An electron microscopic study of peripheral nerve damage in mice induced by repeated subacute
exposure to endrin
by James John Walker
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Biological Sciences
Montana State University
© Copyright by James John Walker (1984)
Abstract:
Endrin is a chlorinated hydrocarbon insecticide that has been used worldwide. Although acute endrin
exposure produces neurological symptoms, there is generally a lack of morphological data
documenting its cellular effects along the neuromuscular axis. This study was designed to characterize
the morphological effects of endrin on peripheral nerve, optic nerve and muscle. Mice were given 20
daily intraperitoneal injections of endrin in sesame oil at subacute doses that increased from 1.5 mg/kg
to 4.0 mg/kg. Controls were given the same intraperitoneal volume without endrin. Animals were
sacrificed after 4, 7, 14, and 20 days of exposure and 14 and 92 days after the last injection. Sciatic
nerve, optic nerve, skeletal muscle and cardiac muscle tissue were examined using both light and
electron microscopy. Daily behavioral/neurological tests were used to assess general effects on the
nervous system. Animals exposed to endrin were hyperactive, hypersensitive to stimuli, had difficulty
maintaining their position on a rod and displayed signs of piloerection. Optic nerve, skeletal muscle
and cardiac muscle appeared unaffected as were myelinated nerve fibers from sciatic nerve also
appeared unaffected. However, unmyelinated axons in the sciatic nerve of exposed animals showed
various changes which included axonal swelling, dissolution of microtubules and neurofilaments,
axonal and Schwann cell vesiculation, and axonal vacuolation. Some vesicles were present in scattered
rows along the axon or within the axon while others were present in pockets which often appeared
continuous with the periaxonal space and often invaginated the axolemma. Some such pockets
contained an amorphous material as well as vesicles and some regions within the Schwann cell that had
contained axons appeared completely filled by vesicles or flocculent background debris. The results of
this study indicate that repeated subacute doses of endrin produce morphological alterations in
unmyelinated peripheral nerve fibers and their associated Schwann cells and thus could probably cause
changes in neurological functions such as pain perception and autonomic activities related to
temperature and blood pressure regulation. AN ELECTRON MICROSCOPIC STUDY OF PERIPHERAL NERVE DAMAGE
IN MICE INDUCED BY REPEATED SUBACUTE EXPOSURE TQ ENDRIN
by
J a m e s Jo h n W alk er
A t h e s i s su b m itte d i n p a r t i a l f u l f i l l m e n t
o f th e re q u ire m e n ts f o r th e degree
of
M a s t e r o f S c ie ric e
in
B io lo g ic a l S cien ces
.
I
■r
MONTANA STATE UNIVERSITY
Bozem an, M ontana
S e p tem b e r 1984
APPROVAL
o f a t h e s i s s u b m i t t e d by
JAMES JOHN WALKER
T h i s t h e s i s h a s b e e n r e a d by e a c h member o f t h e
t h e s i s c o m m itte e
a n d h a s b e e n fo u n d to be s a t i s f a c t o r y r e g a r d i n g c o n t e n t , E n g l i s h
u s a g e , fo rm a t, c i t a t i o n s , b i b li o g r a p h i c s t y l e , and c o n s is t e n c y , and i s
r e a d y f o r s u b m i s s i o n t o t h e C o l l e g e o f G r a d u a te S t u d i e s ,
D ate
//
^
C h a i r p e i/s o n ^ G r a d u a te Committei
A pproved f o r th e
24 tW.st IW
D a te
v
7
H e a d ,M a jo r D e p a r tm e n t
A pproved f o r t h e C o l l e g e o f G r a d u a te S t u d i e s
D a te
G r a d u a t e Dean
iii
STATEMENT OF PERMISSION TO USE
I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l l m e n t o f th e
r e q u i r e m e n t s f o r a m a s t e r ' s d e g r e e a t Montana S t a t e U n i v e r s i t y , I
a g r e e t h a t th e L i b r a r y s h a l l make i t a v a i l a b l e
to b o r r o w e r s u n d e r
r u l e s o f th e L i b r a r y . B r i e f q u o t a t i o n s from t h i s
t h e s i s a re a llo w a b le
w i t h o u t s p e c i a l p e r m i s s i o n , p r o v i d e d t h a t a c c u r a t e ac k n o w led g m e n t o f
so u rc e i s m ade.
P e r m i s s i o n f o r e x t e n s i v e q u o t a t i o n from r e p r o d u c t i o n o f t h i s
t h e s i s may be g r a n t e d by my m a j o r p r o f e s s o r , o r i n h i s / h e r a b s e n c e , by
th e D i r e c t o r o f L i b r a r i e s w hen, i n t h e o p i n i o n o f e i t h e r ,
u s e o f th e m a t e r i a l i s , f o r s c h o l a r l y p u r p o s e s .
the m a t e r i a l i n t h i s
Any c o p y in g o r u s e o f
t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a ll o w e d
w i t h o u t my w r i t t e n p e r m i s s i o n .
Date
th e proposed
iv
ACKNOWLEGMENT
I w ould l i k e to e x p r e s s my s i n c e r e a p p r e c i a t i o n and d e e p e s t
r e s p e c t to D r. Dwight P h i l l i p s
f o r h i s g u i d a n c e a n d e n c o u ra g e m e n t
th ro u g h o u t t h i s s tu d y a s bo th an a d v is o r and f r i e n d .
I w ould a l s o
l i k e t o t h a n k D r. Jam es M c M illa n , D r. W il li a m D organ a n d D r. R o b e r t
Moore f o r s e r v i n g a s my g r a d u a t e c o m m it t e e .
A s p e c i a l t h a n k s g o e s t o C a re n and Jimmy f o r
th e ir p a tie n c e ,
u n d e r s ta n d in g , and s u p p o rt and e s p e c i a l l y f o r th e s a c r i f i c e s th ey have
made f o r t h e b e t t e r m e n t o f my g r a d u a t e s t u d i e s .
V
TABLE OF CONTENTS
T I T L E ..............................................................
. . ..
. . . .
APPROVAL PAGE . ....................................
. . . . . .
.
STATEMENT OF PERMISSION TO USE
. . . . . . . . . .
. . .
. . .. .
i . . i
.. . . ..
.. . . . . . .
ii'
. iii
ACKNOWLEDGMENT.................................
iv
TABLE OF CONTENTS . ........................ ............................................... '. . . . . . . .
. v.
LIST OF TABLES
v ii
...................
LIST OF FIGURES. . .
ABSTRACT
.
.
. . . .
., . . . . . . .
. . . . . . . . . . . .
INTRODUCTION........................
LITERATURE REVIEW
. .
..
.
...
. .. .. .
. . \
...
.. . .
.. . .
.. . . ------- .
C hem ical and P h y s i c a l P r o p e r t i e s o f E n d r in
.. Uses o f E n d r in
. . . . . .
. . . . . .
M e ta b o lis m and E l i m i n a t i o n
. . .. . . .
.
T o x ic ity
. . . .................................
. .
C a rc in o g e n ic ity . . . . . . . . . . . . . . .
T e r a t o g e n c i t y and M u t a g e n i c i t y
..
. ..
P h y s i o l o g i c a l E f f e c t s ...........................................
MATERIALS AND METHODS
. . v iii
xi
. .
. ..
I
. ■.
. ..
. 8
. . . . . .
.
.
.
.
8
. 9
.
...
. 12
. . . . 15
. . . .
18
. . . . . 20
20
...............................................
E x p o su re and T i s s u e P r e p a r a t i o n
B ehav! o r a l / N e u r o l o g i c a l T e s t s
RESULTS
.' . .
... . . . . . . . . . . . . . . . . .
. . . .
. ..
. . ,.
. . . . . . . . . .
. . . . . . . . . . .
23
23
25
............................
28
S c i a t i c N e r v e ......................................
O th e r T i s s u e s ................................................................... . . . . .
B e h a v i o r a l / N e u r o l o g i c a l E f f e c t s ...............................................
28
45
49
vi
r
DISCUSSION
57
....................
S c i a t i c Nerve . . . . . . . . . . . . . . . . . . . .
O th e r T i s s u e s
.............................................
B e h a v i o r a l / N e u r o l o g i c a l E f f e c t s ..........................
Summary . ......................................................................................
REFERENCES C I T E D ........................ ................................. ....
. . ............................. 76
57
66
68
74
vii
LIST OF TABLES
I.
A c u te o r a l t o x i c i t y o f s e l e c t e d c h l o r i n a t e d h y d r o c a r b o n
i n s e c t i c i d e s i n r a t s . . .......................................................
v iii
LIST OF FIGURES
1
1 . Summary o f t h e e f f e c t s o f e n d r i n i n t h e dog . . . . . . . .
2. P r o d u c t s o f e n d r i n d e g r a d a t i o n
6
...............................................................
3 . Summary o f t h e d o s in g p r o t o c o l . . . . . . . .
io
..............................
24
4 . L i g h t m i c r o g r a p h o f s c i a t i c n e r v e , a 4 day c o n t r o l
a n im a l
........................................... .... ............................. 3 Q
5 . L i g h t m i c r o g r a p h o f s c i a t i c n e r v e , a 4 day
e x p e r i m e n t a l a n im a l .............................................................................
30
6 . L i g h t m i c r o g r a p h o f s c i a t i c n e r v e , a 20 day c o n t r o l
a n i m a l .......................
30
7 . L i g h t m i c r o g r a p h o f s c i a t i c n e ^ v e , a 20 day ■
e x p e r i m e n t a l a n i m a l ........................ .... . . . ............................. 30
8 . T y p i c a l f i e l d o f u n m y e l i n a t e d an d m y e l i n a t e d n e r v e f i b e r s ,
a 4 day c o n t r o l a n im a l
....................................................................
31
9 . B u n d le o f u n m y e l i n a t e d a x o n s , a 4 day e x p e r i m e n t a l
a n im a l
........................................................................ . . . . . . .
31
10 . U n m y e lin a te d n e r v e f i b e r b u n d l e , a 4 day c o n t r o l a n im a l . .
32
1 1 . T y p i c a l o r a n e l l e s i n u n m y e l i n a t e d a x o n s , a 4 day c o n t r o l
a n i m a l .........................................................................................................
32
1 2 . E xam ples o f p a t h o l o g y i n u n m y e l i n a t e d a x o n s , a 4 day
a n i m a l ...............................................
34
13 . S w o lle n a x o n s a n d v e s i c l e s , a 4 day a n i m a l
35
. . . . . . .
s w e l l i n g an d c o n s t r i c t i o n , a 4 day a n im a l . . , i . .
35
1 5 . A xonal
v e s i c u l a t i o n , a 4 day a n im a l
36
16. A xonal
v e s i c u l a t i o n , a 4 day a n im a l
.
14. A xonal
. . . . . . . . . . .
1 7 . A xonal v e s i c u l a t i o n a n d axolem m al i n v a g i n a t i o n , a 4 day
a n im a l .......................................................................
36
36
ix
1 8 . A xonal v e s l c u l a t l o n , a 7 day a n im a l
. . . . . , .
. .. .
36
19. P e r i a x o n a l v e s i c l e s , a 4 day a n im a l
.. . .........................................
37
2 0 . A xonal v e s i c l e s , a 4 day a n i m a l ........................
39
2 ] .. A xonal and Schwann c e l l p a t h o l o g y , a 4 day a n im a l . . . .
39
2 2 . L a r g e a x o n a l v a c u o l e , a 4 day a n i m a l ................................. .....
40
2 3 . A xonal v a c u o l e c o n t a i n i n g s m a l l v e s i c l e s a 4 day
a n i m a l ..................................... ...................................................................
40
2 4 . A xonal v a c u o l e , a 14 day a n im a l
; ...............................................
40
2 5 . Membranous a x o n a l t r a n s e c t i o n , a 14 day a n im a l . . . . . .
41
26 . S w o ll e n a x o n a l SER, a 4 day a n i m a l ...............................................
41
2 7 . S w o lle n a x o n a l SER, a 20 day a n i m a l ..........................................
41
28« A xonal v a c u o l i z a t i o n an d y e s i c u l a t i o n . a 34 day
a n i m a l ........................
42
2 9 . U n m y e lin a te d and m y e l i n a t e d n e r v e f i b e r s , a 112 day
a n i m a l ........................ ............................................... .... . •....................
42
3 0 . A xonal s w e l l i n g arid p o c k e t s o f v e s i c l e s , a 112 day
a n i m a l ................................................................... • •
.....................
43
3 1 . L i g h t m i c r o g r a p h o f s k e l e t a l m u s c l e , a 20 day c o n t r o l
a n i m a l ....................................................................................................
46
3 2 . L i g h t m i c r o g r a p h o f s k e l e t a l m u s c l e , a 20 day
e x p e rim e n ta l an im a l
................................................
3 3 . Red m u s c l e f i b e r , a 20 day e x p e r i m e n t a l a n i m a l ..................
46
46
34 . L i g h t m i c r o g r a p h o f c a r d i a c m u s c l e , a 20 day c o n t r o l
a n im a l ...............................................
47
3 5 . L i g h t m i c r o g r a p h o f c a r d i a c m u s c l e , a 20 day .
e x p e r i m e n t a l a n i m a l ...............................................
47
3 6 . C a r d i a c m u s c l e , a 20 day e x p e r i m e n t a l a n i m a l ........................ ....
47
X
3 7 . L i g h t m ic r o g ra p h , o f o p t i c n e r v e , a 20 day c o n t r o l
a n im a l
........................................
48
3 8 . L i g h t m i c r o g r a p h o f o p t i c n e r v e , a 20 day e x p e r i m e n t a l
a n im a l ..................................................... . . . . . . . . . . . .
48
3 9 . M y e l i n a t e d a x o n s i n t h e o p t i c n e r v e , a 20 day
e x p e r i m e n t a l a n im a l : . .................................. ................................
48
4 0 . Summary
52
o f th e r e s u l t s o f th e t a i l f l i c k t e s t
.. . . . .
41 . Summary o f t h e r e s u l t s o f t h e r o d t e s t
..
.
. . . . . .
.
.
53
4 2 . Summary
o f c h a n g e s i n a n im a l w e i g h t s .
.;
,
. ....................
.
55
4 3 . Summary
o f a n im a l m o r t a l i t y ...................................... ............................
56
xi
ABSTRACT
E n d r i n i s a c h l o r i n a t e d h y d r o c a r b o n i n s e c t i c i d e t h a t h a s been
u s e d w o r l d w i d e . A lth o u g h a c u t e e n d r i n e x p o s u r e p r o d u c e s n e u r o l o g i c a l
sym ptom s, t h e r e i s g e n e r a l l y a l a c k o f m o r p h o l o g i c a l d a t a d o c u m e n tin g
i t s c e l l u l a r e f f e c t s a lo n g th e n e u ro m u sc u la r a x i s .
T h i s s t u d y was
d e s i g n e d to c h a r a c t e r i z e t h e m o r p h o l o g i c a l e f f e c t s o f e n d r i n on
p e r i p h e r a l n e r v e , o p t i c n e r v e and m u s c l e . Mice w ere g i v e n 20 d a i l y
i n t r a p e r i t o n e a l i n j e c t i o n s o f e n d r i n i n sesam e o i l a t s u b a c u t e d o s e s
t h a t i n c r e a s e d from 1 . 5 mg/kg to 4 . 0 m g /k g . C o n t r o l s w ere g i v e n th e
same i n t r a p e r i t o n e a l volum e w i t h o u t e n d r i n .
A n im als w ere s a c r i f i c e d
a f t e r 4 , 7, 14, an d 20 d ay s o f e x p o s u r e an d 14 a n d 92 d ay s a f t e r th e
la s t in je c tio n .
S c i a t i c n e r v e , o p t i c n e r v e , s k e l e t a l m u s c l e and
c a r d i a c m u s c l e t i s s u e w ere e x a m in ed u s i n g b o t h l i g h t an d e l e c t r o n
m ic ro sc o p y .
D a i l y b e h a v i o r a l / n e u r o l o g i c a l t e s t s w ere u s e d to a s s e s s
g e n e r a l e f f e c t s on t h e n e r v o u s s y s t e m . A n im a ls e x p o s e d to e n d r i n w ere
h y p e r a c t i v e , h y p e r s e n s i t i v e to s t i m u l i , h a d d i f f i c u l t y m a i n t a i n i n g
t h e i r p o s i t i o n on a r o d an d d i s p l a y e d s i g n s o f p i l o e r e c t i o n .
O p tic
n q r v e , s k e l e t a l m u s c l e a n d c a r d i a c m u s c l e a p p e a r e d u n a f f e c t e d a s w ere
m y e l i n a t e d n e r v e f i b e r s from s c i a t i c n e r v e a l s o a p p e a r e d u n a f f e c t e d .
How ever, u n m y e l i n a t e d a x o n s i n t h e s c i a t i c n e r v e o f e x p o s e d a n i m a l s
showed v a r i o u s c h a n g e s w h ic h i n c l u d e d a x o n a l s w e l l i n g , d i s s o l u t i o n o f
m i c r o t u b u l e s a n d n e u r o f i l a m e n t s , a x o n a l a n d Schwann c e l l v e s i c u l a t i o n ,
and a x o n a l v a c u o l a t i o n .
Some v e s i c l e s w ere p r e s e n t i n s c a t t e r e d rows
a lo n g t h e axon o r w i t h i n t h e axon w h i l e o t h e r s w ere p r e s e n t i n p o c k e t s
w h ic h o f t e n a p p e a r e d c o n t i n u o u s w i t h t h e p e r i a x o n a l s p a c e a n d o f t e n
i n v a g i n a t e d t h e axolem ma.
Some s u c h p o c k e t s c o n t a i n e d a n am orphous
m a t e r i a l a s w e l l a s v e s i c l e s and some r e g i o n s w i t h i n t h e Schwann c e l l
t h a t h a d c o n t a i n e d a x o n s a p p e a r e d c o m p l e t e l y f i l l e d by v e s i c l e s o r
f l o c c u l e n t b a c k g ro u n d d e b r i s .
The r e s u l t s o f t h i s s t u d y i n d i c a t e t h a t
r e p e a te d su b a c u te doses o f e n d r in produce m o rp h o lo g ic a l a l t e r a t i o n s in
u n m y e l i n a t e d p e r i p h e r a l n e r v e f i b e r s an d t h e i r a s s o c i a t e d Schwann
c e l l s and th u s c o u ld p ro b a b ly c a u se changes i n n e u r o l o g i c a l f u n c tio n s
s u c h a s p a i n p e r c e p t i o n a n d a u to n o m ic a c t i v i t i e s r e l a t e d t o
te m p e ra tu re and b lo o d p r e s s u r e r e g u l a t i o n .
I
INTRODUCTION
E n d r in i s a c h l o r i n a t e d h y d r o c a r b o n p e s t i c i d e t h a t h a s been u s e d
i n th e U.S. f o r a g r i c u l t u r a l an d p u b l i c h e a l t h p u r p o s e s s i n c e i t s
i n t r o d u c t i o n by th e V e l s i c o l Company i n 1 9 5 1 . I t i s t h e m o s t a c u t e l y
t o x i c h y d r o c a r b o n i n s e c t i c i d e known ( G a i n e s , 1969) and b e l o n g s to a
g ro u p o f s t r u c t u r a l l y r e l a t e d c y c l o d i e n e compounds w hich i n c l u d e s
a l d r i n , c h lo rd a n e , c h lo rd e c o n e . ( k ep o n e), d ie ld r ir i, h e p ta c h lo r ,
i s o d r i n , an d t e l o d r i n .
A lth o u g h a n i m a l s a n d humans a c u t e l y e x p o s e d to
e n d r i n d i s p l a y n e u r o l o g i c a l symptoms w h ic h i n c l u d e h y p e r e x c i t a b i l i t y ,
h y p e rse n sitiv ity
rec tal
to s t i m u l i ,
b rad y c a rd ia , h y p e rte n sio n , in cre ase d
t e m p e r a t u r e , i n c r e a s e d v a s c u l a r r e s i s t a n c e , t r e m o r , and
c o n v u l s i o n s ( T r e o n , 19 5 5 ; Emerson e t a l . , 1 9 6 4 ; Emerson and
Hinshawr, 1965; R e i n s e t a l . , 1 9 6 4 ;
C o b le e t a l . , 1 9 6 7 ; W eeks, 1 9 6 7 ) ,
m o r p h o l o g i c a l d o c u m e n t a t i o n o f su c h n e u r o l o g i c a l symp tom ology i s
g e n e ra lly la c k in g .
S in c e some o f t h e s e Symptoms a r e i n d i c a t i v e o f
c h a n g e s w i t h i n th e p e r i p h e r a l n e r v o u s s y s te m an d s i n c e e x p o s u r e
to
c h l o r d e c o n e h a s been shown to damage u n m y e l i n a t e d p e r i p h e r a l nervef i b e r s ( P h i l l i p s , and E r o s c h e n k o , 1 9 8 2 ) ,
t h e p r e s e n t s t u d y was
u n d e r t a k e n to exam ine p e r i p h e r a l n e r v e from a n i m a l s e x p o s e d to
e n d rin .
W hile e n d r i n i s
to x ic
to a l l a n i m a l s , t h e l e t h a l t o x i c i t y
to one
h a l f o f a g ro u p o f A n im a ls (LD50) v a r i e s d e p e n d in g on s e x , m ethod of
a d m i n is t r a t io n , and s p e c ie s v a r i a t i o n .
The a c u t d o r a l t o x i c i t y o f
e n d r i n a n d a num ber o f r e l a t e d compounds i s sum m arized i n T a b l e I .
T e b le I - A c u te o r a l t o x i c i t y o f s e l e c t e d c h l o r i n a t e d h y d r o c a r b o n
p e s t i c i d e s i n r a t s . ( R e f e r e n c e ; G a i n e s ,1 9 6 9 ) _____________
L ow est dose to k i l l
Compound
LD50(mg/kg)
a r a t (m g /k g ) .
M ales
F e m ale s
M ales F e m ale s
A ld rin
39
60
25
40
C hlordane
335
430
250
350
DDT( t e c h n i c a l )
217
-
150
-4
D ie ld rin
46
E n d r in
18
46
7 .5
30
10
30
6 .0
H e p tach lo r
100
162
50
100
Kepone
125
125
100
125
■
The e x c r e t i o n o f e n d r i n an d i t s m e t a b o l i t e s t a k e s p l a c e p r i m a r i l y v i a
t h e l i v e r , b i l e a n d f e c e s ( C o l e e t al.. , 1 9 6 8 ) .
A n im als e x p o s e d to
c a r b o n f o u r t e e n l a b e l e d e n d r i n e x c r e t e a p p r o x i m a t e l y 50-60% o f t h e
do se i n f e c e s w i t h i n 24 h o u r s ( C o l e e t a l . ,1 9 6 8 ; L u d w ig ,1 9 6 6 , a s c i t e d
i n S o t o , 1967; H u n t e r , 1960, a s c i t e d i n So t o , 1967; B e d f o r d e t
a l . ,1 9 7 5 a ) , w h ile v e ry l i t t l e
e n d rin or i t s m e ta b o lite s a re e x c re te d
i n th e u r i n e ( H u ts o n e t a l . ,1 9 7 5 )
E n d rin does n o t a p p e a r to a c cu m u la te i n th e t i s s u e s o f exposed
a n i m a l s , r a t h e r , a p l a t e a u l e v e l o f s t o r a g e i s r e a c h e d a , f t e r 6 -1 0 d a y s
3
( B r o o k s , 1969; K o r t e , I 967 and 1 9 7 0 , a s c i t e d i n Donos o , 1 9 7 9 ) .
D e t e c t a b l e l e v e l s o f e n d r i n a r e g e n e r a l l y fo u n d o n l y i n a n i m a l s
r e c e i v i n g d o s e s o f 0 .2 5 ppm o r m o r e , i n d i c a t i n g
th a t a th re sh o ld le v e l
o f i n t a k e i s n e c e s s a r y b e f o r e t h e compound c a n be d e t e c t e d ( T e r r i e r e
a l . , 1 9 5 8 ; K i i g e m a g i e t a l . , 1 9 5 8 ; E l y and M o o r e ,1957; Moubry e t
a l.,1 9 6 8 ).
re la tio n sh ip
I n dogs a c u t e l y e x p o s e d t o e n d r i n t h e r e a p p e a r s to be no
b e tw e e n t h e b l o o d c o n c e n t r a t i o n s o f t h e compound and
th o se in th e n o n - f a t t i s s u e s (R ich a rd so n e t a l . , 1 9 6 7 ) .
Humans do n o t t e n d t o a c c u m u l a t e s i g n i f i c a n t q u a n t i t i e s o f
en d rin .
E n d r i n c o u l d n o t be d e t e c t e d i n t h e b l o o d o f o c c u p a t i o n a l l y
e x p o s e d w o r k e r s e x c e p t i n t h o s e who h a d b e e n o v e r e x p o s e d
( J a g e r , 1 9 7 0 ) . The b i o l o g i c a l h a l f l i f e
o f e n d r i n i n human b l o o d i s
a b o u t 24 h o u r s ( J a g e r , 1 9 7 0 ) . Endrin, was d e t e c t e d i n t h e u r i n e and
b l o o d o f p a t i e n t s a c u t e l y p o i s o n e d by i n g e s t i n g c o n t a m i n a t e d b r e a d
a l t h o u g h b lo o d and u r i n e s a m p le s w e re n o r m a l w i t h i n one week a f t e r
p o i s o n i n g ( C u r l e y e t a l . , 1 9 7 0 ; C oble e t a l . , 1 9 6 7 ) .
E n d r i n i s a n e n v i r o n m e n t a l l y p e r s i s t e n t compound.
F o r t y one
p e r c e p t a n o r i g i n a l a p p l i c a t i o n o f t e c h n i c a l e n d r i n p e r s i s t e d i n th e
s o i l f o r up to 14 y e a r s (N a s h a n d W o o le n ,1 9 6 7 ) . E n d r i n i s one o f th e
xN
l e a s t w a t e r s o l u b l e i n s e c t i c i d e s a n d i s e x t r e m e l y p e r s i s t e n t i n an
a q u e o u s e n v i r o n m e n t w i t h a s much a s 80% o f t h e i n i t i a l e n d r i n
c o n c e n t r a t i o n (2 ppm) r e m a i n i n g i n w a te r, s a m p le s a f t e r 16 weeks
( Sharom e t a l . , 1 9 8 0 ) .
4
I t s a c u t e t o x i c i t y a n d : e n v i r o n m e n t a l p e r s i s t e n c e h a v e l e d to
l i m i t a t i o n of e n d r i n 's u se i n th e p a s t s e v e r a l y e a r s .
M a ss iv e f i s h
k i l l s i n t h e M i s s i s s i p p i r i v e r s y s te m i n t h e e a r l y 1 9 6 0 's l e d to th e
i n i t i a l r e s t r i c t e d u s e o f e n d r i n w h ic h c u l m i n a t e d i n t h e E n v i r o n m e n ta l
P r o t e c t i o n A g e n c y 's ban o f i t s u s e e a s t o f t h e M i s s i s s i p p i i n 1979. I t
i s s t i l l u s e d i n t h e P l a i n s S t a t e s f o r t h e c o n t r o l o f cutw orm i n g r a i n
an d i n t h e P a c i f i c N o r t h w e s t f o r t h e c o n t r o l o f v o l e s i n a p p l e
orchards.
Heavy u s e o f e n d r i n i n M ontana i n 1981 l e d to p u b l i c and
g o v e r n m e n ta l c o n c e r n o v e r d e t e c t e d r e s i d u e a c c u m u l a t i o n i n w i l d l i f e „
r e s u l t i n g i n h u n t i n g s e a s o n r e s t r i c t i o n s a n d f u r t h e r r e s t r i c t i o n s on
i ts a g ric u ltu ra l u se.
H i s t o ^ a t h o l o g i c a l d a t a from a n i m a l s e x p o s e d t o e n d r i n i s s c a r c e .
D iffu s e d e g e n e r a tiv e changes have been r e p o r t e d i n l i v e r and k id n ey s
( I r e o n , I 955; R e i n s e t a l . , 1 9 6 4 ; Boyd a n d S t e f e c ,1 9 6 9 ; R e u b e r , 1 9 7 9 ) ,
a d r e n a l g l a n d s ( T r e o n , 1 9 5 5 ; R e u b e f , 1 9 7 9 ) , h e a r t ( T r e o n , 1955;
R e u b e r , 1 9 7 9 ) , s p l e e n ( R e i n s e t a l . ;1 9 6 4 ; Boyd a n d S t e f e c ,1 9 6 9 ;
R e u b e r , 1 9 7 9 ) , b r a i n ( T r e o n , 1 9 5 5 ; Boyd a n d S t e f e c , 1 9 6 9 ; R e u b e r , 1 9 7 9 ) ,
thym us ( Boyd and S t e f e c ,1 9 6 9 ) and l u n g s ( T r e o n , 1955; R e i n s e t
a l . , 1 9 6 4 ; Boyd an d S t e f e e , 1969) o f l a b o r a t o r y a n i m a l s e x p o s e d to
en d rin .
O th er h i s t o p a t h o l o g i c a l changes in c lu d e d l o c a l i r r i t a t i o n o f
th e g a s t r o i n t e s t i n a l t r a c t , c a p i l l a r y v enous c o n g e s tio n i n the b r a i n ,
h e a r t an d l u n g s , a n d d e p l e t i o n o f s e c r e t i o n s i n t h e s a l i v a r y g l a n d s
(Boyd an d S t e f e c , 1 9 6 9 ) ,
N e u r o l o g i c a l sym ptom ology i n e n d r i n e x p o s e d a n i m a l s , a s
5
m a n i f e s t e d by c o n v u l s i o n s , t r e m o r , i n c r e a s e d s a l i v a t i o n , and
h y p e rs e n s itiv ity , has led
to th e b e l i e f
t h a t e n d r i n a c t s c h i e f l y on
t h e n e r v o u s s y s te m (Em erson e t a l . , 1 9 6 4 ; Hinshaw e t a l . , 1 9 6 6 ') . .
A c t i v a t i o n o f th e s y m p a t h e t i c and p a r a s y m p a t h e t i c n e r v o u s System s by
e n d r i n h a s been p r o p o s e d a f t e r s t u d i e s o f a c u t e l y e x p o s e d dogs
(E m erson e t a l . , 1 9 6 4 a n d 1966; R e i n s e t a l . , 1 9 6 4 and 1966; H inshaw e t
a l.,1 9 6 6 ).
B r a d y c a r d i a , c o p i o u s m u c o id s a l i v a t i o n , h y p e r t e n s i o n , and
c o n v u l s i o n s f o l l o w i n g l e t h a l e x p o s u r e t o e n d r i n ( 1 0 m g/kg)
suggest
h y p e r a c t i v i t y o f b o th th e s y m p a t h e t i c and p a r a s y m p a t h e t i c n e r v o u s
s y s te m s (E m erson e t a l . , 1 9 6 4 ) .
E n d r i n e x p o s e d a n i m a l s d e v e lo p
i n c r e a s e d r e n a l r e s i s t a n c e due to p o s s i b l e s y m p a t h o - a d r e n a l
s t i m u l a t i o n and s u b s e q u e n t i n c r e a s e i n c i r c u l a t i n g c a t e c h o l a m i n e s
( R e in s e t a l . , 1 9 6 4 ) a n d i n c r e a s e d v e n o u s r e t u r n due to a m a s s i v e
s y m p a t h e t i c d i s c h a r g e l e a d i n g to th e r e l e a s e o f b l o o d s t o r e s from th e
l i v e r a n d s p l e e n ( Hinshaw e t a l . , 1 9 6 6 ) .
O t h e r p h y s i o l o g i c a l c h a n g e s i n d u c e d by e n d r i n e x p o s u r e i n dogs
in c lu d e , l e f t h e a r t f a i l u r e , a c id o s i s , h y p o x ia, in c re a s e d r e c t a l
tem p e ra tu re , in c re a s e d h e m o c o n c e n tra tio n , in c re a s e d leu k o cy te
c o n c e n t r a t io n , in c r e a s e d p e r i p h e r a l r e s i s t a n c e , d e c re a s e d g lo m e ru la r
f i l t r a t i o n , i n c r e a s e d c e r e b r a l s p i n a l f l u i d p r e s s u r e and i n c r e a s e d
c e r e b r a l v e n o u s p r e s s u r e (Em erson e t a l . , 1 9 6 4 ; E m e rs o n ,1965; Emerson
and H i n s h a w ,1965; R e i n s e t a l . , 1 9 6 6 ; H in sh a w e t a l . , 1 9 6 6 ) .
The
s u g g e s t e d p h y s i o l o g i c a l a c t i o n o f e n d r i n i n t h e dog i s sum m arized i n
F i g u r e I . Most p a s t s t u d i e s d e a l i n g w i t h e n d r i n h a v e exam ined'
ENDRI N
i ntrqvenous
injection .
xc
C 1NS
\^‘
'“
S y m p o t h e ti c
nerves
Lef t V e n t r i c l e
Adrenals
Parasymp a t h e t i c
nerves
V
Heart Failure
v
Increased
venous
return
Mo r k e d
I
Excessive
soli vat ion
Decreased
Cardiac Output
increase
In p l a s m a
catecholamines
C p n v u l s i ons
(Skeletal
Mu s e I p s ) ___
v
v
Bradycardia'
~
%
D e c r e a s e in
e f f ec ti v e gaseous
t r a n s p o r t in lungs
Increased
cardiac
output
hypoxia
D e c r e a s e in
^ vascu la r
resistance
FIGURE I . Summary o f t h e e f f e c t s o f e n d r i n i n t h e d o g .
7
m e t a b o l i s m , h e p a t i c and r e n a l damage, c h a n g e s i n th e c a r d i o v a s c u l a r
sy ste m , r e p r o d u c ti v e a b n o r m a li ti e s and i t s a c u te and c h ro n ic
to x ic ity .
W hile much s p e c u l a t i o n e x i s t s i n th e l i t e r a t u r e c o n c e r n i n g
i t s e f f e c t s on th e mammalian n e r v o u s s y s t e m , l i t t l e m o r p h o l o g i c a l d a t a
e x is ts
to s u p p o r t s u c h s p e c u l a t i o n e v e n th o u g h th e m o st p r o f o u n d
symptoms a r e n e u r o l o g i c a l .
Most r e s e a r c h up to t h i s p o i n t i n v o l v i n g
t h e e f f e c t s o f e n d r i n on t h e n e r v o u s s y s te m h a s been r e l a t e d
to
s t u d y i n g t h e p h y s i o l o g i c a l a s p e c t s o f c o n v u l s i o n s , h y p e r t e n s i o n , an d
b r a d y c a r d i a w h i l e th e m o r p h o l o g i c a l b a s i s f o r th e d e v e lo p m e n t o f su c h
symptoms h a s been i g n o r e d .
s t u d i e s to c h a r a c t e r i z e
a x is.
T hus, t h e r e i s a n e e d f o r m o r p h o l o g i c a l
th e e f f e c t s o f e n d r i n a lo n g th e n e u r o m u s c u la r
B e c a u se p r e v i o u s s t u d i e s i n t h i s l a b o r a t o r y ( P h i l l i p s and
E r o s c h e n k o ,1 9 8 2 ) h a v e shown t h a t s u b a c u t e e x p o s u r e to th e r e l a t e d
compound c h l o r d e c o n e damages u n m y e l i n a t e d p e r i p h e r a l n e r v e f i b e r s , and
b e c a u s e e n d r i n e x p o s u r e h a s been s p e c u l a t e d to c a u s e a u to n o m ic
d y s f u n c t i o n , . i t was o f i n t e r e s t
to d e t e r m i n e t h e e f f e c t s o f e n d r i n on
p e r i p h e r a l n e r v e i n a n i m a l s e x p o s e d to r e p e a t e d s u b a c u t e d o s e s o f th e
compound.
In a d d it io n ,
s t u d i e s o f some b e h a v i o r a l / n e u r o l o g i c a l
p a r a m e t e r s w ere i n i t i a t e d
to d e t e r m i n e i f c h a n g e s s i m i l a r to t h o s e
o b s e r v e d i n c h l o r d e c o n e e x p o s e d a n i m a l s ( P h i l l i p s and E r o s c h e n k o , 1982;
J o r d a n e t a l . , 1 9 8 1 ) and i n a n i m a l s a c u t e l y e x p o s e d to e n d r i n
( T r e o n , 1 9 5 5 ; Emerson e t a l . , 1 9 6 4 ; R e i n s e t a l . , 1 9 6 4 ) o c c u r r e d i n
an im a ls r e c e iv in g
su b acu te doses o f e n d r in .
8
LITERATURE REVIEW
CHEMICAL AND PHYSICAL PROPERTIES OF ENDRIN
E n d r in i s
t h e common name f o r I , 2 , 3 , 4 , 1 0 , 1 0 , - h e x a c h l o r o - 6 , 7 -
e p o x y -1 , 4 , 4 a , 5 , 6 , 7 , 8 , 8 a-o c ta h y d r o - 1 , 4 -e n d p -e n d o -5 , 8 -d im eth an o n a p h th a le n e .
I t i s a compound c o n t a i n i n g a t l e a s t 92% o f t h e
e n d o -e n d o s t e r e o i s o m e r o f d i e l d r i n and i s a n e p o x id e o f i s o d r i n .
e n d r in i s a w h ite c r y s t a l l i n e
Pure
s o l i d t h a t i s s t a b l e i n th e p resen ce of
w e ttin g a g e n t s , e m u l s i f i e r s , a l k a l i n e o x id iz in g a g e n ts and b a s ic
r e a g e n t s b u t d eco m p o ses when t r e a t e d w i t h a c i d s o r h e a t e d above 200 C.
E n d r i n , l i k e o t h e r c y c l o d i e n e compounds i s s o l u b l e i n m o st o r g a n i c
so lv e n ts but i s in so lu b le in w a te r.
I t i s u n l i k e t h e s e compounds i n
t h a t i t h a s a much s m a l l e r p a r t i t i o n c o e f f i c i e n t ( t h e
t e n d e n c y to
d i s t r i b u t e b e tw e e n p o l a r a n d n o n - p o l a r s u b s t r a t e s ) , w hich l i m i t s i t s
r a t e o f d e c o m p o s i ti o n and r e t e n t i o n i n e x p o s e d o r g a n i s m s ( Montana
D e p t. of A g r i c u l t u r e , 1983).
P u re e n d r i n i s s t a b l e i n a i r and l i g h t b u t u n d e r g o e s
i s o m e r i z a t i o n , r e a r r a n g e m e n t , o r d e c o m p o s i t i o n upon e x p o s u r e
to h e a t ,
a c i d s o r u l t r a v i o l e t l i g h t (S o lo w a y , e t a l . , 1 9 6 0 ; B u r t o n and
P o l l a r d , 1974; Rosen e t a l . , 1 9 6 6 ) .
T h e se p r o c e s s e s , p l u s m i c r o b i a l
m e t a b o li s m ( Matsumura e t a l . , 1 9 7 1 ) a r e t h e p r i m a r y m eans o f
d e g r a d a t i o n o f th e p a r e n t compound a n d p l a y a n i m p o r t a n t r o l e i n i t s
en v iro n m en ta l p e r s i s t e n c e ,
The p r i n c i p l e p r o d u c t s o f s u c h breakdown
9
a r e shown i n F i g u r e 2. S o to and Diechmann ( 1 9 6 7 ) s t u d i e d t h e a c u t e
t o x i c i t y o f t h e s e m e t a b o l i c p r o d u c t s i n r a t s and fo u n d t h e e n d r i n
a l d e h y d e to be e s s e n t i a l l y n o n - t o x i c w h i l e th e e n d r i n k e t o n e was
a p p r o x i m a t e l y 1 /4 a s
to x ic a s e n d rin i t s e l f .
S u s c e p t a b i l i t y o f th e
e n d r in a ld e h y d e a p p e are d g r e a t e r i n fe m a le s th an m ale s.
P h o t o c h e m ic a l d e g r a d a t i o n i s a n i m p o r t a n t f a c t o r i n t h e
p e r s i s t e n c e o f e n d r i n on th e s u r f a c e o f p l a n t s .
H arriso n e t a l .,
(1967) showed t h a t a p p l e f o l i a g e s p r a y e d w i t h e n d r i n r e t a i n e d o n ly 13%
of th e i n i t i a l
t r e a t m e n t a f t e r one week and o n l y 2% a f t e r 7 w e e k s .
A
m o n i t o r i n g program i n i t i a t e d by t h e Montana D e p a rtm e n t o f A g r i c u l t u r e
r e v e a l e d t h a t e n d r i n r e s i d u e s on w h e a t f o l i a g e m e a s u re d 2 days a f t e r
a p p l i c a t i o n ' , h a d d e g r a d e d by 99.9% a f t e r 10 weeks a n d t h a t s o i l
r e s i d u e s had d e g ra d e d 67% d u r in g th e same t i m e .
t h e i n s e c t i c i d e were s t i l l
D e te c ta b le l e v e l s of
p r e s e n t 55 w eeks a f t e r a p p l i c a t i o n (M ontana
D e p t, o f A g r i c u l t u r e , 1 9 8 3 ) .
USES OF ENDRIN
E n d r i n h a s been u s e d t h r o u g h o u t t h e U n i t e d S t a t e s a s a n a v i c i d e ,
\
'
an d r o d e n t i c i d e , b u t i t s m a j o r u s e h a s been a s a n i n s e c t i c i d e .
I t was
use d t h r o u g h o u t th e 1 9 5 0 's on c r o p s s u c h a s i n c l u d e d a l f a l f a ,
e g g p la n t ., l e t t u c e , p o t a t o e s , p e p p e r s , t o m a t o e s , s t r a w b e r r i e s , c o r n ,
s o rg h u m , s u g a r b e e t s , c o t t o n and s m a l l g r a i n s ,
I t was u s e d p r i m a r i l y
i n s o u t h e a s t e r n U .S . d u r i n g th e I 9 6 0 ' s a n d 7 0 ' s to c o n t r o l
■
n ENDRiN KEfONE
Cl
Cl
CHO
m ENORlN ALDEHYDE
W ENORIN ALCOHOL
H
I
C
Cl
S DGCHLORIATED ENDRtN KETONE
FIGURE 2 . P r o d u c t s o f e n d r i n d e g r a d a t i o n .
11
le p id o p te ro u s la rv a in c o tto n c ro p s.
I n 1971, o v e r 75% o f th e e n d r i n
u s e d was f o r t r e a t m e n t o f c o t t o n a n d 99% o f t h i s u s e was i n th e
s o u t h e a s t e r n and d e l t a s t a t e s (D onoso e t a l . , 1 9 7 9 ) .
D u rin g th e p e r i o d fro m 1957 to 1 9 6 6 , t h e lo w e r M i s s i s s i p p i r i v e r
s y s te m had t h e h i g h e s t l e v e l s o f e n d r i n c o n t a m i n a t i o n o f an y s u r f a c e
w a t e r s i n th e U . S . ,
f ie ld s in th is a r e a .
p r i m a r i l y a s a r e s u l t o f r u n o f f from c r o p s and
Such c o n t a m i n a t i o n r e s u l t e d i n c o n t i n u a l
r e s t r i c t i o n o f i t s u s e n a t i o n w i d e a l t h o u g h i n 1981 o v e r 2 7 ,0 0 0 l b s .
o f e n d r i n was u s e d f o r th e c o n t r o l o f r o d e n t s i n th e s t a t e o f
W a sh in g to n ( E a t o n , 1982) w h i l e i n th e same y e a r o v e r 2 0 0 ,0 0 0 a c r e s of.
g r a i n was t r e a t e d i n Montana (M ontana D e p t , o f F i s h , W i l d l i f e and
P a r k s ,1983).
M o n it o r in g p ro g ra m s i n v o l v i n g S t a t e an d F e d e r a l a g e n c i e s d u r in g
th e h e a v y u s e i n Montana r e v e a l e d w i d e s p r e a d c o n t a m i n a t i o n o f f i s h and
w i l d l i f e ( p r i m a r i l y d ocum ented i n b i r d s ) .
S everal y e a rs of
c o n t r o v e r s y a n d s t u d i e s by th e Montana D e p a r tm e n t o f F i s h , W i l d l i f e
a n d P a r k s h a v e f i n a l l y c u l m i n a t e d i n a r e c e n t ( J u l y , 1983) Montana
D e p a r tm e n t o f A g r i c u l t u r e p r e p a r e d EPA E n v i r o n m e n ta l I m p a c t S t a t e m e n t
w h ic h h a s r e s u l t e d i n th e D e p a rtm e n t o f A g r i c u l t u r e s '
rec om m endation
to " s u s p e n d th e s a l e s an d u s e o f e n d r i n a n d to c a n c e l e n d r i n
r e g i s t r a t i o n s when a l t e r n a t i v e s a r e r e g i s t e r e d by th e E n v i r o n m e n ta l
P r o t e c t i o n Agency " (K. K e l l y , 1 9 8 4 , p e r s o n a l c o m m u n ic a t io n ) .
12
METABOLISM AND ELIMINATION
The m e t a b o l i s m o f e n d r i n i n mammals h a s been w e l l s t u d i e d
( T e r r i e r e e t a l . , 1 9 5 8 ; C o le e t a l . , 1 9 6 8 ; B e d f o rd e t a
l 1975a and
1975 b; So to a n d D e ic h m a n n ,1967; B r o o k s , 1969; H u tso n e t a l . , 1 9 7 5 ) .
H u tso n e t a l .( 1 9 7 5 ) dosed r a t s o f b o th s e x e s f o r 2 w eeks and fo u n d
e n d rin and i t s m ajo r m e t a b o l i t e s i n f e c e s i n th e fo llo w in g p r o p o r tio n s
: e n d r i n , 11%; a n t i - 1 2 - h y d r o x y e n d r i n 83%; s y n - 1 2 - h y d r o x y e n d r i n ,
<0.01%; 3 - h y d r o x y e n d r i n , 5%; 1 2 - k e t o e n d r i n , 1%; and d e l t a - k e t o e n d r i n ,
<0.01%. B e d f o rd e t a l . ( 1 975b) r e p o r t e d t h a t t h e o r a l a d m i n i s t r a t i o n o f
e n d rin
to r a t s r e s u l t e d i n t h e p r o d u c t i o n o f t h r e e m e t a b o l i t e s , a l l o f
w hich w ere more a c u t e l y t o x i c th a n th e p a r e n t compound, w i t h
1 2 - k e t o e n d r i n b e in g t h e m o s t a c u t e l y
to x ic .
O th e r i n v e s t i g a t o r s have
r e p o r t e d t h a t th e m e t a b o l i t e s o f e n d r i n a r e l e s s a c u t e l y t o x i c
e n d rin (K lein e t a l . ,
th a n i s
1 9 6 8 , a s c i t e d i n D o n o s o , l 9 7 9 ) . The g r e a t e r
s u s c e p t i b i l i t y of fem a le s a p p e a rs r e l a t e d
to th e f a c t t h a t th e y
m e t a b o l i z e e n d r i n more s lo w ly t h a n do m a l e s ( K o r t e , 1 9 6 7 , a s c i t e d i n
Brooks 1969) and te n d n o t to e x c r e t e 1 2 - k e t o e n d r i n ( H u ts o n e t
a l.,1 9 7 5 ).
The a c c u m u l a t i o n o f e n d r i n i n t h e r a t v a r i e s w i t h t h e s e x (H u tso n
e t a l.,1 9 7 5 ).
T hese a u t h o r s fo u n d t h a t t h e t i s s u e r e s i d u e r e t e n t i o n
was h i g h e r i n f e m a l e s and was composed p r i m a r i l y o f u n m e ta b o I i zed
e n d r i n w h i l e 1 2 - k e t o e n d r i n was th e m ain t i s s u e r e s i d u e fo u n d i n
m ales.
They fo u n d 1 2 - k e t o e n d r i n was t h e m a j o r m e t a b o l i t e i n th e f a t ,
l i v e r and k i d n e y s o f m a l e s w h i l e u n m e t a b o l i z e d e n d r i n was t h e m ain
••
13
com ponent i n f a t a n d k i d n e y s o f f e m a l e s .
were d e t e c t e d i n
E n d r i n and 1 2 - k e t o e n d r i n
t h e l i v e r o f f e m a l e s a l t h o u g h 70% o f t h e o r i g i n a l
r a d i o a c t i v e l y l a b e l e d compound c o u l d n o t be a c c o u n t e d f o r i n t h e i r
a n a ly sis.
The m e t a b o l i c f a t e o f e n d r i n i n th e r a b b i t i s m a r k e d l y d i f f e r e n t
from t h a t i n
th e r a t .
B e d f o rd e t a l . (1 9 7 5 a ) r e p o r t e d t h a t a lm o s t h a l f
o f th e a d m i n i s t e r e d dose o f e n d r i n i n th e r a b b i t i s e x c r e t e d i n th e
u r i n e , com pared to o n ly 2% i n t h e r a t .
e x c re tio n
T h i s s t u d y a l s o found
to be a l m o s t c o m p l e te w i t h i n 24 h o u r s and t h a t t h e m a jo r
f e c a l com ponent was u n c h a n g ed e n d r i n .
The m e t a b o l i t e s o f t h e two
s p e c ie s i s s im ila r a lth o u g h 1 2 -k eto e n d rin i s n o t a s i g n i f i c a n t •
e x c re tio n product in
th e r a b b i t .
E n d r i n t e n d s to r e a c h a s t e a d y s t a t e c o n d i t i o n i n t h e t i s s u e s o f
re p e a te d ly exposed a n im a ls .
R ats fe d c a rb o n f o u rte e n l a b e l e d e n d rin
f o r 12 days te n d e d to r e a c h a p l a t e a u l e v e l o f s t o r a g e a f t e r 9 to 10
d a y s ( B r o o k s , 1969) a l t h o u g h i n a n o t h e r s t u d y , o r a l a d m i n i s t r a t i o n of
e n d r i n to r a t s r e s u l t e d i n a s t e a d y s t a t e o f s t o r a g e a f t e r a b o u t 6
d a y s ( K o r t e ,1967 a n d 1 9 7 0 , a s c i t e d i n Donoso e t a l . ,
1979). S te e rs ,
la m b s , a n d h o g s f e d e n d r i n f o r 12 w eeks ha d d e t e c t a b l e l e v e l s w i t h i n
t h e t i s s u e s o n ly i n t h o s e a n i m a l s r e c e i v i n g a dose h i g h e r t h a n 0,25
ppm ( T e r r i e r e e t a l . , 1 9 5 8 ) .
I n a n i m a l s a l l o w e d to r e c o v e r f o r 6
w e e k s, s t e e r s h a d a 60% r e d u c t i o n i n t h e e n d r i n c o n t e n t i n
w h i l e no d e t e c t a b l e l e v e l s w e re fo u n d i n la m b s and h o g s .
th e ir fa t
In a s im ila r
s t u d y K iig e m a g i e t a l . (1958) f e d d a i r y cows e n d r i n and r e p o r t e d
14
d e t e c t a b l e c o n c e n t r a t i o n s i n m i l k o n l y from a n i m a l s r e c e i v i n g d o s e s o f
0 .2 5 ppm an d g r e a t e r .
They a l s o n o t e d t h a t i n c r e a s e d d i e t a r y l e v e l s
h a d no e f f e c t on th e m i l k s e c r e t i o n c o n c e n t r a t i o n o f e n d f i n .
O th e r
s tu d ie s a ls o in d ic a te th a t a th re sh o ld le v e l of in ta k e i s n ecessary
b e f o r e m e a s u r a b l e am o u n ts o f th e compound c a n be d e t e c t e d ( E ly and
M oore,1 9 5 7 ; Moubry e t a l . , 1 9 6 8 ) .
R i c h a r d s o n e t a l . ( 1 967) d e m o n s t r a t e d
i n dogs t h a t , e x c l u d i n g f a t t h e r e i s no r e l a t i o n s h i p b e tw e e n b lo o d
c o n c e n t r a t i o n s o f e n d r i n and t h o s e i n th e t i s s u e s .
Most e n d r i n a n d i t s m e t a b o l i t e s a r e e x c r e t e d i n t h e f e c e s ( C o le
e t a l .,1 9 6 8 ) , w h ile very l i t t l e
e n d rin or i t s m e ta b o lite s a re e x c re te d
i n t h e u r i n e (H u tso n e t a l . , 1 9 7 5 ) .
Cole e t a l .( 1 9 6 8 ) a d m i n i s t e r e d
c a r b o n f o u r t e e n l a b e l e d e n d r i n i n t r a v e n o u s l y to r a t s w i t h and w i t h o u t
b ile f i s t u la s .
They fo u n d t h a t o v e r 50% o f t h e d a i l y d o s e was
e x c r e t e d w i t h i n 24 h o u r s and t h a t 90% o f t h e e x c r e t e d compound was i n
th e f e c e s .
I n t h e same s t u d y , i n a n i s o l a t e d p e r f u s e d l i v e r , 50% ■
p e r c e n t o f th e l a b e l e d e n d r i n was e x c r e t e d i n th e b i l e w i t h i n one
hour.
I n p i g s f e d e n d r i n , 47% o f th e a d m i n i s t e r e d compound was
r e c o v e r e d from u r i n e , f e c e s , s to m a c h , g u t , l i v e r , f a t , a n d b lo o d
w i t h i n 24 h o u r s w h i l e 94% h a d b e e n r e c o v e r e d a f t e r 6 d a y s ( H u n te r
i 9 6 0 , a s t i t e d i n Spto and D iechm ann, 1 9 6 7 ) . I n t u b a t e d r a t s f e d e n d r i n
f o r 8 d a y s e x c r e t e d 60-70% o f e a c h d a i l y d o s e i n th e f e c e s (Ludw ig,
1966, a s c i t e d i n So to and D eichm ann, .1967) • The f i r s t d a y , 30% o f t h e
amount e x c r e t e d c o n t a i n e d p u r e e n d r i n a n d 70% was m e t a b o l i t e s .
C e s s a t i o n o f d o s in g r e s u l t e d i n 93% o f t h e amount e x c r e t e d a s
15
m e ta b o lite s.
He. a l s o r e p o r t e d t h a t o f t h e am ount e x c r e t e d i n u r i n e ,
82% c o n s i s t e d o f m e t a b o l i t e s a n d 18% was e n d f i n .
R a b b i t s g i v e n two
o r a l d o s e s o f c a r b o n f o u r t e e n l a b e l e d e n d r i n 14 d a y s a p a r t e x c r e t e d
37.3% o f t h e f i r s t dose v i a t h e u r i n e a n d 49.6% i n f e c e s d u r i n g days
1 - 1 3 . The s e c o n d d o s e was e l i m i n a t e d i n a s i m i l a r f a s h i o n ( B e d f o r d e t
a l . ,1 9 7 5 a ).
S u b s e q u e n t r e c o v e r y o f 96.7% o f t h e r a d i o a c t i v i t y i n
u r i n e a n d f e c e s was a c h i e v e d by day 4 9 .
TOXICITY
Numerous s t u d i e s h a v e e x a m in e d t h e t o x i c i t y o f e n d r i n i n
la b o r a to r y a n im a ls.
T r e o n (1 9 5 5 ) s t u d i e d t h e t o x i c i t y o f e n d r i n i n a
num ber o f l a b o r a t o r y a n i m a l s u t i l i z i n g v a r i o u s m odes o f
7
(
a d m in istra tio n .
C o n t i n u a l a p p l i c a t i o n o f e n d r i n ( d r y pow der) to t h e
s k i n o f r a b b i t s f o r 24 h o u r s r e s u l t e d i n d e v e lo p m e n t o f c o n v u l s i o n s i n
th e m ost s e v e r l y p o iso n e d a n im a ls , w h ile i n t e r m i t t e n t a p p l i c a t i o n f o r
2 h o u r s on e a c h o f 5 days p e r week c a u s e d d e a t h i n a n i m a l s r e c e i v i n g
as l i t t l e
a s 19 a p p l i c a t i o n s .
Symptoms d i s p l a y e d by t h e s e a n i m a l s
i n c l u d e d c o n v u l s i o n s , t r e m o r s , an d f a c i a l t w i t c h i n g .
O ral
a d m i n i s t r a t i o n o f I mg o f e n d r i n on e a c h o f 5 d a y s p e r week t o r a b b i t s
a n d r a t s o f b o th s e x e s r e s u l t e d i n a b d o m in a l d i s t e n t i o n i n r a b b i t s ,
and h y p e r s e n s i t i v i t y
to s t i m u l i i n r a t s .
In a n o th e r e x p e rim e n t, th e
same a u t h o r f e d r a t s e n d r i n a t v a r i o u s d o s e s o v e r a p e r i o d up t o 2
y e a rs and r e p o r t e d c o n v u ls io n s and h y p e r s e n s i t i v i t y
to s t i m u l i i n
16
th o se a n im a ls fe d d i e t s w ith th e h i g h e s t c o n c e n t r a t io n o f e n d r i n .
B e d f o r d e t a l . , (1 9 7 5 a ) a l s o r e p o r t e d s i g n s o f t o x i c i t y i n r a t s s u c h a s
a t a x i a f o l l o w e d by t o n i c c o n v u l s i o n s .
T r e o n (1 9 5 5 ) a l s o f o u n d t h a t
d o g s g i v e n l e t h a l i n j e c t i o n s o f e n d r i n r e g u r g i t a t e d t h e i r f o o d , became
le th a rg ic ,
s a l i v a t e d , d ev e lo p e d r e s p i r a t o r y d i s t r e s s and c e n t r a l
n e r v o u s s y s te m symptoms t h a t i n c l u d e d h y p e r s e n s i t i v i t y t o s t i m u l i ,
'
I
tre m o rs, tw itc h in g , and se v ere c o n v u ls io n s .
S e i z u r e s d e v e lo p e d i n m ic e f o l l o w i n g i n t r a v e n o u s i n j e c t i o n s o f
e n d r i n (W alsh an d F i n k , 1 9 7 2 ) . G ra v e s (1 9 6 5 ) g a v e i h t r a p e r i t o n e a l
i n j e c t i o n s o f e n d r i n t o m ic e a n d r e p o r t e d no m o r t a l i t y a t d o s e s o f I
o r 2 m g/kg b u t a l l a n i m a l s d i e d a t e x p o s u r e l e v e l s o f 10 m g/kg o r
m o re .
S i m i l a r r e s u l t s w ere r e p o r t e d i n b a t s e x p o s e d t o v a r i o u s
c o n c e n t r a t i o n s o f e n d r i n o v e r a 28 day p e r i o d (L u c k e n s a n d
D a v i s , 1 9 6 5 ) . They fo u n d c o m p l e te m o r t a l i t y a t a do se o f 12 m g /k g .
A n im als r e c e i v i n g 50 m g/kg d e v e l o p e d t r e m o r s w i t h i n one h o u r an d d i e d
by 2 2 /3 h o u r s a f t e r e x p o s u r e .
E n d rin i n a l s o e x tr e m e ly t o x ic to b i r d s and f i s h .
Q u a i l and
p h e a s a n t s d e v e lo p symptoms o f i n t o x i c a t i o n w i t h i n 48 t o 72 h o u r s a f t e r
b ein g g iv e n e n d r in i n t h e i r d i e t s ( D e W itt,1 9 5 6 ). I n i t i a l l y
th ese b ird s
d i s p l a y e d a l a c k o f m u s c u la r, c o o r d i n a t i o n , o c c a s i o n a l t r e m o r s , and
s t i f f - l e g g e d , h e s i t a t i n g movement i n w a l k i n g .
L a t e r t h e y made
s p a sm o d ic l e a p s arid w e n t i n t o v i o l e n t c a r t w h e e l s .
In a n o th e r stu d y
( D e W i t t i 1 9 5 5 ) , i t was r e p o r t e d t h a t q u a i l d e v e lo p e d s e v e r e t r e m o r s ,
l o s s o f m u s c u l a r c o o r d i n a t i o n a n d " e x t r e m e n e r v o u s n e s s " w i t h i n two
17
h o u r s a f t e r b e in g f e d e n d r i n .
A ll b i r d s e x p o s e d ' to e n d r in i n t h i s
g r o u p d i e d w i t h i n 48 h o u r s .
Numerous s t u d i e s h a v e docum ented t h e t o x i c e f f e c t s o f e n d r i n on
fish .
S ig n s of i n t o x i c a t i o n i n c lu d e i n e f f e c t i v e f e e d in g (G ra n t and
Mer h l e , 1 9 7 0 ) swimming i n w h i r l i n g p a t t e r n s when d i s t u r b e d
( H e r m a n u t z ,1 9 7 8 ) , h y p e r s e n s i t i v i t y
to s u d d e n n o i s e ( G r a n t , 1976; A r g y le
e t a l.,1 9 7 3 ), re s p ira to ry d if f ic u ltie s ,
! ■
s lu g g is h n e s s ( J o h n s o n ,1 9 6 8 ),
a n d h y p e r e x c i t a b i l i t y ( G r a n t an d M e h r l e , ! 9 7 3 ) .
Symptoms o f e n d r i n p o i s o n i n g i n humans h a v e b e e n w e l l
d o c u m e n ted .
in itia l
W hile d e a t h h a s b e e n r e p o r t e d o n l y i n e x tr e m e c a s e s , t h e
symptoms a f t e r l e s s s e v e r e e x p o s u r e s i n c l u d e h e a d a c h e ,
w e a k n e s s , n a u s e a , a n d a b d o m in a l d i s c o m f o r t w h i l e i n d i v i d u a l s more
s e v e r e l y p o i s o n e d o f t e n e x h i b i t c o n v u l s i o n s , u n c o n s c i o u s n e s s , and
f r o t h i n g a t t h e m o u th ( W e e k s ,1 9 6 7 ; C oble e t a l . , 1 9 6 7 ) .
A c u te ly
p o i s o n e d i n f a n t s d i s p l a y e d e a r l y symptoms s u c h a s t o n i c a n d c l o n i c
c o n v u lsio n s,
s li g h t trism u s, u n co n scio u sn ess, ta c h y c a rd ia , an e le v a te d
body t e m p e r a t u r e , r e s p i r a t o r y d i s t r e s s an d c y a n o s i s ( J a c o b z i n e r and
R a y b i n , 1959; Hayden e t a l . , 1 9 6 5 ) .
D u r in g t h e n e x t s e v e r a l d a y s t h e s e
i n d i v i d u a l s d e v e lo p e d symptoms s u c h a s v o m i t i n g , d i f f i c u l t y i n
'
s w a l l o w i n g , c o n v u l s i o n s , l i m b s i n a s t a t e o f p e rm a n e n t c o n t r a c t i o n an d
p u p ila ry d i l a t i o n .
N e u r o l o g i c a l e x a m i n a t i o n o f one c h i l d r e v e a l e d
s i g n s o f " d i f f u s e e n c e p h a l o p a t h y w i t h f o c a l m o to r p a r i e t a l d i s c h a r g e
on t h e r i g h t s i d e r e s u l t i n g i n d e c e r e b r a t e r i g i d i t y a n d b r a i n stem
s i g n s a n d v a s o m o t o r i n s t a b i l i t y a n d h y p e r t e n s i o n due t o i n v o l v e m e n t o f
18
t h e c a r d i o v a s c u l a r c e n t e r s i n t h e m e d u l l a an d p o h s" ( J a c o b z i n e r and
R a y b in ,1 9 5 9 ). A h y p e r a c tiv e s t a r t l e r e a c t i o n and th e a l t e r n a t i n g
e x t e n s i o n a n d f l e x i o n o f t h e u p p e r l im b l e d t h e s e i n v e s t i g a t o r s to
s p e c u l a t e e x t e n s i v e d i e n c e p h a l o n i n v o l v e m e n t a l t h o u g h m ovem ents due to
b a s a l g a n g l i a i n v o l v e m e n t w e re n o t n o t e d .
F o u r o u t b r e a k s o f a c u t e p o i s o n i n g fro m e n d r i n c o n t a m i n a t e d b r e a d
w e re r e p o r t e d i n S a u d i A r a b i a i n 1967 (W eeks, 1 9 6 7 ) . W i t h i n h o u r s a f t e r
i n g e s t i n g c o n ta m in a te d b r e a d , i n d i v i d u a l s d ev elo p ed abdom inal p a in ,
n a u s e a , v o m i t i n g , l e t h a r g y , m e n t a l c o n f u s i o n , u n c o n s c i o u s n e s s and
co n v u lsio n .
hours.
I n many i n s t a n c e s im p ro v e m e n t was r a p i d , o f t e n w i t h i n 2 -5
Seven p e o p l e d i e d i n one i n c i d e n t w i t h i n ,12 h o u r s a f t e r t h e
o n s e t o f symptoms.
S i m i l a r i n c i d e n t s i n E g y p t ( C o b le e t a l , 1 9 6 7 ) a n d
W ales ( D a v i e s and L e w i s , 1956, a s c i t e d i n W e e k s ,1967) h a v e b e e n
d e s c r ib e d , a l l in v o lv in g in g e s t io n of e n d rin c o n ta m in a ted b r e a d .
Symptoms o f i n t o x i c a t i o n i n c l u d e d c o n v u l s i o n s , f a c i a l c o n t o r t i o n s , •
f r o t h i n g a t t h e m o u th , l e t h a r g y , h e a d a c h e s , m e n t a l c o n f u s i o n , b e a t i n g
t h e h e a d on t h e f l o o r , h y p e r a c t i v e r e f l e x e s an d p e r i o d s o f
s e m i c o n s c i o u s n e s s . . R e c o v e ry i n t h e s e p o i s o n i n g s was r a p i d , g e n e r a l l y
o c c u r r i n g w i t h i n 1-7 d a y s , d e p e n d in g on t h e s e v e r i t y o f e x p o s u r e .
CARCINOGENICITY
E n d r i n h a s b e e n r e p o r t e d t o be c a r c i n o g e n i c to r a t s
( R e u b e r , 1 9 7 9 ) . R a t s e x p o s e d t o c o n c e n t r a t i o n s a s low a s 0 . 1 ppm
19
d ev elo p ed s i g n i f i c a n t in c id e n c e s o f ca rc in o m a s i n l i v e r , l u n g ,
t h y r o i d , mammary g l a n d , a n d a d r e n a l c o r t e x .
su sc e p tib le
F e m ale s t e n d e d to be more
to th e d e v e lo p m e n t o f n e o p la s m s i n e n d o c r i n e a n d
re p ro d u c tiv e organs.
I n o t h e r s t u d i e s o f m a le and f e m a le r a t s fe d
e n d r i n i n c o n c e n t r a t i o n s v a r y i n g fro m I t o 100 ppm o v e r a p e r i o d of 2
y e a r s no i n c r e a s e d i n c i d e n c e o f tu m o r s was r e p o r t e d i n e x p e r i m e n t a l
a n i m a l s ( T r e o n , 1 9 5 6 , a s c i t e d i n J a g e r , l 9 7 0 ) . S i m i l a r s t u d i e s i n m ice
and dogs w ere i n c o n c l u s i v e ( R e u b e r , 1 9 7 9 ) . R e u b e r ( 1 9 7 9 ) s t a t e s
th at
" s u f f i c i e n t d o c u m e n t a t i o n i s a v a i l a b l e on q u a l i t a t i v e e x t r a p o l a t i o n o f
a n im a l d a t a t h a t one m u st c o n c lu d e t h a t f i n d i n g o f c a r c i n o g e n i c i t y i n
one mammalian s p e c i e s s h o u l d be deemmed to h a v e r e l e v a n c e i n o t h e r
mammalian s p e c i e s - i n c l u d i n g m a n " .
R e u b e r 's f i n d i n g s (1 9 7 9 ) h a v e n o t b e e n a c c e p t e d by t h e
E n v i r o n m e n t a l P r o t e c t i o n A g e n c y 's C a r c in o g e n A s s e s s m e n t Group who
r e v i e w e d s t u d i e s o f the U .S . Food an d Drug A d m i n i s t r a t i o n ,
N a tio n a l Cancer I n s t i t u t e ,
t h e U n i v e r s i t y o f C i n c i n n a t i and t h e
U n i v e r s i t y o f M iam i. T h i s g r o u p c o n c l u d e d t h a t " . . . .
e v id e n c e i s
th e
th e w e i g h t o f
t h a t e n d r i n i s u n l i k e l y to be a human c a r c i n o g e n "
( A l b e r t , 1 9 7 8 , - a s c i t e d i n Montana D e p t , o f A g r i c u l t u r e , 1983) . E tid rin
i s a l s o n o t on th e l i s t o f c a r c i n o g e n s r e p o r t e d by t h e W orld H e a l th
O r g a n i z a t i o n ( S p e n c e r , 1 9 8 1 , a s c i t e d i n Montana D e p t, o f
A g r i c u l t u r e , 1983).
20
TERATOGENICITY and MUTAGENICITY
E n d r in h a s b e e n shown to be t e r a t o g e n i c i n h a m s t e r s a n d m ic e a s
w e l l a s c a u s i n g w e i g h t r e d u c t i o n and a s i g n i f i c a n t i n c r e a s e i n f e t a l
m o r t a l i t y ( O tto le n g h i e t a l . ,1 9 7 3 ).
A n o m a lie s i n t h e o f f s p r i n g o f
e n d r i n t r e a t e d h a m s t e r s i n c l u d e d open eye,, webbed f o o t , a n d c l e f t
p a la te .
In a n o t h e r s t u d y , C h e r n o f f e t a l . (1 9 7 9 a a n d 1979b) r e p o r t e d
fu sed r i b s , m en in g o en cep h alo celes, reduced s k e le t a l o s s i f i c a t i o n ,
i n c r e a s e d m o r t a l i t y an d f e t a l w e i g h t r e d u c t i o n .
In th e o f f s p r i n g of
e x p o s e d m i c e , t h e r e was no e f f e c t on s u r v i v a l o r w e i g h t a n d o n l y a few
i n c i d e n c e s o f c l e f t p a l a t e , open e y e , m i c r o c e p h a l y and e x e n c e p h a l y
■
(O tto le n g h i e t a l.,1 9 7 3 ) .
E n d r i n h a s been shown to be m u ta g e n ic i n r a t s f o l l o w i n g
t e s t i c u l a r i n j e c t i o n s ( D i k s h i th a n d D a t t a , 1 9 7 3 ) . They fo u n d
chrom osom al c h a n g e s t h a t i n c l u d e d f r a g m e n t a t i o n and t h e f o r m a t i o n o f
s i n g l e a n d d o u b le b r i d g e s w i t h a c e n t r i c f r a g m e n t s , u n e q u a l
d i s t r i b u t i o n o f a n a p h a s e chrom osom es, a n d t r a n s f o r m a t i o n o f c h r o m a t i n
i n t o a n am orphous lu m p .
PHYSIOLOGICAL EFFECTS
P h y s i o l o g i c a l s t u d i e s i n v o l v i n g e n d r i n e x p o s e d a n i m a l s h a v e been
w e l l docum ented and w h i l e l i t t l e
d a t a i s a v a i l a b l e c o n c e r n i n g i t s mode
o f a c t i o n w i t h i n th e n e r v o u s s y s te m , p h a r m a c o l o g i c a l s t u d i e s i n d i c a t e
••• •
th at i t
'
'••••.
i s n o t a c h o l i n e s t e r a s e i n h i b i t o r ( C o l v i n and P h i l l i p s , 1 9 6 8 ).
Ryan and S h a n k la n d (1 9 7 1 ) e x p l o r e d t h e s y n e r g i s t i c a c t i o n of DDT
and e n d r i n on th e g i a n t a x o n s o f th e c o c k r o a c h c e n t r a l n e r v o u s
s y s te m .
They fo u n d t h a t when g i v e n i n d i v i d u a l l y n e i t h e r compound
p r o d u c e d p h y s i o l o g i c a l a b n o r m a l i t i e s o v e r a p e r i o d o f 3-5 h o u r s .
However p r e t r e a t m e n t o f t h e a x o n s w i t h DDT f o l l o w e d by e x p o s u r e
e n d r i n l e d to i n s t a b i l i t y
co n d u c tio n .
to
f o l l o w e d by c o m p l e t e b l o c k a d e o f a x o n a l
They c o n c l u d e d t h a t e n d r i n a l o n e h a d no t o x i c a c t i o n on
th e a x o n a l membrane o f t h e c o c k r o a c h and t h e t h a t a p p a r e n t s y n e r g i s t i c
a c t i o n o f t h e s e compounds c a n n o t be a t t r i b u t e d
to e i t h e r compound
a lo n e .
Jo y ( 1 976) s t u d i e d t h e c o n v u l s i v e p r o p e r t i e s o f a number o f
c h lo r in a te d hydrocarbon i n s e c t i c i d e s in
s y s te m .
th e c a t c e n t r a l n e rv o u s
A n im a ls i n j e c t e d w i t h e n d r i n a t d o s e s o f 1-2 mg/kg d e v e lo p e d
s p o n t a n e o u s s e i z u r e s w i t h i n 5 -1 5 m i n u t e s a n d a t h i g h e r d o s e s d e v e lo p e d
s e iz u r e s in 0 .5 - 2 .0 m in u te s.
Hypo t e n s i o n , w h ic h was f o l l o w e d by
h y p e r t e n s i o n a n d c a r d i a c a r r y t h m i a s were o b s e r v e d i n some a n i m a l s .
F o l lo w in g a d m i n i s t r a t i o n o f e n d r i n , a n y ty p e o f s e n s o r y s t i m u l a t i o n ,
e sp ec ially ta c tile
o r a u d i t o r y , w ould evoke a s e i z u r e .
The a u t h o r
S p e c u l a t e d t h a t th e c h l o r i n a t e d h y d r o c a r b o n compounds a c t d i r e c t l y on
t h e c e n t r a l n e r v o u s s y s te m a n d t h a t t h e y do n o t h a v e to be c o n v e r t e d
to a n a c t i v e m e t a b o l i t e to p r o d u c e t o x i c e f f e c t s ( J o y , 1 9 7 6 ) .
22
S e i z u r e s w ere p r o d u c e d i n a l l p a r t s o f t h e p i g e o n t e l e n c e p h a l o n
a f t e r i n t r a v e n o u s a d m i n i s t r a t i o n o f e n d r i n ; ( R e v z i n , 1 9 6 6 ) . The
e c t o s t r i a t u m a p p e a r e d t o be p a r t i c u l a r l y s e n s i t i v e a n d b e c a u s e i t i s a
v i s u a l p r o j e c t i o n a r e a i n b i r d s , i t i s p ro b a b le t h a t such ex p o su re s
c o u ld r e s u l t i n v i s u a l d e f i c i t s .
23
MATERIALS AND METHODS
EXPOSURE AND TISSUE PREPARATION
Ten week o l d , m a l e , ICR m ic e ( C h a r l e s R i v e r ) w i t h a n . a v e r a g e
w e i g h t o f 34 gram s ( r a n g e 2 8 . 5 - 3 9 . 4 ) w ere d i v i d e d i n t o c o n t r o l and
e x p erim en tal g ro u p s.
E x p e r i m e n t a l a n i m a l s w ere g i v e n d a i l y
i n t r a p e r i t o n e a l i n j e c t i o n s o f e n d r i n i n sesam e o i l ( 0 . 2 ml) a t d o s e s
w hich i n c r e a s e d from 1 .5 mg/kg on d a y s 0 - 3 ,
and f i n a l l y
t o 2 . 0 mg/kg on d a y s 4 - 1 1 ,
to 4 . 0 mg/kg on da y s 1 2 - 1 9 . T h i s d o s in g p r o t o c o l ( F i g u r e
3 ) was e m ployed i n a n a t t e m p t t o m a i n t a i n a maximum s u b l e t h a l
e x p o s u r e , i n a n i m a l s t h a t may h a v e a d a p t e d to t h e p r e v i o u s l e v e l o f
exposure.
The i n i t i a l d a i l y d o s e o f 1 .5 mg/kg r e p r e s e n t s
a p p r o x i m a t e l y 27% o f t h e a c u t e LD50 f o r m ic e w h i l e t h e f i n a l d a i l y
d o se r e p r e s e n t s a p p r o x i m a t e l y 71% o f t h e a c u t e LD50 ( G r a v e s , 1 9 6 4 ) . The
c u m u l a t i v e dose i n t h o s e a n i m a l s r e c e i v i n g 20 c o n s e c u t i v e i n j e c t i o n s
was 54 mg/kg ( a p p r o x i m a t e l y 10 t i m e s t h e a c u t e L D 50). The s o l u t i o n s
w e re p r e p a r e d w i t h e n d r i n (99% p u r i t y )
s u p p l i e d by t h e V e l s i c o l
Company ( C h i c a g o ) . C o n t r o l a n i m a l s r e c e i v e d t h e same volum e o f sesam e
o i l w ith o u t e n d rin .
F o u r e x p e r i m e n t a l a n d two c o n t r o l a n i m a l s were
s a c r i f i c e d a f t e r 4, 7, 1 4 , a n d 20 d a y s o f e x p o s u r e ( d o e )
and a f t e r
14 a n d 92 days o f r e c o v e r y ( d o r ) .
E a c h a n im a l was w e ig h e d on a beam b a l a n c e i m m e d i a t e l y p r e c e d i n g
sa c rific e .
The a n i m a l s w e re a n e s t h e t i z e d w i t h e t h e r a n d , f o l l o w i n g
Daily d o s e ( m g /k g )
6
4 -
I
3 -
2
}
I
-
IZ
F
I
0
1
2
3
4
5
6
7
I
8
*
9
T
I
I
I
I
to
■ 7
r
r
I
i
-r
10 11 12 1.3 14 15 16 17 .18 19
Days
FIGURE 3 . D o sin g P r o t o c o l . A rrow s i n d i c a t e d a y s o f s a c r i f i c e .
25
la te r a l in c isio n s,
t h e r i b c a g e was r e f l e c t e d s u p e r i o r l y .
The
p e r i c a r d i a l s a c was o p e n e d a n d a 21 g a u g e n e e d l e was i n s e r t e d i n t o
l e f t v e n tric le .
th e
The r i g h t a t r i u m was c u t a n d t h e a n im a l was p e r f u s e d
w i t h 3% g l u t a r a l d e h y d e i n 0 . IM p h o s p h a t e b u f f e r (pH 7 . 3 ) a t a p r e s s u r e
o f 8 0 -1 0 0 mm Hg f o r 5 m i n u t e s .
S c ia tic n erv e, o p tic n erv e, g lu te a l
and c a r d i a c m u s c l e w ere re m o v e d , p l a c e d i n f i x a t i v e a n d d i c e d i n t o 2
m illim e te r cubes.
F o l l o w i n g 2—3 h o u r s ' i n c o l d f i x a t i v e ,
was w ashed i n b u f f e r an d s t o r e d o v e r n i g h t .
th e t i s s u e
The n e x t day t h e t i s s u e
was p o s t - f i x e d i n b u f f e r e d 1% osmium t e t r o x i d e , t h e n d e h y d r a t e d i p a
g r a d e d e t h a n o l s e r i e s an d p r o p y l e n e o x i d e a n d embedded i n S p u r r ’ s
re sin .
T i s s u e s e c t i o n s 1 - 2 urn ( m i c r o m e t e r s )
t h i c k w ere c u t from
s e v e r a l b l o c k s o f e a c h a n im a l and s t a i n e d w i t h b a s i c t o Iru i d i n e b l p e
(M eek, 1976) f o r l i g h t ; m i c r o s c o p i c e x a m i n a t i o n a n d s u b s e q u e n t
o rie n ta tio n of th in s e c tio n s.
L i g h t m i c r o g r a p h s Were t a k e n w i t h an
Olympus FM-6 ca m e ra m o u n te d on a n Olympus BH-2 l i g h t m i c r o s c o p e .
T h in
s e c t i o n s w ere c u t w i t h g l a s s k n i v e s on a n LKB U l t r a t o m e I I I , m ounted
on u n c o a t e d c o p p e r g r i d s ,
s ta in e d w ith a lc o h o lic u ra n y l a c e ta te
( W e a k l e y , 1981) and l e a d c i t r a t e ( V e n a b l e a n d C o g g e s h a l l ,1 9 6 5 ) and
e x a m in ed a n d p h o t o g r a p h e d w i t h a Z e i s s 9S -2 e l e c t r o n m i c r o s c o p e .
BEHAVIORAL/NEUROLOGICAL TESTS
B e h a v i o r a l / n e u r o l o g i c a l t e s t s w e re p e r f o r m e d w i t h i n a 30 m in u te
tim e sp a n i m m e d i a t e l y p r e c e d i n g t h e d o s i n g o f t h e c o n t r o l and
26
e x p e r i m e n t a l a n i m a l s , e x c l u d i n g t h e days, i n which t h o s e a n i m a l s were
to be s a c r i f i c e d .
The t e s t s w ere d e s i g n e d to a s s e s s c h a n g e s i n
th e
a n i m a l ' s b e h a v i o r a p p r o x i m a t e l y 24 h o u r s a f t e r t h e p r e v i o u s
in je c tio n .
The t e s t s w ere p e r f o r m e d d a i l y d u r i n g th e 20 d a y s o f
d o s in g ( d a y s 0 - 1 9 ) and t h e r e a f t e r a t I ,
r.
8,
12, 14, 22, 29, a n d 92 d o
Each a n im a l was rem oved from t h e i r c a g e a n d t e s t e d w i t h m in im a l
space r e s t r i c t i o n s .
Each a n im a l was t e s t e d and o b s e r v e d a s f o l l o w s :
GAIT: The g a i t o f i n d i v i d u a l a n i m a l s was v i s u a l l y e v a l u a t e d a s
t h e y moved a b o u t a wooden s u r f a c e .
A n im a ls w ere o b s e r v e d f o r any
c h a n g e s i n g a i t su c h a s s p l a y i n g o f e i t h e r t h e f r o n t o r h i n d l i m b s and
fo r sig n s of u n s te a d in e s s a n d /o r im m o b ility .
TREMOR: A n im als w ere o b s e r v e d f o r a n y s i g n o f t r e m o r .
TAIL FLICK: T h i s t e s t was u s e d to e v a l u a t e e a c h a n i m a l ' s r e s p o n s e
to n o x i o u s th e r m a l s t i m u l i ( J a n s s e n e t a l . , 1 9 6 3 ) .
A p p r o x i m a t e l y one
h a l f o f l e n g t h o f t h e a n i m a l s t a i l was d i p p e d i n a w a t e r b a t h
m a i n t a i n e d a t a t e m p e r a t u r e o f 5 6 - 5 6 . 5 C an d th e tim e r e q u i r e d f o r th e
a n i m a l to remove i t s
t a i l c o m p l e t e l y from t h e w a te r was r e c o r d e d to
th e n e a r e s t t e n t h o f a s e c o n d .
STARTLE RESPONSE: The r e a c t i v i t y o f e a c h a n im a l to a b l a s t o f a i r
in to
t h e i r f a c e was c a t e g o r i z e d a s b e in g h y p o a c t i v e , n o r m a l o r
h y p e ra c tiv e .
Each a n im a l was t e s t e d u s i n g a n a e r o s o l c a n o f
c o m p r e s s e d a i r , a d j u s t e d t o th e h i g h s e t t i n g and d i r e c t e d w i t h an
e x te n s io n tu b e.
27
ACTIVITY: A s u b j e c t i v e e v a l u a t i o n o f a c t i v i t y w as made b a s e d on
v i s u a l o b s e r v a t i o n o f e a c h i s o l a t e d a n im a l, a s t h e y moved a b o u t a
wooden s u r f a c e .
A g e n e r a l d e s c r i p t i o n tia s made o f e a c h a n i m a l ( f r o m
e x t r e m e l y h y p o a c t i v e to e x t r e m e l y h y p e r a c t i v e ) .
ROD TEST: T h i s t e s t c h e c k e d f o r t h e a n i m a l ' s a b i l i t y
to m a in ta in
i t s p o s i t i o n when p l a c e d on a f i b e r g l a s s r o d and was a n i n d i r e c t
m easure of b a la n c e and g r i p s t r e n g t h .
A u n ifo rm f i b e r g l a s s r o d 4 3 .0
cm lo n g an d 8 . 0 mm i n d i a m e t e r was s u s p e n d e d d i a g o n a l l y a c r o s s a box
2 9 .0 cm from t h e b o t to m .
The a n i m a l s w ere h e l d by ttye t a i l and
lo w e re d o n t o t h e r o d s u c h t h a t a l l 4 paws g r i p p e d t h e r o d s e c u r e l y and
t h e a n im a l a p p e a r e d s t a b l e .
The a n im a l was q u i c k l y r e l e a s e d a n d th e
tim e i n t e r v a l b e tw e e n r e l e a s e and c o m p l e te s e p a r a t i o n fro m t h e r o d was
r e c o r d e d to t h e n e a r e s t t e n t h o f a s e c o n d .
28
RESULTS
SCIATIC NERVE
M y elin a te d p e r i p h e r a l n e rv e f i b e r s , m y e lin , and m y elin pro d u cin g
Schwann c e l l s i n e n d r i n e x p o s e d a n i m a l s a p p e a r e d s i m i l a r t o t h o s e i n
c o n t r o l a n i m a l s a t t h e l i g h t ( F i g u r e s ■4—7 ) and e l e c t r o n m i c r o s c o p i c
l e v e l s ( F i g u r e 8 ) a n d r e s e m b l e d t h o s e d e s c r i b e d a s n o r m a l i n th e
l i t e r a t u r e ( P e t e r s e t a i . , 1 9 7 6 ; Landon a n d H a l l , 1 9 7 6 ) . I n b o t h c o n t r o l
a n d e x p e r i m e n t a l t i s s u e , p r e p a r a t i v e a r t i f a c t was commonly o b s e r v e d a s
f o c a l i n t e r p e r i o d s w e l l i n g s w i t h i n t h e m y e l i n ( F i g u r e s 8 and 9 ) .
U n m y e lin a te d p e r i p h e r a l n e r v e f i b e r s from c o n t r o l a n i m a l s
a p p e a r e d s i m i l a r t o n o rm a l f i b e r s d e s c r i b e d i n t h e l i t e r a t u r e (O c h o a ,
1976; P e t e r s e t a l . , 1 9 7 6 ) .
T hese f i b e r s w ere fo u n d t h r o u g h o u t th e
e n t i r e p r o s s - s e c t i o n a l a r e a o f t h e s c i a t i c n e r v e an d t h e a c t u a l number
o f u n m y e l i n a t e d a x o n s a lw a y s o u tn u m b e r e d t h e m y e l i n a t e d f i b e r s .
The
u n m y e l i n a t e d a x o n s w ere g e n e r a l l y 0 . 5 - 0 . 6 urn i n d i a m e t e r w i t h an
a x o n a l membrane (axolem m a) 7 -8 nm t h i c k .
The a x o n a l c y t o p l a s m
( a x o p l a s m ) c o n t a i n e d a n a c c u m u l a t i o n o f m i c r o t u b u l e s ( 2 0 - 2 5 urn i n
d i a m e t e r ) , n e u r o f i l a m e n t s ( 1 0 urn i n d i a m e t e r ) , m i t o c h o n d r i a , and
s p a r c e sm ooth e n d o p la s m ic r e t i c u l u m ( F i g u r e s 8 , 10, 1 1 ) . %he
m i t o c h o n d r i a w ere o f t e n t h i n a n d e l o n g a t e i n l o n g i t u d i n a l s e c t i o n an d
a l m o s t ro u n d i n c r o s s s e c t i o n ( F i g u r e s 8 & 1 0 ) . They w e re o f t e n
s e v e r a l m ic ro m e te rs lo n g and c o n ta in e d c r i s t a e
th a t ty p ic a lly
29
p a r a l l e l e d th e l e n g t h o f th e ax o n .
The sm ooth e n d o p la s m ic r e t i c u l u m
was l o c a t e d b e tw e e n th e. m i c r o t u b u l e s a n d n e u r o f i l a m e n t s a n d was
u s u a l l y r e p r e s e n t e d by a fe w i r r e g u l a r v e s i c l e s o r t u b u l e s t h a t a l s o
p a r a l l e l e d th e l e n g t h of th e axon.
W hile v e s i c l e s i n u n m y e l i n a t e d
axons a re n o t t y p i c a l l y d e s c rib e d i n th e l i t e r a t u r e o th e r th an n e a r
t h e axon t e r m i n a l , o c c a s i o n a l v e s i c l e s w ere o b s e r v e d i n u n m y e l i n a t e d
f i b e r s fro m c o n t r o l a n i m a l s i n t h i s s t u d y .
Such v e s i c l e s w ere ro u n d
to h e x a g o n a l i n s h a p e , 0 . 1 - 0 . 2 urn i n d i a m e t e r , a n d t h e y g e n e r a l l y
o c c u rre d in groups of 2 or 3.
U n m y e li n a te d p e r i p h e r a l a x o n s w ere i n v e s t e d by Schwann c e l l
c y t o p l a s m , a n d i t was n o t uncommon t o f i n d 10 -1 5 a x o n s p e r Schwann
c e l l ( F i g u r e s 8 & 1 0 ) . The Schwann c e l l c y to p l a s m c o n t a i n e d a v a r i e t y
o f c e l l u l a r o r g a n e l l e s , m o s t o f w h ic h w ere n e a r t h e p e r i n u c l e a r a r e a . .
T y p i c a l l y m i c r o t u b u l e s , n e u r o f i l a m e n t s , a n d m i t o c h o n d r i a w e re s e e n
e lsew h ere i n tr a n s v e r s e s e c t i o n s .
A b a s a l l a m in a e n c l o s e d t h e Schwann
c e l l ( F i g u r e s 10 and 1 1 ) . C o l l a g e n o u s f i b e r s w ere f r e q u e n t l y
e n c o u n t e r e d a s p a r t o f t h e e n d o n e u riu m b e tw e e n t h e Schwann c e l l
b u n d le s of u n m y elin a te d axons ( F ig u r e s 8 ,
10, 1 1 ) .
C hanges w ere a p p a r e n t i n u n m y e l i n a t e d n e r v e f i b e r s fro m e n d r i n
e x p o s e d a n i m a l s and w ere m o s t o b v i o u s a f t e r 4 days o f e x p o s u r e .
At
t h a t tim e a p p r o x i m a t e l y 40% o f t h e u n m y e l i n a t e d a x o n a l p r o f i l e s
e x a m in ed showed m o r p h o l o g i c a l c h a n g e s t h a t commonly i n c l u d e d
d i s s o l u t i o n o f m ic r o tu b u le s and n e u r o f ila m e n ts , ax o n al s w e llin g and
v e s i c l e ac cu m u la tio n .
I t w as n o t u n u s u a l t o f i n d a l l o f t h e s e c h a n g e s
30
FIGURES 4 - 7 . L i g h t m i c r o g r a p h s o f s c i a t i c n e r v e t i s s u e from 4 a n d 20
day c o n t r o l an d e x p e r i m e n t a l a n i m a l s show ing f i e l d s o f m y e l i n a t e d ( * )
a n d u n m y e l i n a t e d ( u) a x o n s . x l 5 0 0 . FIGURE 4 . 4 day c o n t r o l a n i m a l .
FIGURE 5. 4 day e x p e r i m e n t a l a n i m a l .
Some s w e l l i n g c a n be s e e n i n
u n m y e l i n a t e d a x o n s ( a r r o w s ) . FIGURE 6 . 20 day c o n t r o l a n i m a l . FIGURE
7 . 20 day e x p e r i m e n t a l a n i m a l . N ote t h e s w e l l i n g i n some o f t h e
u n m y elin a te d axons ( a r r o w s ) .
31
FIGURES 8 & 9 . E l e c t r o n m i c r o g r a p h s o f s c i a t i c n e r v e from 4 day
c o n t r o l an d e x p e r i m e n t a l a n i m a l s show ing f i e l d s o f m y e l i n a t e d ( * ) and
u n m y e l i n a t e d ( u ) a x o n s a n d t h e i r a s s o c i a t e d Schwann c e l l s ( s ) . A r e a s
o f i n t e r p e r i o d s w e l l i n g ( p) c a n be s e e n i n some m y e l i n s h e a t h s .
FIGURE 8 . C o n t r o l a n i m a l .
x l 6 , 4 0 0 . FIGURE 9 . E x p e r i m e n t a l a n i m a l .
N o te t h e s w o l l e n ax o n ( a ) a n d t h e p o c k e t s o f v e s i c l e s ( o p e n a r r o w s ) .
x l8 ,2 0 0 .
32
FIGURES 10 & 1 1 . E l e c t r o n m i c r o g r a p h s o f s c i a t i c n e r v e from 4 day
c o n t r o l a n im a ls . A norm al a c c u m u la tio n o f m ic r o t u b le s ( t ) ,
n e u r o f i l a m e n t s ( f ) , a n d m i t o c h o n d r i a (m) c a n be s e e n w i t h i n t h e
a x o p la s m . N o te t h e Schwann c e l l c y to p l a s m ( c ) be tw e en t h e a x o n s an d a
b a s a l l a m in a e n c i r c l i n g t h e Schwann c e l l ( b ) . FIGURE 1 0 . X 3 5 ,0 0 0 .
FIGURE 1 1 . s = Schwann c e l l n u c l e u s . x 4 0 , 0 0 0 .
33
o c c u r r i n g w i t h i n t h e same axon o r a s s o c i a t e d g r o u p o f a x o n s . S i m i l a r
c h a n g e s w ere o b s e r v e d , a l t h o u g h l e s s f r e q u e n t l y , a f t e r 7, .1 4 , a n d 20 d
o e a n d a f t e r 14 d o r ,
c o n tro ls.
At 92 d o r , m o s t a x o n s a p p e a r e d s i m i l a r to
T hese v a r i a t i o n s i n u l t r a s t r u c t u r e c o u l d n o t be d e t e c t e d a t
th e l i g h t m i c r o s c o p i c l e v e l e x c e p t f o r o c c a s i o n a l s w e l l i n g o f
u n m y elin a te d axons ( F ig u r e s 4 - 7 ) .
The d i s s o l u t i o n o f m i c r o t u b u l e s a n d n e u r o f i l a m e n t s w as f r e q u e n t l y
o b s e r v e d i n a l l e x p e r i m e n t a l a n i m a l s s a c r i f i c e d up t o 14 d o r .
The
n o rm a l a r r a n g e m e n t o f t h e m i c r o t u b u l e s a n d n e u r o f i l a m e n t s w i t h i n t h e .
a x o p la sm was r e p l a c e d by a n i r r e g u l a r d i s p e r s i o n o f a g g r e g a t e d
f i l a m e n t o u s m a t e r i a l t h r o u g h o u t t h e a x o p la s m o r a l o n g t h e p e r i p h e r y o f
t h e axon ( F i g u r e s 9, 1 2 - 1 7 ) . T hese f i l a m e n t o u s " a g g r e g a t e s " c o u l d n o t
be s p e c i f i c a l l y i d e n t i f i e d a s t o t h e i r o r i g i n n o r was i t p o s s i b l e to
d e te rm in e i f
t h e r e was a s p e c i f i c s e q u e n c e t o t h e d i s s o l u t i o n ( t u b u l e s
versu s fila m e n ts ).
c o n ta in v e ry l i t t l e ,
I t was n o t uncommon f o r m a r k e d ly s w o l l e n a x o n s to
i f a n y , o f t h i s m a t e r i a l ( F i g u r e s 1 2 , 13, 1 7 ) .
A xonal s w e l l i n g was a c o n s i s t e n t f i n d i n g i n e x p e r i m e n t a l a n i m a l s
fro m 4 d o e
t o 14 d o r / a n d was m o st o b v i o u s a f t e r 14 d o e ( F i g u r e s
24 a n d 2 5 ) . The s w e l l i n g was o f t e n a c c o m p a n ie d by d i s s o l u t i o n o f
m i c r o t u b u l e s a n d n e u r o f i l a m e n t s ( F i g u r e s 1 2 - 1 4 , 1 7 , 19) a n d f r e q u e n t l y
i n v o l v e d nu m ero u s a x o n s e n s h e a t h e d w i t h i n t h e same Schwann c e l l
( F i g u r e s 9 , 12, 1 3 ) . I n some c a s e s , l o n g i t u d i n a l s e c t i o n s r e v e a l e d
a lte rn a tin g
s w o l l e n a n d c o n s t r i c t e d r e g i o n s w i t h i n t h e same a xon w i t h
th e c o n s t r i c t i o n s o f te n c o n ta in in g dense a c c u m u la tio n s o f fila m e n to u s
34
FIGURE 1 2 . S c i a t i c n e r v e from a 4 day e x p e r i m e n t a l a n im a l showing
v a r i o u s e x a m p le s o f p a t h o l o g y i n u n m y e l i n a t e d a x o n s . N ote t h e
d i s s o l u t i o n o f m ic r o tu b u le s and n e u r o f ila m e n ts w ith in th e sw o lle n
a x o n s ( a ) , t h e a c c u m u l a t i o n s o f v e s i c l e s ( v ) and t h e s w o l l e n SER (o p en
a r r o w s ) . P a r t o f one axon shows a c o n s t r i c t e d r e g i o n w i t h a h i g h
d e n s i t y o f m i c r o t u b u l e s an d n e u r o f i l a m e n t s ( a r r o w ) . x 2 0 , 000.
35
FIGURES 13 & 1 4 . S c i a t i c n e r v e from 4 day e x p e r i m e n t a l a n i m a l s .
FIGURE 1 3 . N ote t h e s w o l l e n a x o n s ( a ) an d a c c u m u l a t i o n s o f v e s i c l e s ,
x l 6 ,0 0 0 .
FIGURE 1 4 . A s w o l l e n a x o n ( a ) an d a d j a c e n t c o n s t r i c t i o n
(a rro w ) w ith a dense a c c u m u la tio n o f m ic r o tu b u l e s and n e u r o f i l a m e n t s .
x 2 8 ,0 0 0 .
36
FIGURES 1 5 - 1 8 . S c i a t i c n e r v e from e x p e r i m e n t a l a n i m a l s show ing
e x a m p le s o f v e s i c u l a t i o n a s s o c i a t e d w i t h u n m y e l i n a t e d a x o n s . N ote t h e
a c c u m u l a t i o n s o f v e s i c l e s a n d t h e i n v a g i n a t e d axolemma ( a r r o w s ) . FIGURE
1 5 . 4 day a n i m a l .
X 2 4 ,0 0 0 . FIGURE 1 6 . 4 day a n i m a l . x 3 2 , 0 0 0 . FIGURE
1 7 . 4 day a n i m a l .
x 4 0 , 0 0 0 . FIGURE 1 8 . 7 day a n i m a l . x 2 8 ,0 0 0 .
37
FIGURE 1 9 . S c i a t i c n e r v e from a 4 day e x p e r i m e n t a l a n i m a l . N ote th e
l o c a t i o n o f t h e v e s i c l e s ( t r i a n g l e s ) , t h e membranous t r a n s e c t i o n o f
t h e a xon ( a r r o w ) a n d th e a x o n a l v e s i c l e ( o p e n a r r o w ) . x l 6 , 4 0 0 .
I
38
m a t e r i a l ( F i g u r e s 12 , 14» 2 1 ) . Many o f t h e s w o l l e n r e g i o n s w e re a s
l a r g e a s 2 . 5 - 3 . 0 um i n d i a m e t e r ( F i g u r e s 12 , 13, 1 6 , 2 4 , 2 5 ) .
Membrane bound v e s i c l e s w e re o f t e n o b s e r v e d i n a s s o c i a t i o n w i t h
a ffe c te d axons.
P o c k e ts o r a g g re g a te s o f d e n s e ly a c cu m u la te d v e s i c l e s
w ere o f t e n s e e n i n t h e Schwann c e l l c y t o p l a s m o r i n t h e a d j a c e n t
p e r i a x o n a l s p a c e ( F i g u r e s 9, 12, 13, 1 5 - 1 9 , 2 1 , 25 , 2 6 , 2 8 ) . Such
p o c k e t s o f v e s i c l e s f r e q u e n t l y i n v a g i n a t e d i n t o t h e axolemma a n d / o r
Schwann c e l l membrane ( F i g u r e s 9 , 12 , 13 , 1 5 - 1 7 , 2 1 , 2 8 , 3 0 ) . The
num ber o f s u c h v e s i c l e s i n t h e s e p o c k e t s r a n g e d from a s few a s 3-4 t o
a s many a s 40 o r m o re .
The v e s i c l e s w e re 0 . 1 - 0 . 2 um i n d i a m e t e r ,
ro u n d , o v a l, o r hexagonal i n s h a p e , and c o n ta in e d an e l e c t r o n l u c id
m a t r i x ( F i g u r e s 1 2 - 1 7 ) . An o c c a s i o n a l v e s i c l e c o n t a i n e d a c o r e
m a te r ia l o f in te r m e d ia te e l e c t r o n d e n s ity ( F igp^es 9,
15, 18, 2 8 ) .
O t h e r s h a d a c l e a r c o r e an d w ere i n v e s t e d by a t h i c k e l e c t r o n dense
" h a l o " o f m a t e r i a l ( F i g u r e s 15 and 1 6 ) . The p o c k e t s o f v e s i c l e s
o c c u r e d s i n g l y ( F i g u r e s 9 , 1 5 - 1 7 ) , a s s e v e r a l p o c k e t s w i t h i n t h e same
axon ( F i g u r e 1 3 ) , o r a s m u l t i p l e s w i t h i n t h e same a s s o c i a t e d g ro u p o f
a x o n s ( F i g u r e s 12 , 15 , 1 6 ) . Some a c c u m u l a t i o n s o f t h e s e v e s i c l e s
a lm o st c o m p le te ly f i l l e d
t h e a x o n a l p r o f i l e o r a r e a t h a t a p p e a r e d to
h a v e p r e v i o u s l y c o n t a i n e d a n axon ( F i g u r e 1 8 ) . I n o t h e r e x a m p le s t h e
v e s i c l e s w ere p r e s e n t i n row s b e s i d e t h e a x o n b u t a p p e a r e d t o be
l o c a t e d e i t h e r w i t h i n t h e p e r i a x o n a l s p a c e o r w i t h i n t h e Schwann c e l l
c y to p l a s m ( F i g u r e s 19, 21» 2 5 ) . T hese v e s i c l e s w ere a l s o 0 . 1 - 0 . 2 um i n
d iam e te r b u t had an e le c tr o n l u c i d c o re o c c a s io n a lly c o n ta in in g
39
FIGURES 20 & 2 1 . S c i a t i c n e r v e from 4 day e x p e r i m e n t a l a n i m a l s .
FIGURE 2 0 . An e x a m p le o f a n a c c u m u l a t i o n o f a x o n a l v e s i c l e s .
x36 ,0 0 0 .
FIGURE 2 1 . Numerous e x a m p le s o f e n d r i n i n d u c e d c h a n g e s
( a r r o w s ) w i t h i n a g r o u p o f a s s o c i a t e d a x o n s . x 2 8 ,0 0 0 .
40
FIGURES 2 2 - 2 4 . S c i a t i c n e r v e from e x p e r i m e n t a l a n i m a l s show ing v a r i o u s
e x a m p le s o f v a c u o l a t i o n o f u n m y e l i n a t e d a x o n s . FIGURE 2 2 . 4 day
a n im a l.
N ote t h e s m a l l v e s i c l e s w i t h i n t h e v a c u o l e ( a r r o w ) x 2 8 ,0 0 0 .
FIGURE 2 3 . 4 day a n i m a l . N ote t h e a c c u m u l a t i o n o f v e s i c l e s w i t h i n t h e
v a c u o l e an d a d j a c e n t axon ( a ) . x 2 4 , 0 0 0 .
FIGURE 2 4 . 14 day a n i m a l .
N ote t h e c l e a r v a c u o l a t e d a r e a ( c ) w h ic h a p p e a r s to d i s p l a c e t h e axon
( a ) . The axolemma a p p e a r s i n v a g i n a t e d ( a r r o w ) . x 2 0 , 000.
41
FIGURES 2 5 - 2 7 . S c i a t i c n e r v e from e x p e r i m e n t a l a n i m a l s . FIGURE 2 5 . 14
day a n i m a l . N ote t h e t r a n s e c t i o n o f t h e a x o n a l p r o f i l e a n d t h e
i n v a g i n a t i o n o f t h e axolemma ( a r r o w ) . A s m a l l v a c u o l e ( c ) w h ic h
c o n t a i n s v e s i c l e s ( open a r r o w s ) a n d o t h e r d e b r i s c a n be s e e n c ro w d in g
t h e axon p r o f i l e . x l 5 , 5 0 0 . FIGURE 2 6 . 4 day a n i m a l .
N ote th e
s w o l l e n m em branous p r o f i l e ( a r r o w ) w i t h i n t h e a x o n . x 2 4 , 0 0 0 . FIGURE
2 7 . 20 day a n i m a l .
N ote t h e s w o l l e n m em branous p r o f i l e ( a r r o w ) w i t h i n
a n a p p a r e n t a x o n a l a r e a . x 3 6 , 000.
42
FIGURES 28 & 2 9 . S c i a t i c n e r v e from e x p e r i m e n t a l a n i m a l s . FIGURE 2 8 .
34 day a n i m a l . N ote t h e c l e a r v a c u o l a t e d a r e a c o n t a i n i n g s m a l l
v e s i c l e ( c ) w i t h i n t h e a r e a p r e v i o u s l y o c c u p i e d by t h e a x o n .
The axon
a p p e a r s to be d i s p l a c e d ( a r r o w ) . An a d j a c e n t a x o n ( a ) i s s w o l l e n and
t h e axolemma i s i n v a g i n a t e d by a p o c k e t o f v e s i c l e s ( o p e n a r r o w ) .
x l6 ,4 0 0 .
FIGURE 2 9 . 112 day a n i m a l . The u n m y e l i n a t e d ( u ) and
m y e l i n a t e d ( * ) a x o n s g e n e r a l l y a p p e a r n o rm a l e x c e p t f o r a n a c c a s i o n a l
c l e a r v e s i c l e . x 2 4 ,0 0 0 .
43
FIGURE 3 0 . S c i a t i c n e r v e fro m a 112 day e x p e r i m e n t a l a n i m a l . N ote
t h a t m o s t u n m y e l i n a t e d a x o n s a p p e a r n o rm a l a l t h o u g h two a x o n s ( a ) a r e
s w o l l e n a n d a r e i n v a g i n a t e d by p o c k e t s o f v e s i c l e s ( a r r o w s ) . x 2 0 ,0 0 0 .
44
i s o l a t e d a c c u m u l a t i o n s o f e l e c t r o n d e n s e m a t e r i a l ( F i g u r e s 19 and 2 5 ) .
The v e s i c l e s w i t h i n t h e axon w ere e n c o u n t e r e d l e s s f r e q u e n t l y t h a n
th o se i n o th e r l o c a t io n s .
The v e s i c l e s w e re o f t e n o f i r r e g u l a r shape
w ith a th ic k rin g of in te rm e d ia te e le c tr o n d e n s ity su rro u n d in g a c le a r
c o r e ( s i m i l a r to t h o s e p r e v i o u s l y d e s c r i b e d ) .
They w e re a p p r o x i m a t e l y
0 . 2 - 0 . 4 urn i n d i a m e t e r a n d o c c u r r e d s i n g l y o r i n g r o u p s ( F i g u r e s 9,
2 0 , 23, 2 4 ) . I t was n o t uncommon f o r a p a r t i c u l a r ax o n o r g r o u p o f
a x o n s to e x h i b i t v a r i o u s c o m b i n a t i o n s o f t h e s e t y p e s o f v e s i c l e
a c c u m u l a t i o n s ( F i g u r e s 9 and 2 1 ) .
O c c a s i o n a l l y l a r g e membrane bound c l e a r v a c u o l e s w e re o b s e rv e d ,
w ith in th e axon.
Such v a c u o l e s o f t e n o c c u p i e d a l a r g e p a r t o f th e
a x o n a l p r o f i l e , commonly d i s p l a c i n g much o f t h e ax o n ( F i g u r e s 2 2 - 2 4 ) .
I n some i n s t a n c e s t h e s e v a c u o l e s c o n t a i n e d s m a l l c i r c u l a r v e s i c l e s
(F ig u re 2 3 ), a c c u m u la tio n s o f i r r e g u l a r l y shaped v e s i c l e s , o r o th e r
d e b r i s o f unknown o r i g i n ( F i g u r e s 24 a n d 25) w i t h i n a c l e a r ,
a p p a re n tly w a te ry , m a tr ix .
The s o u r c e o f t h e membrane e n c l o s i n g su c h
v a c u o l e s c o u l d n o t be d e t e r m i n e d , b u t i n some c a s e s i t a p p e a r e d to
i n c l u d e t h e axolemma ( F i g u r e s 24 a n d 2 5 ) .
Som etim es a t h i n "membrane" a p p e a r e d t o t r a v e r s e t h e e n t i r e
s w o l l e n a x o n a l p r o f i l e ( F i g u r e s 19 an d 2 5 ) and a p p e a r e d t o be a n
i n f o l d i n g o f t h e axolemma.
S w e l l in g o f t h e smooth e n d o p l a s m i c
r e t i c u l u m ( F i g u r e 12) and m embranous p r o f i l e s ( F i g u r e s 26 and 2 7 ) w ere
i n f r e q u e n t l y e n c o u n te re d i n a f f e c t e d axons.
The a p p e a r a n c e o f th e
m i t o c h o n d r i a i n u n m y e l i n a t e d a x o n s fro m e x p e r i m e n t a l a n i m a l s a p p e a r e d
45
sim ila r
to c o n t r o l s i n a l l
t i s s u e s e x a m in e d ( F i g u r e s 9 ,
1 4 -1 6 )
By 14 d o r much o f t h e t i s s u e a p p e a r e d t o r e s e m b l e t h a t from
c o n t r o l a n i m a l s a l t h o u g h c h a n g e s s i m i l a r to t h o s e e n c o u n t e r e d a t
e a r l i e r t i m e s w ere s t i l l p r e s e n t .
T h e se i n c l u d e d a x o n a l s w e l l i n g ,
d i s s o l u t i o n o f m i c r o t u b u l e s a n d n e u r o f i l a m e n t s and t h e a p p e a r a n c e o f
a c c u m u l a t i o n s o f membrane bound v e s i c l e s .
I n one i n s t a n c e a n a r e a
t h o u g h t to h a v e p r e v i o u s l y c o n t a i n e d a n axon was c o m p l e t e l y d e v o id o f
a x o p la s m ic m a t e r i a l a n d c o n t a i n e d o n l y s m a l l v e s i c l e s t h a t a p p e a r e d to
be e x t r u d i n g from th e a r e a ( F i g u r e 2 8 ) . I t i s p o s s i b l e t h a t t h e
axolemma was i n f o l d e d i n t h i s a r e a due t o t h e p r e s e n c e o f a l a r g e
v a c u o le th a t h a s d isp la c e d th e axon.
By 92 d o r ,
m o st t i s s u e from t h e e x p e r i m e n t a l a n i m a l s was
s i m i l a r to t h a t i n c o n t r o l s ( F i g u r e 29) a l t h o u g h i s o l a t e d i n s t a n c e s o f
a x o n a l s w e l l i n g a n d v e s i c l e a c c u m u l a t i o n w ere s t i l l p r e s e n t ( F i g u r e
3 0 ) . I t c o u l d n o t be d e t e r m i n e d i f p e r m a n e n t a x o n a l d e g e n e r a t i o n had
t a k e n p l a c e a l t h o u g h t h e r e a p p e a r e d to be no i n d i c a t i o n o f s u c h .
!
I
■
‘
■
'
'
OTHER TISSUES
L i g h t and e l e c t r o n m i c r o s c o p i c e x a m i n a t i o n o f s k e l e t a l ( g l u t e a l )
m u s c l e , c a r d i a c m u s c le a n d o p t i c n e r v e from a n i m a l s a f t e r 4 a n d 20 d o
e r e v e a l e d no o b v i o u s m o r p h o l o g i c a l c h a n g e s when c o m pared t p t h a t o f
c o n tro l tis s u e (F ig u re s 3 1 -39).
46
FIGURE 3 1 - 3 3 » L i g h t a n d e l e c t r o n m i c r o g r a p h s o f s k e l e t a l m u s c l e from
20 day a n i m a l s . N ote t h e r e s e m b le n c e o f t h e r e d ( r ) an d w h i t e (w)
m u s c l e f i b e r s i n e x p e r i m e n t a l t i s s u e t o t h o s e i n t h e c o n t r o l s . FIGURE
3 1 . C o n t r o l a n i m a l . xlSOO. FIGURE 3 2 . E x p e r i m e n t a l a n i m a l . x l 5 0 0 .
FIGURE 3 3 . Normal a p p e a r i n g t i s s u e fro m a n e x p e r i m e n t a l a n i m a l .
Note
t h e d e n s e a c c u m u l a t i o n o f m i t o c h o n d r i a (m) and m y o f i l a m e n t s ( a r r o w s )
i n t h i s r e d m uscle f i b e r .
x 2 6 ,6 0 0 .
47
FIGURES 3 4 - 3 6 . L i g h t an d e l e c t r o n m i c r o g r a p h s o f c a r d i a c m u s c l e from
20 day a n i m a l s . N ote t h e r e s e m b le n c e o f t h e e x p e r i m e n t a l t i s s u e to
th a t of c o n tr o ls .
Numerous b l o o d v e s s e l s ( b) c a n be s e e n t h r o u g h o u t
th e f i e l d .
FIGURE 3 4 . C o n t r o l a n i m a l . N ote t h e d e n s e a c c u m u l a t i o n o f
m i t o c h o n d r i a w i t h i n t h e m u s c l e b u n d l e s ( a r r o w s ) , x l 500. FIGURE 3 5 .
E x p e rim en ta l a n im a l.
x l 5 0 0 . FIGURE 3 6 . E x p e r i m e n t a l a n i m a l .
The
m ito c h o n d ria (m ), m y o fila m en ts (o ) and t - t u b u le s (a rro w s) a p p e a r
n o rm a l an d g l y c o g e n i s p r o m i n e n t . x 3 3 , 300.
48
FIGURES 3 7 - 3 9 . L i g h t a n d e l e c t r o n m i c r o g r a p h s o f o p t i c n e r v e from 20
day a n i m a l s . The f i e l d c o n t a i n s n u m ero u s m y e l i n a t e d a x o n s ( * ) and
g l i a l c e l l p r o c e s s e s ( open a r r o w s ) . FIGURE 3 7 . C o n t r o l a n i m a l .
x l 5 0 0 . FIGURE 3 8 . E x p e r i m e n t a l a n i m a l . x ! 5 0 0 . FIGURE 3 9 .
E x p e rim en ta l a n im a l.
Some p r e p a r a t i v e a r t i f a c t c a n be s e e n i n t h e
fo rm o f i n t e r p e r i o d s w e l l i n g i n t h e m y e l i n s h e a t h s ( a r r o w s ) . x 3 6 ,0 0 0 .
49
BEgAVIORAL/NEUROLOGICAL EFFECTS
E x p e r i m e n t a l a n i m a l s a p p e a r e d h y p e r a c t i v e w i t h i n 24 h o u r s a f t e r
t h e f i r s t i n j e c t i o n an d t h i s symptom c o n t i n u e d t h r o u g h o u t t h e e x p o s u r e
p h a s e o f t h e e x p e r i m e n t a n d f o ? o v e r 2 w eeks i n t o t h e r e c o v e r y p h a s e .
H y p e r a c t i v i t y was m o s t o b v i o u s 4-5 h o u r s a f t e r e a c h i n j e c t i o n and
t e n d e d t o d i m i n i s h s l i g h t l y o v e r tim e u n t i l t h e n e x t i n j e c t i o n .
Such
b e h a v i o r was c h a r a c t e r i z e d by a n i m a l s r u n n i n g c o n s t a n t l y a b o u t t h e
c a g e a n d r e g u l a r l y c l i m b i n g t h e w i r e c a g e l i d a n d m oving b a c k and
f o r t h o r c i r c l i n g on i t w h i l e h a n g in g u p s id e - d o w n .
In a d d itio n ,
d u r i n g t h e tim e t h e a n i m a l s w ere b e i n g i n j e c t e d o r w e i g h e d , a n i m a l s
c lim b e d o u t o f t h e c a g e a t e v e r y o p p o r t u n i t y .
H ow ever, when t h e
a n i m a l s w ere p l a c e d i n a n i s o l a t e d s e t t i n g , u s u a l l y f o l l o w i n g h a n d l i n g
o r a f t e r t h e i r i n i t i a l r e s p o n s e to a s t a r t l e s t i m u l u s , t h e y e x h i b i t e d
a h y p o a c tiv e and tim id b e h a v io r.
s t a t i o n a r y f o r 1 0 -1 5 s e c o n d s .
Such a n i m a l s o f t e n t e n d e d to r e m a in
I t w as n o t uncommon f o r a n i m a l s to
r e m a in t h i s way u n t i l f o r c e d to m ove, a t w h ic h tim e t h e y e x h i b i t e d
o n ly a tim id e x p lo r a to r y b e h a v io r.
F r e q u e n tly th e a n im a ls d is p la y e d a
Change i n p o s t u r e c h a r a c t e r i z e d by a h u n c h in g o f t h e i r t h o r a c i c s p i n e ,
b o th w h ile s t a t i o n a r y , and w h ile w a lk in g .
T his s t a t i o n a r y a c t i v i t y
a n d p o s t u r a l c h a n g e was o b v i o u s a f t e r 6 d o e and was s t i l l e v i d e n t
a f t e r 14 d o r .
P i l o e r e c t i o n was a p p a r e n t i n , 35% o f t h e e x p e r i m e n t a l a n i m a l s by
16 d . o . e . a n d a s i m i l a r p e r c e n t a g e o f a n i m a l s r e m a in e d so. t h r o u g h o u t
th e rem a in d e r o f th e e x p o su re p e r i o d .
50
The g a i t o f e x p e r i m e n t a l a n i m a l s a p p e a r e d s i m i l a r to c o n t r o l s
u n t i l 13 d o e when some a n i m a l s e x h i b i t e d a g e n e r a l s p l a y i n g o f t h e
h in d lim b s.
T h i s was m o s t o b v i o u s a t 17 d o e when a p p r o x i m a t e l y 65%
o f t h e a n i m a l s w ere i n v o l v e d , b u t d e c r e a s e d t o 28% by 18 d o e , 23% a t
20 d o e a n d c o n t i n u e d t o d e c l i n e u n t i l t h e g a i t a p p e a r e d n o rm a l i n
a l l a n i m a l s by 14 d o r .
g a i t a b n o rm a lity .
C o n tro l a n im a ls n ev er d is p la y e d any s ig n s o f
An e r e c t t a i l p o s t u r e was o b s e r v e d i n e x p o s e d
a n i m a l s a n d was c o n s i s t e n t l y n o t e d b e tw e e n I a n d 15 d o e .
The t a i l
i n t h e s e a n i m a l s w as s t i f f a n d se ld o m c o n t a c t e d t h e g r o u n d w h e r e a s th e
t a i l o f c o n t r o l a n i m a l s was f l a c c i d a n d i n c o n t a c t w i t h t h e w a lk in g
su rface.
T re m o rs o r c o n v u l s i o n s w e re n e v e r o b s e r v e d i n e i t h e r c o n t r o l o r
ex p erim en tal a n im a ls.
h icc u p s.
Two a n i m a l s d e v e l o p e d w h a t a p p e a r e d t o be
I n one a n im a l t h e s e " h i c c u p s " w ere o b v i o u s a t 19 d o e , 12 d
o r a n d 29 d o r ,
a t w h ic h tim e i t was n o t e d t h a t t h e a n i m a l a p p e a r e d
v e r y w ith d r a w n an d h y p o a c t i v e .
The o t h e r a n im a l e x h i b i t e d s i m i l a r
b e h a v i o r a f t e r 14 d o r a n d d e v e l o p e d s h a k e s o f s h o r t d u r a t i o n
im m ed ia te ly fo llo w in g th e " h ic c u p " ,
C o n t r o l a n i m a l s showed v e r y l i t t l e
re s p o n s e to s t a r t l e s t i m u l i ,
commonly o n l y r e a c t i n g w i t h a s l i g h t b l i n k i n g o f t h e i r e y e s .
The
number o f a n i m a l s e x p o s e d t o e n d r i n t h a t d i s p l a y e d a h y p e r a c t i v e
s t a r t l e r e a c t i o n i n c r e a s e d d u rin g th e e x p o s u re s .
t h e a n i m a l s w e re h y p e r - r e s p o n s i v e a n d by 17 d o e ,
A fter I d o e
88% w e re
31% o f
51
h y p er-resp o n siv e.
T h a t p e r c e n t a g e d e c l i n e d t o 25% by 29 d o r ,
f i n a l l y a l l a n i m a l s r e s e m b l e d c o n t r o l s by 92 d o r .
an d
I n e x tre m e
i n s t a n c e s a p u f f o f a i r d i r e c t e d a t a n i m a l s i n t h e i r op en c a g e s c a u s e d
t h e a n i m a l s to jump c o m p l e t e l y o u t o f t h e 6 i n c h h i g h c a g e .
The r e s u l t s o f t h e t a i l f l i c k t e s t a r e sum m arized i n F i g u r e 40 .
T h e re a p p e a r s t o be no s i g n i f i c a n t o r c o n s i s t e n t t r e n d t o t h e d a t a .
Only a f t e r 8 ,
12 a n d 20 d o e
(S tu d e n t t - t e s t )
was t h e r e a s t a t i s t i c a l l y
sig n ific a n t
d i f f e r e n c e b e tw e e n c o n t r o l s a n d e x p e r i m e n t a l s an d i n
a l l t h r e e c a s e s t h e e x p e r i m e n t a l a n i m a l s r e s p o n d e d m ors q u i c k l y t h a n
c o n tro ls.
R e s u l t s o f t h e r o d t e s t a r e sum m arized i n F i g u r e 4 1 . The
i n a b i l i t y o f e x p e r i m e n t a l a n i m a l s to m a i n t a i n t h e i r p o s i t i o n on t b e
f i b e r g l a s s r o d was a c o n s i s t e n t f i n d i n g .
The num ber o f a n i m a l s
i m m e d i a t e l y f a l l i n g o f f t h e r o d w as much g r e a t e r i n t h e e x p e r i m e n t a l
g r o u p t h a n i n c o n t r o l s fro m I t o 4 d o e
a n d t h e n a g a i n on d ay s 8 - 2 0 .
A f t e r day 1 3 , none o f t h e c o n t r o l s f e l l o f f i m m e d i a t e l y .
By day 15
no n e o f t h e e x p e r i m e n t a l a n i m a l s c o u l d m a i n t a i n t h e i r p o s i t i o n on t h e
r o d a l t h o u g h some c o u l d s t i l l g r a s p t h e r o d a n d h a n g t h e r e u n t i l
fin a lly fa llin g
o ff.
By day 16, 56% o f t h e e x p e r i m e n t a l a n i m a l s f e l l
o f f th e ro d im m e d ia te ly .
p l a c e d on t h e r o d .
Most e x p e r i m e n t a l a n i m a l s t r e m b l e d ' when •
By 14 d o r ,
m ost o f th e e x p e rim e n ta l an im a ls
c o u l d m a i n t a i n t h e i r b a l a n c e on t h e r o d f o r 2 - 3 s e c o n d s b e f o r e f a l l i n g
o f f a n d by 29 d o r a l l a n i m a l s t e s t e d h a d no t r o u b l e m a i n t a i n i n g
t h e i r p o s i t i o n on t h e r o d f o r up t o 10 s e c o n d s w hich was t h e d u r a t i o n
2 .4
2.2
2
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Z S ZN ZN
ZN
ZS
ZN ZN ZN z s ZN
Z S Z N ZN ZN ZN Z S ZN
Z S ZN
ZN ZN Z S Z N
Controls
FIGURE 4 0 . Summary o f
th e
sig n ific a n t d iffe re n c e .
7
Z
Z
Z
Z
Z
Z^
ZS
ZS
ZS
ZS
ZS
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ZS
ZN
ZN
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ZN
11
g
te st.
12
13
14
15
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ZN
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16
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r N
ZN
ZN
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ZS
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ZN
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ZS
ZS
ZS
ZS
ZS
ZS
17
y sE
- xperinnentals
* in d ic a te s a s t a t i s t i c a l l y
% falling o ff im m ediately
9
IX /
\
C ontrols
FIGURE 4 1 . Summary o f t h e r o d
a n im a ls f e l l o f f im m ed ia te ly .
10
|
te st.
I I
\
12
13
14
15
16
17
IS
Ex pe r i me n t a l s
No c o n t r o l d a t a i n d i c a t e s t h a t no
19
2 0
21
54
o f th e t e s t .
The p e r c e n t o f c h a n g e i n th e a n i m a l s w e i g h t o v e r t h e 112 day
s t u d y p e r i o d i s d e p i c t e d i n F i g u r e 4 2 . The e x p e r i m e n t a l a n i m a l s do n o t
a p p e a r to g a i n w e i g h t a s r a p i d l y a s t h e c o n t r o l s a l t h o u g h t h e o n ly
s t a t i s t i c a l l y s i g n i f i c a n t (S tu d e n t t - t e s t )
d i f f e r e n c e was on day 20.
T h i r t y - f i v e e x p e r i m e n t a l a n d 15 c o n t r o l a n i m a l s w ere u s e d a t the
s t a r t o f th e s t u d y .
One c o n t r o l a n i m a l , r e p r e s e n t i n g 6.7% o f th e
c o n t r o l a n i m a l s d i e d on day 8 o f th e s t u d y .
E ig h t e n d r i n exposed
a n i m a l s (23%) d ie d d u r in g t h e 20 days o f e x p o s u r e , 5 b e tw e en days 17
a n d 2 0 . T h e re was no m o r t a l i t y i n an y a n i m a l s a f t e r
r e g im e was f i n i s h e d .
th e e x p o s u r e
On day 12, a p p r o x i m a t e l y 4 h o u r s a f t e r
i n c r e a s i n g t h e do sa g e fro m 2 .0 mg/kg t o 4 .0 m g/kg, two a n i m a l s d i e d .
F i g u r e 43 shows th e num ber o f a n i m a l s t h a t d i e d a s a p e r c e n t a g e o f
those re m a in in g .
The g r e a t e s t m o r t a l i t y o c c u r r e d on day 1 8 , when 19%
o f th e a n im a ls d ie d .
P o stm o rte m e x a m i n a t i o n o f dead a n i m a l s r e v e a l e d
no s i g n o f d e a t h due to h e m o rrh a g e c a u s e d by i n j e c t i o n .
I 30
I IO
% o f Initial Weight
I OO
40 -
II2
I/
/ \
C ontrols
FIGURE 4 2 . Animal w e i g h t c h a n g e s .
d iffe re n c e .
Day s
IN Xl E x p e r i m e n t a l s
* in d ic a te s a s t a t i s t i c a l l y
sig n ific a n t
17 16
15 -
% Died
14 12
-
10
-
Days of Exposure
FIGURE 4 3 . Animal m o r t a l i t y .
57
DISCUSSSION
SCIATIC NERVE
E le c tr o n m ic ro s c o p ic s t u d i e s o f p e r i p h e r a l n e rv e i n a n im a ls
e x p o s e d to r e p e a t e d s u b a c u t e d o s e s o f e n d r i n r e v e a l a p a t t e r n o f
d e g e n e r a t i v e c h a n g e s i n u n m y e l i n a t e d a x o n s c h a r a c t e r i z e d by a x o n a l
s w e l l i n g , d i s s o l u t i o n o f m i c r o t u b u l e s a n d n e u r o f i l a m e n t s , a n d th e
a p p e a r a n c e o f v e s i c l e s w i t h i n t h e a x o n a n d a d j a c e n t Schwann c e l l
c y to p la sm .
E n d rin does n o t a p p e a r to c o n tin u o u s ly accu m u late
s i g n i f i c a n t l y w i t h in th e t i s s u e s o f c h r o n i c a l l y exposed a n im a ls (C o le
e t a l . , 1 9 6 8 ) b u t r a t h e r t e n d s to r e a c h p l a t e a u s t o r a g e l e v e l s
( B r o o k s ,1 969). T h is r e s u l t s i n a l i m i t i n g upper l e v e l o f a c c u m u la tio n
t h a t d e c l i n e s w i t h tim e when e x p o s u r e c e a s e s .
A c c o r d in g to Donoso e t
a l .( 1 9 7 9 ) " i t a p p e a r s t h a t , i n i t i a l l y , e n d r i n i s r a p i d l y a b s o r b e d , b u t
a " c o p i n g " m echanism i s d e v e l o p e d t h a t may a l l o w m e t a b o l i s m o r
e x c re tio n o f e n d rin , r e s u l t i n g in a c o n c e n tra tio n drop.
A gradual
i n c r e a s e i n e n d r in f o llo w s , u n t i l th e c o n te n d in g p r o c e s s e s o f u p tak e
a n d e x c r e t i o n r e a c h t h e i r r e s p e c t i v e l e v e l s and a n e q u i l i b r i u m i s
a tta in e d ."
The g r e a t e r am ount o f damage o b s e r v e d a f t e r 4 d a y s o f
e x p o s u r e , may i n d i c a t e t h a t t h e a n i m a l s h a v e h a v e n o t y e t r e a c h e d s u c h
an e q u ilib r iu m and t h a t th e l e v e l s o f e n d rin w ith in th e t i s s u e s
e x c e e d s t h e p l a t e a u l e v e l t h a t w i l l be r e a c h e d l a t e r .
At s u c h a t i m e ,
the c o n c e n t r a t i o n o f th e to x in w i t h in th e t i s s u e s r e a c h e s
a
le v e l th at
58
w i l l n o t be e x c e e d e d u n l e s s t h e d a i l y .d o s e s i s m a r k e d l y i n c r e a s e d .
A
f u r t h e r i n d i c a t i o n o f s u c h a d a p t a t i o n t o a new l e v e l o f e x p o s u r e was
o b s e r v e d a s a h i g h e r f r e q u e n c y o f a x o n a l s w e l l i n g o b s e r v e d on day 14 ,
s h o r t l y a f t e r t h e i n c r e a s e . i n dose from 2 . 0 mg/kg t o 4 . 0 mg/kg on day
12 .
P e r i p h e r a l n e r v e damage, i n d u c e d by c h e m i c a l o r p h y s i c a l m eans i s
o f t e n a c c o m p a n ie d by a x o n a l s w e l l i n g (Dyck an d H o p k i n s , 1 9 7 2 ; Hendelman
and M i r e , 1968; Sima an d R o b e r t s o n , 1979; B ra y e t a l . , 1 9 7 2 ; B ehse e t
a l.,1 9 7 5 ;
S c h l a e p f e r a n d H a s l e r , 1 9 7 9 b ) . The f o c a l d i l a t i o n s and
c o n s t r i c t i o n s r e p o r t e d h e r e a p p e a r n e a r l y i d e n t i c a l to t h o s e r e p o r t e d
by M ire e t a l . ( 1 9 7 0 )
d e p riv a tio n .
fo llo w in g n e rv e t r a n s e c t i o n and n u t r i t i o n a l
P r e v i o u s m o r p h o l o g i c a l s t u d i e s by t h o s e a u t h o r s s u g g e s t
t h a t a n i o n i c i m b a la n c e i n v o l v i n g t h e i o n i c s o d iu m - p o ta s s iu m ATPase
may l e a d t o a b n o rm a l a c c u m u l a t i o n o f a x o n a l sodium i o n s an d w a t e r
l e a d i n g to a x o n a l s w e l l i n g ( Hendelman and M i r e , 1 9 6 8 ; M ire e l
a l.,1 9 7 0 ).
T h i s ATPase s y s te m i s e n e r g y d e p e n d e n t an d r e q u i r e s ATP a s
a su b stra te
to a c h i e v e t h e e x t r u s i o n o f sodipm from th q a x o n .
T h e r e f o r e , a n y p r o c e s s t h a t i n h i b i t s t h e p r o d u c t i o n o f ATP c o u l d l e a d
to s w e llin g .
The s w e l l i n g r e p o r t e d i n t h e p r e s e n t s t u d y c o u l d v e r y
w e l l be t h e r e s u l t o f s u c h a n i o n i c i m b a l a n c e .
f o u n d , i n a x o n s grown i n c u l t u r e ,
M ire e t a l , ( 1 9 7 0 )
t h a t 90-100% o f t h e s w e l l i n g
d is a p p e a re d w i t h in 30-90 m in u te s f o llo w in g e i t h e r r e f e e d in g w ith
n o r m a l medium, s u p p l e m e n t i n g t h e medium w i t h g l u c o s e , o r o p e n in g t h e
c u ltu re
to room a i r .
They s u g g e s t t h a t t h e s e m a n i p u l a t i o n s may l e a d
59
to p r o d u c t i o n o f ATP w i t h i n th e axon r e s u l t i n g i n i n c r e a s e d a c t i v i t y
o f t h e i o n i c s o d iu m - p o ta s s iu m A T Pase. I n a n o t h e r s t u d y ( W e b s t e r and
Am es,1965) d e p r i v a t i o n o f oxygen a n d g l u c o s e
r e s u l te d in o rg a n e lle sw e llin g
t h a t was r e v e r s i b l e o n l y i f
d e p r i v a t i o n was n o t p r o l o n g e d .
c h a n g e s may be r e l a t e d
to i s o l a t e d r a b b i t r e t i n a
su c h
Those a u t h o r s s u g g e s t t h a t such
to th e a v a i l a b i l i t y o f h i g h e n e r g y p h o s p h a te
bonds t h a t may be r e s p o n s i b l e f o r t h e a c t i v e m a i n t e n a n c e o f o s m o l a r i t y
a c r o s s th e m em brane.
reticu lu m ,
T h e . p e r i o d i c s w e l l i n g o f th e smooth e n d o p la s m ic t
t h e s w o l le n membranous p r o f i l e s a n d t h e p r e s e n c e o f
" w a te r y " v a c u o l e s o b s e r v e d i n t h i s s t u d y may be t h e r e s u l t o f su c h
changes i n o s m o la rity r e f l e c t i n g
a v a ila b ility
t h e p o s s i b l e d e c r e a s e i n ATP
i n a n i m a l s e x p o s e d to e n d r i n .
T r a n s e c tio n or c ru s h o f p e r i p h e r a l n e rv e can r e s u l t i n th e
decreased a c t i v i t y
o f i o n i c s o d iu m - p o ta s s iu m ATPase ( B a c h l a r d .and
S i l v a , 1 966). M ito c h o n d ria l ( o lig o m y c in - s e n s i t i v e ) io n ic
m agnesium -ATPase i n t h e r a t b r a i n . i s i n h i b i t e d by e n d r i n ( M e h ro ta e t
a l . , 1 9 8 2 ) and t h e r e l a t e d compound c h l o r d e c o n e ( J o r d a n e t a l * , 1 9 8 1 ) .
M ehrota e t a l . ( 1 982) s u g g e s t t h a t su c h i n h i b i t i o n c o u l d i m p a i r
. ,
a v a i l a b i l i t y o f ATP to t h e i o n i c s o d iu m - p o ta s s iu m ATPase, t h e r e b y
l i m i t i n g i t s n o rm a l f u n c t i o n and r e s u l t i n g i n a n a b n o rm a l sodium
g r a d i e n t a c r o s s t h e m em brane.
One c a n s p e c u l a t e t h a t t h e a x o n a l
s w e l l i n g o b s e r v e d i n t h i s s t u d y c o u l d v e r y w e l l be t h e r e s u l t o f such
im p a irm en t.
The h i g h d e g r e e o f s w e l l i n g on day 14 c o u l d i n d i c a t e a
p e a k i n th e a n i m a l ’ s i n a b i l i t y
to p r o d u c e a d e q u a t e am o u n ts o f ATP. The
60
a n i m a l s may s u b s e q u e n t l y a d a p t t o a new l e v e l o f : t o x i c i t y , a n d a s th e
e n d r in l e v e l i n the t i s s u e s c o n c o m ita n tly d ro p s, th e d e g re e of
i n h i b i t i o n o f t h e sodium pump d e c l i n e s .
am ount o f s w e l l i n g
T h i s c o u l d d e c r e a s e i n th e
. C o l v i n an d P h i l l i p s (1 9 6 8 ) s p e c u l a t e d t h a t e n d r i n
a n d i t s a n a l o g u e s b i n d t o t h e l i p i d —r i c h s t r u c t u r a l c o m p o n e n ts o f
m i t o c h o n d r i a a n d c e r t a i n o t h e r o r g a n e l l e s a n d t h a t t h i s b i n d i n g may
a f f e c t t h e c a t a l y t i c a c t i v i t y o f enzym es a s s o c i a t e d w i t h t h e s e
lip id -r ic h fra c tio n s.
D i s s o l u t i o n o f m i c r o t u b u l e s and n e u r o f i l a m e n t s i s one o f th e
e a r l i e s t s i g n s o f a x o n a l dam age, e i t h e r f o l l o w i n g m e c h a n i c a l i n j u r y
( S c h l a e p f e r an d H a s l e r , 1 9 7 9 a ; M ire e t a l . , 1 9 7 0 ; Dyck an d H o p k i n s , 1972;
Bray e t a l . , 1 9 7 2 ) , s y s t e m i c n e u r o p a t h y ( B e h s e ,1 9 7 5 ; Sima e t a l . , 1 9 7 9 )
o r c h e m i c a l e x p o s u r e ( P h i l l i p s an d E r o s c h e n k o , 1 9 8 2 ) . T h e se o r g a n e l l e s '
a r e r e p l a c e d by a n a m o r p h o u s , f l o c c u l e n t a n d g r a n u l a r m a t e r i a l
( S c h l a e p f e r a n d H a s l e r , 1979b; S c h l a e p f e r an d M ic k o , 1 9 7 8 ) . Clum ping o f
t h e a x o p l a s m i c c o n t e n t s (Dyck a n d H o p k i n s , 1 9 7 2 ) , a n d g r a n u l a r
d i s i n t e g r a t i o n o f a x o p la s m w ere a c c o m p a n ie d by s w e l l i n g
( S c h l a e p f e r , 1 9 7 7 ) . The r e s u l t s p r e s e n t e d h e r e a r e c o n s i s t e n t w i t h
th ese f in d in g s .
■;
The d i s s o l u t i o n o f m i c r o t u b u l e s a n d n e u r o f i l a m e n t s i s
t h o u g h t to
i n v o l v e i n c r e a s e d membrane p e r m e a b i l i t y t o c a l c i u m a n d s u b s e q u e n t
a x o p l a s m i c a l t e r a t i o n ( S c h l a e p f e r ,1 9 7 9 ; S c h l a e p f e r an d M ic k o ,1978;
S c h la e p f e r and H a s l e r , 1979a) v i a a c a lc iu m a c t i v a t e d p r o t e s a s e
( S c h l a e p f e r , 1 9 7 9 ) . Any p r o c e s s t h a t c o u l d l e a d t o t h e l o s s o f
61
s e l e c t i v e p e r m e a b i l i t y o f t h e axolemma c o u l d r e s u l t i n s u c h
breakdow n.
The s t r u c t u r a l a n d c h e m i c a l d i s r u p t i o n o f n e u r o f i l a m e n t s
i s a c c e l e r a t e d by e n e r g y d e p r i v a t i o n o r i n c r e a s e d c a l c i u m
c o n c e n t r a t i o n s i n c u l t u r e ( S c h l a e p f e r ,1 9 7 9 ).
D e g e n e r a t i v e a x o p la s m ic c h a n g e s a r e m ore w i d e s p r e a d i n s m a l l
m y e lin a te d and u n m y e lin a te d f i b e r s th an i n l a r g e m y e lin a te d f i b e r s
a f t e r i n c u b a t i o n i n m e d ia c o n t a i n i n g c a l c i u m ( S c h l a e p f e r a n d
E a s i e r , 1 9 7 9 a ) . T hese a u t h o r s a l s o fo u n d t h e s m a l l e r f i b e r s to be more
p r o n e to breakdow n i n d u c e d by c a l c i u m i n d i c a t i n g t h e p o s s i b i l i t y o f a
h ig h er c o n c e n tra tio n of axonal p ro te a s e s .
They s p e c u l a t e t h a t t h e s e
p r o t e a s e s e x i s t i n a n i n a c t i v e f o rm , a n d a r e a c t i v a t e d by r e l e a s e o f
c a l c i u m from w i t h i n t h e a x o n o r by i n f l u x a c r o s s t h e axolem m a,
p o s s ib ly as a r e s u lt, o f d e f i c ie n c ie s i n the m e ta b o lic c o n tr o l of
c a lc iu m p e r m e a b i l it y .
S t u d i e s i n v o l v i n g s i m i l a r p a t h o l o g i e s h a v e shown t h a t t h e
d isa p p e a ra n c e o f m ic ro tu b u le s p re c e d e s th e l o s s of n e u ro f ila m e n ts
( S c h l a e p f e r ,19 7 4 a n d 1 9 7 7 ) . The p r o g r e s s i o n o f s u c h d i s s o l u t i o n c o u l d
n o t be d e te r m in e d , i n t h e p r e s e n t s t u d y , p e r h a p s b e c a u s e o f t h e tim e
l a p s e b e tw e e n t h e d ay s o f s a c r i f i c e .
Empty a r e a s , d e v o i d o f a x o p l a s m i c m a t e r i a l , an d t h o u g h t t o h a v e
p r e v i o u s l y c o n t a i n e d a n a x o n , w ere o c c a s i o n a l l y o b s e r v e d w i t h i n th e
Schwann c e l l c y t o p l a s m o f e x p e r i m e n t a l a n i m a l s .
S o m etim es t h e s e a r e a s
c o n t a i n e d o n l y a few s c a t t e r e d v e s i c l e s w h i l e o t h e r s w ere a l m o s t
62
c o m p le te ly f i l l e d w ith v e s i c l e s .
Behse e t a l . ( 1 9 7 5 ) r e p o r t e d th e
a p p e a r a n c e o f em pty a x o n a l a r e a s i n t h e Schwann c e l l c y t o p l a s m i n
s u r a l n e r v e b i o p s i e s from p a t i e n t s w i t h s y s t e m i c n e u r o p a t h y .
referred
to t h e s e a r e a s a s "em pty Schwann c e l l s u b - u n i t s " .
They
These
a u t h o r s s u g g e s t e d t h a t s u c h em pty a r e a s may be due to e i t h e r th e
s u r v i v a l o f t h e Schwann c e l l a f t e r t h e l o s s o f t h e u n m y e l i n a t e d a x o n s
o r t o t h e p r o l i f e r a t i o n o f t h e r e m a i n i n g Schwann c e l l s .
S im ila r
f i n d i n g s w ere r e p o r t e d by Dyck an d H o p k in s (1972) f o l l o w i n g c r u s h
i n j u r y o f u n m y elin a te d f i b e r s .
They f o u n d one t h i r d o f t h e
u n m y e l i n a t e d f i b e r s w ere r e p r e s e n t e d by em pty s p a c e s w h ic h showed a
w atery ap p earan ce.
d isa p p e a re d .
The axolemma was d i s c o n t i n u o u s o r ha d
The f i n d i n g s r e p o r t e d i n t h e p r e s e n t s t u d y c o u l d i n f a c t
be a r e s u l t o f a x o n a l l o s s a l t h o u g h t h e low f r e q u e n c y o f s u c h c h a n g e s
m akes i n t e r p r e t a t i o n d i f f i c u l t .
A com prehensive m o rp h im e tric a n a l y s i s
i n v o l v i n g c o u n t s o f u n m y e l i n a t e d f i b e r s from e x p e r i m e n t a l a n i m a l s
c o u l d c o n f i r m su c h s p e c u l a t i o n . ,
One o f t h e m o st c o n s i s t e n t o b s e r v a t i o n s i n s c i a t i c n e r v e from
a n i m a l s e x p o s e d t o e n d r i n was t h e a p p e a r a n c e o f v e s i c l e s i n
u n m y e l i n a t e d a x o n s , a d j a c e n t p e r i a x o n a l s p a c e s , o r w i t h i n t h e Schwann
c e l l c y to p la sm .
The o r i g i n a n d f a t e o f t h e s e v e s i c l e s i s n o t a p p a r e n t
from t h i s s t u d y , s i n c e i t c o u l d n o t be d e t e r m i n e d i f t h e v e s i c l e s w ere
m oving i n t o t h e axon o r b e i n g e x t r u d e d fro m i t .
P h i l l i p s an d
Eroschenko (1 9 8 2 ) have r e p o r t e d s i m i l a r p o c k e ts o f v e s i c l e s in
u n m y elin a te d axons f o llo w in g c h lo rd e c o n e e x p o su re and o b s e rv e d t h a t
63
some o f t h e s e a x o n s c o n t a i n e d p o c k e t s o f v e s i c l e i s w i t h i n t h e Schwann
c e l l c y t o p l a s m w hich h a d i n v a g i n a t e d t h e axolem ma.
S p e n c e r a n d Thomas
(1 9 7 4 ) d e s c r i b e d Schwann c e l l c y t o p l a s m i c i n v a g i n a t i o n s i n p h y s i c a l l y
damaged m y e l i n a t e d f i b e r s ,
t h e s e i n v a g i n a t i o n s w ere i n v e s t e d by a n
i n f o l d i n g o f t h e axolemma a n d e n c l o s e d e l e c t r o n l u c i d a r e a s o f
a x o p la s m i n a "honeycom b" c o n f i g u r a t i o n w h ic h t h e y te r m e d " Schwann
c e ll/a x o n n e tw o rk s".
They r e p o r t e d l a r g e c l u s t e r s o f c l e a r a n d d e n s e
c o r e v e s i c l e s s u r r o u n d e d by a m i c r o f i l a m e n t m eshw ork a n d s p e c u l a t e d
t h a t t h e c y t o p l a s m i c i n v a g i n a t i o n s a r e a m eans by w h ic h t h e axon
s e q u e s t e r s an d p h a g o c y t o s e s d e g e n e r a t e o r u n d e s i r a b l e a x o p la s m ic
d e b r i s su c h a s u n u s u a l o r a b n o rm a l o r g a n e l l e s ( v e s i c l e s o r d e n s e
b o d ies).
A s i m i l a r h y p o t h e s i s h a s b e e n p r e s e n t e d by S c h l a e p f e r and
H a s l e r ( 1 9 7 9 b ) , who o b s e r v e d t h a t t h e g r a n u l a r a x o p la s m ic
d i s i n t e g r a t i o n c o i n c i d e d w i t h t h e a p p e a r a n c e o f f l o c c u l a r , an d
g r a n u l a r m a t e r i a l o f m o d e r a te e l e c t r o n d e n s i t y w i t h i n t h e
e n d o n e u riu m .
Such c h a n g e s w e re a t t r i b u t e d t o a p r o c e s s o f
e x t e r n a l ! z a t i o n o f a x o n a l breakdow n p r o d u c t s i n d e g e n e r a t i n g n e r v e
fib e rs.
F u r th e r e v id e n c e r e l a t i n g
t h e .m o r p h o l o g i c a l c h a n g e s to such
e x t e r n a l ! z a t i o n h a s b e e n r e p o r t e d by Sima an d R o b e r t s o n ( 1 9 8 2 ) , who
fo u n d i r r e g u l a r c o n g l o m e r a t e s o f m em branous p r o f i l e s i n s w o l l e n
u n m y e l i n a t e d a x o n s fro m m u t a n t d i a b e t i c m i c e .
e x t r u s i o n o f ax o p la sm ic d e b r is i n t o
They a l s o r e p o r t e d t h e
t h e Schwann c e l l c y t o p l a s m o r
a d ja c e n t p e ria x o n a l s p a c e , and i n l o n g it u d in a l s e c t i o n s o bserved th e
i n v a g i n a t i o n o f t h e Schwann c e l l c y t o p l a s m i n t o t h e a x o p la s m fo rm in g
"honeycom bed Schwann c e l l axon c o m p l e x e s " .
V e s i c l e s ( sm ooth membrane
64
p r o f i l e s ) h a v e b e e n o b s e r v e d by Wood and E n g e l ( 1 9 7 6 ) i n t h e i n n e r
Schwann c e l l c y t o p l a s m o f 4 - 6 day o l d n e o n a t a l r a t s d u r i n g a c t i v e
s c i a t i c nerve m y e lin a tio n .
They f o u n d t h a t t h e s e p r o f i l e s w ere
u s u a l l y e n c l o s e d w i t h i n a l a r g e r sm ooth membrane s t r u c t u r e a n d
o c c a s i o n a l l y t h e e n t i r e com plex c o u l d be s e e n f u s i n g w i t h t h e
axolem m a.
They s p e c u l a t e d t h a t t h e s e v e s i c l e s may be i n v o l v e d i n a n
e x c h a n g e o f membrane m a t e r i a l b e tw e e n t h e Schwann c e l l an d th e
axolem ma.
T hese a u t h o r s p o i n t o u t t h a t s u c h v e s i c l e s w e re s e e n a t
o t h e r s t a g e s o f e a r l y d e v e lo p m e n t b u t w e re n e v e r o b s e r v e d i n s c i a t i c
n e r v e fro m a d u l t a n i m a l s .
The v e s i c l e s . o b s e r v e d i n t h e p r e s e n t s t u d y c o u l d r e p r e s e n t
a c c u m u l a t i o n s o f a b n o rm a l a x o p la s m ic m a t e r i a l l e a v i n g t h e a x o n , f l u i d ,
o r e v e n a s a n a c c e l e r a t e d m eans o f n o r m a l membrane e x c h a n g e .
At no
p o i n t was t h e r e a n y e v i d e n c e o f e n d o n e u r i a l d e p o s i t i o n o f a x o p la s m ic
d e b ris.
V e s i c l e s w ere o b s e r v e d w i t h i n t h e a x o n , a l o n g t h e a x o n , i n
t h e Schwann c e l l c y t o p l a s m , a n d i n t h e p e r i a x o n a l s p a c $ i n d i c a t i n g
t h e r e may h a v e b e e n some ty p e o f e x t e r n a l i z a t i o n ( o r i n t e r n a l i z a t i o n )
o f t h e s e v e s i c l e s fro m t h e axon b u t t h e d i r e c t i o n a l i t y o f such
movement c o u l d n o t be v e r i f i e d .
T h e se v e s i c l e s c o u l d i n f a c t p l a y a
f o i e i n membrane e x c h a n g e i n s w o l l e n a x o n s w here m ore membrane would
be n e e d e d t o a c c o m o d a te t h e e n l a r g e d ax o n o r th e y c o u l d p r o v i d e a
m eth o d o f f l u i d re m o v a l fro m su ch a x o n s .
The c l e a r c o r e v e s i c l e s
c o n t a i n i n g a " h a l o " o f m a t e r i a l o f i n t e r m e d i a t e e l e c t r o n d e n s i t y w ere
m o s t o f t e n o b s e r v e d w i t h i n t h e axon a n d may be a c c u m u l a t i o n s o f f l u i d
65
d e s t i n e d f o r re m o v a l from t h e a x o n .
V e s i c l e s s i m i l a r to t h e a x o n a l
v e s i c l e s o b s e r v e d i n t h i s s t u d y h a v e b e e n fo u n d i n d e n d r i t e s a n d
n u c l e i o f s u b s t a n t i a n i g r a n e u r o n s i n m ic e e x p o s e d to c h l o r d e c o n e (Van
N a t t a a n d P h i l l i p s , i n p r e p a r a t i o n ) and i n t h e d e v e l o p i n g c u n e a t e
n u c l e i o f th $ r a t (D a v id an d N a t h a n i e l , 1 9 7 8 ) .
T h in c y t o p l a s m i c i n v a g i n a t i o n s o f t h e axolemma s i m i l a r to t h o s e .
o b s e r v e d i n u n m y e l i n a t e d n e r v e f i b e r fro m e x p e r i m e n t a l a n i m a l s h a v e
been p r e v i o u s ly r e p o r t e d i n d e g e n e ra tin g m y e lin a te d n e rv e f i b e r s
( S p e n c e r a n d Thomas, 1974) p o s s i b l y a s a m eans o f s e q u e s t r a t i o n o f
a x o n a l d e b r i s by t h e a d j a c e n t Schwann c e l l .
Such i n v a g i n a t i o n s h a v e
a l s o b e e n r e p o r t e d i n m y e l i n a t e d f i b e r s fro m a n i m a l s e x p o s e d t o e t h y l
n - b u t y l k e t o n e an d m e t h y l e t h y l k e t o n e ( 0 ' Donoghue e t a l . , 1 9 8 4 ) .
In
t h e p r e s e n t s t u d y , axolem m al i n v a g i n a t i o n s d i f f e r e d fro m t h o s e
r e p o r t e d by S p e n c e r an d Thomas a n d O'Donoghue e t a l .
p r i m a r i l y by t h e
d e g r e e o f i n v a g i n a t i o n s i n c e t h e y w ere o c c a s i o n a l l y s e e n t r a v e r s i n g
th e e n t i r e a x o n a l p r o f i l e , a l t h o u g h t h e y w e re n e v e r o b s e r v e d t o h a v e
a c t i v e l y e n c l o s e d a x o p la s m ic c o n t e n t s a s h a s b e e n p r o p o s e d by t h e s e
a u th o rs.
R a t h e r , t h e y a r e m o s t l i k e l y i n v a g i n a t i o n s o f t h e Schwann
c e l l c y t o p l a s m a n d axolem m a, t h a t a s a r e s u l t o f t h e p l a n e o f
s e c t i o n in g , a p p e ar o n ly a s a t h in c y to p la sm ic s t a l k .
The l a r g e membrane bound v a c u o l e s o c c a s i o n a l l y o b s e r v e d w i t h i n
t h e a r e a n o r m a l l y o c c u p i e d by t h e u n m y e l i n a t e d a x o n s o f f e n f i l l e d a
s i g n i f i c a n t a r e a o f t h e a x o n a l p r o f i l e a n d a p p e a r e d t o d i s p l a c e th e
a x o n fo one s i d q .
O th er r e p o r t s o f v a c u o le s s i m i l a r to th o se o b se rv e d
66
i n t h i s s tu d y have n o t been found i n th e l i t e r a t u r e .
Large v a c u o le s
h a v e b e e n o b s e r v e d i n t h e n e u r o p i l o f t h e c e n t r a l n e r v o u s s y s te m i n
p a t i e n t s w i t h C r e u t z f e l d t - J a k o b d i s e a s e ( F o n c i n , 1967; H i r a n o e t
a l . , 1 9 7 2 ; B ignam i a n d F o r n o , 1970; L a m b ert e t a l . , 1 9 7 1 ; G onata1S e t
a l.,1 9 6 5 ).
T hey a r e d e s c r i b e d a s c o n t a i n i n g a n e l e c t r o n l u c i d
m a t e r i a l w h ic h o f t e n c o n t a i n e d s m a l l e r v e s i c l e s o r membrane f r a g m e n t s
and i s bounded by a membrane t h a t i s f r e q u e n t l y i n t e r r u p t e d .
V a c u o l i z a t i o n h a s a l s o b e e n r e p o r t e d by M a r t i n e z e t a l . (1 9 7 8 ) i n
u n m y e l i p a t e d an d s m a l l m y e l i n a t e d a x o n s fro m b i o p s i e d s u r a l n e r v e o f
e m p lo y e e s i n v o l v e d i n t h e m a n u f a c t u r i n g o f k e p o n e .
The v a c u o l e s
o b se rv e d in th e p r e s e n t s tu d y a r e p o s s i b l y a c c u m u la tio n s o f a w a te ry
f l u i d i n th e p e r i a x o n a l sp ace t h a t r e s u l t s i n th e d is p la c e m e n t o f the
ax;on o r more l i k e l y , f i l l s
t h e s p a c e p r e v i o u s l y o c c u p i e d by a
d e g e n e r a t i n g a x o n , o n l y a p o r t i o n o f w h ic h i s v i s i b l e i n some
p ro file s.
T h i s may i n d i c a t e a t r a n s i e n t p h a s e o f a x o n a l d e g e n e r a t i o n
w hich f i n a l l y r e s u l t s i n a n em pty a r e a w i t h i n t h e Schwann c e l l
cy to p la sm .
'
OTHER TISSUES
The f a c t t h a t s k e l e t a l an d c a r d i a c m u s c l e from e x p e r i m e n t a l
a n i m a l s a p p e a r s s i m i l a r t o t i s s u e s fro m c o n t r o l a n i m a l s d o e s n o t r u l e
o u t th e f a c t t h a t s u b t l e b io ch e m ic a l or t r a n s i e n t m o rp h o lo g ic a l
c h a n g e s c o u l d be o c c u r r i n g w i t h i n t h e s e t i s s u e .
A lte ra tio n s in
67
s k e l e t a l m u s c l e w h ic h i n c l u d e d c h a n g e s i n m i t o c h o n d r i a l u l t r a s t r u c t u r e
and l i p i d and g ly co g e n s t o r e s have been r e p o r t e d i n c h lo rd e c o n e
e x p o s e d m ic e ( P h i l l i p s a n d E r o s c h e n k o , 1 9 8 4 ) . H ow ever, a n i m a l s e x p o s e d
to kep o n e d e v e lo p s e v e r e t r e m o r s a n d g a i t d i f f i c u l i t e s r e s u l t i n g i n
e x tr e m e e n e r g y d e p l e t i o n w h ic h may a c c o u n t f o r t h e m o r p h o l o g i c a l
changes o b served i n th e se a n im a ls.
S in ce a n im a ls e x p o sed to e n d rin
w ere n e v e r o b s e r v e d t o d i s p l a y s u c h sym p to m o lo g y , t h e e n e r g y b u r d e n i n
t h e s e a n i m a l s may n o t be a s g r e a t a n d t h u s one m ig h t n o t e x p e c t to s e e
such c h a n g es.
The o p t i c n e r v e i n a l l e x p e r i m e n t a l a n i m a l s a p p e a r e d s i m i l a r to
th a t in c o n tro l a n im a ls.
I f e n d r i n d o e s n o t damage m y e l i n a t e d
p e r i p h e r a l n e r v e f i b e r s t h e n one m ig h t n o t e x p e c t to s e e c h a n g e s i n
t h e o p t i c n e r v e w h ic h i s c o m p r i s e d a l m o s t e n t i r e l y o f m y e l i n a t e d
f i b e r s , a l t h o u g h a more c o m p r e h e n s i v e e x a m i n a t i o n o f s u c h t i s s u e i s
needed fo r v e r i f i c a t i o n of th e s e f in d i n g s .
i
M i t o c h o n d r i a from a l l e x p e r i m e n t a l t i s s u e s sa m p le d r e s e m b l e d '
th o se of c o n tr o l s d e s p it e r e p o r t s o f b io c h e m ic a l changes i n
I
m i t o c h o n d r i a i n e n d r i n e x p o s e d a n i m a l s ( P a r d i n i e t a l . , 1 9 7 1 ; M e h ro ta
e t a l.,1 9 8 2 ).
T h is i n c o n t r a s t to r e p o r t s o f m ito c h o n d r ia l changes i n
c h l o r d e c o n e e x p o s e d m i c e , c h a r a c t e r i z e d by p a r a c r y s t a l l i n e i n c l u s i o n s
i n m i t o c h o n d r i a o f u n m y e l i n a t e d f i b e r s , en d u l t r a s t r u c t u r e a l t e r a t i o n s
i n s k e l e t a l m u s c l e ( P h i l l i p s an d E r o s c h e n k o ,1 9 8 2 ; P h i l l i p s and
E ro sch e n k o ,1984).
68
BEHAVIORAL/NEUROLOGICAL EFFECTS
I t i s g e n e r a ll y b e lie v e d t h a t e n d rin s ti m u la te s c e n t r a l nervous
s y s te m a c t i v i t y .
B e h a v io ra l ch an g es r e p o r t e d h e re a p p e a r to co n firm
such a h y p o t h e s i s .
N e u r o l o g i c a l sym ptom ology d i s p l a y e d i n t h e
e x p e r im e n ta l a n im a ls i n t h i s s tu d y , i n c l u d in g h y p e r a c t i v i t y and
h y p e rse n sitiv ity
to s t i m u l i h a v e b e e n w e l l d ocum ented i n t h e
l i t e r a t u r e and such c h an g es have been c o n s i s t e n t f i n d i n g s i n a v a r i e t y
o f e x p e r i m e n t a l a n i m a l s i n c l u d i n g mammals, b i r d s , a n d f i s h .
H y p e r s e n s i t i v i t y t o s t a r t l e s t i m u l i was one o f t h e e a r l i e s t s i g n s
of ex p o su re to e n d r in .
S i m i l a r f i n d i n g s h a v e been r e p o r t e d i n
c h l o r d e c o n e e x p o s e d r a t s ( R e i t e r a n d K i d d , 1978; J o r d a n e t a l . , 1 9 8 1 )
Snd mjLce ( P h i l l i p s a n d E r o s c h e n k o , 1982) . J a c o b z i n e r a n d R a y b in (1 9 5 9 )
r e p o r t e d a n e x a g g e r a t e d s t a r t l e r e s p o n s e d i s p l a y e d by a c h i l d a c u t e l y
e x p o s e d t o e n d r i n a n d t h e y f e l t t h a t i t was i n d i c a t i v e o f e x t e n s i v e
i n v o lv e m e n t p f t h e d i e n c e p h a l o n .
J o y ( 1 9 7 6 ) fo u n d i n r a t s e x p o s e d
to
e n d r i n t h a t s e n s o r y s t i m u l i o f a n y m o d a l i t y w ould e l i c i t s e i z u r e
a c tiv ity .
H i s s t u d y showed t h a t p e r i p h e r a l n e r v e s t i m u l a t i o n r e s u l t e d
i n t h e e n h a n c e m e n t o f a c e n t r a l n e r v o u s s y s te m r e s p o n s e to s e n s o r y
s t i m u li a t bo th th e so m ato sen so ry and s e n s o r im o to r c o r t e x r e s u l t i n g i n
a t h r e e to f i v e f o l d i n c r e a s e i n c o r t i c a l m o to r o u t f l o w t h a t c o u l d
p o s s i b l y c o n t r i b u t e to th e r e s p o n s e to s t a r t l e s t i m u l i se en i n exposed
a n im a ls.
A s t u d y i n v o l v i n g a n i m a l s e x p o s e d to d i e l d r i n showed t h a t
su c h c h a n g e s a r e r e l a t e d to a n i n c r e a s e d p o s t s y n a p t i c r e s p o n s e o f t h e
69
c o r t i c a l c e l l s and th e a u th o r s p e c u la te s t h a t e n d rin a c t s i n a s im i la r
f a s h io n ( J o y , 1974, a s c i t e d i n J o y , 1 9 7 6 ). Jo rd a n e t a l . ,(1981)
s u g g e s t e d t h a t a s i m i l a r s t a r t l e r e s p o n s e i n r a t s e x p o s e d to
c h l o r d e c o p e was i n d i c a t i v e o f a a b n o rm a l a c t i v i t y i n t h e a u d i t o r y ,
r e t i c u l a r a n d / o r m o to r s y s te m ( d y s f u n c t i o n o r a b n o rm a l e x c ^ t a t i o p ) and
p o s s i b l y g e n e r a l i z e d c e n t r a l n e r v o u s s y s te m i n v o l v e m e n t .
P i l o e r e c t i o n was n o t a n o t i c a b l e e f f e c t i n e x p o s e d a n i m a l s u n t i l
somewhat l a t e r d u r i n g e x p o s u r e .
S im ila r f i n d i n g s have n o t been
r e p o r t e d i n t h e l i t e r a t u r e , a l t h o u g h m ic e e x p o s e d t o k e p o n e become
p i l o e r e c t e d s h o r t l y a f t e r i n j e c t i o n (Van N a t t a a n d P h i l l i p s , i n
p re p a ra tio n ) .
I t h a s b e e n r e p o r t e d t h a t k e p o n e e x p o s u r e l e a d s to
h y p e r - r e s p o n s iv e n e s s of u n m y elin a te d p e r i p h e r a l n e rv e f i b e r s
to
s t i m u l a t i o n (R . W e l l e r , J . M c M illa n a n d D. P h i l l i p s , p e r s o n a l
c o m m u n ic a t io n ) .
I t h a s a ls o been s u g g e ste d t h a t e n d r in ex p o su re
r e s u l t s i n s y m p a t h e t i c n e r v o u s s y s te m s t i m u l a t i o n (E m e rso n e t
a l . , 1 9 6 4 ; H inshaw e t a l . , 1 9 6 6 ) .
I f s ti m u la ti o n of u n m y elin a te d n e rv e
f i b e r s and th u s p o s tg a n g lio n ic sy m p a th e tic f i b e r s does o ccu r i n e n d rin
e x p o s e d a n i m a l s , t h e n i t i s l i k e l y t h a t su c h s t i m u l a t i o n w ould l e a d to
p ilo e re c tio n .
E n d r i n c a u s e s damage t o u n m y e l i n a t e d a x o n s w h ic h i n c l u d e s e n s o r y
o r a u to n o m ic p o s t g a n g l i o n i c f i b e r s .
C o n s e q u e n tl y s u c h damage c o u l d
r e s u l t i n im pairm ent; o f a u to n o m ic f u n c t i o n s r e l a t e d t o t e m p e r a t u r e
r e g u l a t i o n , b l o o d p r e s s u r e m a i n t e n a n c e , a n d a number o f o t h e r v i s c e r a l
fu n ctio n s.
P h y s i o l o g i c a l m a n i f e s t a t i o n s o f su c h im p a ir m e n t h a v e been
70
d ocum ented i n dogs and i n c l u d e , h y p e r t h e r m i a , h y p e r t e n s i o n ,
b r a d y c a r d i a a n d i n c r e a s e d s a l i v a t i o n (Em erson e t a l . , 1 9 6 3 ; R e i n s e t
a l . , 1 9 6 4 a n d 1 9 6 6 ; Em erson a n d H in s h a w ,1 9 6 5 ; E m e rs o n ,1 9 6 5 ) . Damage t o
th ese p a r t i c u l a r f i b e r s c o u ld a ls o r e s u l t i n changes in r e f l e x
re s p o n s e s and s e n s a tio n s r e l a t e d
to n o x io u s s t i m u l i .
I t i s p robable
t h a t many o f t h e s e c h a n g e s c o u l d be be s u b t l e a n d may a f f e c t humans o r
a n i m a l s i n d o s e s w e l l b e lo w t h o s e t h a t w ould p r o d u c e g r o s s b e h a v i o r a l
o r m o r p h o l o g i c a l sym ptom s.
Even i f
su c h s u b t l e c h a n g e s w e re d e t e c t e d ,
t h e c a u s e o f s u c h c h a n g e s w ould n o t l i k e l y be s u s p e c t e d .
R e g a r d in g
su ch s u b t l e c h a n g e s , a w i l d l i f e s t u d y by t h e Montana D e p a r tm e n t o f
F i s h W i l d l i f e an d P a r k s (1 9 8 3 ) s u g g e s t s t h a t " p e s t i c i d e r e l a t e d c a u s e s
f o r su c h c h a n g e s c o u l d i n c l u d e r e p r o d u c t i v e i m p a i r m e n t , i n c r e a s e d
n e o n a ta l m o r t a l i t y and p h y s io l o g i c a l o r b e h a v io r a l changes t h a t can
a f f e c t su rv iv a l" .
G a i t d i s t u r b a n c e s due t o e x p o s u r e t o e n d r i n , i n t h i s s t u d y , were
m in im a l a n d a l l a n i m a l s a p p e a r e d t o h a v e r e c o v e r e d by 14 d o r .
T h is
i s i n c o n t r a s t to t h e s e v e r e g a i t d i f f i c u l t i e s r e p o r t e d i n m ice
e x p o s e d t o c h l o r d e c o n e w hich i n c l u d e d , w i d e - b a s e d s p l a y i n g , i n s t a b i l i t y
and i n c o n s i s t e n t p lacem en t o f th e e x t r e m i t i e s ( P h i l l i p s and
E r o s c h e n k o ,1 9 8 2 ; J o r d a n e t a l . , 1 9 8 1 ; E g l e e t a l . , 1 9 7 9 ) .
A ta x ia h as
b e e n r e p o r t e d i n b i r d s (D eW i.tt, 1955) and r a t s ( B e d f o r d e t a l . , l ? 7 5 )
exposed to e n d rin .
R e s u l t s o f th e t a i l f l i c k t e s t a r e i n c o n c l u s i v e s i n c e a
s ta tistic a lly
s i g n i f i c a n t d i f f e r e n c e b e tw e e n c o n t r o l s a n d
71
e x p e f ! m e n t a l s o c c u r r e d o n l y on 3 d a y s .
I t is lik e ly
t h a t an y c h a n g e
i n p a i n p e r c e p t i o n due to damage to u n m y e l i n a t e d s e n s o r y n e r v e f i b e r s
w ould n o t a f f e c t t a i l w i t h d r a w l s i n c e su c h damage w ould m o st l i k e l y
r e s u l t p r i m a r i l y i n c h a n g e s i n v o l v i n g se c o n d p a i n s e n s a t i o n
( P r i c e , 1972).
R e s u l t s o f th e r o d t e s t w ere i n t e r e s t i n g
b u t d i f f i c u l t to
i n t e r p r e t s i n c e a number o f f a c t o r s c o u l d i n f l u e n c e
n o ted .
the r e s u l t s
A s i g n i f i c a n t c h a n g e o c c u r r e d i n th e a b i l i t y o f th e
e x p e r i m e n t a l a n i m a l to m a i n t a i n i t s e l f on t h e rod»
Few a n i m a l s c o u ld
s t a n d on th e r o d a t a l l by 8 d o e and t h o s e t h a t d i d o f t e n t r e m b le d
m a r k e d ly b e f o r e f i n a l l y f a l l i n g o f f .
I t i s necessary
to p o i n t o u t
t h a t th e a n i m a l s t o t a l r e c o r d e d tim e on t h e r o d d id n o t n e c e s s a r i l y
r e f l e c t the a n im a ls a b i l i t y
to m a i n t a i n i t s
p o s i t i o n once p l a c e d
t h e r e , s i n c e many t im e s th e a n im a l w o u ld f a l l o f f t h e r o d i m m e d i a te l y
o n ly to g r a s p th e r o d a n d hang from i t ,
o f a much l o n g e r r e c o r d e d t i m e .
th u s r e s u l t i n g i n a ssig n m en t
The b a s i s f o r t h i s c h a n g e i n b e h a v i o r
c a n n o t be d e t e r m i n e d from t h i s s t u d y a l t h o u g h one c o u l d s p e c u l a t e a s
to th e r e a s o n s f o r s u c h c h a n g e s .
D istu rb a n c es in v e s ti b u la r fu n c tio n ,
m u s c le c o o r d i n a t i o n a n d / o r m u s c le s t r e n g t h ,
sensory-m otor d y sfu n c tio n
or t i m i d i t y c o u l d a c c o u n t f o r th e a n i m a l s i n a b i l i t y
rod.
to s t a y on th e
L oss o f e q u i l i b r i u m h a s been r e p o r t e d i n f i s h e x p o s e d to e n d r i n
( M o u n t , 1962, a s c i t e d i n G r a n t and M e r h l e ,1 9 7 3 ) b u t n o t i n o t h e r
a n im a ls.
A t a x i a h a s b e e n r e p o r t e d i n some a n i m a l s e x p o s e d to e n d r i n
( D e W i t t , 1955; B e d f o r d e t a l . , 1 9 7 5 ) w h i l e m u s c l e w ea k n e ss, was a
72
c o n s i s t e n t f i n d i n g i n a c u t e l y e x p o s e d humans ( J e n k i n s a n d T o o l e , 1964;
C oble e t a l . , 1 9 6 7 ) .
C o n v u l s i o n s an d to a l e s s e r e x t e n t ,
trem o rs, a re c o n s is te n t
symptoms o f a c u t e e n d r i h e x p o s u r e r e p o r t e d i n t h e l i t e r a t u r e .
The
a b s e n c e o f t h e s e symptoms i n t h i s s t u d y c o u l d be t h e r e s u l t o f s p e c i e s
v a ria tio n ,
dose a n d r o u t e o f e x p o s u r e , o r d u r a t i o n o f e x p o s u r e .
Anim al w e i g h t i n c o n t r o l an d e x p e r i m e n t a l a n i m a l s i n c r e a s e d up to
14 d o r a l t h o u g h t h e w e i g h t o f t h e e x p e r i m e n t a l s h a d d e c r e a s e d
s l i g h t l y by 92 d o r .
The d i f f e r e n c e i n w e i g h t g a i n b e tw e e n t h e
c o n t r o l s a n d e x p e r i m e n t a l s was s t a t i s t i c a l l y d i f f e r e n t o n l y on day 2 0 .
How ever, t h e e x p e r i m e n t a l a n i m a l s c o n s i s t e n t l y g a i n e d l e s s w e i g h t t h a n
c o n t r o l s but; a t ' a r a t e n o t s t a t i s t i c a l l y d i f f e r e n t ,
p o s s i b l y due to
th e h i g h amount o f a q f l v i t y d i s p l a y e d , b y t h e s e a n i m a l s .
Some o f t h e
w e i g h t g a i n i n b o t h g r o u p s o f a n i m a l s was l i k e l y due t o t h e f a c t t h a t
t h e s e a n i m a l s a r e s t i l l g ro w in g a l t h o u g h a r e s i d u a l a c c u m u l a t i o n o f
sesam e o i l ,
e v i d e n t upon s a c r i f i c e o f a l l a n i m a l s UP t o 14 d o
a c c o u n t f o r some o f t h i s i n c r e a s e .
r c o u ld
B l u s ( 1 9 7 8 ) fo u n d t h a t shrew s
e x p o s e d t o e n d r i n l o s t a n a v e r a g e o f 19-27% o f t h e i r w e i g h t .
On t h e
o t h e r h a n d T re b n ( 1 9 5 5 ) r e p o r t e d t h a t p r o l o n g e d f e e d i n g o f e n d r i n to
r a t s r e s u l t e d i n a w e i g h t g a i n e q u a l to o r g r e a t e r t h a n t h a t o f th e
c o n t r o l s e x c e p t i n m a l e s f e d t h e h i g h e s t do se (2 5 p p m ) .
A s i n g l e dose
o f e n d r i n a s h i g h a s 10 mg/kg h a d no e f f e c t on t h e w e i g h t o f p r e g n a n t
h a m s t e r s a l t h o u g h a n i m a l s r e c e i v i n g d o s e s a b o v e 0 . 7 5 m g /k g / d a y f o r 10
c o n s e c u t i v e d a y s a l,l h a d a r e d u c t i o n i n w e i g h t ( C h e r n o f f e t
73
a l.,1 9 7 9 ).
The r a p i d m e t a b o l i c f a t e o f e n d r i n i n e x p o s e d a n i m a l q c o u l d
e x p l a i n why t h e s e v e r i t y o f some symptoms d i m i n i s h w i t h t i m e .
I t may
a l s o a c c o u n t f o r t h e c h a n g e s i n sym ptom ology f o l l o w i n g a c h a n g e i n t h e
do se an d a l s o t h e a p p a r e n t r e c o v e r y o f e x p o s e d a n i m a l s f o l l o w i n g
c e s s a t i o n of d o sin g .
I t a p p e a r s t h a t c y c lo d ie n e s can e x i s t i n th e
b r a i n f o r p e r i o d s o f tim e w i t h o u t p r o d u c i n g t o x i c sym ptom s, b u t a t a
l a t e r tim e t h e same c o n c e n t r a t i o n may p r o d u c e s e v e r e sym ptom s.
T h is
s u g g e s t s a tim e d e p e n d e n t m echanism w i t h i n t h e c e n t r a l n e r v o u s sy ste m
t h a t d e t e r m i n e s when s u c h symptoms w i l l ,become a p p a r e n t (W a lsh an d .
F i n k , 1 9 7 2 ) . The s l i g h t d i m u n i t i o n o f h y p e r a c t i v i t y a n d a p r o n o u n c e d
s t a r t l q resp o n se pver a
hour p e r i o d fo llo w in g i n j e c t i o n p ould
r e s u l t fro m d e c r e a s i n g I q v e l s o f e n d r i n i n e x p o s e d a n i m a l s .
The g a i t
c h a n g e s on day 1 3 , one day f o l l o w i n g a n i n c r e a s e i n t h e d o s e ( f r o m 2 . 0
to 4 . 0 m g/kg) and t h e i r s u b s e q u e n t g r a d u a l d e c r e a s e a f t e r da y 17 c o u l d
i n d i c a t e t h a t th e a n im a ls had "ad a p te d "
to th e p re v io u s d o s e , th u s
e s t a b l i s h i n g a p l a t e a u l e v e l w i t h i n t h e t i s s u e s b u t t h e n ha d to
r e a d a p t t o t h e new d o s e .
The i n a b i l i t y o f a n i m a l s t o m a i n t a i p t h e i r
b a la n c e d u rin g th e ro d t e s t c o n tin u e d th ro u g h o u t th e d o sin g p o r ti o n o f
th e e x p e rim e n t.
S i g n i f i c a n t r e c o v e r y d i s p l a y e d by a n i m a l s i n t h i s
t e s t and t h e g r a d u a l d i s a p p e a r a n c e o f t h e o t h e r o b s e r v e d symptoms
o c c u r r e d o v e r t h e 2 - 4 w eeks f o l l o w i n g c e s s a t i o n o f e x p o s u r e i n d i c a t i n g
a r a p id tu rn o v e r and e lim in a tio n o f e n d rin i n th e se a n im a ls .
T o t a l a n im a l m o r t a l i t y i n t h i s e x p e r i m e n t was 23%. T h e re w ere no
74
o b v i o u s i n d i c a t i o n s f o r t h e c a u s e o f d e a t h , n o r w ere t h e r e o v e r t
p r e m o r t a l symptoms i n d i c a t i n g a n y o f t h e a n i m a l s w ere n e a r d e a t h .
The
i n c i d e n c e o f m o r t a l i t y was c l o s e l y a s s o c i a t e d w i t h t h e c h a n g e i n t h e
4ose c o n c e n t r a t i o n .
m g /k g .
T h e re y a s no m o r t a l i t y a t a d o s e o f 1 .5 o r 2 .0
R a i s i n g t h e d o se t o 4 . 0 mg/kg on d a y 12 r e s u l t e d i n t h e d e a t h
o f two a n i m a l s w i t h i n 4 h o u r s a f t e r i n j e c t i o n an d t h e a d d i t i o n a l de-ath
o f 6 more a n i m a l s o v e r t h e n e x t 7 d a y s .
I t was a p p a r e n t ■t h a t th o se ;
a n i m a l s t h a t c o u l d t o l e r a t e a c h a n g e i n d o s e d i d s o by a, p r o c e s s o f
a d a p t a t i o n , p r o b a b l y t h r o u g h r a p i d m e t a b o l i s m to l e s s t o x i c
i n t e r m e d i a t e s an d s u b s e q u e n t e l i m i n a t i o n w h ic h u l t i m a t e l y r e s u l t e d i n
t h e i r s u r v i v a l u n t i l th e g e rm in a tio n o f th e e x p e rim e n t.
SQMjflARY
T h is s tu d y d e m o n s tra te s t h a t e n d r in does produce s p e c i f i c
m o rp h o lo g ic a l changes i n u n m y elin a te d f i b e r s o f p e r i p h e r a l n e rv e
a l t h o u g h t h e m ec h a n ism s o f s u c h c h a n g e r e m a in t o be e l u c i d a t e d .
T h e re
i s a n e e d f o r f u r t h e r s t u d i e s to s u b s t a n t i a t e t h e a p p a r e n t s p e c i f i c i t y
of e n d r i n 's t o x i c i t y
to u n m y e l i n a t e d f i b e r s i n o t h e r a r e a s o f th e
p e r i p h e r a l n e r v o u s s y s te m a n d p o s s i b l y w i t h i n t h e c e n t r a l n e r v o u s
s y s te m a n d po f u r p h e p c h a r a c t e r i z e t h e damagq o c c u r r i n g w i t Q i n t h e s e
fib e rs,
SucQ Eitpdi^p c o u l d i n c l u d e e x a m i n a t i o n o f s p e c i f i c
p o p u l a t i o n s o f u n m y e l i n a t e d f i b e r s ( s e n s o r y a n d a u to n o m ic ) fro m a
npm ber q f l e g a t i o n s tq d e t e r m i n e i f
t h e s e e f f e c t s are; I q c a l i z e d o r
75
w i d e s p r e a d a n d to i n s p e c t t h e d o r s a l r o o t a n d a u to n o m ic g a n g l i a o f
fib e rs
to v e r i f y p o s s i b l e i n v o l v e m e n t o f t h e p e r i k a r y a .
In a d d it io n ,
i t w ould be o f i n t e r e s t t o i n v e s t i g a t e t h e e f f e c t s o f e n d r i n i n
n e o n a t a l a n i m a l s s i n c e a n o t h e r n e u r o t o x i c compound, c a p s a i c i n , h a s
b e e n shown to s e l e c t i v e l y i n d u c e d e g e n e r a t i o n i n u n m y e l i n a t e d n e r v e s
o f n e o n a t a l r a t s ( J a n c s o e t e l . , 1977; J a n c s o a n d K i r a l y , 1 9 8 1 ; Nagy e t
a l . , 1 9 8 0 ; N a g y ,1 9 8 2 ) . S t u d i e s a r e a l s o n e e d e d t o a s c e r t a i n t h e
t h r e s h o l d do se w h ic h w i l l i n d u c e i n i t i a l m o r p h o l o g i c a l a n d / o r
b e h a v io ra l changes i n exposed a n im a ls,
Knowledge o f s u c h s p e c i f i c i t y
c o u l d l e a d t o t;he u s e o f e n d r i n a s a v a l u a b l e t o o l i n n e u r o l o g i c a l
research .
I
76
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