a t e pyruvate transaminase
a f t e r s u b j e c t i n g r a b b i t s t o p r e c o r d i a l b e t a i r r a d i a t i o n of 500
-
4000 R .
S i m i l a r l y , Mochalova -e t a l . (1969) found sharp i n c r e a s e s i n l a c t a t e
dehydrogenase, a l d o l a s e and c r e a t i n e k i n a s e i n blood serum of r a b b i t s
exposed t o l o c a l i z e d i r r a d i a t i o n of t h e h e a r t .
within
These i n c r e a s e s occured
60 hours of exposure and were accompanied by marked morpholo-
g i c a l changes i n t h e myocardial t i s s u e .
The study of radiation-induced leakage of enzymes from t h e h e a r t
should, i n a d d i t i o n , y i e l d valuable information on t h e p o s t - i r r a d i a t i o n
i n t e g r i t y of c e l l u l a r and s u b - c e l l u l a r membranes.
D i f f e r e n t i a l per-
m e a b i l i t y of c e l l u l a r membranes c o n t r o l s enzyme leakage i n t o t h e plasma.
Under normal p h y s i o l o g i c a l conditions t h e c e l l membrane r e t a i n s biol o g i c a l l y a c t i v e m a t e r i a l i n high c o n c e n t r a t i o n s , t h e maintenance of
which r e q u i r e s a considerable expenditure of energy ( ~ e s s1963).
Retention of b i o l o g i c a l l y a c t i v e molecules may be a r e s u l t of a c t i v e
t r a n s p o r t , t h e s i z e and geometry of t h e membrane pores, and t h e elect r o s t a t i c charge on t h e membrane s u r f a c e .
It i s t h e r e f o r e c l e a r t h a t
p a t h o l o g i c a l m a n i f e s t a t i o n s of a l t e r a t i o n s i n permeability characteri s t i c s may r e s u l t e i t h e r from i n t e r r u p t i o n of t h e energy supply t o t h e
membrane a s a r e s u l t of metabolic l e s i o n s (Schade 1953; WE 1959; Binkley
1961) o r from d i r e c t damage t o t h e s t r u c t u r a l components of t h e membrane.
The enzyme-release hypothesis proposed by Bacq and Alexander (1961)
suggests t h a t many of t h e d e l e t e r i o u s e f f e c t s of r a d i a t i o n on c e l l s
a r e caused by damage t o t h e macromolecules c o n s t i t u t i n g t h e c e l l u l a r
and s u b - c e l l u l a r membranes, r e s u l t i n g i n t h e f a i l u r e of t h e i r s e l e c t i v e
c h a r a c t e r i s t i c s , with t h e consequent leakage of i o n s , c o f a c t o r s and
o t h e r b i o l o g i c a l m a t e r i a l out of t h e c e l l and i t s o r g a n e l l e s .
Although
much e f f o r t has been devoted t o f i n d i n g t h e exact causes of t h e s e a l t e r a t i o n s i n membrane p e r m e a b i l i t i e s of i r r a d i a t e d systems, no c l e a r l y def i n e d s o l u t i o n i s a t hand.
Much of t h e evidence accumulated t o date
i m p l i c a t e s l i p i d p e r o x i d a t i o n , and although many workers have-reported
--
permeability a l t e r a t i o n s r e s u l t i n g from i n v i t r o peroxide formation
( w i l l s and Wilkinson 1967; W i l l s 1 9 7 0 ) ~no c l e a r c u t evidence h a s y e t
been o b t a i n e d t h a t l i p i d peroxides a r e produced i n s i g n i f i c a n t amounts
i n i r r a d i a t e d animals ( ~ e a d1961).
OBJECTIVES OF THE STUDY
The p r e s e n t i n v e s t i g a t i o n i s an attempt t o determine t h e e f f e c t s
of t h e r a p e u t i c l e v e l s of i o n i z i n g r a d i a t i o n on t h e c a r d i a c muscle of
rats.
The p r i n c i p l e o b j e c t i v e i s t o e s t a b l i s h whether r a d i a t i o n damage
does cause s i g n i f i c a n t serum e l e v a t i o n s i n c e r t a i n key enzymes.
t h e enhancement,
nf
sprilm
P I ~ Z ~ertiyities
P
m q he r 2 1 3 t c d
t~
G
I f so,
ZGGZ~;;;I-
t a n t decrease i n t h e t i s s u e l e v e l s of t h e enzymes.
One of t h e biochemical i n d i c a t o r s i n a c u t e r a d i a t i o n i n j u r y i s an
e l e v a t i o n of serum l a c t a t e dehydrogenase (LDH) a c t i v i t y following t o t a l body exposure ( ~ o r ei t a l . 1968).
LDH, found i n most organs, e n t e r t a i n s
an important f u n c t i o n i n carbohydrate metabolism and under normal condit i o n s remains a t a f a i r l y c o n s t a n t l e v e l .
Glutamate o x a l o a c e t a t e t r a n s -
aminase (GOT) and c r e a t i n e k i n a s e ( C K ) a r e b o t h found i n r e l a t i v e l y high
c o n c e n t r a t i o n s i n c a r d i a c muscle.
GOT, involved i n p r o t e i n metabolism
and CK i n t h e replenishment of t h e phosphate moiety of ATP f o r muscular
c o n t r a c t i o n , p l a y important r o l e s a s biochemical markers f o r t h e c l i n i c a l
d e t e c t i o n of myocardial i n j u r y ( ~ r e y f u s-e t a l . 1960; Schmidt e t a l . 1965)
and were t h e r e f o r e s e l e c t e d a s p o t e n t i a l i n d i c a t o r s of t h e degree of
r a d i a t i o n i n j u r y t o t h e c a r d i a c muscle.
S i n c e it i s known t h a t t h e ongoing metabolism of t h e c e l l i n f l u e n c e s
t h e development of major p o s t - i r r a d i a t i o n l e s i o n s , t h e e f f e c t of e x e r c i s e
i n p r e v i o u s l y t r a i n e d animals was a l s o i n v e s t i g a t e d t o determine i f r a d i a t i o n damage i s modified by t h i s s t r e s s c o n d i t i o n .
I n a d d i t i o n , l i p i d per-
o x i d a t i o n i n h e a r t t i s s u e w a s measured i n v i v o , i n an attempt t o c o r r e l a t e
s t r u c t u r a l damage of t h e c e l l membranes t o a l t e r a t i o n s i n p e r m e a b i l i t y .
The f u n c t i o n of vitamin E a s a p o t e n t i a l r a d i o p r o t e c t i v e . agent was a l s o
examined.
The s p e c i f i c o b j e c t i v e s a r e l i s t e d below.
1. Does i r r a d i a t i o n of r a t c a r d i a c muscle r e s u l t i n i n c r e a s e d
serum l e v e l s of t h e t h r e e metabolic enzymes, l a c t a t e deh y d r n p n a s e ; cree-tine k i f i s c e , zcfi g>~t=+n ~ x ~ ~ z c c t ~ ~ c
+
,
,
,
,
,
:
.
.
.
?
.
9
"I
LWI.,OILlllllalJG.
2. Is such an i n c r e a s e accompanied by decreases of t h e enzyme
i n t h e t a r g e t organ o r i s it caused by i n c r e a s e d leakage
r a t e s due t o e l e v a t i o n s of t h e enzyme w i t h i n t h e t i s s u e ?
3. Does p o s t - i r r a d i a t i o n e x e r c i s e i n t r a i n e d animals a l t e r
t h e degree of enzyme leakage w i t h r e s p e c t t o i r r a d i a t e d
s e d e n t a r y animals? Does e x e r c i s e t h e r e f o r e appear harmf u l o r beneficial i n exercise-trained, irradiated subjects?
4. Does t h e r a p e u t i c r a d i a t i o n induce -i n vivo l i p i d peroxidat i o n i n c a r d i a c muscle? I f s o , can t h e changes i n enzyme
e f f l u x i n p o s t - i r r a d i a t e d s u b j e c t s b e r e l a t e d t o t h e degree
of p e r o x i d a t i o n ?
5. Does vitamin E , a s a d i e t a r y supplement, a f f e c t t h e posti r r a d i a t i o n serum enzyme p a t t e r n ?
CHAPTER I1
LITERATURF: REVIEW
For t h e sake of c l a r i t y , t h e following d i s c u s s i o n w i l l be divided
i n t o f o u r sub-sections.
The f i r s t s e c t i o n w i l l p r e s e n t contemporary
c l i n i c a l and experimental works involving enzymological determinations
of r a d i a t i o n damage, t h e second s e c t i o n w i l l cover important works rel a t i n g r a d i a t i o n i n j u r y t o a l t e r a t i o n s i n membrane p e r m e a b i l i t y , and
t h e t h i r d s e c t i o n w i l l propound evidence of t h e p a r t i c i p a t i o n of autoc a t a l y t i c l i p i d p e r o x i d a t i o n i n membrane damage.
Section four w i l l
review l i t e r a t u r e i m p l i c a t i n g vitamin E a s a b i o l o g i c a l l i p i d a.ntr.inxidant and i t s p o s s i b l e involvement a s a n a t u r a l r a d i o p r o t e c t i v e agent.
<
Enzymological Considerations
Experimental and c l i n i c a l i n v e s t i g a t i o n s have r e v e a l e d t h a t s t u d i e s
of enzyme a c t i v i t i e s a r e u s e f u l i n d i c a t o r s of t h e e f f e c t s of i o n i z i n g
r a d i a t i o n on l i v i n g systems.
A s such, "the e s t i m a t i o n of enzymes i n
t i s s u e homogenates, i n t h e i n t e r c e l l u l a r f l u i d , i n t h e serum, and i n
e f f u s i o n s , adds considerably t o our knowledge of t h e e f f e c t s of i o n i z i n g
r a d i a t i o n on t h e homothermal organism. 1 1 1
Ludewig and Chanutin ( 1950) r e p o r t e d t h a t serum a l k a l i n e phosphat ase
l e v e l s decreased following whole-body i r r a d i a t i o n of r a t s .
I n a study of
u r i n a r y deoxyribonuclease , Kowlessar and c o l l a b o r a t o r s (1953) observed an
~ K . N . Karcher. Enzymological examinations : An i n d i c a t i o n of
r a d i a t i o n e f f e c t s i n experiments and c l i n i c a l p r a c t i c e , I n : Biochemical
I n d i c a t o r s of Radiation I n j u r y i n Man, proceedings of t h e I n t e r n a t i o n a l
Atomic Energy Agency, D.H. H i l l ( e d i t o r ) , Unipub, New York, 1971, p . 277.
i n c r e a s e i n t h e u r i n e a c t i v i t y i n r a t s exposed t o 700 r a d s of whole-body
irradiation.
S i m i l a r l y , Roth and E i c h e l (1959) observed a g e n e r a l l o s s
of r i b o n u c l e a s e a c t i v i t y from mitochondria1 , n u c l e a r and microsomal
p a r t i c l e s of r a t s p l e e n , w i t h a concomitant i n c r e a s e i n t h e cytoplasmic
activity.
I n a biochemical and e l e c t r o n microscopic study of i r r a d i a t e d
mitochondria i s o l a t e d from r a t l i v e r , Okada and Peachey (1957) p r e s e n t e d
evidence i n support of t h e hypothesis t h a t t h e noted i n c r e a s e i n deoxyr i b o n u c l e a s e I1 a c t i v i t y i s c a u s a l l y r e l a t e d t o s t r u c t u r a l damage i n t h e
mitochondria.
Kawasaki and Sakurai (1969), i n a s t u d y of i r r a d i a t e d
mouse l i v e r mitochondria, noted an i n c r e a s e i n ATPase a c t i v i t y w i t h a
p a r a l l e l decrease i n o x i d a t i v e phosphorylation w i t h i n t h r e e t o s i x hours
a f t e r whole-body exposure t o s u b l e t h a l X-radiation.
Experiments on -E coli
by B i l l e n and co-worker~ (1953) demonstrated a l o s s of ATP from t h e organi s m s i n t o t h e media following exposure t o r a d i a t i o n .
The r e s u l t s in-
d i c a t e d t h a t i n j u r e d c e l l s r e l e a s e more enzymes i n t o t h e f l u i d t h a n c e l l s
killed instantly.
The damaged c e l l s continue t o produce m e t a b o l i t e s b u t
l o s e t h e m a t e r i a l through t h e i n j u r e d membrane, whereas dead o r r u p t u r e d
c e l l s can r e l e a s e only t h e i r immediate c o n t e n t s .
Various workers have i n v e s t i g a t e d t h e e f f e c t of r a d i a t i o n on
a l t e r a t i o n s i n t h e a c t i v i t y of numerous enzymes i n c o n c e r t .
Wills and
Wilkinson i n 1966 noted a r e l e a s e of a c i d phosphatase, c a t h e p s i n and
B-glucuronidase from r a t l i v e r lysosomes following i r r a d i a t i o n i n v i t r o .
Concomitant w i t h t h e r e l e a s e of enzymes, formation of l i p i d peroxides
was noted, i n d i c a t i n g t h a t l i p i d p e r o x i d a t i o n may mediate r u p t u r e of
t h e lysosome membrane, allowing t h e r e l e a s e of m a t e r i a l .
Although
Klein-Szanto and C a b r i n i (1970) found i n c r e a s e s i n t h e mean enzyme conc e n t r a t i o n s of NADP cytochrome c r e d u c t a s e and glucose-6-phosphate
de-
hydrogenase, l a c t a t e dehydrogenase and s o r b i t o l dehydrogenase concentrat i o n s decreased i n i r r a d i a t e d epidermis.
It w a s suggested t h a t t h e
r a d i a t i o n response might r e s u l t from widespread metabolic adjustments.
I n a r e c e n t c l i n i c a l i n v e s t i g a t i o n Muggia and co-workers (1970) s t u d i e d
t h e serum responses of a s e r i e s of enzymes i n twenty-one p a t i e n t s r e c e i v i n g
i n c i d e n t a l c a r d i a c exposure i n comparison t o twenty-four p a t i e n t s r e c e i v i n g
non-cardiac i r r a d i a t i o n .
The serum l a c t a t e dehydrogenase (LDH) and g l u t a -
mate o x a l o a c e t a t e transaminase (GOT) l e v e l s d i d not appear u s e f u l i n
d e t e c t i n g radiation-induced myocardial i n j u r y , due t o t h e i r frequent
inherent abnormalities.
Serum c r e a t i n e k i n a s e ( C K ) however, appeared
e l e v a t e d i n 33% of t h e p a t i e n t s r e c e i v i n g c a r d i a c i r r a d i a t i o n and may
r e f l e c t r a d i a t i o n damage t o t h e myocardium.
et al.
Hori and Nishio (1962) and Hori -
(1964) found t h a t i n r a b b i t s
and mice exposed t o 300 R of X-rays, plasma LDH a c t i v i t y reached a peak
e l e v a t i o n i n about s i x h o u r s , r e t u r n i n g t o normal by 24 hours.
Transient
i n c r e a s e s were found two and seven days a f t e r i r r a d i a t i o n and have a l s o
been noted by Kgrcher and Kato
(1964). I n a l a t e r study Hori -e t a l . (1968)
found i n c r e a s e d plasma LDH w i t h i n hours of whole body exposure of mice t o
X-radiation of 600
-
900 R.
The e x t e n t of e l e v a t i o n appeared p r o p o r t i o n a l
t o i r r a d i a t i o n exposure i n t h e range below 900 R.
Analysis of v a r i o u s
organs showed a marked l o s s of LDH only i n thymus and s p l e e n .
Radio-
r e s i s t a n t organs showed no s i g n i f i c a n t changes, supporting t h e contention
t h a t i n c r e a s e d plasma LDH l e v e l s a r e d e r i v e d from p o s s i b l e c e l l d e s t r u c t i o n
and membrane p e r m e a b i l i t y changes i n r a d i o s e n s i t i v e t i s s u e s .
Further
support f o r t h e c e l l d e s t r u c t i o n hypothesis was obtained by Takamori
et al.
-
(1969) using i s o l a t e d mouse thymocytes and e r y t h r o c y t e s .
Numerous r e p o r t s of e l e v a t i o n s i n serum glutamate o x a l o a c e t a t e
transaminase l e v e l s a s a r e s u l t of exposure t o i o n i z i n g r a d i a t i o n have
appeared.
Milch and Albaum (1956) showed t h a t serum GOT l e v e l s r o s e
w i t h i n a few hours following whole-body exposure of r a b b i t s , and P e t e r s e n
e t al.
--
(1957) found t h a t serum GOT v a l u e s i n r a b b i t s were 223% of normal
f i v e hours a f t e r exposure t o
644 r a d s .
Out of
1 4 enzymes s t u d i e d , Albaum
(1960) found GOT t o b e t h e b e s t e a r l y index of r a d i a t i o n in.jurv.
However,
d a t a from Brent -e t a l . (1958) suggested t h a t t h e response i s v a r i e d
between s p e c i e s .
Although serum transaminase l e v e l s i n r a b b i t s r o s e
s h a r p l y on t h e f i r s t p o s t - i r r a d i a t i o n day, no s i g n i f i c a n t e l e v a t i o n s
could be found i n r a t s e i t h e r a f t e r whole-body exposure o r f o c a l irr a d i a t i o n of t h e l i v e r w i t h 5,000 r a d s .
Becker -e t a l . (1962), i n
c o n t r a s t , r e v e a l e d s t r i k i n g b u t t r a n s i e n t e l e v a t i o n s of serum GOT
following total-body i r r a d i a t i o n of Sprague-Dawley r a t s .
Elevation
occured between t h e t h i r d and n i n t h hours and appeared t o be r e l a t e d
t o c e l l destruction i n radiosensitive tissues.
From t h e l i t e r a t u r e it i s apparent t h a t c e l l u l a r r a d i a t i o n damage
does e l i c i t changes i n t h e enzyme p a t t e r n s w i t h i n c e l l s and o r g a n e l l e s
which can cause s i g n i f i c a n t a l t e r a t i o n s of t h e serum enzyme l e v e l s .
Although c e l l death and d i s i n t e g r a t i o n have been a t t r i b u t e d a s t h e
11
r g i s o n d l $ t r e " f o r p o s t - i r r a d i a t i o n e l e v a t i o n s i n serum enzymes, it
must be emphasized t h a t many of t h e s t u d i e s noted above involved wholebody exposure i n which r a d i a t i o n damage would r e f l e c t i n j u r y t o t h e most
r a d i o s e n s i t i v e organs.
I n systems such a s t h e s p l e e n and thymus c e l l
d i s r u p t i o n may, i n f a c t , p l a y a c r u c i a l r o l e i n t h e m a n i f e s t a t i o n of
radiation injury.
However, biochemical and h i s t o l o g i c a l examinations
have a l s o confirmed t h e view t h a t r a d i a t i o n administered i n t h e r a p e u t i c
l e v e l s does cause a l t e r a t i o n s i n membrane p e r m e a b i l i t i e s i n s u b c e l l u l a r
o r g a n e l l e s a s w e l l a s changes i n t h e c e l l u l a r membrane ( ~ g r c h e r1971),
which could a l s o account f o r t h e noted e l e v a t i o n s of enzymes i n t h e blood.
Conceivably, both mechanisms a r e f u n c t i o n i n g and t h o s e c e l l s t h a t a r e not
l e t h a l l y damaged continue t o f u n c t i o n i n t h e i r damaged s t a t e , l e a k i n g out
b i o l o g i c a l l y a c t i v e m a t e r i a l from t h e cytoplasm.
Evidence of Radiation I n j u r y t o Membranes
A number of i n v e s t i g a t i o n s have r e v e a l e d t h a t changes occur i n
membrane p e r m e a b i l i t i e s a f t e r exposure t o i o n i z i n g r a d i a t i o n ( ~ c h r e k
1948; L e s s l e r 1959; Barer and Joseph 1960; Novack and Shapiro 1960).
I n 1959 Noyes and Smith observed q u a n t i t a t i v e changes i n r a t l i v e r
mitochondria following whole-body i r r a d i a t i o n .
I t was concluded t h a t
r a d i a t i o n damage i n v o l v e s a p r e f e r e n t i a l fragmentation of small mitochondria, l e a d i n g t o t r a n s i e n t depression i n t h e t o t a l mitochondria1
count.
S i m i l a r a l t e r a t i o n s were r e p o r t e d by Bahr i n 1971.
Hugon and
co-workers ( 1 9 6 5 ) , i n a study of r a d i a t i o n damage t o t h e duodenal c r y p t s
i n mice, noted expansion, c l a r i f i c a t i o n and r u p t u r e of t h e mitochondrial
m a t r i x i n stem c e l l s w i t h i n 30 minutes of exposure, and P o r t e l a
etc.
15
(1963) proposed t h a t one of t h e primary e f f e c t s of r a d i a t i o n exposure i s
damage t o t h e membrane o r g a n i z a t i o n a t t h e l e v e l of t h e mitochondrial
cristae.
Although S c a i f e and Alexander (1961) found no evidence t o support
t h e i d e a of a l t e r a t i o n i n t h e p e r m e a b i l i t y of mitochondria1 membranes,
t h e i r conclusion c o n t r a s t s w i t h f i n d i n g s of Mukerjee and Goldfeder (1972),
who noted i n c r e a s e d p r o t e i n and RNA s y n t h e s i s i n mouse l i v e r mitochondria
s u b j e c t e d t o 2,000 R of X-radiation.
Enhanced s y n t h e s i s was explained by
i n c r e a s e d t r a n s p o r t of RNA and p r o t e i n p r e c u r s o r s due t o changes i n t h e
mitochondrial membrane p e r m e a b i l i t y following exposure.
e t a 1.
--
Work by Desai
(1964) r e v e a l e d t h a t lysosomal p r e p a r a t i o n s exposed t o i n c r e a s i n g
doses of gamma r a d i a t i o n showed r e l e a s e of i n d i c a t o r enzymes, w i t h each
enzyme having a d i f f e r e n t p a t t e r n of r e l e a s e and i n a c t i v a t i o n .
Such
leakage was a t t r i b u t e d t o damage and l y s i s of t h e lysosomal membrane.
R e l a t e d damage and a l t e r a t i o n s i n t h e endoplasmic r e t i c u l u m of h e p a t i c
parenchymal c e l l s of mice were observed by Hendee and Alders (1968)
w i t h i n two minutes a f t e r exposure t o gamma r a d i a t i o n .
The r a p i d develop-
ment of t h e s e changes suggests t h a t membrane damage may be c l o s e l y r e l a t e d
t o i n i t i a l radiation injury.
A study of t h e mechanism of radiation-induced
enhancement of sodium accumulation i n e r y t h r o c y t e s ( ~ h a p i r oand Kollman
1968) r e v e a l e d t h a t membrane s u l f h y d r y l groups appear t o be t h e major
t a r g e t i n r a d i a t i o n a l t e r a t i o n of c e l l p e r m e a b i l i t y .
There was no d i r e c t
evidence suggesting t h e appearance of a membrane h o l e , b u t i n s t e a d ,
r a d i a t i o n damage appeared t o induce a very s p e c i f i c ~ h e m i c a l ~ c h a n gien
t h e membrane.
I n c o n t r a s t , Sutton and Rosen's study i n 1968 of t h e e f f e c t
$
of X-rays on t h e e l e c t r i c a l conductance of phospholipid b i l a y e r s
produced d a t a which was c o n s i s t e n t w i t h a model i n which r a d i a t i o n
exposure induces t h e formation of temporary h o l e s through t h e membrane,
l a r g e enough t o permit t h e f r e e d i f f u s i o n of i o n s p r e s e n t .
Petkau (1971),
u s i n g a model l i p i d membrane system, r e p o r t e d a scavenging of superoxide
i o n s by t h e system a f t e r r a d i a t i o n t r e a t m e n t .
It was e v i d e n t a l s o t h a t
radiation-induced r e a c t i o n s i n t h e aqueous phase were somehow i n f l u e n c e d
by t h e o r g a n i z a t i o n of t h e membrane phospholipids.
Much of t h e r e c e n t u l t r a s t r u c t u r a l and cytochemical evidence
f u r t h e r e l u c i d a t e s t h e mode of r a d i a t i o n damage t o membranes.
Various
workers have independently shown a l t e r a t i o n s i n u n i t membrane c h a r a c t e r i s t i c s w i t h marked breaks i n membranes o f t e n occuring i n e a r l y s t a g e s
following i r r a d i a t i o n ( ~ o l f e d e rand M i l l e r 1963 ; Hugon e t a l . 1965;
Nair and Bhakthan 1969; Bhakthan and Dharamraj 1972).
I n vivo
--
and
in
v i t r o i r r a d i a t i o n of c e l l o r g a n e l l e s has f u r n i s h e d evidence of l o s s of
i n t e g r i t y and changes i n t h e physico-chemical
c h a r a c t e r i s t i c s of t h e
l i p o p r o t e i n membranes ( ~ e s a -ei t a l . 1964; Zyss and Michalska-Kaszczynska
1972)
. E l e c t r o n microscopic
s t u d i e s ( ~ h i d o n i1967) of i r r a d i a t e d primate
l i v e r r e v e a l e d a l o s s of i n t e g r i t y and i n t e r n a l rearrangement of t h e
component macromolecules i n v a r i o u s s t r u c t u r a l membranes.
Mitochondria1
o b s e r v a t i o n s r e v e a l e d c o n s i s t e n t a l t e r a t i o n s , d i s t o r t i o n and s w e l l i n g ,
w i t h an apparent l e a c h i n g of m a t r i x m a t e r i a l .
S i m i l a r observations by
Hugon and c o l l a b o r a t o r s (1965) support t h e enzyme r e l e a s e t h e o r y a s
proposed by Bacq and Alexander (1961).
Other morphological and arch-
i t e c t u r a l s t u d i e s a r e c o n s i s t e n t w i t h t h e s e views ( ~ c h e r e rand Vogell
.
1958; Bencosme -e t a 1 1962; Morgenroth and Themann 1964)
.
There i s a c l e a r r e l a t i o n s h i p between r a d i a t i o n i n j u r y and
a l t e r a t i o n s i n s t r u c t u r e and p e r m e a b i l i t y of o r g a n e l l e and c e l l membranes.
The e x t e n t t o which damage t o membranes i s a r e s u l t of i n i t i a l r a d i a t i o n
i n j u r y o r i s due t o establishment of metabolic l e s i o n s i s a s y e t unresolved.
However, t h e r e has accumulated a wealth of evidence suggesting t h a t
radiation-induced l i p i d p e r o x i d a t i o n may be r e s p o n s i b l e f o r t h e observed
l o s s of membrane i n t e g r i t y .
L i p i d P e r o x i d a t i o n and Damage t o Membranes
A / General Considerations
Lipid peroxidation i s t h e deterior-
a t i v e o x i d a t i o n of p o l y u n s a t u r a t e d l i p i d s and involves a r e a c t i o n of
molecular oxygen o r neighbouring f r e e r a d i c a l s w i t h t h e a c t i v a t e d
a l l y l i c hydrogens on t h e alpha methylene carbons of t h e u n s a t u r a t e d
l i p i d double bond ( ~ e m o p o u l o s1973; Tappel 1973).
Because of t h e i r
unpaired e l e c t r o n s , l i p i d f r e e r a d i c a l s , once formed, w i l l r e a c t
v i g o r o u s l y , i n t i t i a t i n g a chain propagation of hydrogen a b s t r a c t i o n s
and a d d i t i o n r e a c t i o n s t h a t can proceed a u t o c a t a l y t i c a l l y a s long a s
there are available active s i t e s .
Termination of t h i s chain r e a c t i o n
can, however, occur by t h e binding of t h e f r e e r a d i c a l s formed o r v i a
quenching by a n t i o x i d a n t s .
I n g e n e r a l , u n s a t u r a t e d f a t t y a c i d s occur p r i n c i p a l l y i n phosphol i p i d s which a r e i n h e r e n t components of biomembranes.
Because peroxida-
t i o n of t h e f a t t y a c i d s of such l i p i d s i s i n f l u e n c e d not only by t h e i r
degree of u n s a t u r a t i o n b u t a l s o by t h e n a t u r e of t h e moiety e s t e r i f i e d
t o t h e phosphate, it i s c l e a r t h a t t h e c o i t e n t of t h e u n s a t u r a t e d l i p i d ,
degree of u n s a t u r a t i o n and t h e s u b s t i t u t i o n p r o c e s s p r e s e n t , p l a y an
important r o l e i n determining t h e likelyhood of p e r o x i d a t i o n damage t o
s
b i o l o g i c a l membranes ( ~ e m o ~ o u l o1973).
Many r e p o r t s have documented
t h e c a t a l y t i c e f f e c t of m e t a l c a t i o n s ( c a s h -e t a l . 1966), especially
.
i n o r g a n i c i r o n , on membrane l i p i d p e r o x i d a t i o n ( ~ t t o l e n g h iet a 1 1955 ;
Hunter -e t a l . 1963; W i l l s 1969). Bernheim (1963) suggests t h a t i n i r o n a s c o r b a t e mediated p e r o x i d a t i o n t h e p r o c e s s i s i n i t i a t e d by f e r r o u s i o n s ,
which i n t h e p r o c e s s a r e oxidized t o f e r r i c i o n s .
t h e n reduces t h e s e back t o t h e f e r r o u s s t a t e .
by Skrede and Christopherson
Ascorbate o x i d a t i o n
It was f u r t h e r progosed
(1966) t h a t i n t e r m e d i a t e s i n t h i o l oxida-
t i o n may b e involved.
The importance of haematin compounds, mainly haemoglobin, myog l o b i n and cytochrome 2, a s b i o c a t a l y s t s of l i p i d p e r o x i d a t i o n h a s a l s o
been e s t a b l i s h e d .
Haematin compounds may, i n f a c t , be t h e primary endo-
genous c a t a l y s t s f o r l i p i d o x i d a t i o n i n animal t i s s u e s ( ~ a p p e 1953b)
l
.
Biomembranes such a s occur i n mitochondria, endoplasmic reticulum and
lysosomes, c o n t a i n r e l a t i v e l y l a r g e amounts of u n s a t u r a t e d l i p i d s .
Although t h e a c t i v e a l l y l i c hydrogens of t h e l i p i d a r e b u r i e d i n t h e
hydrophobic r e g i o n of t h e membrane and a r e t h u s afforded p r o t e c t i o n
(~emopoulos1 9 7 3 ) , t h e c l o s e a s s o c i a t i o n w i t h t h e s e known b i o c a t a l y s t s
arouses i n t e r e s t i n t h e l a b i l i t y of such membranes t o p o s s i b l e damage by
l i p i d peroxidation.
I n a s e r i e s of i n v e s t i g a t i o n s Hunter and co-workers (1963,
1.964,
1964) s t u d i e d t h e e f f e c t s of f e r r o u s ion-induced l i p i d p e r o x i d a t i o n i n
r a t l i v e r mitochondria.
It was found t h a t changes i n t h e p e r m e a b i l i t y
and s t r u c t u r a l c h a r a c t e r i s t i c s of t h e membrane accompanied t h e o n s e t of
l i p i d p e r o x i d a t i o n , l e a v i n g l i t t l e doubt t h a t p e r m e a b i l i t y a l t e r a t i o n s
and s w e l l i n g were c a u s a l l y r e l a t e d t o l i p i d peroxide formation.
McKnight
and c o l l a b o r a t o r s i n 1965 found t h a t f e r r o u s i o n p e r o x i d a t i o n of l i p i d s
l e d t o a l o s s of mitochondria1 p r o t e i n and l i p i d i n t o t h e surrounding
medium.
The r e l e a s e d m a t e r i a l was thought t o c o n s i s t of t h e bulk of
i n t e r c r i s t a l s u b s t a n c e , t o g e t h e r w i t h s o l u b i l i z e d p r o t e i n s and elements
of t h e e l e c t r o n t r a n s p o r t chain.
Similar alterations i n the integrity
of p e r o x i d i z i n g microsomes were noted by Bidlack and Tappel (1973).
Loss
-
of membrane bound NADPH cytochrome c r e d u c t a s e a c t i v i t y and r e l e a s e of
p r o t e i n were found t o b e r e l a t e d t o a l t e r a t i o n s i n t h e l i p o p h i l i c region
of t h e membrane.
Evidence of i n c r e a s e d p e r m e a b i l i t y i n b r a i n microsome p r e p a r a t i o n s
induced by l i p i d peroxide formation has a l s o been observed ( ~ o b i n s o n1965)
as documented by changes i n t u r b i d i t y of t h e system -i n v i t r o , and was
accompanied by an i n c r e a s e i n t h e r e a c t i v e s u l f h y d r y l groups i n t h e
membrane.
Compounds which bound t h e s u l f h y d r y l groups were found t o
i n c r e a s e p e r m e a b i l i t y and a c c e l e r a t e l i p i d p e r o x i d a t i o n .
This suggests
t h a t i n i t i a t i o n of p e r o x i d a t i o n may involve t h e a l t e r a t i o n of a r e l a t i o n s h i p between t h e s e membrane p r o t e i n s and t h e double bonds of t h e phosphol i p i d s , unmasking t h e double bonds and i n i t i a t i n g s t r u c t u r a l changes.
A s i m i l a r r e l a t i o n s h i p between p r o t e i n s u l f h y d r y l m o i e t i e s and u n s a t u r a t e d
l i p i d s h a s been proposed by S c o t t e t a l .
(1.964) t o account f o r s i m i l a r
changes occuring a f t e r i r r a d i a t i o n .
P e r o x i d a t i o n damage t o membrane p r o t e i n s and enzymes h a s been w e l l
described.
P r o t e i n s and enzymes i n aqueous s o l u t i o n s , when s u b j e c t e d t o
l i p i d p e r o x i d a t i o n , undergo polymerization, chain s c i s s i o n and chemical
changes ( ~ o u b a land Tappel 1966a, 1966b).
I n an attempt t o determine t h e
mechanism of damage t o p r o t e i n by l i p i d p e r o x i d a t i o n Desai and Tappel
(1963) found c o n s i d e r a b l e damage t o cytochrome c a s measured by i t s
decreased s o l u b i l i t y .
Linkages between l i n o l e n i c a c i d and t h e p r o t e i n
were found and i d e n t i f i e d a s mainly peroxy o r e t h e r .
This l i p i d - p r o t e i n
l i n k a g e phenomenon was a l s o observed by Chio and Tappel (1969b) i n a
s t u d y of peroxide i n a c t i v a t i o n of r i b o n u c l e a s e A.
Loss of enzyme a c t i v i t y
was concomitant w i t h t h e formation of i n t r a and inter-molecular crossl i n k a g e s between t h e peroxide degradation p r o d u c t , malonaldehyde and
ribonuclease A.
C r o s s l i n k i n g a p p a r e n t l y involves r e a c t i o n of malon-
aldehyde w i t h t h e primary amino groups of amino a c i d s and p r o t e i n s t o
form a S c h i f f base product RN = CH
-
CH = CH
- NH - R which
displays
f l u o r e s c e n c e maxima i n t h e 450 t o 470 nm region ( c h i 0 and Tappel 1969a).
This polymerization of u n s a t u r a t e d l i p i d s w i t h a v a i l a b l e p r o t e i n s may
be one of t h e main mechanisms of i r r e p a r a b l e damage t o enzymes and
membranes.
V i s u a l i z e t h e molecular havoc t h a t occurs when enzymes
c r o s s - l i n k w i t h t h e i r molecular neighbours i n such a random
d e s t r u c t i v e r e a c t i o n ! The normal p r e c i s i o n arrangement of
p r o t e i n s and enzymes i n s u b c e l l u l a r membranes and o r g a n e l l e s
would be badly d i s r u p t e d and t h e i r b i o l o g i c a l a c t i v i t e s -
would be l o s t o r impaired. 2
The p o s s i b i l i t y t h a t i o n i z i n g
B/ R a d i o l o g i c a l Considerations
r a d i a t i o n may induce a u t o x i d a t i o n of l i p i d s i n t h e l i v i n g organism h a s
occupied i n v e s t i g a t o r s i n t e r e s t e d i n t h e e f f e c t s of r a d i a t i o n exposure
on animals.
;n s t u d i e s w i t h t i s s u e homogenates and i s o l a t e d systems it
has been observed t h a t i n exposure t o i o n i z i n g r a d i a t i o n peroxides a r e
formed t h a t can cause e x t e n s i v e d e s t r u c t i o n of l i p i d s ( ~ a u g a a r d1968).
However, t h e a b i l i t y of t h e l i v i n g organism t o d e s t r o y peroxides h a s
made t h e search d i f f i c u l t and of many attempts t o f i n d peroxides i n
l i v i n g organisms, few have been s u c c e s s f u l .
Propagative o x i d a t i o n of l i p i d s induced by i r r a d i a t i o n i s unique
only i n t h e d e t a i l s of i n i t i a t i o n and t e r m i n a t i o n ( ~ e a d1961).
Promo-
t i o n of a u t o x i d a t i o n by r a d i a t i o n may proceed v i a d i r e c t f r e e r a d i c a l
formation of t h e t a r g e t molecule o r through e x t r a c t i o n of a l l y l i c
hydrogens from f r e e r a d i c a l s formed i n t h e c e l l water.
INITIATION :
RH
PROPAGATION : R
.A
+ 0
ROO*
TERMINATION : R
or Re
>-.
>
R*
B
+ RH >- C
>-.
+ H * >-
+ R
D
+
H*
ROO
AUTOCATALYTIC
ROOH + R *
R :R
R:H
from Harmon 1968
I n t h e above diagram r e a c t i o n A i s i n i t i a t e d by e i t h e r d i r e c t o r
i n d i r e c t a c t i o n of i o n i z i n g r a d i a t i o n , forming a l i p i d f r e e r a d i c a l which
2 ~L.. Tappel
P r o c . , 3 2 ( 8 ) : 1870
L i p i d P e r o x i d a t i o n Damage t o C e l l Components, Fed.
1874, 1973
-
combines w i t h oxygen ( r e a c t i o n B ) t o form a l i p i d peroxide r a d i c a l .
This
s p e c i e s can t h e n r e a c t with neighbouring l i p i d s t o form l i p i d peroxide
and newly formed f r e e r a d i c a l s which can s e r v e t o c a t a l y z e t h e propagat i o n sequence a g a i n , u n l e s s t e r m i n a t e d by f r e e r a d i c a l binding o r hydrogen
quenching ( r e a c t i o n D)
.
Work by Dubouloz and c o l l a b o r a t o r s (1950, 1952) has shown t h a t
l i p i d peroxides a r e formed i n r a t s k i n following X - i r r a d i a t i o n .
However,
t h e f a c t t h a t t h e peroxides were not d e t e c t e d u n t i l t h e f o u r t h day suggests
t h a t t h e y could not have been primary products of i r r a d i a t i o n .
Barber
and Ottolenghi ( 1 9 5 7 ) ~ i n demonstrating t h e p r o t e c t i v e a c t i o n of EDTA
i n i r r a d i a t e d mitochondria, showed t h a t t h i s p r o t e c t i v e a b i l ' t y was
p r o p o r t i o n a l t o t h e i n h i b i t i o n of peroxide formation.
An i n t e r e s t i n g
suggestion was t h a t r a d i a t i o n may r e l e a s e c a t i o n s , known f o r t h e i r
c a t a l y t i c p r o p e r t i e s , from normal b i o l o g i c a l c h e l a t i n g a g e n t s , t h u s
i n i t i a t i n g t h e chain propagation.
E a r l i e r experiments on mice by Horgan
and P h i l p o t (1954, 1955) i n d i c a t i n g t h a t a u t o c a t a l y t i c peroxide product i o n may be o p e r a t i n g i n t h e p o s t - i r r a d i a t e d animal were supported by
Bernheim and c o l l a b o r a t o r s (I-956), who found t h a t exposure of r a b b i t s
t o 1400 r a d s of X-radiation produced s i g n i f i c a n t e l e v a t i o n s i n peroxide
v a l u e s i n t h e bone marrow.
I n add:-tion, evidence of p a r t i a l d e s t r u c t i o n
o f a n t i o x i d a n t s s u p p o r t s t h e c o n t e n t i o n t h a t a n t i o x i d a n t d e s t r u c t i o n may
be involved i n r a d i a t i o n i n j u r y .
A l t e r a t i o n of t h e membrane l i p i d s
l e a d i n g t o i n c r e a s e d s e n s i t i v i t y t o p e r o x i d a t i o n , o r an i n c r e a s e d c a t a l y t i c a b i l i t y of b i o l o g i c a l pro-oxidants such a s non-haem i r o n , have a l s o
been proposed by W i l l s (1970) a s a l t e r n a t e modes inducing p e r o x i d a t i o n .
S t u d i e s by W i l l s and Wilkinson (1967) on t h e r a d i a t i o n e f f e c t i n
s u b c e l l u l a r f r a c t i o n s of r a t l i v e r -i n v i t r o provide f u r t h e r evidence of
r a d i a t i o n induced l i p i d p e r o x i d a t i o n .
The a u t h o r s suggested t h a t t h e
formation of l i p o i d a l peroxides may be r e s p o n s i b l e f o r t h e primary biochemical l e s i o n i n i r r a d i a t e d animals.
S i m i l a r r e s u l t s were obtained by
W i l l s (1970) i n i r r a d i a t e d microsomal systems i n which p o s t - i r r a d i a t i o n
peroxide formation produced e x t e n s i v e degradation of t h e microsomal l i p i d ,
accompanied by a r e l e a s e of malonaldehyde o r r e l a t e d di-aldehydes.
Although Bacq and co-workers d e t e c t e d peroxides i n t h e xylene ,
e x t r a c t s of mouse and r a t depot f a t following i r r a d i a t i o n (1951)~ t h e
extremely small v a l u e s obtained do n o t g i v e conclusive evidence t h a t
i n j u r y i n l i v i n g systems i n i t i a t e s -i n v i v o p e r o x i d a t i o n of l i p i d s .
Furthermore, only n e g l i g i b l y small i n c r e a s e s i n l i p i d peroxides occured
i n vivo
--
i n v a r i o u s organs of r a t s i n response t o whole-body i r r a d i a t i o n ,
i n c o n t r a s t t o s i g n i f i c a n t amounts of peroxides formed i n incubated
t i s s u e homogenates ( ~ c h i ie t al. 1 9 6 8 ) .
These r e s u l t s i n d i c a t e t h a t
--
l i p i d p e r o x i d a t i o n i n v i t r o does not r e p r e s e n t t h e amount formed
--
in
--
viva, and makes t h e e x t r a p o l a t i o n of i n v i t r o evidence t o t h e i n vivo
s i t u a t i o n v e r y tenuous indeed.
Although t h e r e i s no c l e a r c u t evidence t h a t peroxides a r e formed
i n damaging amounts i n i r r a d i a t e d animals, it i s p o s s i b l e t h a t even
extremely small amounts of peroxides may produce a c o n s i d e r a b l e radiol o g i c a l e f f e c t , a s was shown by Bernheim and co-workers (1952), s o t h a t
t h e q u e s t i o n of radiation-induced peroxide damage remains open.
Vitamin E:
Antioxidant o r Metabolic I n t e r m e d i a t e ?
The b i o l o g i c a l f u n c t i o n of vitamin E has been a t o p i c of i n t e n s i v e
r e s e a r c h f o r over 30 y e a r s and t o d a t e t h e r e i s s t i l l no agreement on i t s
t m e biochemical r o l e .
the literature.
Two schools of thought have, however, dominated
The a n t i o x i d a n t h y p o t h e s i s , which has r e c e i v e d t h e
widest s u p p o r t , contends t h a t vitamin E a c t s s o l e l y a s a p h y s i o l o g i c a l
l i p i d a n t i o x i d a n t , a c t i v e i n t e r m i n a t i n g t h e a u t o c a t a l y t i c propagation
of f r e e r a d i c a l i n t e r m e d i a t e s .
The second hypothesis c o n s i d e r s t h a t
a-tocopherol has some o t h e r bi ol ogi c a l r o l e ( s ! dfs t i c c t l y s e p a r z t z f s ~ x
i t s possible antioxidant a c t i v i t y .
Although Dam (1957) has s m a r i z e d evidence t h a t vitamin E f u n c t i o n s
i n vivo a n t i o x i d a n t , t h e r e i s much accumulated evidence
e n t i r e l y a s an -s u g g e s t i n g t h a t v i t a m i n E does not prevent a u t o x i d a t i o n of l i p i d s in
vivo reen en
-
et al.
1967; Diplock et a l . 1968).
Recent experimental work
--
h a s been p r e s e n t e d by Diplock e t a l . (1971) which i n d i c a t e s t h a t t h e
f u n c t i o n of vitamin E may b e t o p r o t e c t a reduced form of selenium from
bio-oxidation.
Another i d e a contends t h a t , i n s t e a d of f u n c t i o n i n g a s an
a n t i o x i d a n t , vitamin E may f i l l a p h y s i o l o g i c a l r o l e i n membrane s t a b i l i z a t i o n by v i r t u e of s p e c i f i c physico-chemical i n t e r a c t i o n s between i t s
p h y t y l s i d e chain and t h e f a t t y a c i d a c y l chains of polyunsaturated l i p i d s .
D i f f e r e n t conclusions were drawn, however, by McCay and co-workers (1972)
i n a study involving l i p i d degradation i n membranes r e s u l t i n g from f r e e energy changes i n e l e c t r o n t r a n s p o r t systems.
It was found t h a t a-tocopherol
preserved membrane i n t e g r i t y by scavenging f r e e r a d i c a l s formed a s a
r e s u l t of e l e c t r o n t r a n s p o r t and t h u s afforded p r o t e c t i o n a g a i n s t
p e r o x i d a t i v e chain cleavage by v i r t u e of i t s a n t i o x i d a t i v e c a p a b i l i t i e s .
I n c o n t r a s t , Levander and Morris (1971) suggest t h a t a-tocopherol may
play a r o l e i n t h e maintenance of i n t e r - c e l l u l a r c a t i o n g r a d i e n t s .
To
add t o t h e confusion, t h e r e i s now a l a r g e body of evidence suggesting
t h a t vitamin E plays a key r o l e i n t h e r e g u l a t i o n of haem b i o s y n t h e s i s ,
presumably by c o n t r o l l i n g t h e mechanisms of induction-repression a t t h e
enzymatic s t e p s i n t h e s y n t h e s i s of o-aminolevulinic a c i d (ALA) and
yorphobilinogen (PBG)
a air
1972).
I n an e f f o r t t o f i n d common ground
between t h e two viewpoints, Nair (1972) has proposed t h a t t h e r e g u l a t i o n
of haem b i o s y n t h e s i s by a-tocopherol i n h e r e n t l y c o n t r o l s t h e conjugation
of haem with t h e apoprotein of c a t a l a s e , which i n t u r n c a r r i e s out t h e
c a t a l y t i c scavenging of peroxides formed by t h e b i o l o g i c a l oxidation of
lipids.
Hence, a metabolic f u n c t i o n of vitamin E i s proposed which i s
manifested i n i t s a n t i o x i d a n t a c t i v i t y .
However, Horwitt s t a t e s t h a t
Since t h e mammalian organism w i l l s u r v i v e i n t h e absence
of a-tocopherol i f some o t h e r a n t i o x i d a n t
i s subjected,
it i s most i l l o g i c a l t o continue t o a t t a c h any major import a n c e t o r e p o r t s about a-tocopherol being an i n t e g r a l p a r t
of a c r i t i c a l enzyme system.3
...
Although many workers d i s a g r e e with t h e b i o l o g i c a l antioxidant
concept of vitamin E
reen en -e t al.
--
1967; Diplock e t a l . 1968; Green 1972;
Lucy 1972; Schwarz 1972; Diplock and Lucy 1973), many workers have produced considerable evidence i n support of t h e proposal.
An i n v e s t i g a t i o n
3 ~ . Horwitt,
~ .
Role of vitamin E , selenium, and polyunsaturated
f a t t y acids i n c l i n i c a l and experimental muscle d i s e a s e . FED PROC.
24: 68 - 72, 1965.
by Tappel (1952a) of t h e o x i d a t i o n of u n s a t u r a t e d f a t t y a c i d s c a t a l y z e d
by haematin compounds r e v e a l e d t h a t t h e r e a c t i o n could be i n h i b i t e d by
vitamin E and o t h e r a n t i o x i d a n t s , i n d i c a t i n g t h a t a-tocopherol may
f u n c t i o n -i n vivo t o i n h i b i t haematin-catalyzed o x i d a t i o n of f a t .
In
a d d i t i o n , chenges i n c a t a l y t i c and s t r u c t u r a l p r o t e i n s due t o vitamin E
d e f i c i e n c y a r e thought by Tappel (1955) t o r e s u l t from t h e r e a c t i o n of
l i p i d peroxide products w i t h t h e p r o t e i n , and support t h e c o n t e n t i o n t h a t
v i t a m i n E f u n c t i o n s p r i n c i p a l l y a s an a n t i o x i d a n t .
It i s l i k e l y t h a t
vitamin E a c t s a s a chain-breaking a n t i c x i d a n t , i n h i b i t i n g t h e f r e e r a d i c a l p e r o x i d a t i o n v i a hydrogen a b s t r a c t i o n o r through t h e formation
of a l i p i d peroxy r a d i c a l - t o c o p h e r o l complex (Tappel 1972).
However,
i n s t u d i e s of a- tocopherol o x i d a t i o n p r o d u c t s i s o l a t e d from animal ti ssue ,
t h e a b s t r a c t i o n of hydrogen appears t o be t h e dominant p r o c e s s (Tappei
A s t u d y of t h e e f f e c t s of r a d i a t i o n and peroxidation-induced
r a d i c a l s on a-tocopherol showed t h a t r a d i a t i o n of vitamin E i n isooctane
produced many products.
However, 5-exomethylenetocopher-6-one,
derived
from t h e e x t r a c t i o n of two hydrogen atoms, appeared t o be t h e major
product formed
( ~ n a p pand Tappel 1961).
The conversion of a-tocopherol
t o t h i s a r r a y of degradation products i s c o n s i s t e n t w i t h observed p a r t i a l
d e s t r u c t i o n of t o c o p h e r o l i n l i v i n g t i s s u e s ( ~ e a d1961)
.
Furthermore,
t h e dehydrogenation of vitamin E t o t h e conjugated ketone product from
r a d i a t i o n suggests t h a t t h e "modus operandi" of r a d i a t i o n damage may
involve d e s t r u c t i o n of t h e a n t i o x i d a n t i n l i v i n g t i s s u e , allowing unc o n t r o l l e d l i p i d p e r o x i d a t i o n t o ensue.
Such has been proposed by
27
Myers and Bide (19661, who suggested t h a t radiation-induced d e s t r u c t i o n
of t h e membrane a n t i o x i d a n t i s c a u s a l l y r e l a t e d t o t h e formation of l i p i d
peroxides i n e r y t h r o c y t e membranes.
I n a d d i t i o n , t h e i r r a d i a t i o n of blood
from mice p l a c e d on v a r i o u s d i e t s supplemented w i t h t h e vitamin ( p r i n c e
1973) has shown t h a t tocopherol f o r t i f i c a t i o n not only r e v e r s e s haemolytic
e f f e c t s i n t o c o p h e r o l d e f i c i e n t e r y t h r o c y t e s b u t a l s o decreases haemolysis
and l o s s of potassium from t h e c e l l s .
It was furthermore apparent t h a t
d i e t a r y l e v e l s of a n t i o x i d a n t may be an important f a c t o r i n t h e radios e n s i t i v i t y of e r y t h r o c y t e s .
-
Wills (1970) found vitamin E t o be an e f f e c t i v e i n h i b i t o r of i n
v i t r o p e r o x i d a t i o n of i r r a d i a t e d r a t l i v e r microsomes, and i n a l a t e r
s t u d y Dawes and W i l l s (1972) showed t h a t supplementation of t h e d i e t
w i t h v i t a m i n E depressed t h e r a t e of p e r o x i d a t i o n i n t i s s u e homogenates
of mice following exposure t o i o n i z i n g r a d i a t i o n .
It was concluded t h a t
t h e a n t i o x i d a n t c o n c e n t r a t i o n s a t t h e c e l l u l a r and s u b c e l l u l a r l e v e l p l a y
a major r o l e i n t h e r e g u l a t i o n of p o s t - i r r a d i a t i o n p e r o x i d a t i o n of l i p i d s .
Although many i n v e s t i g a t i o n s -i n v i t r o support t h e a n t i o x i d a n t
h y p o t h e s i s , t h e l a c k of conclusive evidence of -i n v i v o peroxide formation
o r t h e presence of peroxides i n t i s s u e s ( ~ c h w a r z1972) has s e v e r e l y
hampered s t u d y of t h e a n t i o x i d a n t a c t i v i t y of vitamin E i n t h e i n t a c t ,
l i v i n g organism.
Perhaps confusing t h e i s s u e i s t h e f a c t t h a t
2 vivo
p e r o x i d a t i o n of p o l y u n s a t u r a t e d l i p i d s i s suppressed by many o t h e r
~ e l Strong evidence of
mechanisms i n a d d i t i o n t o vitamin E ( ~ a ~ 1972).
t h e a n t i o x i d a n t a b i l i t y of vitamin E i n i n t a c t , l i v i n g systems w i l l be
r e q u i r e d i n t h e f i n a l resolvement of t h i s controversy.
CHAPTER I11
RADIATI ON-INDUCED ENZYME EFFLUX
FROM RAT HEART:
I . SEDENTARY ANIMALS
INTRODUCTION
One of t h e biochemical i n d i c a t o r s of a c u t e r a d i a t i o n i n j u r y i s an
i'ncrease i n serum l e v e l s of l a c t a t e dehydrogenase (LDH).
This has been
--
documented by Hawrylewicz and B l a i r (I-966), Hori e t a l . (1968, 1970) and
Kgrcher ( 1 9 7 1 ) ~ who have independently shown t r a n s i e n t e l e v a t i o n s of
t o t a l LDH and t h e v a r i o u s isoenzymes following i r r a d i a t i o n .
I n addition,
research i n v o l v i n g s e r u m g l 1 ~ t ~ a r n a t . oxal
e
o a c e t , a t . e t s r a n s a m in a s e ( G O T ) a s a
r a d i o b i o l o g i c a l marker has shown t h a t i n some mammalian s p e c i e s subl e t h a l exposures l e d t o s i g n i f i c a n t , t r a n s i e n t i n c r e a s e s i n serum
a c t i v i t i e s ( ~ i l c hand Albaum 1956; P e t e r s e n -e t a l . 1957; Brent -e t al.
e t a l . 1962; Almonte -e t a l . 1965).
1958; Becker -e t al.
--
The d a t a from Brent
(1958) s u g g e s t s t h a t t h e response i s v a r i e d between s p e c i e s .
I r r a d i a t e d r a b b i t s e x h i b i t e d s i g n i f i c a n t e l e v a t i o n s of serum GOT w i t h i n
twenty-four hours of t r e a t m e n t , while r a t s s u b j e c t e d t o e i t h e r wholebody o r l o c a l i r r a d i a t i o n of t h e l i v e r d i d not show any s u b s t a n t i a l GOT
e l e v a t i o n a f t e r one day.
Although l i t t l e i s known about t h e p o t e n t i a l
of c r e a t i n e k i n a s e ( C K ) a s a r a d i o b i o l o g i c a l marker, observable serum
e l e v a t i o n s occured i n p a t i e n t s ( p o s t - t r e a t m e n t ) r e c e i v i n g i n c i d e n t a l
c a r d i a c i r r a d i a t i o n ( ~ u g g i a1970).
Since CK i s l o c a t e d i n s k e l e t a l and
c a r d i a c muscle i n high c o n c e n t r a t i o n , i t s appearance i n t h e blood may
-
indicate radiation injury t o these tissues.
\
Much of t h e work t o d a t e has involved t h e enzymological e f f e c t s of
whole-body i r r a d i a t i o n , where c e l l death and d i s i n t e g r a t i o n of radios e n s i t i v e t i s s u e s may c o n t r i b u t e g r e a t l y t o t h e observed serum e l e v a t i o n .
L i t t l e work, however, has been published o u t l i n i n g t h e e f f e c t s of l o c a l i z e d i r r a d i a t i o n of r a d i o r e s i s t a n t t i s s u e s , where t h e r a p e u t i c doses do not
involve massive c e l l d i s r u p t i o n .
The p r e s e n t s t u d y w a s undertaken t o
i n v e s t i g a t e p o s s i b l e changes i n serum l e v e l s of LDH, GOT and CK a s
p o s s i b l e i n d i c a t o r s of r a d i a t i o n i n j u r y t o t h e h e a r t , and t o attempt t o
c o r r e l a t e t h e s e changes w i t h a l t e r a t i o n s i n t i s s u e l e v e l s of t h e enzyme.
I r r a d i a t i o n Procedure
S p r a m e Dawley, male r a t s of 150 t o 200 grams were p l a c e d under
l i g h t e t h e r a n a e s t h e s i a twenty-four hours p r i o r t o i r r a d i a t i o n and marked
l a t e r a l l y a c r o s s t h e t o p of t h e s c a p u l a t o d e f i n e t h e t a r g e t a r e a .
At
t h e time of i r r a d i a t i o n t h e animals were p l a c e d i n t o v e n t i l a t e d p l e x i g l a s s
t u b e s t o r e s t r i c t movement.
The f i e l d exposure a r e a was a d j u s t e d t o cover
3 cm p o s t e r i o r t o t h e s c a p u l a markings, a s t h i s was found t o be optimal i n
minimizing r a d i a t i o n damage t o p e r i p h e r a l t i s s u e s and organs, while allowi n g i r r a d i a t i o n of t h e e n t i r e h e a r t .
Rats were i r r a d i a t e d i n groups of
f o u r , u s i n g an Eldorado 8 ( ~ t o m i cEnergy of Canada) t h e r a p e u t i c c o b a l t
unit.
The dose r a t e was 281 r a d s p e r minute a t t h e s u r f a c e and exposure
time was c o r r e c t e d f o r an absorbance l o s s of 15.7% a t an average depth of
3 cm t o g i v e an a c u t e dosage of 2,000 r a d s .
S h a m - i r r a d i a t e d - c o n t r o l s were
t r e a t e d i n an i d e n t i c a l manner t o t h e experimental animals.
A l l animals
were t r e a t e d i n accordance w i t h t h e g u i d e l i n e s e s t a b l i s h e d i n Care of
Experimental Animals
-A
Guide f o r Canada, published by t h e Canadian
Council on Animal Care, Ottawa.
Sampling Procedure
I n o r d e r t o minimize serum t u r b i d i t y t h e r a t s were f a s t e d f o r s i x
A t pre-determined i n t e r v a l s r a t s were p l a c e d
hours p r i o r t o s a c r i f i c e .
under mild e t h e r a n a e s t h e s i a and an i n c i s i o n was made a c r o s s t h e abdomen.
Blood was withdrawn from t h e i n f e r i o r vena cava u s i n g a non-heparinized
s y r i n g e and immediately t r a n s f e r r e d t o s e a l e d r ~ n t . r i f i l g et.ll'hes -
S%cnl
r--u
were allowed t o c l o t f o r 30 minutes a t 2 5 ' ~ and were t h e n p l a c e d on i c e
f o r approximately 30 minutes u n t i l c e n t r i f u g i n g a t 2,000 x g f o r 1 5
minutes ( ~ i s n i e w s k a1970).
Serum was c o l l e c t e d and d i v i d e d i n t o i n d i v i d u a l v i a l s f o r t h e
enzyme a s s a y s .
A l l samples f o r l a c t a t e dehydrogenase (LDH) determina-
t i o n s were s t o r e d a t 4
- 6 ' ~ and
assays were run w i t h i n 40
-
48 hours.
LDH samples were n o t f r o z e n due t o known i n a c t i v a t i o n of t h e enzyme
( ~ o l i m a nand Van Den Berg 1 9 7 1 ) .
Samples f o r c r e a t i n e k i n a s e ( C K ) and
glutamate o x a l o a c e t a t e transaminase (GOT) determinations were f r o z e n i n
l i q u i d n i t r o g e n and s t o r e d on dry i c e u n t i l assayed.
It has been shown
t h a t CK and GOT a c t i v i t y l e v e l s remain s t a b l e f o r s e v e r a l weeks when s t o r e d
'
at - 2 0 ' ~ (Lederle D i a g n o s t i c s , American Cyanamid Co., B u l l e t i n 27819).
Heart Muscle P r e p a r a t i o n
Rat h e a r t s were e x i s e d , trimmed of f a t t y and connective t i s s u e
and plunged i n t o i c e - c o l d 0.15 M K C 1 .
q u i c k l y b l o t t e d dry and weighed.
A f t e r r i n s i n g , t h e h e a r t s were
The t i s s u e was f i n e l y minced w i t h
s c i s s o r s and washed f i v e times i n ice-cold 0.15 M K C 1 and twice i n a
m u l t i p l e s a l t s b u f f e r modified from Chappell and P e r r y (1954).
This
medium contained 0 . 1 M K C 1 , 0.005 M MgS04 and 0.001 M ethylenediamine t e t r a a c e t a t e (EDTA) i n 0.05 M TRIS H C 1 b u f f e r , pH 7.4.
The minced
muscle was t h e n homogenized i n two volumes of t h e above medium using
a ground-glass, Ten-Broeck t y p e homogenizer, and a p p r o p r i a t e l y d i l u t e d
t o make a 10% homogenate.
Homogenate samples were c e n t r i f u g e d a t
650 x g f o r f i v e minutes a f t e r which t h e s u p e r n a t a n t was c o l l e c t e d and
t h e o p e r a t i o n r e p e a t e d t o remove c e l l u l a r d e b r i s ( ~ r n s t e rand Nordenbrand 1967).
The r e s u l t a n t s u p e r n a t a n t was c e n t r i f u g e d a t 6,000 x g
f o r 1 5 minutes t o o b t a i n a crude mitochondrial p e l l e t , which was resuspended i n f r e s h b u f f e r and c e n t r i f u g e d a s b e f o r e .
Although h i g h e r
c e n t r i f u g e speeds i n c r e a s e t h e product y i e l d , it was found t h a t 6,000
x g o f f e r e d considerably p u r e r f r a c t i o n s .
This has been independently
shown by Holton -e t a1 (1957) and Hedman (1965).
Supernatant r e s u l t i n g
from t h e f i r s t mitochondrial f r a c t i o n was c o l l e c t e d , d i v i d e d i n t o ind i v i d u a l v i a l s and s t o r e d i n t h e same manner a s were serum samples.
The washed mitochondrial p e l l e t was r i n s e d t h r e e t i m e s i n 0.15 M K C 1
and suspended i n 1 . 0 m l of t h i s s o l u t i o n .
The f i n a l mitochondrial
suspension was f r o z e n i n l i q u i d n i t r o g e n and s t o r e d on dry i c e u n t i l
I n t h e above procedure a l l manual o p e r a t i o n s were c a r r i e d out on
i c e and c e n t r i f u g a t i o n s were performed a t
c e n t r i f u g e using a JA-20,
OOC
i n a Beckman model 521
fixed-angle r o t o r .
Enzyme Assays
C r e a t i n e k i n a s e assays employed t h e forward r e a c t i o n o r Rosalki
technique ( ~ o s a l k i1967) using t h e following r e a g e n t s ( g i v e n a s f i n a l
c o n c e n t r a t i o n s i n t h e assay medium) :
M magnesium s a l t , 1.45 x
loa2
M c r e a t i n e phosphate, 1.1 x
M PIPES b u f f e r , 11.5 x
5 x
M glucose, 9 . 1 x
M ADP, 8.67 x
M AMP, 1 . 8 x
M reduced
I
M NAIF, 3
g l u t a t h i o n e , 5 .O x
TTT p e r a s s a y
n f hexnkinase, 1 IU p e r
assay of g l ~ c o s e - 6 - ~ h o s p h a t edehydrogenase ( ~ lr ela g e n t s were prepackaged by Calbiochem, San ~ i e g o ) . Serum assays were run using f r e s h l y
thawed, u n d i l u t e d samples.
Homogenate samples were d i l u t e d t o 0.2%
a f t e r thawing, u s i n g modified Chappell-Perry medium.
Measurement of
background u t i l i z e d NADP, u s i n g c r e a t i n e phosphate-free c o n t r o l s o l u t i o n s ,
(Calbiochem) was pre-determined f o r both serum and homogenate p r e p a r a t i o n s ,
and was found t o be n e g l i g i b l e .
Since t u r b i d i t y e r r o r s f o r both assays
were a l s o found n e g l i g i b l y s m a l l , a s s a y s were c a l i b r a t e d a g a i n s t a
d i s t i l l e d water blank a t 340 nm and run f o r twelve minutes a t 3 0 ' ~ by
a d d i t i o n of 10
-
20
vl
of enzyme s o l u t i o n t o 3.0 m l of assay mixture.
L a c t a t e dehydrogenase a s s a y s were performed u s i n g t h e r e v e r s e
r e a c t i o n developed by Wroblewski and LaDue (1955) i n which t h e s u b s t r a t e ,
p y r u v a t e , i s reduced t o l a c t a t e .
The assay system contained 0.08 M
potassium phosphate b u f f e r , pH 7 . 5 , 7 . 5 x
lo-'
M sodium pyruvate, and
1.1 x
lom4 M
NADH.
Prepared r e a g e n t s w e r e purchased from Calbiochem.
Serum was assayed i n u n d i l u t e d form; homogenate samples were d i l u t e d t o
1.0%
w i t h modified c h a p p e l l - ~ e r r y b u f f e r
.
Background e r r o r was pre-
determined, u s i n g a s u b s t r a t e - f r e e c o n t r o l s o l u t i o n of 1.1 x
( ~ a l b i o c h e m )i n 0.08 M potassium phosphate b u f f e r , pH 7.5.
lo-'
M NADH
The a n a l y s i s
showed no d e t e c t i b l e u t i l i z a t i o n of NADH, i n d i c a t i n g no need f o r p a r a l l e l
c o n t r o l runs.
T u r b i d i t y e r r o r s i n serum and homogenate were found t o be
n e g l i g i b l y s m a l l , allowing assays t o be c a l i b r a t e d a t 340 nm, a g a i n s t a
d i s t i l l e d water b l a n k .
adding 1 0
-
Assays were run f o r 2
-
3 minutes a t 30•‹c, a f t e r
20 p1 of enzyme s o l u t i o n t o 3.0 m l of assay mixture.
Glutamate o x a l o a c e t a t e transaminase was determined by t h e u l t r a v i o l e t method of Karmen (1955) on serum, homogenate and h e a r t mitochond r i a l fractions.
pH 7.4, 4.0 x
The assay system contained 0 . 1 M phosphate b u f f e r ,
M L-aspartate, 1 . 2 x
loe2
M NADH, 0.25 mg malate
dehydrogenase p e r m l , 0.25 mg l a c t a t e dehydrogenase p e r m l and 0.25 M
a-oxoglutarate.
Prepared r e a g e n t s were purchased from Boehringer
Mannheim Corporation.
To 3.2 m l of assay mixture was added 0.5 m l of
enzyme s o l u t i o n , and t h e degradation of NADH was follwed at 366 nm f o r
4 t o 6 minutes a t 25'~.
Serum was assayed i n u n d i l u t e d form; homogen-
a t e samples, however, were d i l u t e d w i t h b u f f e r t o 0.1%.
Due t o t h e
impermeability of b i o l o g i c a l membranes to-many s u b s t r a t e s and c o f a c t o r s
( ~ y c o c kand Nahorski 1 9 7 1 ) , mitochondrial samples were r u p t u r e d by
a d d i t i o n of 1%
TRITON X -100 i n 0 . 1 M t r i s - a c e t a t e b u f f e r , pH 6.8
( ~ b o o dand Alexander 1957; G r e v i l l e and Chappel 1959; Wainio 1 9 7 0 ) .
Although t h i s assay employs an ammonia-free system, p o s s i b l e contamina-
ti on by g l u t m a t e dehydrogenase ( GLDH ) i n homogenate and m i tochondri a 1
p r e p a r a t i o n s was checked.
Enzyme a n a l y s i s by t h e method of Schmidt (1965)
r e v e a l e d only n e g l i g i b l e l e v e l s of t h e enzyme i n r a t h e a r t .
This has been
--
f u r t h e r shown by Kochakian e t a.. ( 1 9 5 9 ) , who found attempts t o determine
GLDH l e v e l s i n mice and r a t h e a r t s y i e l d e d q u e s t i o n a b l e r e s u l t s .
Back-
ground degradation of NADH, using s u b s t r a t e - f r e e c o n t r o l s f o r t h e GOT
system, i n d i c a t e d an average e r r o r of l e s s t h a n 3% i n serum.
No de-
t e c t a b l e background a c t i v i t y was found i n homogenate and mitochondrial
preparations.
Due t o considerable serum t u r b i d i t v . s m g l e s were c a l j -
b r a t e d a g a i n s t a serum blank c o n t a i n i n g 0.50 m l u n d i l u t e d serum i n 3.2
m l of 0 . 1 M phosphate b u f f e r , pH 7.4.
Because t u r b i d i t y e r r o r s f o r
homogenate and mitochondrial samples were n e g l i g i b l e , t r a n s m i t t a n c e was
c a l i b r a t e d a g a i n s t a blank of 0 . 1 M phosphate b u f f e r .
P r o t e i n Determination
I n o r d e r t o r e l a t e t h e c a l c u l a t e d mitochondrial enzyme a c t i v i t i e s
t o t h e c o n c e n t r a t i o n of mitochondria, determinations of t o t a l mitochondrial
p r o t e i n were o b t a i n e d u s i n g t h e b i u r e t method ( ~ a ~ 1957).
n e
0.8 m l of m i t o c h o n d r i a l suspension, 0.6
-
-
0.2 m l of 5% deoxycholate
( ~ a c o b set a l . 1956) was added t o remove t u r b i d i t y .
4
To 0.4
A f t e r t e n minutes
--
m l of b i u r e t r e a g e n t ( ~ o r n a l el t a l . 1949) was added and t h e mixture
was allowed t o s t a n d f o r 30 minutes a t room temperature.
Absorbance
v a l u e s were recorded a t 550 nm a g a i n s t a blank c o n t a i n i n g
4
reagent w i t h 1 . 0 m l of d i s t i l l e d water.
ml of b i u r e t
P r o t e i n c o n c e n t r a t i o n s were
o b t a i n e d by r e f e r e n c e t o a c a l i b r a t i o n curve e s t a b l i s h e d by s e r i a l
d i l u t i o n of bovine serum albumin.
I
Data Analysis
Reaction o r d e r k i n e t i c s f o r each enzyme system were p r e v i o u s l y
determined and r e a c t i o n r a t e s were followed on a Beckman DB-GT s p e c t r o photometer f i t t e d w i t h a P h i l l i p s model PM 8100 f l a t - b e d r e c o r d e r .
Reaction r a t e d a t a was t h e n f i t t e d t o t h e e q u a t i o n f o r t h e corresponding
o r d e r , and r a t e s were computed by a l e a s t s q u a r e s f i t u s i n g a D i g i t a l
Equipment Company PDP-8 computer.
Mean v a l u e s and i n d i v i d u a l v a r i a n c e s
were determined f o r each sample group a f t e r s u b j e c t i n g t h e s a m ~ l e st o a
Q-test analysis.
A p p l i c a t i o n of t h e F - t e s t f o r an a n a l y s i s of v a r i a n c e
between groups showed no s i g n i f i c a n t d i f f e r e n c e s i n t h e p o p u l a t i o n
v a r i a n c e s , and warranted t h e d e t e r m i n a t i o n o f a pooled e s t i m a t e of
v a r i a n c e ( ~ i x o nand Massey 1957).
T - t e s t s between c o n t r o l and e x p e r i -
mental v a l u e s were r u n f o r a o n e - t a i l e d , d i r e c t i o n a l h y p o t h e s i s , u s i n g
t h e pooled e s t i m a t e of v a r i a n c e ( ~ o w n i eand Heath 1 9 7 0 ) .
RESULTS
Serum Assays
I n a l l c a s e s serum enzyme l e v e l s reached maximal v a l u e s w i t h i n
twenty-four hours f o l l o w i n g t h o r a c i c i r r a d i a t i o n .
s i x t o twelve hours p o s t - i r r a d i a t i o n ,
t r e n d t o r e t u r n t o normal l e v e l s .
Peak e l e v a t i o n s a r o s e
a f t e r which was noted a g e n e r a l
These r e s u l t s a r e i n agreement with
--
o t h e r s t u d i e s i n v o l v i n g whole-body i r r a d i a t i o n ( ~ e c k e re t a l . 1962;
et al.
Almonte et a l . 1965; Hori -e t a l . 1969; Hori -e t al. 1968; ~ a k & n o r i-1970)
.
L a c t a t e dehydrogenase (LDH) serum l e v e l s r o s e 139% above c o n t r o l
l e v e l s s i x hours a f t e r exposure ( ~ i g .1 ) .
t h i s increase t o be significant (p < .05).
S t a t i s t i c a l a n a l y s i s showed
Twelve and twenty-four hours
a f t e r exposure, serum LDH a c t i v i t y decreased r e s p e c t i v e l y t o 48% and 21%
above c o n t r o l l e v e l .
Although t h e twelve hour sample shows s i g n i f i c a n t
e l e v a t i o n , t h e tyenty-four hour l e v e l i s not s t a t i s t i c a l l y d i f f e r e n t
On day t h r e e a second i n c r e a s e i n serum LDH a c t i v i t y
from c o n t r o l l e v e l .
t o 52% above c o n t r o l showed marginal s i g n i f i c a n c e a t t h e 5% l e v e l , and
r e t u r n e d t o normal on days s i x and n i n e .
This g e n e r a l p a t t e r n has a l s o
been observed by Hori and Nishio (1962).
A t h i r d transient increase t o
\
79% above c o n t r o l a c t i v i t y occurred twelve days a f t e r i r r a d i a t i o n .
The
mean LDH a c t i v i t y on day f i f t e e n does not show s i g n i f i c a n t d e v i a t i o n from
the control
able
1).
C r e a t i n e k i n a s e (CK) serum a c t i v i t y r o s e 131% above t h e c o n t r o l
v a l u e s i x Bours a f t e r i r r a d i a t i o n ( p < . 0 5 ) .
A t twelve hours a
68%
e l e v a t i o n was observed ( p < . 0 5 ) , which decreased t o w i t h i n c o n t r o l l e v e l
by twenty-four hours
a able
2).
A secondary e l e v a t i o n t o 110% above
c o n t r o l a c t i v i t y occurred on day t h r e e ( P < . 0 5 ) , b u t r e t u r n e d t o normal
by day s i x .
A t h i r d s i g n i f i c a n t e l e v a t i o n occurred on day twelve which,
a s i n t h e case of t h e serum LDH p a t t e r n , remained e l e v a t e d a t day f i f t e e n .
However, due t o t h e s m a l l sample s i z e of day f i f t e e n , t h e e l e v a t i o n i s not
s i g n i f i c a n t l y d i f f e r e n t from t h e c o n t r o l ( ~ i g .2 ) .
-
TABLE 1
SERUM UVELS OF LACTATE DEHYDROGENASE AT VARIOUS INTERVALS
AFTER EXPOSURE TO 2,000 RADS OF y-RADIATION.
SIGNIFICANT DIFFERENCES BETWEEN CONTROL
AND TEST MEANS WERE DETERMINED BY
t-TEST, USING A POOLED ESTIMATE
OF VARIANCE
Me an
Activity
mU/ml
Group
Number
of
Samples
Control
5
1.31 x
f
u
3.16 x io7
1 2 Hour
6
1.94
lo3
24 Hour
5
1.59 x
lo3
3 Day
4
2.00 x
lo5
6
Day
4
1.27 x
lo3
9 Day
3
1.36 x 103
1 2 Day
3
2.35 x
lo3
1 5 Day
2
1.89 x
lo3
6 I I ~ ~ ~ -
Pooled Variance :
lo3
356561
Pooled Standard Deviation :
597
Significant
Difference
From C o n t r o l
@ p < .05
% Elevation
From Control
Figure 1. Serum l e v e l s of l a c t a t e dehydrogenase
a t v a r i o u s i n t e r v a l s a f t e r ~ ~ ~ m t i. 2~~, Qr Kp
rads nf Y-radiatf cs
.
ACTIVITY l ~ ( m ~ / r nserum)xlo3
l
SERUM LEVELS OF CREATINE KINASE AT VARIOUS INTERVALS
AFTER EXPOSURE TO 2,000 RADS OF GAMMA RADIATION.
SIGNIFICANT DIFFERENCES BETWEEN CONTROL
AND TEST MEANS WRE DETERMINED BY
t-TEST, USING A POOLED ESTIMATE
OF VARIANCE
Group
Number
of
Samples
Me an
Activity
mu/&
Control
77
-...-
u nuur'
1 2 Hour
24 Hour
3 Day
6 Day
9 Day
1 2 Day
1 5 Dw
Pooled Variance:
13812
Pooled Standard Deviation:
118
Significant
Difference
From C o n t r o l
@ p < .05
%
Elevation
From C o n t r o l
Figure 2. Serum l e v e l s of c r e a t i n e k i n a s e a t
v a r i o u s i n t e r v a l s a f t e r exposure t o 2,000
r a d s of Y - r a d i a t i o n .
ACTIVITY mU/mI serum
Assays of serum glutamate o x a l o a c e t a t e transaminase (GOT)
a c t i v i t i e s r e v e a l e d an i n c r e a s e of 54% a t s i x hours p o s t - i r r a d i a t i o n
( p < .05).
Maximal e l e v a t i o n occurred a f t e r twelve h o u r s , showing an
a c t i v i t y i n c r e a s e of 66% ( p < . 0 5 ) .
l e v e l s r e t u r n e d t o normal
able 3 ) .
A f t e r twenty-four hours serum GOT
An unexpected drop i n a c t i v i t y t o
59% of t h e c o n t r o l l e v e l on day s i x was n o t c o n s i s t e n t with t h e d i r e c t i o n a,l h y p o t h e s i s .
However, s i n c e t h e p r o b a b i l i t y v a l u e f o r a non-direction-
a 1 t e s t showed s i g n i f i c a n c e a t t h e 5% l e v e l , a p o s s i b l e r a t i o n a l e f o r
t h i s observed decrease w i l l b e p r e s e n t e d elsewhere i n t h i s c h a p t e r .
Serum GOT l e v e l s f o r days n i n e and twelve following exposure do not
show s i g n i f i c a n t d e v i a t i o n s from c o n t r o l v a l u e s ( ~ i g .3 ) .
However, a
g e n e r a l t r e n d of e l e v a t i o n does occur from day s i x t o day f i f t e e n , showi n g d e f i n i t i v e i n c r e a s e s i n enzyme l e v e l s .
Such secondary i n c r e a s e s , a s
noted i n a l l enzyme systems s t u d i e d , may be i n d i c a t i v e of t h e manifestat i o n of major biochemical l e s i o n s induced by t h e ongoing metabolism of
the injured tissue cells.
Homogenate Assays
L a c t a t e dehydrogenase a c t i v i t y i n h e a r t muscle homogenates
4 ) decreased 36% by twelve hours p o s t - i r r a d i a t i o n ( p
<
.05).
a able
This de-
c r e a s e remained s t a t i s t i c a l l y s i g n i f i c a n t a f t e r twenty-four hours, showi n g a 33% mean l o s s , r e t u r n i n g t o normal a f t e r t h r e e days.
Activity
l e v e l s from t h r e e t o s i x days a f t e r exposure d i s p l a y e d a g e n e r a l dec r e a s i n g t r e n d , however, were not s t a t i s t i c a l l y d i f f e r e n t w i t h r e s p e c t
. .Day n i n e showed a secondary l o s s of
t o t h e c o n t r o l a c t i v i t y ( ~ i4 )~
TABLE
3
SERUM LEVELS OF GLUTAMATE OXALOACETATE TRANSAMINASE
AT VARIOUS INTERVALS AFTER EXPOSURE TO 2,000 RADS
OF +f-RADIATION. SIGNIFICANT DIFFERENCES
BETWEEN CONTROL AND TEST MEANS
WERE DETERMINED BY t-TEST,
USING A POOLED ESTIMATE
OF VARIANCE
---
Group
Number
of
Samples
Me an
Activity
m~/ml
Significant
Difference
From C o n t r o l
ep
-
%
Elevation
From C o n t r o l
< .05
Control
6
Ecxr
1 2 Hour
24 Hour
3 Day
6 Day
9 Day
1 2 Day
1 5 Day
Pooled Variance:
Pooled S t a n d a r d Deviation:
10.3
*Where a c t i v i t y l e v e l s d i f f e r e d s i g n i f i c a n t l y from c o n t r o l l e v e l s and
d i d not a g r e e w i t h t h e d i r e c t i o n a l h y p o t h e s i s , a n o n - d i r e c t i o n a l , twot a i l e d t - t e s t was used t o determine t h e l e v e l o f s i g n i f i c a n c e .
Figure 3. Serum l e v e l s of glutamate oxaloacetate
transaminase a t v a r i o u s i n t e r v a l s a f t e r expo;;-iii.-ct u 2,GOG r-aiis ui' y - r . a r i i a i i u n .
ACTIVITY rnU/m 1 ser urn
HEART HOMOGENATE LEVELS OF LACTATE DEHYDROGENASE AT
VARIOUS INTERVALS AFTER EXPOSURE TO 2,000 RADS
OF -RADIATION. SIGNIFICANT DIFFERENCES
BETWEEN CONTROL AND TEST MEANS WERE
DETERMINED BY t-TEST, USING A
POOLED ESTIMATE OF VARIANCE
Group
Number
of
Samples
Control
3
Me an
Activity
mU/mg
Muscle
6 Hour
12 Hour
24 Hour
3 Day
6 Day
9 Day
1 2 Day
1 5 Day
Pooled Variance :
2414
Pooled Standard Deviation:
49.1
Significant
Difference
From Control
@ p
.05
% Decrease
From Control
Figure 4. Heart homogenate l e v e l s of l a c t a t e
dehydrogenase a t v a r i o u s i n t e r v a l s a f t e r
exposure t o 2,000 r a d s of Y-radiation.
ACTIVITY mU/mg muscle
homogenate a c t i v i t y s i g n i f i c a n t l y d i f f e r e n t from t h e c o n t r o l a t t h e 5%
level.
However, a c t i v i t i e s r e t u r n e d t o normal by day twelve.
C r e a t i n e k i n a s e l e v e l s i n h e a r t homogenates
a able 5 )
reflected
s i m i l a r changes w i t h an observed l o s s of 21% from c o n t r o l l e v e l a t twelve
hours p o s t - i r r a d i a t i o n .
A c t i v i t y l e v e l s a f t e r twenty-four hours r e f l e c t -
e d a 25% mean l o s s which was not s t a t i s t i c a l l y d i f f e r e n t from t h e twelve
hour measurement.
Although enzyme l e v e l s r e t u r n e d t o normal during day
t h r e e , a secondary drop i n homogenate a c t i v i t y , occuring during day s i x
and day n i n e , showed s t a t i s t i c a l l y s i g n i f i c a n t l o s s e s of 17% and 25%
r e s p e c t i v e l y ( ~ i g .5 ) .
Glutamate o x a l o a c e t a t e transaminase (GOT ) l o s s from homogenate
p r e p a r a t i o n s shows a p a t t e r n s i m i l a r t o t h e previous system s t u d i e d .
The mean p o s t - i r r a d i a t i o n a c t i v i t y l o s s a f t e r twelve hours i s 30%
( p < . 0 5 ) , b u t r e t u r n s t o w i t h i n c o n t r o l l i m i t s by twenty-four hours
(p > - 0 5 ) .
Homogenate GOT l e v e l s from day t h r e e t o day f i f t e e n f l u x u a t e
w i t h i n c o n t r o l l i m i t s , and do not d i s p l a y s t a t i s t i c a l l y s i g n i f i c a n t
a l t e r a t i o n s with respect t o control a c t i v i t i e s ( ~ i g 6
. ).
Mitochondria1 Assays
Loss of mitochondria1 GOT a c t i v i t y , induced by an a c u t e radio-
*
l o g i c a l dosage of 2,000 r a d s , i s not s i g n i f i c a n t a t t h e 5% l e v e l of
probability
a able 7 ) .
Although a mean a c t i v i t y l o s s of 36% occurred
a f t e r s i x hours p o s t - i r r a d i a t i o n ,
t h e l a r g e sample v a r i a n c e l i m i t s t h e
p r e c i s i o n of measurement and may obscure t h e s i g n i f i c a n c e of such small
*
alterations ( ~ i g 7
. ).
TABLE 5
HEART HOMOGENATE LEVELS OF CREATINE KINASE AT VARIOUS
INTERVALS AFTER EXPOSURE TO 2,000 RADS OF
y -RADIATION
SIGNIFI CANT DIFFERENCES
BETWEEN CONTROL AND TEST MEANS WERE
DETERMINED BY t-TEST, USING A
POOLED ESTIMATE OF VARIANCE
.
Group
Number
of
Samples
Me an
Activity
mU/m
Muscle
Control
6
Holm
1 2 Hour
24 Hour
3 Day
6 Day
9 Day
1 2 Day
1 5 Day
Pooled Variance :
2712
Pooled Standard Deviation:
52.1
Significant
Difference
From Control
@ p < .05
$ Decrease
From Control
Figure 5. Heart homogenate l e v e l s of c r e a t i n e
k i n a s e at v a r i o u s i n t e r v a l s a f t e r exposure
t o 2,000 r a d s of Y-radiation.
6
CONTROL
24 '
3
DAY INTERVALS
a HOUR INTERVALS
12
HOURS
6
9
DAYS
TABLE 6
HEART HOMOGENATE LEVELS OF GLUTAMATE OXALOACETATE TRANSAMINASE
AT VARIOUS INTERVALS AFTER EXPOSURE TO 2,000 RADS
OF Y-RADIATION. SIGNIFICANT DIFFERENCES
BETWEEN CONTROL AND TEST MEANS WERE
DETERMINED BY t-TEST, USING A
POOLED ESTIMATE OF VARIANCE
-
Group
Control
L TI-..,
w
I L V U I
Number
of
Samples
Me an
Activity
m11/mg
Mus c l e
3
132
-,
9
fif
IUV
3
1 2 Hour
3
24 Hour
3
114
3 Day
3
142
6 Day
3
156
9 Day
2
107
1 2 Day
3
134
1 5 Day
2
149
Pooled Variance:
92.3
414
Pooled Standard Deviation:
20.3
Significant
Difference
From Control
@ p < -05
-
--
% Decrease
From Control
Figure 6. Heart homogenate l e v e l s of glutamate
o x a l o a c e t a t e transaminase a t various i n t e r v a l s
a f t e r exposure t o 2,000 rads of Y-radiation.
ACTIVITY mU/mg muscle
TABLE 7
MITOCHONDRIAL LEVELS OF GLUTAMATE OXALOACETATE TRANSAMINASE
AT VARIOUS INTERVALS AFTER EXPOSURE TO 2,000 RADS OF
Y.-RADIATI ON. SIGNIFICANT DIFFEIiENCES
BETWEEN CONTROL AND TEST MEANS WERE
DETERMINED BY t-TEST, USING A
POOLED ESTIMATE OF VARIANCE
Number
of
Samples
Mean A c t i v i t y
mU/mg
Biuret
Protein
6 Hour
3
0.87 x 10-
1 2 Hour
3
1.09
24 Hour
3
1 . 1 4 x 1 03
3 Day
3
1.64 x lo3
6 Day
3
3
1.34 x 1 0
9 Day
3
1.49 x
lo3
1 2 Day
3
1.11
lo3
1 5 Day
3
1.30 x
lo3
Group
Control
Pooled Variance:
2
lo3
133812
Pooled Standard Deviation:
366
Significant
Difference
From C o n t r o l
@ p < .05
% Decrease
From Control
Figure 7. Mitochondria1 l e v e l s of glutamate
o x a l o a c e t a t e transaminase a t various
i n t e r v a l s a f t e r exposure t o 2,000 ra.ds
of Y-radiation.
A C T I V I T Y ( ~ U / rng biuret p r o t e i n ) ~ 1 0 3
DISCUSSION
Although t h e h e a r t has o f t e n been regarded a s one of t h e most
r a d i o r e s i s t a n t organs of t h e body ( ~ u r c h-e t a l . 1968), t h e onset of
radiation-induced h e a r t d i s e a s e h a s been recognized a s an important
m a n i f e s t a t i o n of t h o r a c i c exposure ( s t e w a r t and Fajardo 1971).
The
p r e s e n t i n v e s t i g a t i o n documents biochemical evidence of i n i t i a l c a r d i a c
i n j u r y i n response t o t h e a c u t e a d m i n i s t r a t i o n of i o n i z i n g r a d i a t i o n t o
t h e t h o r a c i c a r e a of r a t s .
One of t h e biochemical i n d i c a t o r s i n a c u t e r a d i a t i o n i n j u r y i s an
----
--
-1
.L2
G L ~ ; V ~ ~ , L W L ~
uf
i a c i a i e ciehydrogenase a c t l v l t y Sollowing whole-body
~ ~ L . L U I I
exposure ( ~ o r -ei t a l . 1968).
L a c t a t e dehydrogenase, l o c a t e d i n most
organs, has an important f u n c t i o n i n carbohydrate metabolism, and under
normal c o n d i t i o n s remains a t a f a i r l y c o n s t a n t l e v e l .
Glutamate oxalo-
a c e t a t e transaminase and c r e a t i n e k i n a s e a r e both found i n r e l a t i v e l y
h i g h c o n c e n t r a t i o n s i n c a r d i a c muscle.
GOT, involved i n t h e mediation
of p r o t e i n metabolism, and CK i n t h e r e s t o r a t i o n of a v a i l a b l e energy f o r
muscular c o n t r a c t i o n , p l a y important r o l e s a s biochemical markers f o r t h e
e t a l . 1960; Schmidt et
c l i n i c a l d e t e c t i o n of myocardial i n j u r y ( ~ r e y f u s-al.
-
1965) and were t h e r e f o r e s e l e c t e d a s p o t e n t i a l i n d i c a t o r s f o r the
study of r a d i a t i o n i n j u r y t o c a r d i a c muscle.
Serum l e v e l s of a l l t h r e e enzymes r e v e a l e d t r a n s i e n t e l e v a t i o n s
w i t h i n twelve hours of t h o r a c i c i r r a d i a t i o n , r e t u r n i n g t o w i t h i n c o n t r o l
l e v e l s by twenty-four hours.
S i m i l a r response p a t t e r n s have been docu-
--
--
mented i n whole-body exposure ( ~ e c k e re t a l . 1962; Almonte e t a l . 1965;
Takamori -e t a l . 1969; Hori -e t a l . 1970) , i n d i c a t i n g t h a t such p a t t e r n s
appear t o be r e p r e s e n t a t i v e of a g e n e r a l r a d i o l o g i c a l response and a r e
not r e l a t e d t o t h e r a d i o s e n s i t i v i t y of t h e system s t u d i e d .
Lactate
dehydrogenase and c r e a t i n e k i n a s e serum l e v e l s d i s p l a y e d g r e a t e r than
two-fold i n i t i a l e l e v a t i o n s while GOT showed an i n c r e a s e of only 50%
above normal.
remarkable.
Although t h e s e e l e v a t i o n s a r e s i g n i f i c a n t , t h e y a r e not
Because of t h e smallness of t h e changes and t h e sample
v a r i a n c e w i t h i n a p o p u l a t i o n , c l i n i c a l a p p l i c a t i o n of t h e s e systems a s
biochemical markers f o r r a d i a t i o n i n j u r y t o t h e c a r d i a c muscle does not
appear l i k e l y u n l e s s one had p r e - i r r a d i a t i o n serum l e v e l s on t h e s u b j e c t .
i n a d d i t i o n , s t u d i e s by Brent and co-workers (1938j r e v e a l t h a t no
apparent c o r r e l a t i o n e x i s t s between induced serum e l e v a t i o n s and t h e
l i k e l y h o o d of d e a t h .
A l l serum enzyme p a t t e r n s i n d i c a t e a t r e n d toward normality a f t e r
reaching i n i t i a l maximal l e v e l s .
Such a p a t t e r n suggests a p o s s i b l e re-
i n s t a t e m e n t of membrane i n t e g r i t y following a t r a n s i e n t a l t e r a t i o n of
membrane p e r m e a b i l i t y .
However, t h e e f f e c t might e q u a l l y w e l l be
mediated by a lowering of t h e e f f l u x r a t e of enzymatic m a t e r i a l from t h e
c e l l due t o a d e c r e a s e of t h e c o n c e n t r a t i o n g r a d i e n t between c e l l u l a r and
e x t r a c e l l u l a r l e v e l s caused by t h e d e p l e t i o n of t h e c e l l u l a r c o n s t i t u e n t s .
Such f a c t o r s , p o s s i b l y o p e r a t i n g i n c o n c e r t , combined w i t h t h e r a p i d
removal of t h e e x t r a c e l l u l a r enzymes through a c t i v e uptake i n t h e e x t r a c e l l u l a r f l u i d ( ~ c h m i d tet a l . 1965) may account f o r a "pseudo" t r a n /
s i e n t n a t u r e of t h e i n i t i a l e l e v a t i o n s .
Note t h a t t h e r e s t o r a t i o n of
t i s s u e enzyme l e v e l s t o c o n t r o l v a l u e s on day t h r e e (due p o s s i b l y t o
enhanced s y n t h e s i s of t h e enzymes) tends t o r e s t o r e t h e c o n c e n t r a t i o n
gradient.
This could subsequently l e a d t o enhanced leakage and a
secondary e l e v a t i o n of serum LDH and CK i f t h e normal p e r m e a b i l i t y of
t h e c e l l membrane had not y e t been r e s t o r e d .
Although t i s s u e homogenate
GOT l e v e l s r e t u r n t o normal on day t h r e e , t h e serum l e v e l of GOT, u n l i k e
LDH and CK, d i d not i n c r e a s e s i g n i f i c a n t l y above normal a t t h a t time.
This may i n d i c a t e a s i g n i f i c a n t r e s t o r a t i o n of membrane p e r m e a b i l i t y
w i t h r e s p e c t t o GOT.
Some overcompensatory r e p a i r mechanism f o r GOT
p e r m e a b i l i t y , whether a t t h e c e l l u l a r o r hormonal l e v e l , o r an enhanced
i n a c t i v a t i o n of t h e serum enzyme could conceivably account f o r t h e noted
t r a n s i e n t r e d u c t i o n i n serum GOT l e v e l s a s shown i n Figure 3.
Trends i n t h e serum enzyme l e v e l s occuring a f t e r i n i t i a l e l e v a t i o n s
suggest t h e p o s s i b l e m a n i f e s t a t i o n of secondary r a d i a t i o n e f f e c t s r e s u l t i n g from metabolic o r biochemical l e s i o n s i n t h e t a r g e t organ.
However,
a s t r i c t i n t e r p r e t a t i o n of t h e serum e l e v a t i o n s w i t h r e s p e c t t o changes
i n t h e h e a r t must be approached w i t h extreme c a u t i o n s i n c e r a d i a t i o n
damage t o p e r i p h e r a l t i s s u e s cannot be e l i m i n a t e d .
I n a d d i t i o n , physio-
l o g i c a l a l t e r a t i o n s mediated by hormonal a c t i o n may f u r t h e r d i s t o r t t h e
relationship.
The s i g n i f i c a n t decreases i n t h e homogenate l e v e l s of t h e enzymes
p a r a l l e l t h e i n i t i a l serum e l e v a t i o n s and i n d i c a t e t h a t t h e observed
serum e l e v a t i o n s a r e not a r e s u l t of enhanced t i s s u e l e v e l s of t h e enzymes,
b u t r a t h e r a r e a consequence of radiation-induced leakage of b i o l o g i c a l l y
a c t i v e m a t e r i a l o u t of t h e i n j u r e d c e l l s .
Similar a l t e r a t i o n s i n t h e
c e l l u l a r l e v e l s of b i o l o g i c a l l y a c t i v e molecules i n p o s t - i r r a d i a t e d c e l l s
and o r g a n e l l e s have been recorded (0kada and Peachey 1957; Roth and E i c k e l
1959; Kawasaki and Sakurai 1969).
Secondary serum e l e v a t i o n s , however,
do not appear t o be r e l a t e d t o any s i g n i f i c a n t secondary d e p l e t i o n s of
homogenate a c t i v i t i e s .
It i s u n l i k e l y t h a t d i r e c t i n a c t i v a t i o n of t h e
enzymes could account f o r t h e noted i n i t i a l decreases i n t h e homogenate
a c t i v i t i e s s i n c e it has been demonstrated t h a t most enzymes a r e r e l a t i v e l y
r a d i o r e s i s t a n t ( ~ c h l e n k-e t a l . 1946; Ord and Stocken 1953 ; Winstead and
Reece 1 9 7 0 ) , r e q u i r i n g s u b s t a n t i a l l y l a r g e r doses t o i n i t i a t e any signif icant inactivation.
i n vivo i n j u r y t o t h e
It appears, a t t h e biochemical l e v e l , t h a t -mitochondria with 2,000 r a d s of gamma r a d i a t i o n i s not s i g n i f i c a n t a t
t h e 5% confidence l e v e l .
The apparent r e s i s t a n c e of t h e mitochondria1
membrane t o damage, i n comparison b i t h t h e c e l l membrane, may r e s u l t
from t h e double membrane c h a r a c t e r of t h e mitochondria o r physicochemical d i f f e r e n c e s i n t h e s t r u c t u r e of t h e membrane systems.
It i s ,
moreover, probable t h a t t h e GOT concentration g r a d i e n t between mitochondria and cytoplasm i s not n e a r l y a s g r e a t a s t h e cytoplasm-plasma
gradient,
Thus, t h e motive f o r c e and r e s u l t a n t leakage of t h e enzyme
from t h e damaged mitochondria would not r e f l e c t a s a c c u r a t e l y t h e i n j u r y
s u s t a i n e d from r a d i a t i o n damage t o t h e o r g a n e l l e .
\
CHAPTER I V
RADIATION-INDUCED ENZYME EFFLUX FROM RAT HEART:
I1 EXERCISE-TRAINED ANIMALS
INTRODUCTION
Apart from v a r i o u s long-term performance s t u d i e s
.
o ones
e t a l . 1967;
Belksy -e t a l . 1972) s u r p r i s i n g l y l i t t l e i s known about t h e e f f e c t s of
e x e r c i s e on i r r a d i a t e d s u b j e c t s .
S i n c e it i s g e n e r a l l y known t h a t t h e
ongoing metabolism o f a c e l l i s damaged by r a d i a t i o n , it i s l o g i c a l t o
propose t h a t a d d i t i o n a l consequences of r a d i a t i o n exposure may become
apparent under c o n d i t i o n s of p h y s i o l o g i c a l s t r e s s .
The determination
of such consequences would allow an assessment of t h e i r r a d i a t e d animal's
f u n c t i o n a l s t a t u s and may u l t i m a t e l y prove important i n t h e development
of c l i n i c a l t h e r a p e u t i c t r e a t m e n t programs.
The p r e s e n t s t u d y was, t h e r e f o r e , undertaken t o e v a l u a t e t h e e f f e c t
of e x e r c i s e a s an added metabolic s t r e s s i n r a t s s u b j e c t e d t o l o c a l i z e d
thoracic irradiation.
The i n v e s t i g a t i o n involved an a n a l y s i s of serum
LDH, CK and GOT, w i t h r e s p e c t t o a l t e r a t i o n s of t h e s e enzymes i n t h e t a r g e t
organ, a s i n d i c a t o r s of r a d i a t i o n i n j u r y t o t h e h e a r t .
The r e s u l t s com-
p i l e d i n t h i s c h a p t e r were a l s o s t a t i s t i c a l l y compared t o t h e d a t a comp i l e d i n t h e p r e v i o u s c h a p t e r i n an e f f o r t t o r e l a t e t h e e x t e n t of
r a d i a t i o n i n j u r y between e x e r c i s e d and s e d e n t a r y animals.
MATERIAL AND METHODS
Training Procedure
Many i n v e s t i g a t o r s have demonstrated t h a t e x e r c i s e s t r e s s of
u n t r a i n e d s u b j e c t s r e s u l t s i n sharp e l e v a t i o n s of plasma enzymes
( ~ l t l a n dand Highman 1961; Halonen. and Konttinen 1962; Fowler -e t al.
1962). However, such e l e v a t i o n s a r e temporary and r a p i d l y r e t u r n t o
e t a 1 1968).
normal l e v e l s (schlang 1961; Papadopoulos --
Changes i n
serum enzyme l e v e l s a f t e r e x e r c i s e of t r a i n e d and u n t r a i n e d s u b j e c t s
s t u d i e d by Fowler et al. (1962) revealed t h a t serum enzyme e l e v a t i o n s
viduals.
S i m i l a r observations have been made i n man ( ~ u t t a l and
l
Jones
1968) and o t h e r animals
co-workers
e eat on
1966).
Evidence compiled by Garbus and
(1964) i n d i c a t e s t h a t e x e r c i s e t r a i n i n g e f f e c t i v e l y p r o t e c t s
a g a i n s t a r i s e i n serum enzymes.
S i m i l a r conclusions were a l s o drawn
by Highman and A l t l a n d (1963), who revealed t h a t r a t s t r a i n e d f o r s i x
hours d a i l y f o r seventeen t o twenty days showed no s i g n i f i c a n t changes
i n serum enzymes immediately following e x e r c i s e s t r e s s .
I n order t o minimize serum e l e v a t i o n s due t o e x e r c i s e i t s e l f ,
male Sprague-Dawley r a t s of an average weight of 65 grn and an average
age of t h i r t y days were placed on an exhaustive e x e r c i s e t r a i n i n g program.
The age of t h e r a t s was chosen t o correspond a f t e r t h i r t y days of t r a i n i n g
t o t h e average age of t h e animals u t i l i z e d i n t h e previous t e s t s .
After
i n i t i a l o r i e n t a t i o n t h e r a t s were e x e r c i s e d d a i l y on a v a r i a b l e speed
t r e a d m i l l a t speeds between 25
-
40 meters/minute f o r 30
-
50 minutes
u n t i l f a t i g u e was e v i d e n t .
Following t h e l a s t day of t r a i n i n g , twenty-
f o u r hours p r i o r t o i r r a d i a t i o n , t h e animals were p l a c e d under l i g h t
e t h e r a n a e s t h e s i a and marked l a t e r a l l y a c r o s s t h e t o p of t h e s c a p u l a
t o define t h e t a r g e t area.
I r r a d i a t i o n Procedure
Exercised r a t s were i r r a d i a t e d i n an i d e n t i c a l manner a s r e p o r t e d
i n Chapter 111. Sham-irradiated, e x e r c i s e d c o n t r o l animals were t r e a t e d
accordingly.
A s soon a s p o s s i b l e following i r r a d i a t i o n a l l animals
( i n c l u d i n g t h e sham-irradiated c o n t r o l s ) were e x e r c i s e d i n t h e normal
manner and t h e r e a f t e r a t twenty-four hour i n t e r v a l s approximately s i x
t o twelve hours previous t o s a c r i f i c e .
I r r a d i a t e d animals were s a c r i f i c e d a t i n t e r v a l s of s i x , twelve, and
twenty-four hours and t h e n a t d a i l y i n t e r v a l s of t h r e e , s i x , n i n e , and
twelve days.
The blood sampling procedure, p r e p a r a t i o n of h e a r t homo-
genate , m i t o c h o n d r i a l i s o l a t i o n , methods of enzyme a n a l y s i s , mitochondri a 1
p r o t e i n determinations and methods of d a t a a n a l y s i s were a l l c a r r i e d out
i n t h e manner p r e v i o u s l y r e p o r t e d .
RESULTS
E f f e c t s of Y-Radiation on Exercised Rats
Serum Assays
The mean a c t i v i t y of LDH i n serum i n c r e a s e d 238%
from c o n t r o l l e v e l s i x hours a f t e r exposure t o 2,000 r a d s of gamma
radiation.
This e l e v a t i o n was s i g n i f i c a n t beyond t h e .05 confidence
l e v e l and remained s o a f t e r twelve hours p o s t - i r r a d i a t i o n ,
showing a
67
mean i n c r e a s e of 166%
able
1 ~ ) . A f t e r twenty-four hours serum LDH
a c t i v i t y r e t u r n e d t o w i t h i n normal l i m i t s .
Although t h e mean a c t i v i t y
was 90.5% h i g h e r t h a n t h e c o n t r o l v a l u e , t h i s e l e v a t i o n w a s not s t a t i s t i c a l l y d i f f e r e n t a t t h e .05 l e v e l of confidence.
No f u r t h e r s i g n i f i c a n t
d e v i ~ t i o n sabove t h e c o n t r o l value were recorded; however, t h e r e does
occur a secondary e l e v a t i o n between sampling days t h r e e t o twelve ( ~ i ~ u r e
Serum CK a c t i v i t y e x h i b i t e d an i n i t i a l i n c r e a s e t o 182% above t h e
mean c o n t r o l a c t i v i t y s i x hours following exposure ( p < . 0 5 ) .
After
twelve hours t h e serum CK a c t i v i t y of i r r a d i a t e d animals remained
significantly elevated
a able
2 ~ ) b, u t l e v e l s r e t u r n e d t o w i t h i n c o n t r o l
l i m i t s by twenty-four hours ( ~ i g u r e2.A).
This i n i t i a l response i s s t r i k -
i n g l y congarable t o t h e observed e l e v a t i o n s of LDH, a s was r e p o r t e d i n
Chapter 111.
Serum CK l e v e l s remained w i t h i n c o n t r o l l i m i t s on days
t h r e e and s i x , b u t d i d show a marginally s i g n i f i c a n t e l e v a t i o n on day
nine ( p < .05).
Enzyme l e v e l s d i d not show s i g n i f i c a n t e l e v a t i o n s on
day twelve.
The mean GOT a c t i v i t y i n t h e serum of i r r a d i a t e d , e x e r c i s e d r a t s
e x h i b i t e d a moderate i n c r e a s e of 50.6% a t t h e s i x hour i n t e r v a l ( p < . 0 5 ) .
The enzyme a c t i v i t y remained s i g n i f i c a n t l y e l e v a t e d twelve hours a f t e r
exposure, showing a mean i n c r e a s e of 31.3%, b u t r e t u r n e d t o c o n t r o l l e v e l
a f t e r twenty-four hours (p > - 0 5 ) .
Although serum GOT a c t i v i t y of r a t s
sampled t h r e e and s i x days a f t e r i r r a d i a t i o n demonstrated decreases of
31.3% and 25.5% r e s p e c t i v e l y , t h e s e f l u c t u a t i o n s were not s i g n i f i c a n t a t
TABLE 1A
SERUM LEVELS OF LACTATE DEIIYDROGENASE I N EXERCISE-TRAINED RATS
AT VARIOUS INTERVALS AFTER EXF'OSURE TO 2 , 0 0 0 RADS
OF y-RADIATION.
SIGNIFICANT DIFFERENCES
BETWEEN CONTROL AND TEST MEANS WERE
DETERMINED BY t-TEST, USING A
POOLED ESTIMATE OF VARIANCE
Group
Number
of
Samples
Me a n
Activity
mU/ml
Control
6 Hour
12 Hour
24 H o u r
3 Day
6 Day
9 Day
1 2 Day
POOLED VARIANCE:
322717
POOLED STANDARD DEVIATION :
568
Significant
Difference
From C o n t r o l
@ p < .05
% Increase
From C o n t r o l
Figure 1 A . Serum l e v e l s of l a c t a t e dehydrogenase
i n e x e r c i s e - t r a i n e d r a t s a t various i n t e r v a l s
a f t e r exposure t o 2,000 r a d s of Y-radiati on -
ACTIVITY IN (mu /ml serum)xlo3
TABLE 2A
SERUM LEVELS OF CREATINE KINASE I N EXERCISE-TRAINED RATS
AT VARIOUS INTERVALS AFTER EXPOSURE TO 2 , 0 0 0 RADS
OF ?-RADIATION.
SIGNIFICANT DIFFERFNCES
BETWEEN CONTROL AND TEST MEANS WERE
DETERMINED BY t-TEST, USING A
POOLED ESTIMATE OF VARIANCE
Number
Group
0f
Samples
Control
C
V
n u- u r
rr
1 2 Hour
24 H o u r
3 Day
6 Day
9 Day
1 2 Day
POOLED VARIANCE:
3
5284
POOLED STANDARD DEVIATION:
Mean
Activity
mU/ml
Significant
Difference
From C o n t r o l
@ p < .05
% Increase
From C o n t r o l
Figure 2.A. Serum l e v e l s of c r e a t i n e k i n a s e i n
e x e r c i s e - t r a i n e d r a t s a t various i n t e r v a l s
a f ' t e r exposure t o 2,000 r a d s of Y-radiation.
ACTIVITY IN mlJ/ml serum
t h e .05 l e v e l of confidence
able
3 ~ ) . Serum GOT a c t i v i t i e s remained
w i t h i n c o n t r o l l i m i t s f o r t h e remaining sampling p e r i o d s .
However, t h e r e
occurred a demonstrable e l e v a t i n g t r e n d from day t h r e e t o day twelve
(Figure 3 ~ ) .
Homogenate Assays
LDH a c t i v i t y i n prepared h e a r t homogenates of
i r r a d i a t e d animals showed a mean decrease of 32.3% a t s i x hours following
exposure ( p < . 0 5 ) .
Although LDH l e v e l s r e t u r n e d t o normal a t t h e twelve
hour i n t e r v a l , a subsequent l o s s of 19.6% i n a c t i v i t y occurred a f t e r
twenty-four hours
able
4 ~ ) . Rats s a c r i f i c e d t h r e e days following
i r r a d i a t i o n d i d not show s i g n i f i c a n t alt.ernt.ions i n t h e hnmcgenate level
of t h e enzymes.
However, animals sampled on days s i x , n i n e and twelve
showed r e s p e c t i v e l o s s e s of 29.6%, 24.3% and 15.92, which were a l l found
s t a t i s t i c a l l y s i g n i f i c a n t a t t h e 5% l e v e l ( F i g u r e & A ) .
I n c o n t r a s t t o t h e observed l o s s e s of c e l l u l a r LDH, h e a r t homogena t e l e v e l s of CK i n i r r a d i a t e d r a t s d i d not e x h i b i t s i g n i f i c a n t v a r i a t i o n s
from sham-irradiated c o n t r o l 'animals w i t h i n t h e f i r s t t h r e e days
able
5 ~ ) .
Although a temporary l o s s of CK a c t i v i t y was observed on day s i x ( P < . 0 5 ) ,
homogenate l e v e l s r e t u r n e d t o normal on days nine and twelve ( F i g u r e 5 ~ ) .
The mean GOT a c t i v i t i e s of p o s t - i r r a d i a t e d r a t s showed a p a t t e r n
s t r i k i n g l y s i m i l a r t o t h e homogenate CK p a t t e r n discussed above.
No
s i g n i f i c a n t a l t e r a t i o n s i n mean GOT l e v e l s , w i t h r e s p e c t t o t h e c o n t r o l
v a l u e , were found w i t h i n t h e f i r s t t h r e e days
a able
6 ~ ) . Despite a
s i g n i f i c a n t l o s s of 17.5% i n enzyme a c t i v i t y on day s i x , GOT l e v e l s remaine d s t a t i s t i c a l l y i d e n t i c a l t o t h e mean c o n t r o l l e v e l f o r t h e remaining
TABLE 3A
SERUM LEVELS OF GLUTAMATE OXALOACETATE TRANSAMINASE
EXERCISE-TRAINED RATS AT VARIOUS INTERVALS AFTER
EXPOSURE TO 2,000 RADS OF y-RADIATION.
SIGNIFICANT DIFFERENCES BETWEEN
CONTROL AND TEST MEANS WERE
DEliRMINED BY t-TEST,
USING A POOLED
ESTIMATE OF
VARIANCE.
Group
Number
of
Samples
Mean
Activity
mu/ml
Control
6 Hour
1 2 Hour
24 Eiour
3 Day
6 Day
9 Day
1 2 Day
POOLED VAX
POOLED STANDARD DEVIATION:
9.15
Significant
Difference
From q o n t r o l
ti! p < .05
% Increase
From Control
Figure 3A. Serum l e v e l s of glutamate oxaloacetate
transaminase i n e x e r c i s e - t r a i n e d rats a t various
i n t e r v a l s a f t e r exposure t o 2,000 rads of Yradiation.
ACTIVITY IN m ~ / m lserum
TABLE 4~
HEART HOMOGENATE LEVELS OF LACTATE DEHYDROGENASE I N
EXERCISE-TRAINED RATS AT VARIOUS INTERVALS AFTER
EXPOSURE: TO 2,000 RADS OF Y-RADIATION.
S I G N I F I C A N T DIFFERENCES BETWEEN
CONTROL AND T E S T MEANS WERF:
DETERMINED BY t - T E S T ,
USING A P O O U D
ESTIMATE OF
VARIANCE.
Group
Number
of
Samples
Me a n
Activity
d/mg
Muscle
Control
3
622
6 Hour
3
4 21
our
3
548
24 H o u r
3
500
3 Day
3
548
6 Day
3
438
9 Day
3
471
12 Day
3
523
12
POOLED VARIANCE:
2234
POOLED STANDARD DEVIATION :
47.3
Significant
Difference
From C o n t r o l
@ p < .05
% Decrease
From C o n t r o l
+
19.6
Figure 4 ~ . Heart homogenate l e v e l s of l a c t a t e
dehydrogenase i n e x e r c i s e - t r a i n e d rats at.
v a r i o u s i n t e r v a l s a..ft.er e u p s u r e ts 2,000
rads of Y-radiation.
ACTIVITY IN m ~ / m gmuscle
HEART HOMOGENATE LEVELS OF CREATINE KINASE I N EXERCISETRAINED RATS AT VARIOUS INTERVALS AFTER EXPOSURE:
TO 2 , 0 0 0 RADS OF Y-RADIATION.
SIGNIFICANT
DIFFERENCES BETWEEN CONTROL AND TEST
MEANS WERE DETERMINED BY t-TEST,
USING A POOLED ESTIMATE OF
VARIANCE.
Group
Number
of
Samples
Me an
Activity
mU/mg
Muscle
24 Hour
3
734
3 Day
3
807
6 Day
3
646
9 Day
3
745
1 2 Day
3
754
POOLED VARIANCE:
3199
POOLED STANDARD DEVIATION :
56.6
Significant
Difference
From C o n t r o l
@ p < .05
% Decrease
From C o n t r o l
Figure 5A. Heart homogenate Levels of c r e a t i n e
k i n a s e i n e x e r c i s e - t r a i n e d r a t s a t various
i n t e r v a l s a f t e r exposure t o 3,000 rads of
y-radiation.
ACTIVITY IN mU/mg muscle
TABLE 6~
HEART HOMOGENATE LEVELS OF GLUTAMATE OXALOACETATE TRANSAMINASE
I N EXERCISE-TRAINED RATS AT VARIPUS INTERVALS AFTER
EXPOSURE TO 2,000 RADS OF y-RADIATION.
SIGNIFICANT DIFFERENCES BETWEEN
CONTROL AND TEST MEANS WERE
DETERMINED BY t-TEST ,
USING A POOLED
ESTIMATE OF
VARIANCE.
Group
Number
of
Samples
Me an
Activity
mU/mg
Muscle
Control
6 Hour
1 2 Hour
24 Hour
3 Day
6 Day
9 Day
1 2 Day
POOLED VARIANCE:
111.1
POOLED STANDARD DEVIATION:
10.5
Significant
Difference
From Control
@ p < .05
% Decrease
From C o n t r o l
F i g u r e 6 ~ .Heart homogenate l e v e l s of glutamate
o x a l o a c e t a t e transaminase i n e x e r c i s e - t r a i n e d
r u i s ai various i n t e r v a l s a f t e r exposure t o
2,000 r a d s of y - r a d i a t i o n .
ACTIVITY IN m ~ / m gmuscle
sampling i n t e r v a l s .
Mitochondrial Assays
GOT enzyme l e v e l s of h e a r t mitochondria
i s o l a t e d at t h e s t a t e d i n t e r v a l s d i d not show s i g n i f i c a n t a l t e r a t i o n s
u n t i l twenty-four hours a f t e r i r r a d i a t i o n
a able 7 ~ ) .Samples
isolated
at twenty-four hours demonstrated a 36.8% nean l o s s w i t h r e s p e c t t o t h e
mean c o n t r o l v a l u e ( p < . 0 5 ) .
Mitochondrial enzyme l e v e l s r e t u r n e d t o
normal by day t h r e e and showed no f u r t h e r s i g n i f i c a n t d e v i a t i o n s ( ~ i g u r e
7A).
The E f f e c t of E x e r c i s e T r a i n i n g on Sham-irradiated Control Rats
i n o r d e r t o determine t h e e f f e c t of t h e e x e r c i s e t r a i n i n g procedure
on t h e enzyme l e v e l s i n serum, h e a r t homogenate and h e a r t mitochondria of
sham-irradiated c o n t r o l animals, t h e mean enzyme l e v e l s of t h e e x e r c i s e d
samples were s t a t i s t i c a l l y compared t o t h e l e v e l s of sedentary c o n t r o l s .
To o b t a i n a pooled v a r i a n c e between t h e e x e r c i s e d and sedentary groups,
v a r i a n c e s p r e v i o u s l y pooled f o r each enzyme system were s u b j e c t e d t o a
t e s t of homogeniety, using t h e f ratj.0.
Degrees of freedom were d e t e r -
mined u s i n g t h e t o t a l population u t i l i z e d i n c a l c u l a t i n g t h e o r i g i n a l
pooled v a r i a n c e s i n each system.
The r a t i o s obtained c l e a r l y showed t h a t
t h e v a r i a n c e s w i t h i n - e x e r c i s e d and s e d e n t a r y groups were homogeneous and
were t h e r e f o r e pooled t o o b t a i n a s i n g l e v a r i a n c e f a c t o r .
Mean enzyme
l e v e l s were s u b j e c t e d t o a t w o - t a i l e d t - t e s t f o r a non-directional
h y p o t h e s i s and s i g n i f i c a n c e l i m i t s were s e t a t t h e 5% l e v e l of confidence.
Serum LDH and CK l e v e l s demonstrated s l i g h t r e d u c t i o n s as a consequence of e x e r c i s e t r a i n i n g .
However, t h e decreased a c t i v i t i e s were not
TABLE 7A
MITOCHONDRIAL LEVELS OF GLUTAMATE OXALOACETATE TRANSAMINASE
I N EXERCISE-TRAINED RATS AT VARIOUS INTERv ALS AFTER
EXPOSURE TO 2,000 RADS O F Y-RADIATION.
S I G N I F I C A N T DIFFERENCES BETWEEN
CONTROL AND T E S T MEANS WERE
DETERMINED BY t - T E S T ,
USING A POOLED
ESTIMATE O F
VARIANCE.
Group
Number
of
Samples
Me an
Activity
mU/mg.
Biuret
Protein
Control
6 Hour
12 H o u r
24 H o u r
3 Day
6 Day
9 Day
12 D a y
POOLED VARIANCE :
56836
POOLED STMJDARD DEVIATION:
238
Significant
Difference
From C o n t r o l
@ p < .05
% Decrease
From C o n t r o l
Figure 7A. Mitochondria1 l e v e l s of glutamate
o x a l o a c e t a t e transaminase i n e x e r c i s e - t r a i n e d
rats at v a r i o u s i n t e r v a l s a f t e r exFosure t.o
2,000 r a d s of Y-radiation.
ACTIV ITY IN ( m ~ / m gbiuret protein)x103
s i g n i f i c a n t l y d i f f e r e n t from sedentary v a l u e s a t t h e .05 l e v e l of
a able 8 ~ ) . Serum
confidence
GOT, s i m i l a r l y , d i d not e x h i b i t s i g n i f i c a n t
changes between t h e groups ( 2 >~ . 0 5 ) .
Although h e a r t homogenate l e v e l s
of GOT and CK d i d n o t demonstrate any s i g n i f i c a n t a l t e r a t i o n s , homogenate
LDH of t h e e x e r c i s e d animals d i d e x h i b i t a 36.1% mean i n c r e a s e i n a c t i v i t y
(2p < .05).
The mean a c t i v i t y of mitochondria1 GOT i n e x e r c i s e d animals
was not s i g n i f i c a n t l y d i f f e r e n t from t h a t of s e d e n t a r y animals.
Comparison of Radiation-Induced A l t e r a t i o n s i n Enzyme Concentrations i n
Serum, Heart Homogenate and Heart Mitochondria i n Exercised and Sedentary
Rats.
-r
A n
ur&r
i u u b i a i a cunridence i n t e r v a l s f o r t h e percentage changes
i n enzyme a c t i v i t i e s p r e v i o u s l y r e p o r t e d , t h e pooled s t a n d a r d d e v i a t i o n s
f o r t h e previous d a t a were d i v i d e d by t h e known c o n t r o l a c t i v i t i e s i n
each t a b l e and m u l t i p l i e d by 100.
The r e s u l t a n t f a c t o r s r e p r e s e n t e d t h e
s t a n d a r d d e v i a t i o n ( s ) f o r t h e recorded percentage changes.
The r e s u l t i n g
v a r i a n c e s ( s 2 ) were s u b j e c t e d t o an f r a t i o t e s t and were found t o b e
homogeneous.
The v a r i a n c e s of t h e e x e r c i s e d and sedentary groups were
t h e r e f o r e pooled t o o b t a i n a s i n g l e pooled v a r i a n c e .
Percentage i n c r e a s e s
were s u b j e c t e d t o a n o n - d i r e c t i o n a l , t w o - t a i l e d t - t e s t s e t a t t h e 5% l i m i t
of confidence.
Serum Enzyme E l e v a t i o n s
S i g n i f i c a n t i n c r e a s e s i n t h e percentage
e l e v a t i o n of serum LDH i n e x e r c i s e d , i r r a d i a t e d r a t s occurred a t s i x and
twelve hours following exposure i n comparison with i r r a d i a t e d , sedentary
animals
a able
9 ~ ) . No s i g n i f i c a n t d i f f e r e n c e s i n LDH e l e v a t i p n s between
TABLE 8 A
ENZYME LEVELS OF SHAM-IRRADIATED, EXERCISED AND SEDENTARY CONTROLS.
A COMPARISON OF EXERCISE-INDUCED CHANGES I N ENZYME ACTIVITIES OF
SERUM ( s ) , HEART HOMOGENATE ( H ) , AND HEART MITOCHONDRIAL ( M )
FRACTIONS.
Assay
System
S-LDH
S-CK
S-CZT
,
Sedentary Level
E x e r c i s e d Level
Pooled
S
1,310
mu/ml
714
mu/ml
586
208
mu/ml
152
mU/ml
104
.
Ian 0
c
-7
1-1
ulu 1 HIJ.
-TT
zh. 6 i ~ / n i l
9.88
H-LDH
457
mU/mg
622
mU/mg
48.3
H-CK
690
mu/mg
757
mU/mg
54.4
H-GOT
132
m~/mg
160
mU/mg
16.3
M-GOT
1,310
1,080
mU/mg
mU/mg
313
Significant
Difference
8 2p < .05
+
-
Mean enzyme l e v e l s were s u b j e c t e d t o a t w o - t a i l e d t - t e s t f o r a nond i r e c t i o n a l h y p o t h e s i s . S i g n i f i c a n c e l i m i t s were s e t a t t h e .05
l e v e l of confikence.
TABLE gA
COMPARISON OF ALTERATIONS I N SERUM ENZYME LEVELS OF
POST-IRRADIATED SEDENTARY AND EXERCISED RATS.
PostIrradiation
Time
%
Enzyme
System
Activity
in
Sedentary
Rats
% Activity
in
Exercised
Rats
Sig n i f i cant
Difference
@ 2p < .05
LDH
CK
GOT
1 2 Hours
LDH
CK
GOT
LDE
CK
GOT
3 Days
LDH
CK
GOT
6 Days
LDH
CK
GOT
LDH
CK
GOT
103
(3)
131
(3)
(3)
74.0
216
111
91.2
(3)
(3)
(3)
1 2 Days
LDH POOLED STANDARD DEVIATION:
CK POOLED STANDARD DEVIATION:
GOT POOLED STANDARD DEVIATION:
58.4
53.5
19.5
F i g u r e s i n p a r e n t h e s i s r e p r e s e n t t h e number of r a t s sampled,
Values a r e expressed a s p e r c e n t a c t i v i t y w i t h r e s p e c t t o c o n t r o l
v a l u e s of 100%.
-
e x e r c i s e d and s e d e n t a r y animals occurred during t h e remaining sampling
intervals.
Although no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s i n serum CK
e l e v a t i o n s between e x e r c i s e d and s e d e n t a r y groups occurred during t h e
f i r s t twenty-four h o u r s , a temporary d e c r e a s e was found i n e x e r c i s e d
animals s a c r i f i c e d a f t e r t h r e e days ( 2 p < . 0 5 ) .
No f u r t h e r d i f f e r e n c e s
i n serum e l e v a t i o n s were found d u r i n g t h e remaining sampling times.
P o s t - i r r a d i a t i o n serum GOT e l e v a t i o n s showed no s i g n i f i c a n t
d i f f e r e n c e s between s e d e n t a r y and e x e r c i s e d animals a t t h e 5% l e v e l of
confidence.
Heart Homogenate Enzyme Levels
Although t h e percentage decrease
of LDH enzyme a c t i v i t y i n t h e s i x hour post-exposure samples d i d n o t d i f f e r
s i g n i f i c a n t l y between e x e r c i s e d and s e d e n t a r y animals, t h e r e was an i n c r e a s e d percentage a c t i v i t y i n e x e r c i s e d r a t s a t twelve hours ( 2 p < . 0 5 ) ,
s u g g e s t i n g a more r a p i d recovery p r o c e s s t o t h e i n i t i a l enzyme l o s s .
Except f o r a temporary decrease i n t h e LDH a c t i v i t y of t h e e x e r c i s e d
samples on day s i x , no f u r t h e r s i g n i f i c a n t d i f f e r e n c e s a r e apparent
a able
10~).
Homogenate CK a c t i v i t i e s i n t h e e x e r c i s e d r a t s g e n e r a l l y remained
s i g n i f i c a n t l y h i g h e r i n most p o s t - i r r a d i a t i o n samples and, i n c o n t r a s t t o
t h e t r a n s i e n t leakage demonstrated i n i r r a d i a t e d s e d e n t a r y r a t s , d i d not
show an i n i t i a l l o s s of a c t i v i t y ( ~ a b l e s5 & 5 ~ ) .
The mean a c t i v i t y of GOT i n h e a r t homogenates was g e n e r a l l y g r e a t e r
i n e x e r c i s e d r a t s d u r i n g t h e i n i t i a l twenty-four hour p o s t - i r r a d i a t i o n
TABLE: 1OA
COMPARISON OF ALTERATIONS I N HOMOGENATE ENZYME LEVELS OF
POST-IRRADIATED SEDENTARY AND EXERCISED RATS.
- --
PostIrradiation
Time
6 Hours
%
Enzyme
Systern
Activity
in
Sedentary
Rats
LDH
CK
GOT
79.9
1 2 Hours
LDH
CK
GOT
63.7
79.1
70.0
(3)
(3)
(3)
24 Hours
T,DH
CK
GOT
66.8
(3)
75.3
(3)
(3)
3 Days
6 Days
9 mas
1 2 Days
LDH
CK
GOT
LDH
CK
GOT
-
--
--
-
% Activity
in
Exercised
Rats
-
--
Significant
Difference
@ 2p < .05
(3)
86.1 ( 2 )
80.4 ( 3 )
86.8
(3)
101
89.3
(3)
(3)
108
93.1
83.1
118
(3)
(3)
(3)
LDH
CK
GOT
79.2
(3)
LDH
CK
GOT
103
91.2
102
75.1
81.2
LDH POOLED STANDARD DEVIATION:
CK POOLF,D STANDARD DEVIATION:
GOT POOLED STANDARD DEVIATION:
(3)
(2)
(3)
(3)
(3)
9.28
7.55
11.85
Figures i n p a r e n t h e s i s r e p r e s e n t t h e number of r a t s sampled.
Values a r e expressed a s percent a c t i v i t y with r e s p e c t t o c o n t r o l
values of 100%.
.
TABLE 11A
COMPARISON OF ALTEFULTIONS I N MITOCHONDRIAL GOT LEVELS OF
POST-IRRADIATED SEDENTARY AND EXF,RCISED RATS.
PostIrradiation
Time
% Activity
% Activity
in
Sedentary
Rats
in
Exercised
Rats
Significant
Difference
@ 2p < .05
6 Hours
1 2 Hours
24 Hours
3 Days
125
(3)
111
(3)
6 Days
102
(3)
122
(3)
9 Days
113
(3)
90.7
(3)
(3)
84.3
(3)
1 2 Days
84.4
POOLED STANDARD DEVIATION:
25.1
Values a r e expressed a s p e r c e n t a c t i v i t y w i t h r e s p e c t t o c o n t r o l
v a l u e s of 100%.
F i g u r e s i n p a r e n t h e s i s r e p r e s e n t t h e number of r a t s sampled.
-
i n t e r v a l , although t h e d e v i a t i o n s a r e s t a t i s t i c a l l y s i g n i f i c a n t only a t
t h e twelve hour i n t e r v a l .
Unlike t h e temporary decrease i n sedentary
animals noted a f t e r 1 2 hours
a able 6 ,
Chapter I I I ) , t h e r e was no s i g n i f i -
cant l o s s of homogenate GOT a c t i v i t y i n i r r a d i a t e d , e x e r c i s e d r a t s
a able
6 ~ ) . Except f o r a temporary decrease i n a c t i v i t y on day s i x , t h e homogena t e GOT a c t i v i t y of e x e r c i s e d r a t s d i d n o t d i f f e r s i g n i f i c a n t l y from t h e
post-exposure v a l u e s of s e d e n t a r y animals f o r t h e remaining sampling
intervals.
Heart Mitochondria1 Enzyme Levels
Mean GOT enzyme l e v e l s i n
mitochondria i s o l a t e d from t h e h e a r t s of e x e r c i s e d and sedent.alry rats
exposed t o r a d i a t i o n showed no s i g n i f i c a n t d i f f e r e n c e s f o r a l l sampling
p e r i o d s involved
a able
11~).
DISCUSSION
The l a r g e b u t t r a n s i e n t serum enzyme e l e v a t i o n s e x h i b i t e d by LDH
and CK r e v e a l e d a s i g n i f i c a n t p o s t - i r r a d i a t i o n response which was maximal
s i x hours following r a d i a t i o n exposure, r e t u r n i n g t o c o n t r o l l e v e l s by
twenty-four hours.
Such a p a t t e r n suggests a r a p i d b u t temporary leakage
of b i o l o g i c a l m a t e r i a l o u t of t h e damaged t a r g e t t i s s u e s .
Similar biolo-
g i c a l responses have been r e p o r t e d i n s t u d i e s i n v o l v i n g whole-body exposure
--
--
Takamori e t a l . 1969; Hori e t a l . 1970) , and i n t h e previous study conducted i n t h i s l a b o r a t o r y involving t h o r a c i c i r r a d i a t i o n of s e d e n t a r y male
r a t s ( ~ a c ~ i l l i aand
m Bhakthan, submitted f o r p u b l i c a t i o n ) .
Although serum
LDH showed no f u r t h e r s t a t i s t i c a l l y s i g n i f i c a n t e l e v a t i o n s during t h e
remaining sampling p e r i o d s , both LDH and CK were, i n g e n e r a l , e l e v a t e d
beyond day t h r e e (F'igure 3 , Chapter I11 and Figure 3 ~ ) i,n d i c a t i n g t h e
p o s s i b l e development of secondary i n j u r y processes, a s was suggested i n
t h e previous work.
The small i n c r e a s e i n t h e post-exposure serum GOT
a c t i v i t i e s of e x e r c i s e d r a t s i s a l s o s i m i l a r t o t h e response noted prev i o u s l y i n sedentary animals, and i n d i c a t e s enhanced leakage of t h e enzyme from t h e damaged t i s s u e s .
The r e s u l t a n t r e t u r n t o normal i n serum
GOT l e v e l s a f t e r twenty-four hours i n d i c a t e s t h e p o s s i b l e re-instatement
of membrane i n t e g r i t y .
Although no f u r t h e r s i g n i f i c a n t serum GOT eleva-
t i o n s occurred during t h e remaining sampling i n t e r v a l s , an e l e v a t i n g trend
s i m i l a r t o t h e LDH and CK p a t t e r n s occurred from day t h r e e t o day twelve.
This evidence adds f u r t h e r support t o t h e suggested development of seconda r y r a d i a t i o n e f f e c t s , which may include t h e development of major biochemical l e s i o n s o r metabolic a l t e r a t i o n s .
The p a t t e r n of temporary reductions i n LDH a c t i v i t y of h e a r t
homogenates w i t h i n t h e twenty-four hour p e r i o d following r a d i a t i o n exposure i n d i c a t e s an i n i t i a l leakage of enzyme m a t e r i a l from t h e t a r g e t
organ, which i n v e r s e l y p a r a l l e l s t h e observed t r a n s i e n t serum e l e v a t i o n s .
Such a r e l a t i o n s h i p has been previously observed i n sedentary animals and
lends f u r t h e r support t o t h e suggestion t h a t r a d i a t i o n induces changes i n
membrane p e r m e a b i l i t y , r e s u l t i n g i n an e f f l u x of b i o l o g i c a l m a t e r i a l o u t
of t h e i n j u r e d c e l l .
Although samples taken twelve hours and t h r e e days
following exposure do not demonstrate any marked reduction i n LDH enzyme
a c t i v i t y , all remaining sampling times e x h i b i t s i g n i f i c a n t l o s s of t h e
enzyme.
The l a c k of normalization a f t e r twelve days following exposure
suggests t h a t t h e permeability of t h e damaged membrane with r e s p e c t t o
LDH has not been r e s t o r e d .
An a l t e r n a t e , b u t l e s s l i k e l y , suggestion i s
t h a t r a d i a t i o n exposure may have reduced o r a l t e r e d t h e normal metabolic
requirements f o r t h e enzyme, r e s u l t i n g i n a decreased c e l l u l a r l e v e l .
S i m i l a r l o s s e s of b i o l o g i c a l l y a c t i v e m a t e r i a l from i r r a d i a t e d systems
have been independently observed (0kada and Peachey 1957; Kawasaki and
Sakurai 1969).
I n c o n t r a s t t o t h e LDH e f f e c t s , homogenate l e v e l s of CK
and GOT i n i r r a d i a t e d , e x e r c i s e d r a t s d i d not e x h i b i t s i g n i f i c a n t decreases
i n a c t i v i t y w i t h i n t h e f i r s t twenty-four hours.
These observations c o n t r a s t
w i t h t h e r e s u l t s obtained with sedentary r a t s ( c h a p t e r
demonstrable l o s s e s i n homogenate a c t i v i t i e s occurred.
III), i n which
The d a t a suggest
t h a t exposure of e x e r c i s e d r a t s t o 2,000 r a d s of gamma r a d i a t i o n does not
cause s i g n i f i c a n t a l t e r a t i o n s i n t h e permeability of t h e s e enzymes i n t h e
t a r g e t organ.
It may be t h a t e x e r c i s e s t r e s s p r o t e c t s a g a i n s t t h e l o s s
of b i o l o g i c a l m a t e r i a l from t h e damaged c e l l .
This proposal i s , however,
i n disagreement with t h e observed leakage of homogenate LDH and, a s w e l l ,
t h e noted enhancement i n serum enzyme l e v e l s of CK and GOT.
A second and
more p l a u s i b l e suggestion i s t h a t e x e r c i s e , a s a p h y s i o l o g i c a l s t r e s s , may
induce s p e c i f i c metabolic adjustments such a s increased s y n t h e s i s of p r o t e i n s .
Recent evidence of s t i m u l a t e d s y n t h e s i s of nuclear base p r o t e i n s following
exposure of i s o l a t e d r a b b i t lymphocytes t o 1,000 R of X-radiation has been
r e p o r t e d ( ~ h o s h1971)
.
The observed temporary l o s s of mitochondria1 GOT from samples i s o l a t e d
twenty-four hours a f t e r i n vivo exposure suggests t h a t r a d i a t i o n may
induce t r a n s i e n t m o d i f i c a t i o n s i n t h e membrane p e r m e a b i l i t y of h e a r t
mitochondria i n e x e r c i s e - s t r e s s e d r a t s , r e s u l t i n g i n a leakage of t h e
enzyme o u t of t h e s u b c e l l u l a r o r g a n e l l e and i n t o t h e cytoplasm.
Evidence
of secondary p e r m e a b i l i t y changes could not be found.
Sham-irradiated e x e r c i s e d r a t s , s a c r i f i c e d a f t e r t h i r t y days of
e x h a u s t i v e t r a i n i n g , do n o t e x h i b i t s i g n i f i c a n t d i f f e r e n c e s i n serum
l e v e l s of t h e t h r e e enzymes w i t h r e s p e c t t o t h e s e d e n t a r y sham-irradiated
animals.
The r e s u l t s i n d i c a t e t h a t t h e animals were w e l l adapted, physio-
l o g i c a l l y , t o t h e t r a i n i n g procedure and suggest t h a t t h e r e were no f a l s e
serum enzyme e l e v a t i o n s i n t h e i r r a d i a t e d animals due t o e x e r c i s e .
Similar
r e s u l t s i n d i c a t i n g t h e p r o t e c t i v e e f f e c t of e x e r c i s e t r a i n i n g a g a i n s t a
r i s e i n serum enzymes have been r e p o r t e d
arbus us
-e t al.
1964).
Although
exercise-induced m o d i f i c a t i o n s i n h e a r t homogenate l e v e l s of CK and GOT
were n e g l i g i b l e , t h e s i g n i f i c a n t l y i n c r e a s e d a c t i v i t y of t i s s u e LDH i n
t h e t r a i n e d animals i n d i c a t e s a metabolic a d a p t a t i o n t o t h e imposed
physiological s t r e s s .
I n comparing t h e r a d i a t i o n response of e x e r c i s e d and sedentary r a t s ,
s i g n i f i c a n t i n c r e a s e s i n t h e percentage e l e v a t i o n of serum LDH i n e x e r c i s e d
animals were noted w i t h i n t h e f i r s t twenty-four hours following exposure.
The d a t a suggests t h a t e x e r c i s e , a s an added pos t - i r r a d i a t i o n s t r e s s ,
f u r t h e r modifies t h e a l t e r e d p e r m e a b i l i t y of t h e damaged t a r g e t c e l l s ,
r e s u l t i n g i n an enhanced e f f l u x i n response t o t h e i n i t i a l i n j u r y .
Such
d i f f e r e n c e s may, however, be p a r t i a l l y a t t r i b u t a b l e t o an i n c r e a s e i n t h e
cell-serum enzyme g r a d i e n t caused by t h e noted enhancement of t h e c e l l u l a r
enzyme l e v e l a s a r e s u l t of t h e t r a i n i n g procedure.
I n contrast, the
d a t a f o r serum CK and GOT r e v e a l t h a t no s i g n i f i c a n t enhancement i n
post-exposure serum e l e v a t i o n occurred during t h e e n t i r e sampling p e r i o d ,
suggesting t h a t t h e metabolic s t r e s s of e x h a u s t i v e e x e r c i s e i n i r r a d i a t e d
animals d i d not compound t h e i n i t i a l r a d i o l o g i c a l i n j u r y noted i n sedent a r y r a t s w i t h r e s p e c t t o t h o s e enzymes.
I n a d d i t i o n , it i s i n t e r e s t i n g
t o n o t e t h a t u n l i k e LDH, homogenate l e v e l s of CK and GOT d i d not i n c r e a s e
s i g n i f i c a n t l y as a r e s u l t of t h e t r a i n i n g procedure, and would n o t , t h e r e f o r e , c o n t r i b u t e t o any s i g n i f i c a n t i n c r e a s e i n t h e enzyme c o n c e n t r a t i o n
gradient.
Hence, it appears t h a t although t h e r e i s evidence of enhanced
enzyme e f f l u x due t o p o s t - i r r a d i a t i o n e x e r c i s e s t r e s s , t h i s response i s
not a g e n e r a l c h a r a c t e r i s t i c of a l l enzymes s t u d i e d and may, i n s t e a d ,
r e f l e c t i n c r e a s e d cell-serum enzyme g r a d i e n t s of s p e c i f i c components as
a r e s u l t of t h e e x e r c i s e t r a i n i n g i t s e l f .
The s i g n i f i c a n t d i f f e r e n c e noted a t t h e twelve hour sampling
i n t e r v a l between homogenate LDH l e v e l s of e x e r c i s e d and s e d e n t a r y s u b j e c t s
i n d i c a t e s an enhanced recovery p r o c e s s t o t h e i n i t i a l enzyme l o s s may
occur i n t h e e x e r c i s e - t r a i n e d animals.
This proposal i s , however, very
tenuous, based on t h e a v a i l a b l e d a t a , and i s meant p r i m a r i l y a s a suggestion f o r further investigation.
Unlike homogenate LDH, t h e c e l l u l a r l o s s
of CK g e n e r a l l y remains s i g n i f i c a n t l y r e t a r d e d i n e x e r c i s e d s u b j e c t s with
respect t o t h e sedentary i r r a d i a t e d r a t s .
The d a t a i n d i c a t e t h a t p o s t -
exposure e x e r c i s e of t r a i n e d animals markedly i n h i b i t s t h e l o s s of CK
from t h e h e a r t observed i n sedentary groups.
This occurrence-does not
*
n e c e s s i t a t e t h e p r e s e r v a t i o n of membrane i i l t e g r i t y , which would be
c o n t r a r y t o t h e observed serum e l e v a t i o n s , b u t i n d i c a t e s , i n s t e a d , t h a t
e x e r c i s e may s t i m u l a t e p r o t e i n s y n t h e s i s a s a p o s s i b l e r e s u l t of a l t e r e d
metabolic p r i o r i t i e s due t o t h e s t r e s s c o n d i t i o n .
Recent evidence of
GOT homogenate l e v e l s i n e x e r c i s e d s u b j e c t s a l s o d i s p l a y s a s i m i l a r
r e t a r d a t i o n of i n i t i a l enzyme l o s s from t h e damaged t a r g e t organ.
Although a s i g n i f i c a n t decrease i n t h e enzyme a c t i v i t y of e x e r c i s e d
animals occurred on day s i x , t h i s t r a n s i e n t decrease could not b e
c o r r o b o r a t e d i n l a t e r samples.
Mitochondria1 l e v e l s of GOT, although showing a temporary reduct i o n i n e x e r c i s e d animals twenty-four hours a f t e r exposure, do not d i f f e r
s i g n i f i c a n t l y between e x e r c i s e d and s e d e n t a r y animals.
I t can b e con-
cluded t h a t e x e r c i s e , as a metabolic s t r e s s i n animals r e c e i v i n g 2,000
r a d s of gamma r a d i a t i o n t o t h e t h o r a c i c r e g i o n , does not cause any
remarkable a l t e r a t i o n s i n t h e p e r m e a b i l i t y of t h e mitochondria1 membrane
w i t h r e s p e c t t o t h e enzyme system i n s t u d y .
CHAPTER V
RADIATION-INDUCED ENZYME EFFLUX FROM RAT HEART:
111. EFFECT OF DIETARY SUPPLEFENTATION WITH
VITAMIN E ON LIPID PEROXIDE FORMATION
INTRODUCTION
I n s t u d i e s w i t h t i s s u e homogenates and i s o l a t e d systems it h a s been
observed t h a t exposure t o i o n i z i n g r a d i a t i o n can induce t h e formation of
*
p e r o x i d e s , known t o cause e x t e n s i v e d e s t r u c t i o n of l i p i d s ( ~ a u ~ a a r1968)
d
.
However, t h e a b i l i t y of l i v i n g organisms t o d e s t r o y peroxides has made t h e
search f o r radiation-catalyzed l i p i d oxidation d i f f i c u l t .
Evidence of in
v i t r o l i p l a peroxide formation a s a r e s u l t of r a d i a t i o n exDosure has heen
documented (wills and Wilkinson 1967; Wills 1 9 7 0 ) .
I n a d d i t i o n , -i n vivo
s t u d i e s by Bacq and co-worker~ (1951) d e t e c t e d peroxides i n e x t r a c t s of
mouse and r a t depot f a t following i r r a d i a t i o n .
However, t h e extremely
small v a l u e s obtained d i d not f u r n i s h conclusive evidence t h a t r a d i a t i o n
--
exposure i n l i v i n g systems i n i t i a t e s i n vivo p e r o x i d a t i o n of l i p i d s .
Recent evidence by Wills (1970) suggests t h a t vitamin E may a c t
a s an e f f e c t i v e i n h i b i t o r a g a i n s t p e r o x i d a t i o n i n i r r a d i a t e d r a t - l i v e r
microsomes.
It i s expected t h a t vitamin E may a c t as a chain-breaking
l i p i d a n t i o x i d a n t , i n h i b i t i n g f r e e - r a d i c a l p e r o x i d a t i o n v i a hydrogen
a b s t r a c t i o n o r through t h e formation of a l i p i d - t o c o p h e r o l complex
( ~ a p p e l1972).
--
Although many i n v i t r o s t u d i e s support t h e a n t i o x i d a n t
h y p o t h e s i s , t h e l a c k of conclusive evidence of e i t h e r peroxide formation
i n vivo
-9
o r presence of peroxides i n t i s s u e s ( ~ c h w a r z1972) has s e v e r e l y
hampered t h e s t u d y of t h e a n t i o x i d a n t a c t i v i t y of vitamin E and i t s
r e l a t i o n s h i p t o f r e e - r a d i c a l induced peroxide formation i n t h e l i v i n g
organism.
The p r e s e n t i n v e s t i g a t i o n attempted t o f u r n i s h evidence of
in
vivo peroxide formation i n i r r a d i a t e d r a t h e a r t and r e l a t e such damage
t o t h e e s t a b l i s h e d leakage of b i o l o g i c a l l y a c t i v e m a t e r i a l o u t of t h e
damaged t a r g e t organ.
I n a d d i t i o n , t h e study attempted t o r e l a t e t h e
degree of p o s t - i r r a d i a t i o n c a r d i a c damage t o t h e d i e t a r y l e v e l of
a-tocopherol administered p r i o r t o t h e i r r a d i a t i o n procedure.
MATERIAL AND METHODS
Male Sprague-Dawley r a t s of an average weight of 300 grn were
s e l e c t e d a t random and d i v i d e d i n t o two groups.
The f i r s t group (normal
d i e t ) was f e d s t a n d a r d P u r i n a l a b chow, known t o c o n t a i n
a-tocopherol ( 1 I U = 1 mg) p e r kgm of d i e t
.'
66 mg of
The second group ( f o r t i f i e d
d i e t ) was f e d powdered l a b chow f o r t i f i e d w i t h 13.2 gm of D-L,
01a c e t a t e ( 2 5 0 I U = 1 p) p e r kg of d i e t ,
a-tocopher-
The v i t a m i n was purchased
from N u t r i t i o n a l Biochemicals Corporation, Ohio.
This gave a tocopherol
c o n c e n t r a t i o n f a c t o r of approximately 50 times t h e endogenous l e v e l ,
known t o cause s i g n i f i c a n t v a r i a t i o n s i n t h e r a t e of -in vitro lipid
peroxide format ion i n l i v e r homogenates of i r r a d i a t e d mice ( ~ a w e sand
Wills 1972).
Both groups were f e d and watered ad l i b i t u m f o r two weeks
'D. S h e l t o n , Ralston-Purina Co.
communication.
, St.
Louis ; through p e r s o n a l
prior t o irradiation.
I r r a d i a t i o n Procedure
A l l animals were prepared f o r i r r a d i a t i o n a s described previously
(chapter 111).
The r a t s were i r r a d i a t e d i n groups of f o u r using an
.
Eldorado 8 gamma u n i t ( ~ t o m i cEnergy of ~ a n a d a ) The dose r a t e was
c a l c u l a t e d t o be 266 rads/minute a t t h e s u r f a c e and exposure time was
c o r r e c t e d f o r an absorbance of 15.7% t o give an estimated dosage of
2,000 r a d s t o t h e h e a r t .
Sham-irradiated c o n t r o l s f o r each group were
t r e a t e d i n an i d e n t i c a l manner t o t h e experimental animals, excluding
t t c i u a i exposure.
At pre-determines i n t e r v a l s of s i x , twelve and twenty-
f o u r hours following exposure, r a t s were s e l e c t e d and placed under l i g h t
e t h e r anaesthesia f o r s a c r i f i c e .
Heart Muscle P r e p a r a t i o n f o r T h i o b a r b i t u r a t e Analysis
I m e d i a t e l y a f t e r e x t r a c t i n g t h e blood, h e a r t s were excised,
trimmed of f a t t y and connective t i s s u e and r i n s e d i n ice-cold 0.15 M KC1.
A f t e r quickly b l o t t i n g d r y , t h e t i s s u e w a s plunged i n t o l i q u i d n i t r o g e n
u n t i l f r o z e n , weighed, wrapped i n t i n f o i l and s t o r e d on dry i c e .
Samples
were analyzed w i t h i n t h r e e t o f i v e days.
T h i o b a r b i t u r a t e Analysis f o r L i p i d Peroxides
Heart t i s s u e was thawed a t room temperature, c u t i n t o a f i n e mince
and placed i n t o 2.0 m l of 0.15 K C 1 per gm of t i s s u e .
K C 1 replaced t h e
u s u a l sucrose medium, s i n c e it has been determined t h a t carbohydrates
i n t e r f e r e with t h i s a n a l y s i s ( w i l l s 1967)
.
The t i s s u e was homogenized
i n a manual ground-glass b l e n d e r and t o t h e r e s u l t i n g homogenate was
added 1 . 0 m l of 10% t r i c h l o r o a c e t i c a c i d p e r m l of suspension.
After
a g i t a t i n g , t h e mixture was c e n t r i f u g e d f o r f i v e minutes and t h e res u l t a n t s u p e r n a t a n t was c a r e f u l l y e x t r a c t e d and f i l t e r e d through Whatman
GF/A g l a s s paper t o remove r e s i d u a l m a t e r i a l .
To 4.0 m l of t h e f i l t r a t e
was added 1 . 0 m l of 1%
t h i o b a r b i t u r a t e and t h e s o l u t i o n was heated a t
1 0 0 f~o r~ t e n minutes (Tappel and Zalkin 1959)
.
The r e s u l t a n t colour
was measured a t 530 nm on a Beckman DB-GT spectrophotometer a g a i n s t a
s o l u t i o n blank c o n t a i n i n g 1 . 0 m l of water p e r
4
m l of s u p e r n a t a n t .
Peroxide v a l u e s ( TBA v a l u e s ) a r e expressed as m o l e s of malonalaehvde
p e r l i t r e of s u p e r n a t a n t , using t h e molar e x t i n c t i o n c o e f f i c i e n t of
1.56 x
lo5
M-l.
cm-1
determined by Sinnhuber et a l . (1958).
I
RESULTS
Serum Enzyme Analysis
Serum LDH l e v e l s of r a t s f e d t h e normal d i e t d i d not r e v e a l a
s i g n i f i c a n t e l e v a t i o n u n t i l twelve hours a f t e r exposure
a able
1 ~ ) The
.
twelve hour i n t e r v a l , however, e x h i b i t e d a mean a c t i v i t y i n c r e a s e of 73%
( p < .05).
Serum LDH l e v e l s r e t u r n e d t o normal a f t e r twenty-four hours
(I?igure 1 ~ ) Although
.
t h e animals maintained on t h e t o c o p h e r o l - f o r t i f i e d
d i e t e x h i b i t e d t h e same g e n e r a l p o s t - i r r a d i a t i o n p a t t e r n (F'igure 2 ~ ) a, t
no time was t h e mean serum e l e v a t i o n s t a t i s t i c a l l y d i s t i n c t from t h e shami r r a d i a t e d c o n t r o l s of t h a t group
percentage e l e v a t i o n s
able
2 ~ ) . Furthermore, t h e mean
fu able 3 ~ comparing
)
t h e i r r a d i a t i o n response
TABLE 1 B
POST-IRRADIATION SERUM LACTATE DEHYDROGENASE LEVELS I N RATS
MAINTAINED ON STANDARD PURINA LAB CHOW. SIGNIFICANT
DIFFERENCES BETWEEN CONTROL AND TEST MEANS
WERE DETERMINED BY t-TEST, USING A
POOLdD ESTIMATE OF VARIANCE.
Me an
Activity
mU/ml
Serum
Significant
Difference
From Control
@ P < .05
Group
Number
of
Samples
Control
6
1.35 x
6 Hour
5
1-47 x
1 2 Hour
4
2.34 x 10
24 Hour
4
1.13 x
POOLED VARIANCE:
lo3
2n3
3
300022
POOLED STANDARD DEVIATION :
548
lo3
% Increase
From
Control
F i g u r e 1B, P o s t - i r r a d i a t i o n serum l a c t a t e dehydrogenase l e v e l s i n r a t s maintained on
s t a n u a r d Yurina l a b chow.
ACTIVITY IN ( m ~ / m lserum)xlo3
TABLE 2B
POST-IRRADIATION SERUM LACTATE DEHYDROGENASE LEVELS I N
RATS MAINTAINED ON VITAMIN E - F O R T I F I E D LAB CHOW
FOR TWO WEEKS P R I O R TO IRRADIATION.
S I G N I F I C A N T DIFFERENCES BETWEEN
CONTROL AND T E S T MEANS WERE
DETERMINED BY t - T E S T ,
USING A P O O U D
ESTIMATE OF
VARIANCE.
Me an
Activity
mU/ml
Serum
Group
Number
of
Samples
Control
6
611
5
736
12 H o u r
4
860
24 H o u r
4
528
6
HOW
POOLED VARIANCE :
94564
POOLED STANDARD DEVIATION:
308
Significant
Difference
From C o n t r o l
@ p < .05
% Increase
From
Control
Figure 2B. P o s t - i r r a d i a t i o n serum l a c t a t e dehydrogenase l e v e l s i n rats maintained on
vitamin E - f o r t i f i e d 1a.h chow f o r twz u z c k s
prior t o irradiation.
ACTIVITY IN mlJ/ml serum
TABLE 3B
-
COMPARISON OF ALTERATIONS I N SERUM LACTATE DEHY DROGENASE
LE:VELS BETWEEN RATS FE3 STANDARD LAB CHOW AND
TOCOPHEROL-FORTIFIED DIETS.
PostIrradiation
Time
POOLED VARIANCE :
% Activity
Normal
Diet
%
Activity
TocopherolFortified
Diet
Significant
Difference
@ p < .05
2098
POOLED STANDARD DEVIATION :
45 .8
Figures i n p a r e n t h e s i s r e p r e s e n t t h e number of r a t s sampled.
Standard d e v i a t i o n s , r e p r e s e n t i n g confidence l i m i t s of t h e percentage
e l e v a t i o n s , were c a l c u l a t e d by d i v i d i n g t h e pooled s t a n d a r d d e v i a t i o n s
of t h e serum a c t i v i t e s by t h e c o n t r o l a c t i v i t y and m u l t i p l y i n g t h i s
f a c t o r by 100. The r e s u l t a n t v a r i a n c e s ( s 2 ) were s u b j e c t e d t o an f
r a t i o and were found t o be homogeneous. The v a r i a n c e s of t h e normal
d i e t and vitamin E - f o r t i f i e d d i e t were, t h e r e f o r e , pooled t o o b t a i n a
s i n g l e variance f a c t o r .
between t h e two groups were not s i g n i f i c a n t l y d i f f e r e n t ( p > .05) and
i n d i c a t e t h a t t h e d i e t a r y tocopherol f o r t i f i c a t i o n d i d not s i g n i f i c a n t l y
reduce t h e p o s t - i r r a d i a t i o n leakage of enzyme i n t o t h e serum.
A comparison of serum LDH l e v e l s between sham-irradiated r a t s f e d
normal and f o r t i f i e d d i e t s
able
4B) d i d , however, r e v e a l a s i g n i f i c a n t
r e d u c t i o n i n t h e mean enzyme l e v e l of t h e t o c o p h e r o l - f o r t i f i e d group
(2p < .05).
The mean LDH serum a c t i v i t y of t h e c o n t r o l d i e t group w a s ,
i n f a c t , 121% above t h e mean l e v e l found i n t h e t o c o p h e r o l - f o r t i f i e d
animals.
L i pi d-
Peroxide Determination
- -
Malonaldehyde, a major degradation product of l i p i d peroxides,
allows a s e n s i t i v e e s t i m a t i o n of t h e e x t e n t of p e r o x i d a t i o n i n l i v i n g
t i s s u e s by t h e formation of a coloured complex w i t h t h i o b a r b i t u r a t e .
Although t h e v a l i d i t y of t h i s determination h a s been questioned ( ~ h i l p o t
1 9 6 3 ) , W i l l s and R o t b l a t (1964) and Wills (1966) found t h a t t h e t h i o b a r b i t u r a t e (TBA) method c o r r e l a t e d w e l l w i t h o t h e r contemporary e s t i m a t i o n s
of peroxide determination.
The mean peroxide v a l u e s (TBA v a l u e s , expressed a s malonaldehyde
c o n c e n t r a t i o n ) i n t h e h e a r t homogenate p r e p a r a t i o n s of i r r a d i a t e d , normal
d i e t rats d i d not d i f f e r s i g n i f i c a n t l y from t h e TBA v a l u e s of t h e shamirradiated controls
a able
5 ~ ) . Although a 17.6% i n c r e a s e d i d occur a f t e r
twenty-f our hours ( ~ i g u r e3B ) , t h i s e l e v a t i o n was not s t a t i s t i c a l l y s i g n i ficant.
diet
S i m i l a r l y , i r r a d i a t e d r a t s maintained on t h e t o c o p h e r o l - f o r t i f i e d
able 6B)
d i d not e x h i b i t any evidence of i n c r e a s e d t i s s u e l e v e l s of
TABLE
4~
COMPARISON OF LACTATE DEHYDROGENASE SERUM ACTIVITY BETWEEN
SHAM-IRRADIATED CONTROL RATS FED STANDARD LAB CHOW
AND TOCOPHEROL-FORTIFIED DIETS.
Serum A c t i v i t y
Normal Diet
( m ~ / m lserum)
Serum A c t i v i t y
TocopherolF o r t i f i e d Diet
(mu/ml Serum)
Pooled
S
Significant
Difference
@ 2p < -05
Figures i n p a r e n t h e s i s r e p r e s e n t t h e number of r a t s sampled.
Variances p r e v i o u s l y c a l c u l a t e d f o r each system were s u b j e c t e d t o a t e s t
of homogeniety u s i n g t h e f r a t i o . The r a t i o i n d i c a t e d t h a t t h e v a r i a n c e s
were homogeneous and were, t h e r e f o r e , pooled t o g e t h e r t o o b t a i n a s i n g l e
variance f a c t o r .
TABLE: 5B
-
CONCENTRATIONS OF TBA REACTANTS, EXPRESSED AS NMOLAR VALUES,
I N THE FINAL SUPERNATANT OF HEART HOMOGENATES FROM RATS
FED STANDARD PURINA LAB CHOW BEFORE IRRADIATION.
SIGNIFICANT DIFFERENCES EETWEEN CONTROL AND
TEST MEANS WERE DETERMINED BY t-TEST,
USING A POOLED ESTIMATE OF VARIANCE
Mean Concentration
of Malonaldehyde
(nmoles/l)
Significant
Difference
From Control
@ p < .05
Group
Number
of
Samples
control
6
6 Hour
4
1 2 Hour
6
554
-
24 Hour
6
648
-
POOLED VARIANCE :
6525
POOLED STANDARD DEVIATION:
80.8
% Increase
From
Control
0.54
17.6
Figure 3B. Concentration of TBA r e a c t a n t s , expressed
as nmolar malonaldehyde, i n t h e f i n a l s u p e r n a t a n t
of h e a r t homogenates from r a t s fed s t . ~ n d a r dllh
chow and t o c o p h e r o l - f o r t i f i e d d i e t s b e f o r e i r r a d iation.
am
6 ti0 URS
12 HOURS
24 HOURS
CONTROL DIII1':SHAM - IRRADIATED CONTROL
TOCOPHEROL FORTIFIED DIET: SHAM- IRRADIATED
CONTROL DIE:T: IRRADIATED
CONTROL
TOCOPHEROL FORTIFIED DIET: IR RADIATED
CONTROLS
TABLE 6~
-
CONCENTRATION OF TBA REACTANTS, EXPRESSED A S NMOLAR VALUES,
I N THE FINAL SUPERNATANT OF HEART HOMOGENATES FROM RATS
FED TOCOPHEROL-FORTIFIED DIET. SIGNIFICANT
DIFFERENCES BETWEEN CONTROL AND TEST
MEANS WERE DETERMINED BY t-TEST,
USING A POOLED ESTIMATE
OF VARIANCE.
Group
Numb e r
of
Sample s
Control
6
6 Hour
5
1 2 Hour
6
24 Hour
5
POOLED VARIANCE :
Mean Concentration
of Malonaldehyde
(nmoles/l)
7181
POOLED STANDARD DEVIATION :
84.7
Significant
Difference
From Control
@ p < .05
% Increase
From
Control
COMPARISON OF WONALDEHYDE CONCENTRATIONS I N THE FINAZl
SUPERNATANT OF HEART HOMOGENATES FROM SHAM-IRRADIATED
CONTROL RATS FED NORMAL DIET AND VITAMIN
E-FORTIFIED DIET.
Mean Concentration
Normal Diet
(nmolesll)
Mean Concentration
Vitamin EF o r t i f i e d Diet
(nmoles/l)
Pooled
S
Significant
Difference
@ p < .05
Figures i n p a r e n t h e s i s r e p r e s e n t t h e number of r a t s samples.
Variances determined were found t o be homogeneous and were, t h e r e f o r e ,
pooled t o o b t a i n a s i n g l e v a r i a n c e f a c t o r .
malonaldehyde ( ~ i g u r e3 ~ ) .
Comparison of h e a r t homogenate TBA v a l u e s between sham-irradiated
r a t s fed normal and f o r t i f i e d d i e t s
able 7 ~ r e) v e a l e d
a n o t a b l e re-
duction of 31% i n t h e malonaldehyde t i s s u e l e v e l of t h o s e animals maint a i n e d on t h e vitamin E-enriched d i e t ( 2 p < . 0 5 ) and i n d i c a t e s t h a t d i e t -
ary supplementation of vitamin E may s e r v e t o decrease i n vivo peroxide
f o r m a t i on.
DISCUSSION
The i n i t i a l r a d i a t i o n response of t h e rats maintained on t h p
s t a n d a r d l a b chow d i e t i s n o t i c e a b l y d i f f e r e n t from p r e v i o u s r e s u l t s
obtained i n both e x e r c i s e d and s e d e n t a r y animals.
I n contrast t o the
maximal s i x hour responses noted p r e v i o u s l y , t h e s i x hour response does
not e x h i b i t any s i g n i f i c a n t enzyme e l e v a t i o n .
I n a d d i t i o n , t h e maximal
response a t twelve hours i s n o t i c e a b l y decreased w i t h r e s p e c t t o t h e
maximal e l e v a t i o n s p r e v i o u s l y documented.
Such d i f f e r e n c e s i n serum LDH
e l e v a t i o n s may l i e i n t h e lower r a d i o l o g i c a l dose r a t e used i n t h e p r e s e n t
work.
However, a more l i k e l y reason may be t h e age d i f f e r e n c e s between
t h e animals used i n t h e s t u d i e s .
I n order t o obtain sufficient heart
t i s s u e f o r t h e TBA a n a l y s i s , r a t s employed i n t h e p r e s e n t work weighed
an average of 300 gms and were n e c e s s a r i l y o l d e r t h a n t h e 150
r a t s used p r e v i o u s l y .
- 200 gm
Such a f i n d i n g i n d i c a t e s t h e chronological age of
t h e animal may s i g n i f i c a n t l y a f f e c t t h e r a d i a t i o n response observed.
Although i r r a d i a t e d animals p r e v i o u s l y maintained on t h e tocopherol-
122
enriched d i e t d i d not e x h i b i t any s i g n i f i c a n t serum enzyme e l e v a t i o n s
( a s was observed i n t h e normal d i e t a n i m a l s ) , t h e comparison of percent a g e e l e v a t i o n s between groups d i d not r e v e a l any s i g n i f i c a n t p r o t e c t i v e
a c t i o n of vitamin E on t h e post-exposure response.
Such evidence i n d i c a t e s
t h a t excessive q u a n t i t i e s of vitamin E does not s e r v e a s a r a d i o p r o t e c t i v e
agent i n v i v o .
However, it may b e t h a t d i f f e r e n c e s would have been noted
between v i t a m i n E d e f i c i e n t r a t s and r a t s maintained on a normal d i e t .
Furthermore, i t must b e noted t h a t t h e e l e v a t i o n of serum LDH a s a radiob i o l o g i c a l marker may n o t , a s a r e s u l t of l a r g e v a r i a n c e and r e l a t i v e l y
low response, s e r v e a s a s e n s i t i v e enough i n d i c a t o r of damage.
The s i g n i f i c a n t decrease i n serum LDH l e v e l s i n t h e sham-irradiated
r a t s maintained on vitamin E i n d i c a t e s t h a t t h e d i e t a r y a d m i n i s t r a t i o n of
t o c o p h e r o l decreases t h e normal serum l e v e l of t h e enzyme.
Although t h e
observed decrease may r e s u l t from s t i m u l a t e d i n a c t i v a t i o n of t h e serum
p r o t e i n , a more p l a u s i b l e e x p l a n a t i o n involves a decreased r a t e of enzyme
e f f l u x from t h e t i s s u e s a s a r e s u l t of tocopherol-induced s t a b i l i z a t i o n
of membrane p e r m e a b i l i t y .
This supports t h e proposal of Lucy and Dingle
(1964) , Lucy (1972) and Diplock and Lucy (1973) t h a t v i t a m i n E a c t s as a
stereochemical s t a b i l i z e r of c e l l u l a r membranes, without which membranes
may demonstrate abnormally high p e r m e a b i l i t y .
I n addition, the elevation
o f serum and plasma enzymes i n c l i n i c a l l y - i n d u c e d tocopherol d e p l e t i o n
has proved t o be a u s e f u l means of observing t h e development of v i t a m i n E
d e f i c i e n c y ( ~ k s a n e n1970; T o l l e r s u d 1970; Hyldgaard-Jensen 1973).
Studies
by Boyd (1968) , u s i n g lambs w i t h experimental n u t r i t i o n a l myopathy , showed
t h a t a d m i n i s t r a t i o n of a-tocopherol completely stopped t h e abnormal
leakage of enzymes observed under such c o n d i t i o n s and provide f u r t h e r
s u p p o r t i n g evidence t o t h e proposal of membrane s t a b i l i z a t i o n .
The f a i l u r e t o secure evidence of radiation-induced l i p i d peroxide
format i o n does not r u l e out t h e p o s s i b i l i t y t h a t i o n i z i n g r a d i a t i o n may
i n i t i a t e a u t o c a t a l y t i c peroxide formation -i n vivo.
It has been r e p o r t e d
t h a t t h e amounts of peroxides formed -i n vivo a s a r e s u l t of r a d i a t i o n
i n j u r y a r e very s m a l l ( ~ a -ect ~a l . 1 9 5 1 ) , which suggests t h a t t h e accumul a t i o n of peroxide products w i t h i n twenty-four hours of exposure may have
been below t h e l i m i t s of experimental. s e n s i t i v i t y .
vivo condition
-
Tn a i l d i t i c e , t h e
iz
-
allows f o r t h e ongoing metabolism and d e s t r u c t i o n of per-
oxides and by-products,
s o t h a t malonaldehyde a n a l y s i s of such systems
may not r e f l e c t t h e t r u e accumulation of peroxides i n t h e damaged t i s s u e s .
Dawes and W i l l s (1972) have r e p o r t e d marked i n c r e a s e s i n t h e r a t e of l i p i d
p e r o x i d a t i o n i n incubated l i v e r , kidney and h e a r t homogenates of r a t s exposed t o 500
-
2,000 r a d s of whole-body i r r a d i a t i o n .
I n comparison t o t h e
--
r e s u l t s p r e s e n t l y r e p o r t e d , it becomes e v i d e n t t h a t t h e i n vivo a n a l y s i s
a s compared t o -i n v i t r o i n c u b a t i o n of homogenates may y i e l d v a s t l y d i f f e r ent results.
Such i s supported i n a r e c e n t study on i r r a d i a t e d r a t s by
I c h i i and co-workers
(1968).
It was r e p o r t e d t h a t , i n all t i s s u e s analyzed,
--
t h e amount of TBA chromagen formed i n vivo was n e g l i g i b l e a t a l l p e r i o d s
t e s t e d u n l e s s homogenates were incubated i n v i t r o .
It i s , therefore,
d i f f i c u l t t o support t h e assumption t h a t a n a l y s i s of TBA products
&
v i v o i s a r e a l i s t i c r e p r e s e n t a t i o n of t h e t r u e s i t u a t i o n of peroxide
formation.
Due t o t h e f a c t t h a t radiation-induced peroxide formation
could not b e d e t e c t e d i n e i t h e r d i e t a r y group of r a t s , t h e p r o t e c t i v e
f u n c t i o n of vitamin E a s a l i p i d a n t i o x i d a n t a g a i n s t r a d i a t i o n i n j u r y
could n o t b e e v a l u a t e d .
However, i n a s i m i l a r s t u d y u s i n g an i d e n t i c a l
tocopherol c o n c e n t r a t i o n f a c t o r between c o n t r o l and f o r t i f i e d d i e t s a s
used i n t h i s study
awes
and Wills 1 9 7 2 ) , it was r e p o r t e d t h a t t h e t i s s u e
l e v e l of vitamin E c l e a r l y p l a y s an important r o l e i n determining t h e
r a t e of -i n v i t r o p e r o x i d a t i o n of i r r a d i a t e d r a t t i s s u e s .
Such c o n f l i c t -
i n g evidence, w i t h r e s p e c t t o p e r o x i d a t i o n , s t r e s s e s t h e importance i n
s e l e c t i o n of t h e method of a n a l y s i s .
It t h e r e f o r e appears e s s e n t i a l t o
determine whether t h e -i n vivo t i s s u e a n a l y s i s o r t h e -i n v i t r o incubation
method i s most r e p r e s e n t a t i v e of t h e t r u e e s t i m a t i o n of peroxide format i o n i n t h e i n t a c t organism.
The r e l a t i v e l y h i g h l e v e l s of TBA r e a c t a n t s , expressed a s nrnoles
of malonaldehyde, do n o t r e p r e s e n t t h e a b s o l u t e l e v e l s of l i p i d peroxide
products ( ~ a l k i nand Tappel 1960).
Although t h e TBA r e a c t i o n p r i m a r i l y
measures malonaldehyde formed i n p e r o x i d a t i o n , animal t i s s u e s a r e a l s o
known t o c o n t a i n o t h e r TBA r e a c t a n t s ( ~ i n n h u b e r-e t a l . 1958) i n c l u d i n g
aromatic aldehydes and carbohydrate condensation products which g i v e
r i s e t o a s i g n i f i c a n t background e r r o r .
However, t h e 31% r e l a t i v e de-
c r e a s e i n t h e c o n c e n t r a t i o n of TBA r e a c t a n t s i n t h e h e a r t homogenates of
tocopherol-fortified,
sham-irradiated c o n t r o l animals does i n d i c a t e a
n o t a b l e decrease i n t h e t i s s u e l e v e l of peroxide products.
It i s apparent
t h a t d i e t a r y supplementation w i t h vitamin E e f f e c t i v e l y decreases t h e
h e a r t - t i s s u e l e v e l of t h e s e accumulated TBA r e a c t a n t s .
Although t h i s
could be mediated v i a stimulated metabolic degradation of t h e products,
a more plausable explanation would be t h a t supplementation of normal
d i e t a r y l e v e l s of a-tocopherol e f f e c t i v e l y reduces t h e r a t e of ongoing
l i p i d peroxide formation, leading t o observable reductions i n t h e t i s s u e
l e v e l s of t h e s e TBA chromatic r e a c t a n t s .
Similar comparisons i n t h e
t i s s u e l e v e l s of TBA r e a c t a n t s i n normal and tocopherol-deficient r a t s
have been previously reported ( ~ a l k i nand Tappel 1960; Kitabchi and
W i l l i a m s 1968).
CHAPTER V I
GENERAL CONCLUSIONS
IRRADIATION EFFECTS I N SEDENTARY ANIMALS
I r r a d i a t i o n of c a r d i a c muscle induces a t r a n s i e n t e f f l u x of
b i o l o g i c a l l y a c t i v e m a t e r i a l o u t of t h e t a r g e t organ which c o r r e l a t e s
with an i n c r e a s e of t h i s m a t e r i a l i n t h e serum.
It i s , t h e r e f o r e , pro-
posed t h a t enhanced serum enzyme l e v e l s following r a d i a t i o n exposure
r e s u l t from leakage of t h e m a t e r i a l a c r o s s t h e damaged membrane and a r e
not a consequence of i n c r e a s e d l e v e l s w i t h i n t h e h e a r t muscle.
Subsequent
s t a b i l i z a t i o n of b o t h serum and homogenate enzyme l e v ~ l si n d i c a t e a
p o s s i b l e r e i n s t a t e m e n t of c e l l u l a r i n t e g r i t y following t h e observed
i n i t i a l alterations.
Secondary e l e v a t i o n s of serum enzyme l e v e l s a r e
not accompanied by cytoplasmic l o s s e s and i m p l i c a t e some biochemical
l e s i o n o r metabolic a l t e r a t i o n w i t h i n t h e t a r g e t organ.
Although t h e p o s s i b i l i t y of c e l l u l a r death and d i s r u p t i o n cannot
be discounted, t h e doses normally r e q u i r e d t o produce such e f f e c t s v i a
cytoplasmic i n j u r y a r e much h i g h e r t h a n dosages used a t t h e t h e r a p e u t i c
l e v e l ( ~ l t m a n-e t a l . 1970; Fajardo 1973).
I n a d d i t i o n , t h e s i m i l a r i t y of
serum and homogenate p a t t e r n s f o r L;DH and CK, i n c o n t r a s t t o GOT, suggests
t h a t leakage from t h e myocardium i s not due t o massive c e l l d i s r u p t i o n ,
s i n c e such a c a t a s t r o p h e would cause simultaneous l o s s of a l l b i o l o g i c a l l y
a c t i v e m a t e r i a l from t h e ruptured c e l l and t h e r e b y r e s u l t i n e s s e n t i a l l y
i d e n t i c a l leakage p a t t e r n s f o r t h e enzymes s t u d i e d .
It appears, i n s t e a d ,
t h a t t h e e f f l u x of t h e s e enzymes from t h e damaged c e l l s may be r e g u l a t e d
126
by c e r t a i n " l i m i t i n g " f a c t o r s such a s c o n c e n t r a t i o n g r a d i e n t s , molecular
s i z e and geometry o r e l e c t r o s t a t i c charge.
Whereas LDH and CK appear t o
be c o n t r o l l e d by s i m i l a r f a c t o r s , GOT leakage may be governed by some
o t h e r l i m i t i n g determinants i n f l u e n c i n g i t s membrane p e r m e a b i l i t y as i s
--.
suggested by t h e work of Henley e t a 1
(1959)
.
IRRADIATION EFFECTS I N EXERCISED ANIMALS
S i g n i f i c a n t serum e l e v a t i o n s of LDH, CK and GOT following t h o r a c i c
i r r a d i a t i o n of e x e r c i s e - t r a i n e d r a t s add f u r t h e r support t o t h e c o n t e n t i o n
t h a t r a d i a t i o n i n j u r y causes a l t e r a t i o n s i n c e l l memhrane p e r m e l h i l i t y ,
l e a d i n g t o a leakage of m a t e r i a l o u t of t h e t a r g e t organ.
Similar patterns
r e v e a l i n g r a p i d , t r a n s i e n t e l e v a t i o n of serum enzymes have been p r e v i o u s l y
observed ( ~ a k a m o r i-e t a l . 1969; Hori -e t a l . 1970).
Subsequent e l e v a t i n g
p a t t e r n s i n l a t e r sampling i n t e r v a l s i n d i c a t e t h e p o s s i b l e development of
secondary r a d i a t i o n i n j u r y .
I n c o n t r a s t t o t h e i n i t i a l decrease i n h e a r t homogenate l e v e l s of
CK and GOT observed i n Chapter 111, p o s t - i r r a d i a t i o n l o s s of t h e enzymes
i n h e a r t s e x i s e d from e x e r c i s e d animals was s i g n i f i c a n t l y reduced.
Although it may be suggested t h a t e x e r c i s e s t r e s s i n p o s t - i r r a d i a t e d
animals p r o t e c t s a g a i n s t l o s s of m a t e r i a l from t h e t a r g e t organ, t h e
observed l o s s of homogenate LDH and i n c r e a s e d serum enzyme l e v e l s do n o t
i m p l i c a t e a p r e s e r v a t i o n of membrane i n t e g r i t y .
A more p l a u s i b l e ex-
p l a n a t i o n may be t h a t e x e r c i s e induces s p e c i f i c metabolic adjustments
which c o u n t e r a c t t h e e f f l u x of m a t e r i a l from t h e damaged organ.
Enzyme l e v e l s of GOT i n i s o l a t e d r a t h e a r t mitochondria suggest
t h a t p o s t - i r r a d i a t i o n e x e r c i s e d i d not cause s i g n i f i c a n t a l t e r a t i o n s i n
membrane i n t e g r i t y w i t h r e s p e c t t o i r r a d i a t e d s e d e n t a r y animals.
IRRADIATION EFFECTS I N VITAMIN E-FORTIFIED ANIMALS
Comparison of percentage serum LDH e l e v a t i o n between r a t s f e d
s t a n d a r d P u r i n a l a b chow and tocopherol-f o r t i f i e d d i e t s d i d not r e v e a l
any s i g n i f i c a n t r a d i o p r o t e c t i v e a c t i o n of t h e vitamin.
Although such
evidence suggests t h a t vitamin E does not s e r v e a s a u s e f u l radioprotect i v e a g e n t , it i s f e l t t h a t t h e e l e v a t i o n of serum LDH may not b e a
s e n s i t i v e enough r a d i o b i o l o g i c a l marker.
S i g n i f i c a n t r e d u c t i o n of serum LDH l e v e l s i n sham-irradiated r a t s
maintained on v i t a m i n E i n d i c a t e s t h a t t h e d i e t a r y a d m i n i s t r a t i o n of
t o c o p h e r o l decreases t h e serum enzyme l e v e l found under normal d i e t a r y
conditions.
It i s known t h a t e x t r a c e l l u l a r enzymes e x i s t i n a ''steady-
s t a t e " s i t u a t i o n , t h e i r c o n c e n t r a t i o n i n t h e blood dependent upon t h e
r a t e of e f f l u x from t i s s u e c e l l s and t h e i r r a t e of i n a c t i v a t i o n .
Sig-
n i f i c a n t r e d u c t i o n s i n normal serum l e v e l s must, t h e r e f o r e , ocdur through
t h e a l t e r a t i o n of e i t h e r t h e leakage r a t e o r t h e r a t e of i n a c t i v a t i o n .
Although d i e t a r y supplementation w i t h a-tocopherol may i n d i r e c t l y a l t e r
t h e r a t e of enzyme i n a c t i v a t i o n , a more p l a u s a b l e e x p l a n a t i o n involves a
decrease i n t h e r a t e of c e l l u l a r e f f l u x a s a r e s u l t of induced s t a b i l i z a t i o n of c e l l u l a r membrane p e r m e a b i l i t i e s .
Due t o t h e f a c t t h a t a u t o c a t a l y t i c peroxide formation i n vivo was
not d e t e c t e d , t h e r a d i o p r o t e c t i v e f u n c t i o n of vitamin E a s a l i p i d a n t i oxidant could not be e v a l u a t e d .
However, t h e f a i l u r e t o secure evidence
of peroxide formation does not r u l e o u t t h e p o s s i b i l i t y t h a t i o n i z i n g
r a d i a t i o n may i n i t i a t e p e r o x i d a t i o n of membrane l i p i d s .
It i s q u i t e
conceivable t h a t , because of minute q u a n t i t i e s o r ongoing metabolic
e l i m i n a t i o n , t h e amount formed was below t h e t h r e s h o l d of experimental
sensitivity.
I t was, however, shown t h a t d i e t a r y supplementation with
vitamin E e f f e c t i v e l y reduces t h e normal h e a r t - t i s s u e l e v e l of peroxidation products.
It i s proposed t h a t d i e t a r y supplementation w i t h l a r g e
amounts of a-tocopherol reduces t h e r a t e of ongoing peroxide formation
i n t h e i n t a c t animal, l e a d i n g t o an observable r e d u c t i o n i n t h e t i s s u e
l e v e l of t h e s e compounds.
BIBLIOGRAPHY
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-
ALBAUM, H.G.
Serum enzymes following whole-body r a d i a t i o n i n t h e r a b b i t .
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.
.
ALNONTE, T . J . , J . H . BARNES, S.C. BAUTISTA, B. DELACRUZ, L. LANSANGAN and
P.A. PINEDA. Serum transaminase i n mice a f t e r whole-body X-irradiat i o n and t h e e f f e c t of a chemical p r o t e c t o r . I n t . J . R a d i a t . B i o l .
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APPENDIXES
PAGE
APPENDIX
1
2
Computer program f o r enzyme r e a c t i o n o r d e r a n a l y s i s
and c a l c u l a t i o n of r e a c t i o n r a t e s
.......................
145
Computer programs f o r converting enzyme r e a c t i o n r a t e s
f o r s e m , homogenate and mitochondria1 assays. Rates
a r e converted from micromolar values i n t o m i l l i - u n i t
values
.................................................. 149
3
Computer program f o r c a l c u l a t i n g various s t a t i s t i c a l
parameters, including mean, variance and standard
deviation
..............................................
4
Computer program f o r c a l c u l a t i o n of a pooled e s t i m a t e
of variance
5
Computer program f o r t - t e s t , using a pooled e s t i m a t e
of v a r i a n c e
153
............................................. 155
............................................. 157
Appendix 1. Computer program f o r enzyme r e a c t i o n
o r d e r a n a l y s i s and c a l c u l a t i o n of r e a c t i o n r a t e s .
Appendix 2. Computer programs for converting enzyme
reaction rates for serum, homogenate and mitochondiral assays. Rates are converted from micromolar values into milli-unit values.
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C 53
' TYPE
[F;?
._Sv+p
C 7 3 ' )/OW
I!/ TIiC ASSAY VOLUNE'
T Y P E II/ TNZ? I~IITOCHOCPRIAL VOLUME'
C83 M V 4
C9I
C 103
C 11 I
' ENYER
DIL4
FII?ALLY ENTER TIlR /lEIGllT OF PROTL'IIJ IN /!C.
C 123
rJP+U
[ 131
ACTIV+RATR~ASV~~OO-:-(I.~V~~~~LX~!P)
C 141 ACTIV
v
TIIE DILUTION FACTOR( P E R C E N T ) '
; '14ILLI-UNITS/b!C.
PROTFI!Jt
'
'
Appendix 3. Computer program f o r c a l c u l a t i n g various
s t a t i s t i c a l parameters, i n c l u d i n g mean, variance
ana standard d e v i a t i o n .
Appendix 4. Computer program f o r c a l c u l a t i o n of
a pooled e s t i m a t e of v a r i a n c e .
1 1 T Y P E I# THE NUiIBEHS O F S A N P L E S IN EACH CROUP N+A*R,C,.
2 1 T Y P E IIJ TNF: STANDARD DEVIATIOlJ Ilir ISACII GROUP S + X , Y , Z , .
..
,.
Appendix 5. Computer program f o r t - t e s t , u s i n g a
pooled e s t i m a t e of v a r i a n c e .
V TPOOL
C 1 3 ' T Y P E IIl?AfJ X
'
C l . 5 1 MX+n
C 2 1 ' T Y P E MEAN Y'
C 2 . 5 1 MY40
C 3 1 'TYPE
C 3.5 1
C4I
POOLED V A R I E C C E '
PVARtO
' T Y P E h7UNBER S A M P L E S I N X'
C4.51
NX+n
C 5 1 ' T Y P E NUMBER S A M P L E S IN Y '
C 5 , 5 3 NY+O
C 6 1 NUWMX-MY
C71 DEN+(PVARX((;NX)+(~NY)))*.~
C 8 1 T+UUMtDEfJ
C93 T
v