Biology and Medicine AEC Research and Development Report...

A N L - 6 9 0 6 B i o l o g y and M e d i c i n e ( T I D - 4 5 0 0 , 34th E d . ) A E C R e s e a r c h and D e v e l o p m e n t R e p o r t

A R G O N N E N A T I O N A L L A B O R A T O R Y 9700 South C a s s A v e n u e A r g o n n e , I l l i n o i s 60440

B I O L O G I C A L A N D M E D I C A L R E S E A R C H D I V I S I O N S E I M I A N N U A L R E P O R T

J u l y t h r o u g h D e c e m b e r 1963

June 1964

P r e c e d i n g R e p o r t : A N L - 6 8 2 3 J a n u a r y t h r o u g h June 1963

O p e r a t e d b y The U n i v e r s i t y o f C h i c a g o unde r

C o n t r a c t W - 3 1 - 1 0 9 - e n g - 3 8 w i t h the

U . S . A t o m i c E n e r g y C o m m i s s i o n

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T A B L E O F C O N T E N T S

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R a d i a t i o n m o r t a l i t y in the mouse: K i n e t i c s of i n ju ry a c c u m u l a t i o n du r ing p r o t r a c t e d exposures in the sup ra l e tha l range

S . P h y l l i s S t e a m e r and M a r g a r e t H. Sanderson 7

T h r o m b i in the left a t r i u m R . J . M i c h a e l F r y , K a t h e r i n e H a m i l t o n , and H e r m a n n L i s c o . 1 9

The i n t e s t i n a l f l o r a of the mouse R . J . M i c h a e l F r y , P a t r i c i a A . B r e n n a n , and Thomas E . F r i t z 2 1

P o p u l a t i o n k i n e t i c s of neop la s t i c m a s t c e l l s (P815Y) after X - i r r a d i a t i o n

G . M a r k K o l l m o r g e n 2 2

Ef fec t s of h i b e r n a t i o n on the latent p e r i o d in n o r m a l and X - i r r a d i a t e d ground s q u i r r e l s

B e r n a r d N . J a r o s l o w and Douglas E . S m i t h 2 3

P r o g r e s s r epor t : D i s e a s e s and ca re o f l a b o r a t o r y a n i m a l s

I . D i s e a s e c o n t r o l p r o g r a m R o b e r t J . F l y n n , Thomas E . F r i t z , P a t r i c i a C . B r e n n a n , and C a l v i n M . P o o l e 2 7

II. S a l m o n e l l a c e r r o i s o l a t e d f r o m a b reed ing colony P a t r i c i a C . B r e n n a n , Thomas E . F r i t z , and R o b e r t J . F l y n n 28

III. Studie s on P a s t e u r e l l a pneumot rop i ca P a t r i c i a C . B r e n n a n , Thomas E . F r i t z , and R o b e r t J , F l y n n 28

I V . H e m o r r h a g i c d i a thes i s i n l a b o r a t o r y rodents Thomas E . F r i t z , D a v i d V . T o l l e , R o b e r t J . F l y n n , and C a l v i n M . P o o l e 3 0

V . P n e u m o n i a ep i zoo t i c i n m i c e R o b e r t J . F l y n n , Thomas E . F r i t z , P a t r i c i a C . B r e n n a n , and C a l v i n M . P o o l e 3 2

V I . Rodent b r e e d i n g p r o g r a m T h o m a s E . F r i t z , R o b e r t J . F l y n n , and M e r l i n H . D i p e r t 3 3

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V I I . Dog c a r e p r o g r a m C a l v i n M . P o o l e and R o b e r t J . F l y n n 3 4

V I I I . U s e o f an u l t r a s o n i c uni t in the den ta l c a r e o f r e s e a r c h dogs

C a l v i n M . P o o l e and R o b e r t J . F l y n n 3 5

I X . D o g b r e e d i n g p r o g r a m C a l v i n M . P o o l e and R o b e r t J . F l y n n 3 6

X . N e w s t r a i n s and s p e c i e s : J a p a n e s e q u a i l ( C o t u r n i x c o t u r n i x )

D u r w a r d D . B a n i s t e r , C a l v i n M . P o o l e , R o b e r t J . F l y n n , and T h o m a s E . F r i t z 3 7

T o x i c i t y and m e t a b o l i s m o f r a d i o n u c l i d e s W i l l i a m P . N o r r i s , L o u i s e S . L o m b a r d , C a r l E . R e h f e l d , and R o b e r t J . Toft 41

D r y e m u l s i o n a u t o r a d i o g r a p h y L l o y d J . R o t h , A k e H a n n g r e n , and W i l l i a m P . N o r r i s 6 1

A r a p i d b l o o d c a t a l a s e s c r e e n i n g t echn ique ad jus tab le to any l e v e l o f a c t i v i t y

R o b e r t N . F e i n s t e i n , J u d i t h B . H o w a r d , L a r r y B . B a l l o n o f f , and J o h n E . S e a h o l m

E f f e c t o f p u r o m y c i n o n b l o o d and l i v e r c a t a l a s e R o b e r t N . F e i n s t e i n , J u d i t h B . H o w a r d , and J o h n E . S e a h o l m 70

L a c k o f effect o f exogenous c a t a l a s e a d m i n i s t e r e d d u r i n g p r e g n a n c y on c a t a l a s e content o f p r o g e n y

R o b e r t N . F e i n s t e i n , J o h n E . S e a h o l m , and J u d i t h B . H o w a r d . . • 73

S e a r c h for a n a c a t a l a s e m i c m o u s e R o b e r t N . F e i n s t e i n , J o h n E . S e a h o l m , and J u d i t h B . H o w a r d .

A t t e m p t s a t r a d i a t i o n p r o t e c t i o n by compounds r e l a t e d to 3 - a m i n o - 1 , 2 , 4 - t r i a z o l e

R o b e r t N . F e i n s t e i n and J o h n E . S e a h o l m 78

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O c c a s i o n a l r e s i s t a n c e o f m i c e t o g l u c o s e o x i d a s e i n j e c t i o n R o b e r t N o F e i n s t e i n and J o h n E . S e a h o l m . . . . . . . . . . . . . 8 1

M u t a t i o n i n d u c e d w i t h caf fe ine , u l t r a v i o l e t l i g h t , o r Z - a m i n o p u r i n e H e r b e r t E . K u b i t s c h e k and H a r o l d E . B e n d i g k e i t . . . . . . . . 8 5

C o l l o i d a l p r o p e r t i e s o f p l u t o n i u m i n d i l u t e aqueous s o l u t i o n A r t h u r L i n d e n b a u m and W i l l i a m M „ W e s t f a l l . . . . . . . . . . . 8 7

P r o g r e s s r e p o r t : P l u t o n i u m r e m o v a l . X . D i s t r i b u t i o n and r e m o v a l o f m o n o m e r i c and p o l y m e r i c p l u t o n i u m i n the r a t

M a r c i a W . R o s e n t h a l , J o a n F . M a r k l e y and A r t h u r L i n d e n b a u m . 88

P r o g r e s s r e p o r t : P l u t o n i u m r e m o v a l . X I . R e m o v a l o f p o l y m e r i c p l u t o n i u m f r o m m i c e b y c o m b i n e d t h e r a p y w i t h the c a l c i u m che l a t e and p e n t a e t h y l e s t e r o f D T P A

J o a n F. M a r k l e y . . . 90

R a d i o r e s t o r a t i o n o f a m o e b a e af ter i n j e c t i o n o f u n i r r a d i a t e d p r o t o p l a s m

E d w a r d W 0 D a n i e l s and E v e l y n P . B r e y e r 9 2

T h e m e t a b o l i s m o f m i t o c h o n d r i a l p r o t e i n s . I . The i s o z y m e s o f g l u t a m i c - p y r u v i c t r a n s a m i n a s e

R o b e r t W 0 S w i c k , P a u l a L . B a r n s t e i n , and J o h n L . Stange . . 9 3

E f f e c t o f i so tope exchange on the p r o t o n a t i o n o f D N A L y l e G„ B u n v i l l e . 101

N u c l e a r changes i n ra t l i v e r c e l l s d u r i n g t h i o a c e t a m i d e a d m i n i s t r a t i o n

A . D 0 B a r t o n and E l i z a b e t h A 0 C e r n y . . . . . . . . . . . . . . . . 102

N u c l e a r changes i n r a t k i d n e y c e l l s af ter c a s t r a t i o n and a d m i n i s t r a t i o n o f t e s t o s t e r o n e

A . D , B a r t o n and K a t h l e e n M o T r a c y . . . . . . . . . . . . . . . . 106

E f f e c t o f X - r a d i a t i o n o n o x i d a t i v e p h o s p h o r y l a t i o n i n r a t l i v e r m i t o c h o n d r i a , , II.

J o h n F„ T h o m s o n and S h a r r o n L. N a n c e . . . . . . . . . . 110

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P a g e

P h o s p h o l i p a s e C , l o w p H , and t e m p e r a t u r e effects o n a c i d phospha tase r e l e a s e f r o m r a t l i v e r , s p l e e n and t h y m u s

Y . E . R a h m a n 113

F i n e s t r u c t u r e o f the l acunae and c a n a l i c u l i i n bone F . W a s s e r m a n n and J a m e s A . Y a e g e r . . 118

V a r i o u s a s p e c t s o f f i x a t i v e s fo r u l t r a t h i n s e c t i o n s and r e s o l u t i o n i n e l e c t r o n m i c r o s c o p y

T h e o d o r e N . T a h m i s i a n , C a r o l J . C h r i s t i a n s e n , R o s e m a r i e L . D e v i n e , and B e t t y J e a n W r i g h t 126

P u b l i c a t i o n s 130

R A D I A T I O N M O R T A L I T Y I N T H E M O U S E : K I N E T I C S O F I N J U R Y A C C U M U L A T I O N D U R I N G

P R O T R A C T E D E X P O S U R E S I N T H E S U P R A L E T H A L R A N G E

S . P h y l l i s S t e a m e r and M a r g a r e t H . S a n d e r s o n

R a d i a t i o n dea th af ter 7 - r a y e x p o s u r e s i n the 3 0 - d a y l e t h a l r ange o c c u r s a l m o s t e x c l u s i v e l y be tween 9 and 30 d a y s ; ( l ) i t i s a s s o c i a t e d w i t h f u n c t i o n a l f a i l u r e o f the h e m a t o p o i e t i c s y s t e m and a p p a r e n t l y i n v o l v e s p r i n c i p a l l y a n e m i a and b a c t e r e m i a . (2> 3) W i t h i n c r e a s e in r a d i a t i o n dose ( in the s o - c a l l e d 3 0 - d a y s u p r a l e t h a l r ange) , s u r v i v a l t i m e d e c r e a s e s to a m e a n . v a l u e of about 4.5 days and then r e m a i n s cons tan t fo r a d d i t i o n a l doses as g r e a t a s s e v e r a l thousand roen tgens ; damage to the i n t e s t i n a l e p i t h e l i u m i s the c h a r a c t e r i s t i c s y m p t o m .

( 4 - 6 ) T h e r e i s e v i d e n c e that the i m m e d i a t e

cause o f dea th i s a s s o c i a t e d w i t h e x c e s s i v e l o s s o f w a t e r and e l e c t r o l y t e s and r e s u l t a n t f l u i d i m b a l a n c e . - 12) j n rnost i n s t a n c e s b a c t e r i a l i n f e c t i o n a p p e a r s to be a m i n o r f ac to r c o n t r i b u t i n g to dea th w i t h i n t h i s p e r i o d . 1 2 , 13) E x p o s u r e to doses b e t w e e n the L D 5 0 for the 9-30 day l e t h a l i t y and the L D 5 0

for dea th w i t h i n 4 - 6 days c o m m o n l y r e s u l t s i n a m e a n s u r v i v a l t i m e o f 7 o r 8 days , w i t h s e v e r e b a c t e r e m i a a c o m m o n f i n d i n g . 1 3 , 14)

I n a p r e v i o u s r e p o r t a n e m p i r i c a l m o d e l was p r o p o s e d that d e s c r i b e d the a c c u m u l a t i o n o f l e t h a l i n j u r y d u r i n g r a d i a t i o n , for doses i n the 3 0 - d a y l e t h a l r a n g e . (1) T w o p r o c e s s e s w e r e pos tu l a t ed , the f i r s t dependent on e x p o s u r e t i m e ( r e v e r s i b l e ) and e m b o d y i n g a cons tan t , l i n e a r r e v e r s a l o f r a d i a t i o n e f fec t s , and the s econd independent o f e x p o s u r e t i m e for p e r i o d s t h r o u g h a t l e a s t 24 h o u r s ( i r r e v e r s i b l e ) . M a t h e m a t i c a l d e s c r i p t i o n o f the t i m e - d e p e n d e n t p r o c e s s i s g i v e n b y

w h e r e D / D 0 i s the r a t i o o f a dose ( D ) p r o t r a c t e d o v e r a t i m e p e r i o d t t o the i n s t a n t a n e o u s l y d e l i v e r e d dose ( D 0 ) that p r o d u c e s the s ame m o r t a l i t y , T i s a s p e c i e s p a r a m e t e r that r e l a t e s r a d i o s e n s i t i v i t y and r e v e r s a l rate.,

T h e s tud ie s r e p o r t e d h e r e a r e c o n c e r n e d w i t h a k i n e t i c d e s c r i p t i o n o f r a d i a t i o n i n j u r y a c c u m u l a t i o n a s s o c i a t e d w i t h l e t h a l i t y e x p r e s s e d w i t h i n 9 days af ter e x p o s u r e . T h e d e s c r i p t i v e a n a l y s i s i s l i m i t e d t o s i n g l e p r o t r a c t e d doses o f C o 6 0 7 - r a y s and i s b a s e d on the s a m e m o d e l that was u s e d for 9-30 day l e t h a l i t y . ( l>

M e t h o d s

S i x - w e e k - o l d L A F X m i c e , m a l e and f e m a l e , w e r e ob ta ined f r o m the J a c k s o n M e m o r i a l L a b o r a t o r y , B a r H a r b o r , M a i n e . A t 120 ± 7 days they w e r e e x p o s e d t o 7 - r a y s f r o m one o f t h r ee C o 6 0 s o u r c e s : 5000- , 1000- , o r 1 0 0 - c u r i e . C o n d i t i o n s o f i r r a d i a t i o n and e x p e r i m e n t a l p r o c e d u r e s w e r e a s p r e v i o u s l y d e s c r i b e d . ( - 0 T h e r e s u l t s r e p o r t e d h e r e a r e b a s e d o n s i n g l e doses of 900 to 3200 r , p r o t r a c t e d o v e r 9 d i f f e r en t e x p o s u r e p e r i o d s r a n g i n g f r o m 60 to 1440 m i n . T h e r e w e r e 93 d o s e - e x p o s u r e - t i m e g r o u p s ; m o s t c o n t a i n e d 60-72 a n i m a l s , w i t h e q u a l n u m b e r s o f e a c h sex . A t o t a l o f a p p r o x i m a t e l y 5500 i r r a d i a t e d a n i m a l s i s r e p r e s e n t e d i n the a n a l y s i s . T i m e o f dea th was c o m p u t e d f r o m the b e g i n n i n g o f the e x p o s u r e . G r o s s n e c r o p s i e s w e r e p e r f o r m e d o n r a n d o m l y s e l e c t e d a n i m a l s f r o m e a c h d o s e - t i m e g r o u p . I n s o m e i n s t a n c e s , s egmen t s o f duodenum and i l e u m w e r e e x a m i n e d m i c r o s c o p i c a l l y .

I n g r o u p s g i v e n a n t i b i o t i c t h e r a p y , e ach m o u s e r e c e i v e d 5 mg s t r e p t o m y c i n i n 0.25 m l s a l i n e s u b c u t a n e o u s l y once e a c h day, b e g i n n i n g o n the f i r s t day af ter i r r a d i a t i o n . C o r r e s p o n d i n g c o n t r o l s r e c e i v e d a n e q u i v a l e n t v o l u m e o f s a l i n e o r no th ing . F o r d e t e r m i n a t i o n o f the d a i l y i n c i d e n c e o f b a c t e r e m i a , 200 a n i m a l s f r o m a sepa ra t e s a c r i f i c e s e r i e s w e r e u s e d . A b d o m e n and ches t w e r e opened a s e p t i c a l l y and b l o o d was a s p i r a t e d f r o m the h e a r t w i t h a s t e r i l e s y r i n g e . S o m e b l o o d was s t r e a k e d on the s u r f a c e of a b l o o d aga r p la te and the r e m a i n i n g b l o o d was i n o c u l a t e d in to n u t r i e n t o r b r a i n - h e a r t i n f u s i o n b r o t h . C u l t u r e s w e r e i n c u b a t e d for 2 4 hr ; then a l l b r o t h tubes w e r e s t r e a k e d onto E M B a g a r . I n d i v i d u a l c o l o n i e s w e r e i s o l a t e d f r o m bo th types o f p l a t e s and w e r e i n o c u l a t e d in to v a r i o u s s p e c i a l b r o t h m e d i a for i d e n t i f i c a t i o n o f o r g a n i s m s . N e g a t i v e p la t e s w e r e i n c u b a t e d fo r a n a d d i t i o n a l 2 4 h r . N o a n a e r o b i c c u l t u r e s w e r e m a d e s i n c e i t has been r e p o r t e d that v e r y few a n a e r o b e s a r e found in b l o o d c u l t u r e s . (

C h a r a c t e r i s t i c s o f the P r i n c i p a l S y m p t o m C o m p l e x e s

T h e p a t t e r n o f d a i l y m o r t a l i t y af ter s i n g l e e x p o s u r e s o v e r a w i d e dose r ange r e v e a l s a c l u s t e r i n g o f deaths a r o u n d c e r t a i n c h a r a c t e r i s t i c s u r v i v a l t i m e s . W h e n the m e a n s u r v i v a l t i m e i s about 5 d a y s , the m o r t a l i t y i s c o n c e n t r a t e d w i t h i n 4 - 6 days af ter e x p o s u r e ; af ter dose s i n the 3 0 - d a y l e t h a l r ange , m o s t o f the m o r t a l i t y o c c u r s w i t h i n the p e r i o d 9 -16 d a y s . B e t w e e n these e x t r e m e s , i n g roups e x p o s e d to i n t e r m e d i a t e d o s e s , t h e r e i s a c l u s t e r i n of deaths b e t w e e n 6 and 9 d a y s . M o r t a l i t y in the 4 to 6 -day p e r i o d , d e s c r i b e d a s "acute i n t e s t i n a l dea th , " i s c h a r a c t e r i z e d b y e x t e n s i v e l o s s o f i n t e s t i n a l e p i t h e l i u m . T h e i n t e r m e d i a t e p e r i o d (6-9 days ) i s not a l w a y s r e c o g n i z e d a s a d i s t i n c t en t i ty , but a p p e a r s to be n e i t h e r a la te i n t e s t i n a l n o r an e a r l y m a r r o w dea th .

A f t e r doses o f C o 6 7 - r a y s i n the 4 - t o 6 -day l e t h a l r ange , s e v e r e denuda t ion of i n t e s t i n a l e p i t h e l i u m is found in decedents a t 4 days . By the 6th day e v i d e n c e o f ex t ens ive r e p a i r i s f r equen t ly seen, o r even f a i r l y n o r m a l in tac t m u c o s a m a y be found. Tha t the re had been p r e v i o u s damage to the i n t e s t i n a l m u c o s a i s i n d i c a t e d by the a b n o r m a l a r c h i t e c t u r e . A l t h o u g h l a r g e s egmen t s o f denuded i n t e s t i n a l e p i t h e l i u m can be t o l e r a t e d for l ong p e r i o d s , ^ ) rnost ev idence i n d i c a t e s that s e v e r e f l u i d and e l e c t r o l y t e l o s s f r e q u e n t l y o c c u r s i n the 4 - t o 6 -day p e r i o d and i s i m p o r t a n t i n the c r i t i c a l l e t h a l event . (2 ,7- 11)

D e s p i t e ev idence of e x t e n s i v e r e p a i r of the i n t e s t i n a l m u c o s a on the 6th day, a h igh i n c i d e n c e of b a c t e r e m i a was o b s e r v e d in a n i m a l s that s u r v i v e d to the 6th or 7th day. In o r d e r to a s s e s s the r o l e of i n f e c t i o n in the m o r t a l i t y o c c u r r i n g in the f i r s t 9 days after e x p o s u r e , the effect of p r o t e c t i v e t r e a t m e n t w i t h s t r e p t o m y c i n was tes ted a t r a d i a t i o n l e v e l s a t w h i c h 4 - t o 6 -day m o r t a l i t y p r e d o m i n a t e s and a t l e v e l s a t w h i c h 6- to 9 -day m o r t a l i t y p r e d o m i n a t e s . In a d d i t i o n , the i n c i d e n c e o f p o s i t i v e b l o o d c u l t u r e s in these g roups was d e t e r m i n e d for c o m p a r i s o n w i t h m o r t a l i t y s t ud i e s . A f t e r a 6 0 - m i n e x p o s u r e a t a dose l e v e l w i t h i n the range for i n t e s t i n a l death, the d a i l y i n c i d e n c e of b a c t e r e m i a w i t h i n the f i r s t 4 days d i d not exceed 20%, and s t r e p t o m y c i n t r e a t m e n t r e s u l t e d i n a l m o s t n o change i n m e a n s u r v i v a l t i m e ( f r o m 5.7 to 5.9 days ) . A f t e r a somewha t l o w e r dose , w h e r e 6- to 9 -day m o r t a l i t y p r e d o m i n a t e d , the p r i n c i p a l m o r t a l i t y o c c u r r e d w i t h i n 9 -13 days when s t r e p t o m y c i n t h e r a p y was g i v e n . D a i l y m o r t a l i t y i n t r e a t e d and un t rea ted i r r a d i a t e d g roups i n shown i n T a b l e 1 . I n un t rea ted i r r a d i a t e d g roups , a l m o s t a l l a n i m a l s t e s t ed had p o s i t i v e b l o o d c u l t u r e s b y day 7 . B a c t e r i a w e r e u s u a l l y r e c o v e r e d i n pure c u l t u r e and w e r e p r i n c i p a l l y g r a m - n e g a t i v e rods c o m m o n l y found in the i n t e s t i n a l t r a c t . In s t r e p t o m y c i n - t r e a t e d a n i m a l s , the i n c i d e n c e of b a c t e r e m i a was l e s s than 25%, and the o r g a n i s m s r e p r e s e n t e d w e r e shown to be d r u g - r e s i s t a n t .

T A B L E 1

E f f e c t o f s t r e p t o m y c i n t h e r a p y o n d a i l y m o r t a l i t y a f te r i r r a d i a t i o n

M o r t a l i t y , % M e a n

S t r e p t o m y c i n t h e r a p y * D a y s af ter i r r a d i a t i o n s u r v i v a l

t i m e

N 4 5 6 7 8 9 10 11 12 13 (days)

2800 r * *

0 70 37.1 58.6 4.3 5.7 + 40 5.0 22.5 55.0 10.0 7.5 5.9

1900 r * *

0 108 5.5 3.7 11.1 25.0 26.8 22.2 5.5 7.5 + 54 3.7 7.4 3.7 3.7 7.4 18.5 27.8 22.2 5.5 10.1

* D o s e : 5 m g s t r e p t o m y c i n i n 0.25 m l s a l i n e s o l u t i o n per m o u s e , a d m i n i s t e r e d d a i l y b e g i n n i n g on day 1

* 6 0 - m i n e x p o s u r e

A n a l y s i s

L e t h a l i t y i n the 4 - t o 6 -day p e r i o d

B a s e d on the c o r r e l a t i o n be tween d a i l y d i s t r i b u t i o n o f deaths and c h a r a c t e r i s t i c pa thology, the s i x t h day af ter i r r a d i a t i o n a p p e a r s to r e p r e s e n t the m a x i m u m s u r v i v a l t i m e for e x p r e s s i o n o f the i n t e s t i n a l mode of death and the m i n i m u m t i m e for a l a t e r r a d i a t i o n death a s s o c i a t e d w i t h i n t e r m e d i a t e d o s e s . The t o t a l m o r t a l i t y o c c u r r i n g on day 6 i s , then, the r e s u l t o f these two l e t h a l m e c h a n i s m s . T o m i n i m i z e the e r r o r r e s u l t i n g f r o m o v e r l a p in t i m e o f death, the m o r t a l i t y on the s i x t h day has been p a r t i t i o n e d on the b a s i s of the r e l a t i v e m a g n i t u d e s of the m o r t a l i t y on the f i f th and seven th days . T h e r e i s , o f c o u r s e , a d i f f e r e n c e in the amount of m o r t a l i t y i n th i s p e r i o d depending o n whe ther o r not a l l 6 -day deaths a r e i n c l u d e d . I t c an be d e m o n s t r a t e d , h o w e v e r , that the k i n e t i c r e l a t i o n s of dose and e x p o s u r e t i m e for i n j u r y a c c u m u l a t i o n a re u n a l t e r e d when a n a l y s i s i s b a s e d on the e n t i r e 4 - t o 6 -day m o r t a l i t y . I n c i d e n t a l l y , no deaths o c c u r r e d be fo re the fou r th day in the e n t i r e dose - e x p o s u r e - t i m e s e r i e s r e p o r t e d h e r e .

F e m a l e s showed a c o n s i s t e n t l y g r e a t e r s e n s i t i v i t y than m a l e s ; the d i f f e r e n c e was dependent on e x p o s u r e t i m e . The a v e r a g e shif t i n L D 5 0

was 100 r . C o n s t a n t s of the e x p o s u r e - t i m e effect, h o w e v e r , w e r e not s i g n i f i c a n t l y d i f fe ren t . In the s t a t i s t i c a l a n a l y s i s , m o r t a l i t y data for the m a l e s have been adjus ted for the sex d i f f e r e n c e in s e n s i t i v i t y , and c o m puta t ions a r e b a s e d on c o m b i n e d data .

The pos tu la t e s upon w h i c h the f o l l o w i n g a n a l y s i s i s b a s e d have been p r e v i o u s l y s ta ted for 9 - to 3 0 - d a y l e t h a l i t y . D o s e - m o r t a l i t y r e l a t i o n s for a l l e x p o s u r e p e r i o d s a r e shown a s p r o b i t t r a n s f o r m s i n F i g u r e 1 . In g roups exposed for 360 m i n or l e s s , l e t h a l i t y i s a s s u m e d to

Figure 1

Probit transform of 4- to 6 -day mortal i ty (°Jo)t versus dose, by exposure-t ime group. Dashed l ine , estimated mortal i ty distribution for instantaneously del ivered doses ( L D ^ q = 1642 r, cr = 2 2 8 r). Sol id l ines, theoret ical curves. See explanation in text.

11

exp res s t ime-dependent i n ju ry only . The effect of t ime- independent l e tha l p r o c e s s e s is seen in groups exposed over longer pe r iods , and this effect p redomina tes for p ro t r ac t i ons of 960 and 1440 m i n . The effect of exposure t ime on d o s e - m o r t a l i t y r e l a t ions for exposure per iods through 360 m i n i s used to d e s c r i b e the k ine t i c s of in ju ry accumula t i on for the t ime-dependent effect. The d i s t r i b u t i o n of m o r t a l i t y wi th respec t to dose in groups exposed for 60 m i n is used as the re fe rence point in the e m p i r i c a l d e t e r m i n a t ion of the D ' / D ' ^ Q r a t i o s . * The mean ra t io s for exposure t imes through 360 m i n are g iven i n Tab le 2. The r e l a t i o n between mean D ' / D ' ^ Q r a t io s and exposure t ime ( F i g u r e 2) shows a constant l i nea r r educ t ion of

T A B L E 2

M ean Dy D'^Q r a t ios for m o r t a l i t y w i th in 4 to 6 days after i r r a d i a t i o n

E x p o s u r e t ime , m i n

N u m b e r of groups

M e a n r a t io

Standard dev ia t ion

60 15 1.0080 0.04381 90 13 1.0546 0.06073

1Z0 12 1.0916 0.06309 180 9 1.1349 0.05886 240 10 1.2915 0.08092 360 10 1.3529 0.07107

i 1 1 1 1 1 y—p—r

EXPOSURE TIME (min)

Figure 2. Relation between mean D ' /D 'gO a n d exposure time. The slope of the least-squares line, forced through 1.00 at t = 60 min, is 0.0012854 ± 0.0000761. 1/b = 778 min, £ yb = 61 min .

* T h e notations prime and double prime are used to distinguish variables of the 4-to 6-and 6-to 9-day periods from each other and also from corresponding variables of the 9-30 day period previously described, (i)

d o s e - e f f e c t i v e n e s s w i t h i n c r e a s e i n e x p o s u r e t i m e . T h e c u r v e was f i t t ed b y the me thod of l e a s t s q u a r e s , w i t h the r e s t r i c t i o n that D ' / D ' ^ Q = 1.000 at t = 60 m i n . F r o m the s lope of th i s c u r v e , def ined as l / ( r ! + 60) (where T 1

is the t i m e constant ) , the e s t ima t e of T 1 is d e t e r m i n e d to be 718 i 61 m i n . T h e e s t i m a t e d D ' / D ' Q l i ne ( F i g u r e Z) has a s lope equa l to the r e c i p r o c a l of T ' . The e s t i m a t e d m o r t a l i t y d i s t r i b u t i o n for i n s t a n t a n e o u s l y d e l i v e r e d d o s e s * shown i n F i g u r e 1 has L D 5 0 = 1642 r and d = 228 r . The c u r v e s i n F i g u r e 1, o ther than that for the 60 m i n g roups , a r e t h e o r e t i c a l and a r e b a s e d on cons tan ts of the l e a s t - s q u a r e s l ine of F i g u r e 2 c o r r e s p o n d i n g to each e x p o s u r e t i m e . F r o m the pos tu la te o f a l i n e a r r e v e r s a l ra te , the r a t e o f dose r e d u c t i o n i s cons tant per uni t t i m e d u r i n g i r r a d i a t i o n and independent o f amount o f a c c u m u l a t e d dose . T h e m e a n o f the n o r m a l l y d i s t r i b u t e d r e v e r s a l r a t e s i s 2.29 r / m i n ; that i s , 50% of the a n i m a l s can r e v e r s e the effects w i t h w h i c h m o r t a l i t y in th i s p e r i o d i s a s s o c i a t e d a t a ra te equa l to o r g r e a t e r than 2.29 r / m i n .

To ob ta in an e s t ima te o f s e n s i t i v i t y to t i m e - i n d e p e n d e n t i n j u r y m e c h a n i s m s a lone , the a s s u m p t i o n of independence of effects was a p p l i e d . In g roups exposed for 480, 960 and 1440 m i n , the c o n t r i b u t i o n of the r e v e r s i b l e effect was r e m o v e d f r o m the t o t a l m o r t a l i t y . C o m p u t a t i o n s w e r e b a s e d on the f o r m u l a

R j £ ~ F^j ^ R d , t = p d , t + Q d - p d , t Q d > o r Qd = i_ P ' » (2)

d, t

w h e r e ^ is the t o t a l p r o p o r t i o n of a n i m a l s dy ing f r o m a dose d p r o t r a c t e d o v e r t i m e t , and ^ and a r e the p r o p o r t i o n dy ing of t i m e - d e p e n d e n t and t i m e - i n d e p e n d e n t effects , r e s p e c t i v e l y . ) T h e r e s u l t a n t m o r t a l i t y , a s s u m e d to r e p r e s e n t e x p r e s s i o n o f the t i m e - i n d e p e n d e n t l e t h a l i n j u r y , i s a p p r o x i m a t e l y n o r m a l l y d i s t r i b u t e d w i t h r e s p e c t t o dose ( F i g u r e 3 ) and i s c h a r a c t e r i z e d by the l e a s t - s q u a r e s l ine f i t ted to the p r o b i t t r a n s f o r m for the c o m b i n e d g r o u p s . L e a s t - s q u a r e s cons tan t s a r e L D 5 0 = 2838 t 35 r and 8 = 394 r .

Figure 3

Probit transform of percent morta l i ty , versus dose, for the t ime-independent (irreversible) effect expressed in the 4- to 6-day period. Constants of the least-squares l ine for the combined are L D 5 0 = 2838 ± 35 r; slope = 0.0025395 ± 0.0002991; a - 394 r.

2000 2500 3000 3500

DOSE (r)

* Calcu la ted from the relation D 0 = t « / ( t « + 6 0 ) ( D 6 0 ) , (See Reference 1.)

L e t h a l i t y in the 6- to 9-day p e r i o d

T h e m o r t a l i t y p a t t e r n in the 6- to 9 -day p e r i o d i s l e s s s table than that in the 4- to 6- or the 9- to 3 0 - d a y p e r i o d s and p r o b a b l y r e f l e c t s c o n t a m i n a t i o n f r o m l e t h a l m e c h a n i s m s that a r e p r e d o m i n a n t l y e x p r e s s e d i n the e a r l i e r and l a t e r p e r i o d s . F o r th i s r e a s o n d e t e r m i n a t i o n s o f the c o n stants a r e subjec t to g r e a t e r e r r o r . In spi te o f t h i s , h o w e v e r , the g e n e r a l d e s c r i p t i o n o f the d o s e - e x p o s u r e - t i m e r e l a t i o n s r e v e a l s a p a t t e r n s i m i l a r to that o b s e r v e d for the o ther m o r t a l i t y p e r i o d s . S ince t he re was no a p pa ren t d i f f e r e n c e i n r e s p o n s e be tween m a l e s and f e m a l e s i n th i s p e r i o d , da ta f r o m the two sexes have been c o m b i n e d .

Dea ths i n th i s p e r i o d p r e d o m i n a t e d after r a d i a t i o n doses that w e r e i n t e r m e d i a t e i n the range c o v e r e d i n th i s s tudy. D o s e - m o r t a l i t y r e l a t i o n s (p rob i t t r a n s f o r m s ) for a l l e x p o s u r e t i m e s a r e shown i n F i g u r e 4 . The p r e s e n c e o f t i m e - d e p e n d e n t and t i m e - i n d e p e n d e n t l e t h a l p r o c e s s e s a r e i n d i c a t e d as in the e a r l i e r p e r i o d , but the t i m e effect was of somewha t s m a l l e m a g n i t u d e . A g a i n , the d o s e - m o r t a l i t y d i s t r i b u t i o n for g roups exposed for 60 m i n was u sed as the b a s i c e x p o s u r e t i m e s e r i e s . T a b l e 3 g i v e s the m e a n D n / D n

6 0 r a t i o s for e x p o s u r e t i m e s t h r o u g h 3 60 m i n , and F i g u r e 5 the r e l a t i o n be tween m e a n D n / D , f

6 0 r a t i o s and e x p o s u r e t i m e . F r o m the s lope of the l e a s t - s q u a r e s l i ne the e s t i m a t e of T F L was c a l c u l a t e d to be 963 + 104 r n i n . The e s t i m a t e d D M / D M Q l i ne i n F i g u r e 5 has a s lope equa l t o the r e c i p r o c a l o f T " . T h e e s t i m a t e d m o r t a l i t y d i s t r i b u t i o n for i n s t a n t a n e o u s l y d e l i v e r e d doses shown i n F i g u r e 4 has L D 5 0 = 1385 r and O = 178. A l l c u r v e s , o ther than that for the 6 0 - m i n g roups , a r e t h e o r e t i c a l , b a s e d o n D , ,

/ / D ! , 5 0 v a l u e s i n F i g u r e 5 that c o r r e s p o n d t o each exposu re t i m e . F o r 6-to 9 -day m o r t a l i t y the m e a n o f the n o r m a l l y d i s t r i b u t e d r e v e r s a l r a t e s i s 1.44 r / m i n .

Figure 4. Probit transform of the 6-to 9-day mortali ty (°]o) versus dose, by exposure-time group. Dashed l ine , estimated mortal i ty distribution for instantaneously delivered doses ( L D 5 0 = 1385 r, £ = 178 r). Sol id l ine , theoretical curves. See explanation in text.

T A B L E 3

M e a n D " / D " 6 0 r a t i o s for m o r t a l i t y w i th in 6 to 9 days after i r r a d i a t i o n

E x p o s u r e t ime , m i n

N u m b e r of groups

M e a n ra t io

Standard dev ia t ion

60 9 0.9858 0.062347 90 5 1.0776 0.066890

120 4 1.0655 0.050948 180 8 1.1549 0.057613 240 9 1.1700 0.045604 360 9 1.2814 0.074572

,.b i , ! , j , ! , r

1.5 -

E X P O S U R E T I M E ( m i n )

Figure 5 . Relation between mean D"/D"6o a n d e x P o s u r e time. The slope of the least-squared line, forced through 1.00 at t = 60 min is 0.00097749 ± 0.0000994. 1/b = 1023 min, & yh - 104 min.

T o e s t i m a t e the s e n s i t i v i t y t o t i m e - i n d e p e n d e n t i n j u r y m e c h a n i s m s e x p r e s s e d i n t h i s p e r i o d , the c o n t r i b u t i o n o f the r e v e r s i b l e e f f ec t w a s r e m o v e d f r o m the t o t a l m o r t a l i t y i n g r o u p s e x p o s e d o v e r 480 , 960 a n d 1440 m i n . C o m p u t a t i o n a l p r o c e d u r e w a s the s a m e a s f o r the 4 - t o 6 - d a y p e r i o d . T h e d o s e - m o r t a l i t y r e l a t i o n f o r the r e s u l t a n t t i m e - i n d e p e n d e n t l e t h a l i n j u r y ( F i g u r e 6 ) a p p e a r s t o b e n o r m a l l y d i s t r i b u t e d , a n d i s c h a r a c t e r i z e d b y the l e a s t - s q u a r e s l i n e f i t t e d t o the p r o b i t t r a n s f o r m fo r the c o m b i n e d g r o u p s . C o n s t a n t s a r e L D 5 0 = 2369 ± 35 r , a n d B = 462 r .

1200 1500 2000 2500 3 0 0 0

DOSE (r)

Figure 6. Probit transform of per cent mortal i ty versus dose for the t ime-independent (irreversible) effect expressed in the 6- to 9-day period. Constants of the least-squares l ine for the combined groups are L D 5 Q = 2369 ± 35 r, slope = 0.0021660 ± 0.0001897. £ = 462 r.

D i s c u s s i o n

D i f f i c u l t i e s a s s o c i a t e d w i t h s e p a r a t i o n o f the s e v e r a l m a j o r i n j u r y c o m p l e x e s that f ind l e t h a l e x p r e s s i o n in the acute (30-day) p o s t i r r a d i a t i o n p e r i o d a r e w e l l r e c o g n i z e d . Y e t a k i n e t i c d e s c r i p t i o n o f the a c c u m u l a t i o n of effect for e ach of the p r i n c i p a l m o d e s of i n j u r y i s o f va lue in c h a r a c t e r i z i n g the d i f f e ren t i n j u r y p r o c e s s e s . I t i s h a r d to o v e r c o m e p r o b l e m s s u c h as d i s t i n g u i s h i n g the v a r i o u s modes of acute death and e s t i m a t i n g the extent o f p o s s i b l e i n t e r a c t i o n s in the a c t u a l p r o d u c t i o n of l e t h a l i t y . That d i f f e r e n c e s i n s e n s i t i v i t y and k i n e t i c s do e x i s t i s c l e a r l y evident , and they have been d e s c r i b e d . 15, 16) N e v e r t h e l e s s , adequate i n d i c a t o r s a r e not a v a i l ab le for i d e n t i f y i n g the p r e d o m i n a n t i n j u r y o r i n j u r i e s i n each i n d i v i d u a l a n i m a l that s u c c u m b s to r a d i a t i o n effects , and i n d i r e c t methods o f s e p a r a t i o n a r e r e q u i r e d . E r r o r s i n the m o r t a l i t y v a l u e s for a s p e c i f i c mode o f death, when p a r t i t i o n i s b a s e d o n t i m e a lone , a r i s e f r o m r a n d o m shif ts i n the m o d a l t i m e of death. In g e n e r a l , h o w e v e r , th i s a p p r o a c h y i e l d s a s t a b le r e s p o n s e pa t t e rn , e s p e c i a l l y for the 4 - t o 6 -day p e r i o d . I t a p p e a r s to i n v o l v e a m i n i m a l amount of e r r o r and to p r o v i d e a p r o c e d u r e for s e p a r a t i ng a t l e a s t the m a j o r componen t p r o c e s s e s e x p r e s s e d in the acute m o r t a l i t y .

T h e t i m e - d o s e p a t t e r n o f m o r t a l i t y , t aken together w i t h the t i m e s o f o c c u r r e n c e o f i n t e s t i n a l l e s i o n s and b a c t e r e m i a , suppor t s the t h e s i s that the 4 - to 6 -day m o r t a l i t y i s l a r g e l y d i s t i n c t f r o m that w h i c h o c c u r s l a t e r . T h e s e v e r e l y d a m a g e d i n t e s t i n a l m u c o s a , w i t h which m o r t a l i t y i n

the 4 - to 6 -day p e r i o d i s a s s o c i a t e d , i s f r e q u e n t l y r e p a i r e d af ter 6 d a y s . T h i s i s found to be the case even in deceden t s . I t w o u l d appea r that b r e a k d o w n of the i n t e s t i n a l b a r r i e r due to l o s s o f e p i t h e l i u m i s o f l i t t l e i m p o r t a n c e in the i n i t i a t i o n o f b a c t e r e m i a that d e v e l o p s af ter s e v e r a l d a y s . S m a l l n u m b e r s o f e n t e r i c o r g a n i s m s n o r m a l l y pene t ra te the i n t e s t i n a l l i n i n g but a r e unab le t o m u l t i p l y in the c i r c u l a t i n g b l o o d . (^)

E a r l y f a i l u r e o f n o r m a l body defense m e c h a n i s m s m a y b e the c r i t i c a l f a c to r in d e v e l o p m e n t of the i n f e c t i o n . In m o s t c a s e s o n l y a s i n g l e s p e c i e s of m i c r o o r g a n i s m was i s o l a t e d . T h u s , i t a p p e a r s that m u l t i p l i c a t i o n m u s t o c c u r i n the b l o o d s t r e a m , r e s u l t i n g i n the o v e r w h e l m i n g b a c t e r e m i a . I n dose g r o u p s i n w h i c h m o r t a l i t y was c o n c e n t r a t e d i n the 6-to 9 - d a y p e r i o d , s t r e p t o m y c i n t h e r a p y g r e a t l y r e d u c e d m o r t a l i t y i n th i s p e r i o d . Y e t t h e r e w e r e n o s u r v i v o r s b e y o n d 1 4 days (unpub l i shed r e s u l t s ) . H e m a t o p o i e t i c f a i l u r e , p r o b a b l y c h i e f l y a n e m i a , m a y accoun t for t h i s m o r t a l i t y i n the a b s e n c e o f i n f e c t i o n . S i m i l a r r e s u l t s w e r e r e p o r t e d b y M i l l e r , e t a l . who found a h i g h i n c i d e n c e o f b a c t e r e m i a af ter 6-7 days when the r a d i a t i o n dose was s u f f i c i e n t t o p r o d u c e e x t e n s i v e m o r t a l i t y on days 8-9; s t r e p t o m y c i n t h e r a p y was found to be m o s t e f fec t ive in r e d u c i n g th i s m o r t a l i t y . 17) A f t e r a l o w e r r a d i a t i o n dose ( s l i g h t l y m o r e than the L D 5 0 / 3 0 ) , the h ighes t i n c i d e n c e o f b a c t e r e m i a o c c u r r e d af ter 10-11 days and was n e v e r as s e v e r e as that o b s e r v e d af te r l a r g e r d o s e s . D r u g t h e r a p y was o f m o r e l i m i t e d v a l u e i n r e d u c i n g m o r t a l i t y i n t h i s p e r i o d ; s o m e deaths a l w a y s o c c u r r e d i n sp i te o f t r e a t m e n t .

O u r k i n e t i c m o d e l o f i n j u r y a c c u m u l a t i o n d u r i n g i r r a d i a t i o n has b e e n u s e d p r e v i o u s l y in a s t a t i s t i c a l d e s c r i p t i o n o f the e x p o s u r e t i m e effect fo r 9- to 3 0 - d a y m o r t a l i t y . E x t e n s i o n o f the m o d e l t o o the r m o r t a l i t y p e r i o d s p e r m i t s c o m p a r i s o n o f r e v e r s a l k i n e t i c s for d i f f e r e n t l e t h a l c o m p l e x e s . A r a p i d r e v e r s a l o r r e p a i r o f the r a d i a t i o n effect i s c h a r a c t e r i s t i c o f i n t e s t i n a l i n j u r y . T h i s i s i n d i c a t e d b y a v a l u e o f 719 m i n for the r e v e r s a l cons t an t T 1 , c o m p a r e d w i t h 1419 m i n for the i n j u r y a s s o c i a t e d w i t h 9 - t o 3 0 - d a y m o r t a l i t y ; ' ' fo r the i n t e r m e d i a t e 6- to 9 - d a y m o r t a l i t y , T" i s 963 m i n .

W e w i s h t o e x p r e s s ou r a p p r e c i a t i o n t o S y l v a n u s A . T y l e r for h i s a s s i s t a n c e i n the d e v e l o p m e n t o f the m o d e l and t o M e r l i n H . D i p e r t fo r d i r e c t i o n o f the n u m e r i c a l p r o c e s s i n g o f the da ta .

R e f e r ence s

1 . S t e a m e r , S . P . , and S . A . T y l e r . R a d i a t i o n m o r t a l i t y i n the m o u s e . M o d e l o f the k i n e t i c s o f i n j u r y a c c u m u l a t i o n . I . P r o t r a c t e d dose s i n the 3 0 - d a y l e t h a l r a n g e . R a d i a t i o n R e s . 20, 619-663 (1963).

2 . B o n d , V . P . , M . S . S i l v e r m a n , and E . P . C r o n k i t e . P a t h o g e n e s i s and pa tho logy o f p o s t - i r r a d i a t i o n i n f e c t i o n . R a d i a t i o n R e s . JL, 389 -400 (1954).

17

3 . V o g e l , H . H . , J r . , J . W . C l a r k , C . W . H a m m o n d , D . B . C o o p e r , and C . P . M i l l e r . Endogenous in fec t ion i n m i c e i r r a d i a t e d w i th fast neu t rons o r g a m m a r a y s . P r o c . Soc . E x p t l . B i o l . M e d . 87_, 114- 119 (1954).

4 . W a r r e n , S . L . , and G . H . W h i p p l e . Roen tgen r a y i n t o x i c a t i o n , I . U n i t dose ove r t h o r a x n e g a t i v e - o v e r abdomen l e t h a l . E p i t h e l i u m o f s m a l l in tes t ine s e n s i t i v e to X - r a y s . J . E x p t l . M e d . _35, 187-202 (1922).

5 . Q u a s t l e r , H. The nature of i n t e s t i n a l r a d i a t i o n death. R a d i a t i o n R e s . 4_, 303-320 (1956).

6 . Q u a s t l e r , H . , and M- Z 'ucke r . The h i e r a r c h y o f modes o f r a d i a t i o n death in s p e c i f i c a l l y p ro t ec t ed m i c e . R a d i a t i o n R e s , J_0, 402-409 (1959)

7 . J a c k s o n , K - L . , R . Rhodes , and C . E n t e n m a n . E l e c t r o l y t e e x c r e t i o n i n the r a t after s eve re i n t e s t i n a l damage by X - i r r a d i a t i o n . R a d i a t i o n R e s .

_8, 361-373 (1958).

8 . Swif t , M. N - , and S . T . T a k e t a . E f f ec t on c i r c u l a t i n g b lood v o l u m e o f p a r t i a l s h i e l d i n g o f r a t in tes t ine d u r i n g X - i r r a d i a t i o n . R a d i a t i o n R e s . _8, 516-525 (1958).

9 - S m i t h , J . C- E f f ec t o f s e g m e n t a l in tes t ine s h i e l d i n g on m o r t a l i t y f r o m i n t e s t i n a l r a d i a t i o n . A . M . A . A r c h . P a t h o l . 70.. 94-102 ( i960 ) .

10. T a k e t a , S . T . W a t e r - e l e c t r o l y t e and a n t i b i o t i c t he rapy aga ins t acute (3- to 5-day) i n t e s t i n a l r a d i a t i o n death in the ra t . R a d i a t i o n R e s . 1_6_, 312-326 (1962).

11. C o n a r d , R . A . , E . P . C r o n k i t e , G . B r e c h e r , and C P . A . S t r o m e . E x p e r i m e n t a l t he r apy o f the g a s t r o i n t e s t i n a l s y n d r o m e p r o d u c e d by l e t h a l doses o f i o n i z i n g r a d i a t i o n . J . A p p l . P h y s i o l . % 227-233 (1956).

12. W a r r e n , S . L , , and G . H . W h i p p l e . R o e n t g e n r a y i n t o x i c a t i o n . I . B a c t e r i a l i n v a s i o n o f the b l o o d s t r e a m a s in f luenced b y X - r a y d e s t r u c t i o n o f the m u c o s a l e p i t h e l i u m o f the s m a l l i n t e s t i ne . J . E x p t l . M e d . 38, 713-723 (.1923).

13. G o n s h e r y , L , , R . Q . M a r s t o n , and W . W . S m i t h . N a t u r a l l y o c c u r r i n g in fec t ions i n un t rea ted and s t r e p t o m y c i n - t r e a t e d X - i r r a d i a t e d m i c e . A m . J . P h y s i o l . 172, 359-364 (1953).

14. M i l l e r , C . P . , C . W . H a m m o n d , and M . T o m p k i n s . The r o l e o f i n f e c t i o n i n r a d i a t i o n i n j u r y , J , L a b , C l i n . M e d . 38, 331-343 (1951).

15. L a m e r t o n , L . F . , A , H . P o n t i f e x , N - M . B l a c k e t t , and K - A d a m s , E f f ec t s of p r o t r a c t e d i r r a d i a t i o n on the b l o o d - f o r m i n g o rgans of the ra t . P a r t I : Con t inuous e x p o s u r e . B r i t . J . R a d i o l . 33, 287-301 ( i960) .

18

16. S t o r e r , J . B . Rate o f r e p a i r o f r a d i a t i o n damage in m i c e . S y m p o s i u m on the S h o r t e r - T e r m B i o l o g i c a l H a z a r d s o f a F a l l o u t F i e l d . A t o m i c E n e r g y C o m m i s s i o n , Washington , D . C D e c . 12- 14, 1956. pp. 93 -99 .

17. M i l l e r , C P . , C . W . H a m m o n d , M . T o m p k i n s , and G . S h o r t e r . The t r ea tment o f p o s t - i r r a d i a t i o n in fec t ion w i t h an t i b io t i c s ; A n e x p e r i m e n t a l study on m i c e . J . L a b . C l i n . M e d . 39, 462-479 (1952).

T H R O M B I I N T H E L E F T A T R I U M

R . J . M i c h a e l F r y ; K a t h e r i n e H a m i l t o n , and H e r m a n n L i s c o

In a p r e v i o u s r e p o r t ( l ) a d e s c r i p t i o n was g i v e n of a c a r d i a c l e s i o n found i n i n a c t i v e B A L B / c f ema le b r e e d e r s . T h e l e s i o n c o n s i s t s o f one o r m o r e t h r o m b i , in v a r i o u s s tages o f o r g a n i z a t i o n , in the left a t r i u m . A n u m b e r o f t h r o m b o t i c ep i sodes m a y o c c u r w i t h a c o n s i d e r a b l e t i m e l a p s e be tween e p i s o d e s . T h e r e a p p e a r s to be a c o r r e l a t i o n be tween the n u m b e r o f l i t t e r s and the n u m b e r o f t h r o m b i . F o r th i s r e a s o n and o the r s i t i s b e l i e v e d that the t h r o m b u s f o r m a t i o n i s a s s o c i a t e d w i t h p r e g n a n c y o r p a r t u r i t i o n . T h e s e f ind ings have been c o n f i r m e d i n another c o l o n y , ( w T h e i n c i d e n c e o f t h i s l e s i o n , a p p r o x i m a t e l y 60%, i n r e t i r e d f e m a l e b r e e d e r s , i s s i m i l a r in the two c o l o n i e s . We have a l s o found the s a m e type o f l e s i o n i n v i r g i n f e m a l e and i n m a l e B A L B / c m i c e , h o w e v e r . The f o l l o w i n g b r e e d i n g e x p e r i m e n t was c a r r i e d out to a s c e r t a i n whe ther a genet ic f ac to r was i n v o l v e d . A B A L B / c m a l e was c r o s s e d w i t h a C 5 7 B L / 6 f e m a l e and the F j p r o g e n y w e r e b r e d w i t h the B A L B / c m i c e . T h e p r o g e n y o f each s u c c e s s i v e g e n e r a t i o n w e r e s i m i l a r l y b r e d w i t h m i c e o f the B A L B / c p a r ent s t r a i n . S i x b a c k c r o s s gene ra t i ons w e r e p r o d u c e d , each w i t h a n i n c r e a s e d pe r cen t age of the B A L B / c s t r a i n . The r e s u l t s , shown i n T a b l e 4, i n d i c a t e that w i t h an i n c r e a s i n g pe rcen tage of B A L B / c i n the f ema le m i c e the p r o b a b i l i t y o f t h r o m b u s f o r m a t i o n i n c r e a s e s . T h i s i s o n l y k n o w n to be t rue i f the f e m a l e m i c e r e s u l t i n g f r o m the b a c k c r o s s b r e e d i n g a r e t h e m s e l v e s u s e d for b r e e d i n g . I t i s not known whe ther the genet ic m a k e - u p of the h y b r i d i n f l u e n c e s the i n c i d e n c e o f th i s l e s i o n in the v i r g i n f e m a l e o r

T A B L E 4

I nc idence o f t h r o m b u s i n s u c c e s s i v e b a c k c r o s s gene ra t i ons

G e n e r a t i o n % B A L B / c

N o . f e m a l e s

N o . w i t h t h r o m b i % N o .

m a l e s N o . w i t h t h r o m b i

1 75.0 3 0 0 2 0

2 87.5 9 2 22 7 0

3 93.8 6 2 33 5 0

4 96.9 7 3 43 6 0

5 98.4 10 6 60 6 0

6 99.2 17 13 76 18 1

m a l e m i c e . F u r t h e r m o r e the a s s o c i a t i o n between the genetic fac tor and the pathogenesis of t h rombus f o r m a t i o n is not known. The genetic factor m a y be r e l a t ed to coagula t ion and the changes that o c c u r in this du r ing p regnancy and p a r t u r i t i o n . On the other hand, the a r c h i t e c t u r e of the left a t r i u m m a y be affected.

R e f e r e n c e s

1 . F r y , R . J . M . , K . H a m i l t o n , and H . L i s c o . F e d e r a t i o n P r o c . 1 % 109 (I960).

2 . M e i e r , H . , and W. C Hoag . E x p t l . M e d . Surg . 1% 317-322 ( l 9 6 l ) .

T H E I N T E S T I N A L F L O R A O F T H E M O U S E

R . J . M i c h a e l F r y , P a t r i c i a A . B r e n n a n , and T h o m a s E . F r i t z

I t i s now d e s i r a b l e , i f not e s s e n t i a l , to know the s p e c t r u m and d i s t r i b u t i o n of the i n t e s t i n a l f l o r a of a n i m a l s that w i l l be u s e d for i r r a d i a t i o n and i n t e s t i n a l c e l l g e n e r a l e x p e r i m e n t s .

T h e r e i s c o n s i d e r a b l e v a r i a t i o n i n the n u m b e r and types o f b a c t e r i a i n m i c e m a i n t a i n e d under d i f f e ren t e n v i r o n m e n t s . T h e p r e s e n t i n v e s t i g a t i o n has b e e n c o n c e r n e d w i t h the f o l l o w i n g :

1) E s t a b l i s h i n g the types , d i s t r i b u t i o n , and n u m b e r s ( e s t i m a t e s ) o f b a c t e r i a in the v a r i o u s r e g i o n s o f the s m a l l i n t e s t i n e o f C F # 1 and B C F X m i c e ;

2 ) D e t e r m i n i n g the i n f l uence of v a r i o u s f a c t o r s , s u c h as s t a r v a t i o n or a n t i b i o t i c s , on the d i s t r i b u t i o n of the f l o r a .

T h e r e s u l t s s o far c o n f i r m the r e l a t i o n s h i p b e t w e e n n u m b e r and d i s t r i b u t i o n o f b a c t e r i a and i n t e s t i n a l m o t i l i t y . The upper s m a l l i n t e s t i n e , a l though not s t e r i l e , has a s m a l l e r b a c t e r i a l p o p u l a t i o n that the m o r e d i s t a l r e g i o n s - T h i s i s due, in pa r t , to the f l u s h i n g effect o f the r a p i d pas sage of i n t e s t i n a l con ten t s . The n u m b e r and d i s t r i b u t i o n o f b a c t e r i a i s m a r k e d l y a l t e r e d i n m i c e m a i n t a i n e d o n g l u c o s e o r w a t e r for 2 4 h o u r s p r i o r t o i n v e s t i g a t i o n . In a p r e v i o u s i n v e s t i g a t i o n by one of us , i t was found that n e o m y c i n was e f f ec t ive i n e l i m i n a t i n g the m a j o r i t y o f b a c t e r i a f r o m the c o l o n and l o w e r s m a l l i n t e s t i n e but not f r o m the upper p a r t o f the s m a l l i n t e s t i n e . O u r p r e l i m i n a r y f i nd ings appea r t o c o n f i r m t h i s f i n d i n g .

P O P U L A T I O N K I N E T I C S O F N E O P L A S T I C M A S T C E L L S ( P 8 1 5 Y ) A F T E R X - I R R A D I A T I O N

G . M a r k K o l l m o r g e n

N e o p l a s t i c m a s t c e l l s ( P 8 1 5 Y ) have been g r o w n both i n v i v o ( p e r i t o n e a l c a v i t y o f D B A / z m i c e ) and i n v i t r o af ter e x p o s u r e t o X - i r r a d i a t i o n i n an a t t empt to d e t e r m i n e the dependence o f k i l l i n g and m i t o t i c l a g on dose . X - r a y doses of 62.5 , 125, 250, 500, 750 and 1000 r w e r e g i v e n .

C o n t r o l m a s t o c y t e c u l t u r e s have a d o u b l i n g t i m e of about 15 h o u r s , r e p r o d u c e a s f r e e - l i v i n g c e l l s wi thou t a t t achmen t t o g l a s s s u r f a c e s , and f u l l y r e t a i n t h e i r s t r a i n - s p e c i f i c t u m o r - i n d u c i n g p r o p e r t i e s and c h a r a c t e r i s t i c b i o c h e m i c a l func t ions af ter ex tended p e r i o d s o f r e p r o d u c t i o n i n c u l t u r e . A f t e r an L D 5 0 dose (about 130 r ) , c e l l s i n c u l t u r e r e t u r n to n o r m a l g r o w t h r a t e i n a p p r o x i m a t e l y 4 5 h o u r s . W i t h l a r g e r doses m o r e t i m e i s r e q u i r e d . W h e n c e l l s a r e i r r a d i a t e d and g r o w n i n v i v o , the s i z e o f the i n o c u l u m r e q u i r e d to c a u s e death of the hos t i s i n c r e a s e d as a func t ion of X - r a y dose . F u r t h e r m o r e , the h i g h e r the X - r a y dose , the l o n g e r the s u r v i v a l t i m e o f the hos t a n i m a l .

T h e o b s e r v e d pa t t e rns o f s u r v i v a l and r e g r o w t h a r e b e i n g a n a l y z e d i n t e r m s o f a s t a t i s t i c a l m o d e l that i n c o r p o r a t e s p a r a m e t e r s fo r g e n e r a t i o n t i m e and s e n s i t i v i t y o f s tages i n the g e n e r a t i o n c y c l e .

E F F E C T S O F H I B E R N A T I O N O N T H E L A T E N T P E R I O D I N N O R M A L A N D X - I R R A D I A T E D G R O U N D S Q U I R R E L S

B e r n a r d N . J a r o s l o w and D o u g l a s E „ S m i t h

In e x p e r i m e n t s d e s i g n e d to e x a m i n e the d e t a i l s of the i m m u n e r e -sponses in h i b e r n a t o r s , we have s tud ied the d i s a p p e a r a n c e of I - l a b e l e d bov ine s e r u m a l b u m i n ( B S A ) f r o m the c i r c u l a t i o n o f g r o u n d s q u i r r e l s ( C i t e l l u s t r i d e c e m l i n e a t u s ) .

A l l a n i m a l s r e c e i v e d a s i n g l e i n t r a p e r i t o n e a l i n j e c t i o n o f 10 mg o f I 1 3 1 t r a c e - l a b e l e d B S A o r 1 m l o f I 1 3 1 t r a c e - l a b e l e d n o r m a l g r o u n d - s q u i r r e l s e r u m (method o f T a l m a g e e t a l . )„ ( i ) The r a d i o a c t i v i t y o f the B S A s o l u t i o n was 99% p r e c i p i t a b l e by 5% t r i c h l o r o a c e t i c a c i d .

B l o o d s a m p l e s f r o m the t a i l w e r e c o l l e c t e d o n t a r e d f i l t e r pape r , a i r d r i e d o v e r n i g h t , w e i g h e d , and coun ted i n a w e l l - t y p e s c i n t i l l a t i o n c o u n t e r . A f t e r the counts w e r e c o r r e c t e d fo r b a c k g r o u n d , decay , and we igh t o f s a m p l e they w e r e c o n v e r t e d t o p e r cent o f a c t i v i t y o f the f i r s t s a m p l e t a k e n af ter a n t i g e n i n j e c t i o n and p lo t t ed a g a i n s t t i m e on a s e m i l o g a r i t h m i c s c a l e . The a n t i g e n d i s a p p e a r a n c e ra te ( A D R ) i s c a l c u l a t e d f r o m the s lope o f the l i n e . A s a n i n d i c a t o r o f the r e l a t i v e A D R , a l l o f our data a r e p r e sen ted i n t e r m s o f the h a l f - d i s a p p e a r a n c e t i m e (t j / 2 ) i n d a y s . I n g r o u n d s q u i r r e l s , a n t i g e n d i s a p p e a r a n c e o c c u r s i n th ree d i s t i n c t phases m a r k e d b y s h a r p changes i n A D R . The phases a r e s i m i l a r t o those o b s e r v e d i n r a b b i t s by D i x o n e t a h , (2) who t e r m e d t h e m 1) the e q u i l i b r a t i o n phase , 2) the n o n i m m u n e d i s a p p e a r a n c e phase , and 3) the i m m u n e d i s a p p e a r a n c e phase - m a r k e d by an a c c e l e r a t e d A D R that s i g n i f i e s the end o f the la ten t p e r i o d and the a p p e a r a n c e of an t ibody . P h a s e 1 was not u s u a l l y o b s e r v e d in the p r e s e n t s tud ies b e c a u s e the f i r s t s a m p l e was t aken 1 or 2 days af te r a n t i g e n i n j e c t i o n .

B o t h h i b e r n a t i n g and c o n t r o l s q u i r r e l s w e r e i r r a d i a t e d i n c h a m b e r s m a i n t a i n e d a t 5°C . T h e y w e r e g i v e n a s i n g l e t o t a l - b o d y X - r a y dose of 450 r ( 2 5 0 K V , 1 5 m a ; 0.5 m m C u + 3 m m b a k e l i t e f i l t e r s ; 1.7 m m C u h a l f - v a l u e l a y e r ; 3 5 c m t a r g e t d i s t a n c e ; 128 r / m i n ) .

Re su i t s

In 14 g r o u n d s q u i r r e l s m a i n t a i n e d in q u a r t e r s a t 2 3 ° C , the m e a n tl/z

of t r a c e - l a b e l e d B S A d u r i n g the n o n i m m u n e phase was 9*4 ± 2.7 d a y s . At the end of the l a t en t p e r i o d , w h i c h had a m e a n v a l u e of 7.1 ± 0.3 days , the i m m u n e d i s a p p e a r a n c e phase began and the m e a n tx/2 d e c r e a s e d to 1.8 ± 0.2 d a y s .

Ground s q u i r r e l s were injected w i t h B S A and i m m e d i a t e l y put i n the c o l d r o o m for 14, 28, o r 5 6 d a y s . The A D R d u r i n g th i s p e r i o d was h i g h l y v a r i a b l e and was p r o b a b l y r e l a t e d to the amount o f t i m e spent in h i b e r n a t i o n In th i s and s u c c e e d i n g e x p e r i m e n t s , we u s e d data on ly f r o m those a n i m a l s i n w h i c h the A D R was v e r y s l o w (ti/2 > 87 days) w h i l e in h i b e r n a t i o n , r e g a r d l e s of the n u m b e r of o b s e r v e d spontaneous a r o u s a l s .

The data i n T a b l e 5 i n d i c a t e that the A D R i s s i m i l a r for a l l e x p e r i m e n t a l g roups d u r i n g each phase and that the re w e r e no s i g n i f i c a n t d i f f e r ences be tween g roups (P > 0.05). In a n i m a l s that h i b e r n a t e d for 14 days , the m e a n p o s t h i b e r n a t i o n la tent p e r i o d was s i g n i f i c a n t l y s h o r t e r than the la ten t p e r i o d in c o n t r o l s (Tab le 5), P < 0 .001. A f t e r h i b e r n a t i o n , fo r 28 or 56 days the p o s t h i b e r n a t i o n l a ten t p e r i o d r a n g e d f r o m 0-9 d a y s .

T A B L E 5

A n t i g e n d i s a p p e a r a n c e ( I - B S A ) and the p o s t h i b e r n a t i o n la tent p e r i o d i n g r o u n d s q u i r r e l s af ter a r o u s a l f r o m h i b e r n a t i o n *

T r e a t m e n t g roup** N o . of

a n i m a l s N o n i m m u n e ,

t1/z days t , t t I m m u n e ,

tj/2 days t

P o s t h i b e r n a t i o n la tent p e r i o d ^,

days

1. N o h i b e r n a t i o n 14 9.4 ± 2.7 1.8 ± 0.2 7.1 ± 0.3

2. 14-day h i b e r n a t i o n 10 4.1 ± 0.3 (10) 2.1 ± 0.2 5.7 ± 0.2

3. 2 8 - d a y h i b e r n a t i o n 5 4.4 ± 1.2 (4) 1.8 ± 0.3 7.3 ± 0.8 (4)

4. 56-day h i b e r n a t i o n 8 3.2 + 0.3 (4) 1.6 ± 0.7 5.8 ± 1.1 (4)

5. H i b e r n a t i o n 4 days af ter i n j e c t i o n 8 3.7 ± 0.2 (6) 1.7 ± 0.1 8.0 ± 0.4 (6)

6. H i b e r n a t i o n 6 days af ter i n j e c t i o n 6 2.9 ± 0.2 (2) 1.2 ± 0.2 5.0 ± 0 (2)

* O n l y those a n i m a l s w e r e u s e d in w h i c h the t1/z of a n t i g e n was l o n g e r than 87 days d u r i n g h i b e r n a t i o n .

* * A n i m a l s i n G r o u p s 2-4 w e r e p l a c e d i n the c o l d r o o m i m m e d i a t e l y af ter a n t i g e n i n j e c t i o n , those in G r o u p s 5 and 6 w e r e p l a c e d in the c o l d r o o m for 18 and 14 d a y s , 4 and 6 days af ter an t i gen i n j e c t i o n , r e s p e c t i v e l y .

+ The f i g u r e s r e p r e s e n t the M e a n ± S. E.

^ N u m b e r s in p a r e n t h e s e s i n d i c a t e the n u m b e r o f a n i m a l s that had a m e a s u r a b l e p o s t h i b e r n a t i o n la tent p e r i o d . The r e m a i n d e r , upon a r o u s a l , e l i m i n a t e d a n t i g e n a t the i m m u n e r a t e .

G r o u n d s q u i r r e l s w e r e p l a c e d in the c o l d r o o m 4 and 6 days af ter an t i gen i n j e c t i o n for 18 and 14 days , r e s p e c t i v e l y . In the f o r m e r g roup , the p o s t h i b e r n a t i o n la tent p e r i o d was 0 days in 2 a n i m a l s and e s s e n t i a l l y n o r m a l (6.5 to 9.3 days) in 6 a n i m a l s ; in the l a t t e r g roup , i t was 0 days in 4 a n i m a l s and 5 days in 2 a n i m a l s . I t was , t h e r e f o r e , appa ren t that the events o f the la tent p e r i o d that had o c c u r r e d be fo re e n t r y in to h i b e r n a t i o n w e r e c o m p l e t e l y o r p a r t i a l l y a b r o g a t e d d u r i n g h i b e r n a t i o n in 75% of one g roup and in 33% of the other (see T a b l e 5).

25

S i n c e X - i r r a d i a t i o n a t d i f f e r en t t i m e s be fo re o r af ter an t i gen i n j e c t i o n m a y s u p p r e s s o r i n c r e a s e a n t i b o d y - f o r m i n g c a p a c i t y , (3)

and r a d i a t i o n i n j u r y m a y d e v e l o p d u r i n g h i b e r n a t i o n , (4) we i n v e s t i g a t e d the effect o f h i b e r n a t i o n on r a d i a t i o n - i n d u c e d i n j u r y t o the i m m u n e p r o c e s s e s . F o u r g roups o f h i b e r n a t i n g s q u i r r e l s w e r e t r e a t e d i n the f o l l o w i n g m a n n e r : G r o u p A , 4 a n i m a l s w e r e i n j e c t e d w i t h B S A , a l l o w e d to con t inue i n h i b e r n a t i o n for 14 days , i r r a d i a t e d w i t h 450 r , and then p l a c e d in a r o o m a t 2 3 ° C ; G r o u p B, 7 a n i m a l s w e r e i n j e c t e d w i t h B S A , p l a c e d i n a r o o m a t 23°C , i r r a d i a t e d w i t h 450 r one day l a t e r and r e t u r n e d to a r o o m at 2 3 ° C ; G r o u p C, 8 a n i m a l s w e r e i r r a d i a t e d w i t h 450 r , p l a c e d i n a r o o m a t 23°C and i n j e c t e d w i t h B S A one day l a t e r ; G r o u p D, 8 a n i m a l s w e r e i r r a d i a t e d w i t h 450 r , a l l o w e d to con t inue in h i b e r n a t i o n for 14 d a y s , and then i n j e c t e d w i t h B S A and p l a c e d in a r o o m at 2 3 ° C .

O n l y

Figure 7.

A l l four g r o u p s showed u n i f o r m i n h i b i t i o n o f an t ibody p r o d u c t i o n . 4 of 27 a n i m a l s ( F i g u r e 7), 1 in G r o u p A, 2 in G r o u p B, and 1 in G r o u p C

gave e v i d e n c e of a phase of i m m u n e d i s a p p e a r a n c e . In a l l 4 , the l a ten t p e r i o d was l o n g e r than n o r m a l (12.4, 10.6, 13, and 10 days , r e s p e c t i v e l y ) . E x a m p l e s o f a n e r g i c a n i m a l s a r e shown in F i g u r e 7 . The m e a n n o n i m m u n e t j / 2 i n each o f the g roups was A , 3.6 + 0.5; B , 3.5+ 0.4; C , 3.7 ± 0.6; and D, 3.3 + 0 . 1 d a y s . A c u r i o u s p h e n o m enon was o b s e r v e d in the a n i m a l s o f G r o u p A . T h e r e was 50-70% d e c r e a s e o f c i r c u l a t i n g an t i gen d u r i n g the f i r s t 2 days af ter i r r a d i a t i o n and then the m e a n tx/z r o s e to v a l u e s t y p i c a l f o r a l l g roups ( F i g u r e 7).

To d e t e r m i n e whe the r the ob s e r v e d A D R for the f i r s t few days af ter a r o u s a l f r o m h i b e r n a t i o n was c o m p a r a b l e to that be fore h i b e r n a t i o n , we i n j e c t e d 9 g r o u n d s q u i r r e l s w i t h I 1 3 1 - l a b e l e d n o r m a l g r o u n d - s q u i r r e l s e r u m . F i v e s q u i r r e l s w e r e p l a c e d in h i b e r n a t i o n for 14 days on the f o u r t h day af ter i n j e c t i o n and the r e m a i n i n g four s q u i r r e l s w e r e m a i n t a ined a t 2 3 ° C . V a l u e s of the m e a n tx/2 of the I 1 3 1 - l a b e l e d n o r m a l s e r u m before (3.9 ± 0.6 days) and af ter (3.5 ± 0.3 days) h i b e r n a t i o n and in the n o n h i b e m a t i n g g r o u p (3.3 + 0.2 days) w e r e not s i g n i f i c a n t l y d i f f e r en t .

DAYS AFTER INJECTION OF I 1 3 1 BSA

Antigen disappearance in ground squirrels irradiated with 450 r, 14 days or 1 day, before or after antigen injection. Letters refer to the group. Numbers indicate the r.i/2 for each phase of antigen disappearance. during hibernation in room at 5 C; after arousal from hibernation in room at 23°C

D i s c u s s i o n

A n t i g e n d i sappearance i s m u c h s lower in h ibe rna t ing than in non-h ibe rna t ing ground s q u i r r e l s . That the events of the latent p e r i o d can take place du r ing h ibe rna t i on is shown by the l a c k of a pos th ibe rna t ion latent p e r i o d in some of the a n i m a l s that h ibe rna ted for 56 days .

The n o r m a l pos th ibe rna t ion latent p e r i o d o b s e r v e d in some of the s q u i r r e l s that en tered h ibe rna t i on 4 or 6 days after ant igen in j ec t ion i n dica te that events of the latent p e r i o d , i n i t i a t ed before en t ry into h ibe rna t i on , cou ld be comple t e ly or p a r t i a l l y abrogated in as shor t a p e r i o d as 18 days in h i b e r n a t i o n and that the p r o c e s s e s of induc t ion r e c u r r e d upon a r o u s a l .

X - i r r a d i a t i o n wi th 450 r i nh ib i t ed the syn thes i s of ant ibody in a l l g roups . The pa t te rn of ant igen d i sappearance was the same as that o b s e r v e d in i r r a d i a t e d rabb i t s by D i x o n e t a l . \ ^ ) Those that p roduced ant ibody d i d so only after a delay, a response that is t y p i c a l of i r r a d i a t e d an ima l s . (3 ) It is apparent that a 2 -week h ibe rna t i on p e r i o d i n t e rposed between i r r a d i a t i o n and ant igen in j ec t ion d id not r e s u l t in any detectable r e c o v e r y . I r r a d i a t i o n after induc t ion has o c c u r r e d u s u a l l y does not r e s u l t in the i n h i b i t i o n of ant ibody product ion . (^) I n G r o u p A , the a n i m a l s were r e n d e r e d a n e r g i c when i r r a d i a ted 2 weeks after ant igen in j ec t ion . T h i s suggests that the events of the latent p e r i o d may have gone f u l l c y c l e d u r i n g h ibe rna t ion , SQ that, a t the t ime o f X - i r r a d i a t i o n , induc t ion had not r e c u r r e d . The r a p i d d rop in c i r c u l a t i ng ant igen i m m e d i a t e l y after i r r a d i a t i o n and a r o u s a l in G r o u p A i s undoubtedly an effect of i r r a d i a t i o n and not of a r o u s a l , as can be seen f r o m a c o m p a r i s o n wi th a l l the other g roups , whether in jec ted w i th B S A or w i th n o r m a l s e r u m .

We w i s h to thank M i s s Joan A . S t achura for he r exce l l en t t e c h n i c a l as s i s t ance .

Re fe rences

1 . T a l m a g e , D. W. , H. R . B a k e r , and W. A k e s o n . The s epa ra t i on and a n a l y s i s of l a b e l l e d an t ibod ies . J . Infect. D i s e a s e s 94, 199-212 (1954).

2 . D i x o n , F . J . , S . C . Bukan tz , and G. J . D a m m i n . The effect o f s e n s i t i z a t ion and X - r a d i a t i o n on the m e t a b o l i s m o f I 1 3 1 l a b e l e d p r o t e i n s . S c i ence 1 13, 274-276 (1951).

3 . T a l i a f e r r o , W . H . , L . G . T a l i a f e r r o , and B . N . J a r o s l o w . R a d i a t i o n and i m m u n e m e c h a n i s m s . A c a d e m i c P r e s s , N e w Y o r k , i n p r e s s .

4 . S m i t h , D . E . The effects o f i o n i z i n g r a d i a t i o n i n h i b e r n a t i o n . M a m m a l i a n h ibe rna t ion , ed. C . P . L y m a n and A . R . Dawe, H a r v a r d U n i v e r s i t y P r e s s , C a m b r i d g e , M a s s a c h u s e t t s , I960, pp. 493-506 .

27

P R O G R E S S R E P O R T : D I S E A S E S A N D C A R E O F L A B O R A T O R Y A N I M A L S

I . D i s e a s e C o n t r o l P r o g r a m

R o b e r t J . F l y n n , T h o m a s E . F r i t z , P a t r i c i a C 0 B r e n n a n , and C a l v i n M , P o o l e

A s y s t e m of m o n i t o r i n g the h e a l t h s tatus of a l l b r e e d i n g , s tock , and e x p e r i m e n t a l a n i m a l s has been i n i t i a t e d .

The p r o g r a m o f m i c r o b i o l o g i c m o n i t o r i n g for the p r e s e n c e o f S a l m o n e l l a sp. and P s e u d o m o n a s a e r u g i n o s a w i t h i n the s e v e r a l r a t and m o u s e b r e e d i n g c o l o n i e s i s now w e l l e s t a b l i s h e d 0 W a t e r s a m p l e s a r e c o l l e c t e d f r o m a p p r o x i m a t e l y 25% of the b r e e d i n g cages e a c h week and t e s t e d fo r J? , a e r u g i n o s a ; f e c a l s a m p l e s a r e c o l l e c t e d f r o m a p p r o x i m a t e l y 10% o f the cages each week and c u l t u r e d for S a l m o n e l l a sp. The i n c i d e n c e of J?, a e r u g i n o s a i n f e c t i o n has b e e n v e r y l o w , and the few a n i m a l s that gave a p o s i t i v e tes t fo r th i s o r g a n i s m w e r e r e c e n t a d d i t i o n s f r o m ou ts ide s o u r c e s . T h e C F # l / A n l c o l o n y has been f ree of_P. a e r u g i n o s a i n f e c t i o n for m a n y m o n t h s . S i m i l a r l y , the i n c i d e n c e o f S a l m o n e l l a sp. i n f e c t i o n has been n e a r l y z e r o . H u n d r e d s o f s a m p l e s have been t e s t ed but on ly one S a l m o n e l l a o r g a n i s m has been i s o l a t e d . I t was i d e n t i f i e d as So c e r r o and a b r i e f r e p o r t on i t f o l l o w s .

The i n c i d e n c e o f i n t e s t i n a l p a r a s i t e s r e m a i n s m o d e r a t e , and the e l i m i n a t i o n of these f r o m the s e v e r a l c o l o n i e s w i l l depend upon the s u c c e s s f u l r e p l a c e m e n t o f the b r e e d i n g s tock w i t h c e s a r e a n - d e r i v e d o r o t h e r w i s e p a r a s i t e - f r e e a n i m a l s .

The c o n t r o l o r e l i m i n a t i o n o f e c t o p a r a s i t e s f r o m the r a t and m o u s e b r e e d i n g c o l o n i e s has b e e n ano the r g o a l o f the h e a l t h p r o g r a m . To th i s end, a l l d i s c a r d e d b r e e d e r s a r e now b e i n g e x a m i n e d for m i t e s and l i c e . N o l i c e have b e e n de tec ted , and on ly two a n i m a l s have been found to be i n f e c t e d w i t h m i t e s .

R a t s and m i c e p u r c h a s e d f r o m ou t s ide v e n d o r s a r e s i m i l a r l y r o u t i n e l y s c r e e n e d fo r P a a e r u g i n o s a and S a l m o n e l l a sp. and fo r i n t e r n a l and e x t e r n a l p a r a s i t e s . The r e s u l t s o f these t es t s v a r y ; they depend on the s t r a i n and s o u r c e o f s u p p l y . In a l l c a s e s , h o w e v e r , the p u r c h a s e d a n i m a l s have a h i g h e r i n c i d e n c e o f d i s e a s e than those r a i s e d i n the D i v i s i o n ' s a n i m a l f a c i l i t i e s .

The e f f i c i e n c y o f the w a s h i n g and s a n i t i z i n g o p e r a t i o n i n e l i m i n a t i n g p a t h o g e n i c m i c r o o r g a n i s m s , p a r t i c u l a r l y _P. a e r u g i n o s a , f r o m cages and w a t e r bo t t l e s i s a l s o b e i n g c h e c k e d b y d a i l y t e s t i n g o f s a m p l e cages and f i l l e d b o t t l e s . The r e s u l t s to date i n d i c a t e that no pa thogen ic m i c r o o r g a n i s m s s u r v i v e the p r e s e n t s a n i t i z i n g p r o c e d u r e s .

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I n a d d i t i o n , a l l a n i m a l s that d ie w i t h i n the b r e e d i n g c o l o n i e s a r e n e c r o p s i e d , and an a t t empt i s made to a s c e r t a i n the cause o f dea th . T h e s e p r o c e d u r e s a r e now be ing ex tended t o i n c l u d e a l l s tock a n i m a l s and w i l l b e ex tended , w h e n e v e r f e a s i b l e , t o i n c l u d e e x p e r i m e n t a l a n i m a l s a s w e l l .

The i n f o r m a t i o n that i s be ing a c q u i r e d on spontaneous d i s e a s e s o f the v a r i o u s s t r a i n s w i l l be of g r ea t va lue i n e l i m i n a t i n g e x t r a n e o u s e x p e r i m e n t a l v a r i a b l e s and w i l l a l l o w for be t t e r i n t e r p r e t a t i o n of e x p e r i m e n t a l r e s u l t s .

II. S a l m o n e l l a c e r r o I s o l a t e d f r o m a B r e e d i n g C o l o n y

P a t r i c i a C . B r e n n a n , T h o m a s E . F r i t z , and R o b e r t J . F l y n n

S a l m o n e l l a c e r r o was f i r s t i s o l a t e d i n U r u g u a y f r o m the m e s e n t e r i c l y m p h nodes o f n o r m a l hogs ( i ) and l a t e r f r o m infants s u f f e r i n g f r o m e n t e r i t i s . ( 2 ) i t has been i s o l a t e d i n the U n i t e d States f r o m dogs and f r o m f o w l . ( 3 ' 5 )

W h i l e p e r f o r m i n g r o u t i n e t e s t s o f f e c a l s a m p l e s f r o m the C F # l / A n l b r e e d i n g c o l o n y , a n o r g a n i s m was i s o l a t e d that p r o d u c e d a c i d i n m a n n i t o l and a c i d and gas i n g l u c o s e . N o change was o b s e r v e d i n l a c t o s e o r s u c r o s e . U r e a was not h y d r o l y z e d ; i ndo le was not p r o d u c e d . The o r g a n i s m was m o t i l e , gave a p o s i t i v e m e t h y l r e d test , u t i l i z e d c i t r a t e and p r o d u c e d H 2 S . I t a g g l u t i n a t e d w i t h p o l y v a l e n t S a l m o n e l l a a n t i s e r u m (Di fco ) but f a i l e d to agg lu t ina t e w i t h S a l m o n e l l a g r o u p s p e c i f i c a n t i s e r a , G r o u p s A t h r o u g h I . A p o s i t i v e n i n h y d r i n t e s t W was ob ta ined , and o n that b a s i s the c u l t u r e was s u b m i t t e d t o the C o m m u n i c a b l e D i s e a s e C e n t e r , U . S . P u b l i c H e a l t h S e r v i c e , A t l a n t a , fo r s e r o l o g i c a l i d e n t i f i c a t i o n . I t was i d e n t i f i e d as _S. c e r r o .

We have not been ab le t o r e i s o l a t e j5. c e r r o f r o m th i s c o l o n y . N e i t h e r has any o ther m e m b e r o f the genus been i s o l a t e d s u b s e q u e n t l y f r o m the c o l o n y .

T h e s o u r c e o f t h i s o r g a n i s m and m e t h o d o f i n t r o d u c t i o n a r e m a t t e r s o f s p e c u l a t i o n , but s e v e r a l p o s s i b i l i t i e s e x i s t . T h e s e i n c l u d e the c o m m e r c i a l food, w h i c h i s not s t e r i l i z e d , and c o n t a m i n a t i o n b y the a n i m a l c a r e t a k e r s .

III. S tud ie s o n P a s t e u r e l l a p n e u m o t r o p i c a

P a t r i c i a C . B r e n n a n , T h o m a s E . F r i t z , and R o b e r t J . F l y n n

A h i g h i n c i d e n c e o f c o n j u n c t i v i t i s was o b s e r v e d i n the C F # l / A n l m o u s e c o l o n y d u r i n g the p e r i o d c o v e r e d b y th i s r e p o r t .

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R o u t i n e b a c t e r i o l o g i c s c r e e n i n g o f the a f fec ted a n i m a l s r e s u l t e d i n the c o n s i s t e n t i s o l a t i o n of a g r a m - n e g a t i v e o r g a n i s m that p r o d u c e d a c i d but no gas i n g l u c o s e , l a c t o s e , s u c r o s e and m a l t o s e b r o t h s , and that d i d not f e r m e n t m a n n i t o l . I t d i d not g r o w on e o s i n m e t h y l e n e b lue aga r , and i t was i n d o l and u r e a s e p o s i t i v e and m e t h y l r e d n e g a t i v e . On the b a s i s o f these r e a c t i o n s , the o r g a n i s m was c l a s s i f i e d a s P a s t e u r e l l a p n e u m o t r o p i c a , a c l a s s i f i c a t i o n that was c o n f i r m e d b y the C o m m u n i c a b l e D i s e a s e C e n t e r , A t l a n t a , G e o r g i a .

P . p n e u m o t r o p i c a was f i r s t i s o l a t e d and c h a r a c t e r i z e d by Jawetz.(7> 8 ) H e s h o w e d that the o r g a n i s m was c o m m o n l y la ten t i n a p p a r e n t l y n o r m a l a n i m a l s and that i t was d i f f i c u l t t o d e m o n s t r a t e p a t h o g e n i c i t y wi thou t s e r i a l p a s s a g e t h r o u g h a n i m a l s . J a w e t z and B a k e r ( 9 ) d e s c r i b e d the pa tho logy and p a t h o g e n e s i s o f the l e s i o n s a s s o c i a t e d w i t h i n f e c t i o n s w i t h v i r u l e n t s t r a i n s o f th i s o r g a n i s m .

H o a g , e t a l . ( 1 0 ) s t u d i e d l a ten t i n f e c t i o n s o f P . p n e u m o t r o p i c a i n a m o u s e c o l o n y and i s o l a t e d the o r g a n i s m o n 8 1 o c c a s i o n s f r o m n u m e r o u s t i s s u e s i n s e v e r a l s t r a i n s o f m i c e . T h e y w e r e unab le , h o w e v e r , t o d e m o n s t r a t e any la ten t v i r u l e n c e o r p a t h o g e n i c i t y i n n a t u r a l l y o r e x p e r i m e n t a l l y i n f e c t e d a n i m a l s ,

The b a c t e r i a i s o l a t e d and s t u d i e d by both J a w e t z and H o a g e t a l . w e r e b i o c h e m i c a l l y i d e n t i c a l . The one we i s o l a t e d d i f f e r s , h o w e v e r , i n that i t does not f e r m e n t m a n n i t o l .

H e y l ^ a l s o d e s c r i b e d a n o r g a n i s m i s o l a t e d f r o m m i c e w i t h p n e u m o n i a , w h i c h h e c l a s s i f i e d a s P . p n e u m o t r o p i c a . H i s s t r a i n , too, c o n s i s t e n t l y f a i l e d t o f e r m e n t m a n n i t o l .

S i n c e our s tud ie s onJP . p n e u m o t r o p i c a w e r e begun, we have i s o l a t e d the o r g a n i s m 67 t i m e s f r o m a n u m b e r o f s p e c i e s o f a n i m a l s , i n c l u d i n g s i x d i f f e r e n t s t r a i n s o f m i c e . T h e s e i s o l a t e s w e r e ob ta ined f r o m a v a r i e t y o f t i s s u e s and i n s o m e c a s e s f r o m s e v e r a l t i s s u e s o f the s a m e a n i m a l . The m o s t c o m m o n o c c u r r e n c e has been in the lungs o f m i c e and r a t s (21 c a s e s ) , and i n e a c h c a s e the o r g a n i s m w a s a s s o c i a t e d w i t h p n e u m o n i a . The s econd m o s t c o m m o n s o u r c e was the eyes o f m i c e (16 c a s e s ) ; i n e a c h o f these c a s e s c o n j u n c t i v i t i s was p r e s e n t . O t h e r s o u r c e s o f the o r g a n i s m have been u t e r i and v a g i n a s o f m i c e w i t h m e t r i t i s , l o c a l a b s c e s s e s ( p e r i t o n e a l and p l e u r a l ) in m i c e , the ea r of a dog w i t h o t i t i s and the lung of a h a m s t e r w i t h p n e u m o n i a .

O u r i s o l a t i o n o f the o r g a n i s m f r o m the eyes o f m i c e a f fec ted w i t h c o n j u n c t i v i t i s i s the f i r s t r e p o r t e d f r o m th i s o r g a n . N e l s o n ( 1 2 ) s t u d i e d c o n j u n c t i v i t i s i n a c o l o n y o f m i c e and found that p l e u r o p n e u m o n i a - l i k e o r g a n i s m s ( P P L O ) w e r e c o m m o n l y a s s o c i a t e d w i t h the d i s e a s e . H o w e v e r , h i s a t t e m p t s to r e p r o d u c e the d i s e a s e by d i r e c t con tac t o r by i n s t a l l a t i o n s o f

o c u l a r w a s h i n g s f r o m af fec ted m i c e w e r e l a r g e l y u n s u c c e s s f u l . H i s o b s e r v a t i o n that c o n j u n c t i v a l s m e a r s o f a f fec ted m i c e c o m m o n l y c o n t a i n e d n u m e r ous g r a m - n e g a t i v e b a c i l l i sugges ts that p e r h a p s a s y n e r g i s t i c r e a c t i o n be tween two o r g a n i s m s o r o ther p r e d i s p o s i n g f a c t o r s a r e n e c e s s a r y for the onset o f c l i n i c a l s i g n s . I t i s a l s o p o s s i b l e that the g r a m - n e g a t i v e b a c i l l i that he f a i l e d to iden t i fy w e r e P . p n e u m o t r o p i c a .

S e v e r a l a t t empts to p r o d u c e p n e u m o n i a o r c o n j u n c t i v i t i s w i t h a n e w l y -i s o l a t e d s t r a i n o f P . p n e u m o t r o p i c a , i n c l u d i n g one a t t empt that i n v o l v e d s t r e s s i n g the a n i m a l s by p l a c i n g t h e m in a 4 °C e n v i r o n m e n t , have been u n s u c c e s s f u l . The o r g a n i s m i s now be ing p a s s e d i n 1 8 t o Z l - d a y - o l d m i c e i n an a t t empt t o i n c r e a s e i t s v i r u l e n c e , a s d e s c r i b e d by J awe tz . ( ^ ) The p o s s i b i l i t y that P . p n e u m o t r o p i c a m i g h t r e a c t w i t h o r r e q u i r e another o r g a n i s m i n o r d e r t o e x e r t i t s p a t h o g e n i c i t y i s a l s o b e i n g i n v e s t i g a t e d .

I t s h o u l d be po in t ed out that our s tud ies of th is o r g a n i s m thus fa r have not been of the na tu re of a s u r v e y . In each i n s t a n c e , the i s o l a t i o n of the o r g a n i s m was a t t e m p t e d because o f c l i n i c a l ev idence o f d i s e a s e . We hope to conduc t a s u r v e y in the nea r future to d e t e r m i n e the i n c i d e n c e of th i s b a c t e r i u m in a p p a r e n t l y n o r m a l a n i m a l s , and we expec t t o f i nd that i t e x i s t s in a h i g h p r o p o r t i o n o f t h e m .

I V . H e m o r r h a g i c D i a t h e s i s i n L a b o r a t o r y R o d e n t s

T h o m a s E . F r i t z , D a v i d V . T o l l e , R o b e r t J . F l y n n , and C a l v i n M . P o o l e

The e v e r - p r e s e n t p r o b l e m o f c h r o n i c i n f e c t i o n s i n l a b o r a t o r y r o d e n t s , p a r t i c u l a r l y i n f e c t i o n s o f the r e s p i r a t o r y t r a c t , has p r o m p t e d us to d e v e l o p b r e e d i n g c o l o n i e s f ree o f these i n f e c t i o n s .

F i f t y - f o u r p a i r s o f s p e c i f i c - p a t h o g e n r f r e e r a t s , i n i t i a l l y r a i s e d g e r m -f ree u n t i l wean ing age, w e r e ob ta ined f r o m a c o m m e r c i a l b r e e d e r . T h e s e w e r e set up in b r e e d i n g p a i r s a t 12 weeks o f age . E a c h p a i r s u c c e s s f u l l y c o n c e i v e d a f i r s t l i t t e r w i t h v e r y few l o s s e s a m o n g the young and none a m o n g the a d u l t s .

W h e n the r a t s w e r e about 16 w e e k s of age, one of the m a l e s d i e d of p r o l o n g e d h e m o r r h a g e f r o m a m i n o r l a c e r a t i o n . W i t h i n the nex t four w e e k s , ano the r 30 of the m a l e s had d i e d , and w i t h i n e ight weeks 44 of the 54 w e r e dead . D u r i n g th i s s a m e p e r i o d , t h r ee f e m a l e s a l s o d i e d .

M o s t often the a n i m a l s w e r e not no ted a s s i c k but w e r e found dead in t h e i r c a g e s . T h o s e o b s e r v e d t o b e s i c k showed g e n e r a l i z e d a n e m i a , h e m o r rhage f r o m the c o n j u n c t i v a and e x t e r n a l n a r e s , h e m o r r h a g e o f the s k i n o f the

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face , subcu taneous h e m o r r h a g e s o r h e m a t o m a s i n v a r i o u s a r e a s (but p r e d o m i n a n t l y i n the c e r v i c a l a r ea ) , h e m o r r h a g e into the t u n i c a v a g i n a l i s v i s i b l e t h r o u g h the s c r o t u m , and i n s e v e r a l i n s t a n c e s p o s t e r i o r p a r a l y s i s .

T h e a n i m a l s w i t h p o s t e r i o r p a r a l y s i s c o n s i s t e n t l y had e x t e n s i v e h e m o r r h a g e o f the b r a i n , the s p i n a l c o r d , o r both , but h e m o r r h a g e in these a r e a s was a l s o o b s e r v e d i n a n i m a l s wi thout n e r v o u s i n v o l v e m e n t .

O t h e r l e s i o n s o b s e r v e d a t n e c r o p s y i n c l u d e d m i l d t o m a r k e d h e p a t i c d e g e n e r a t i o n , and h e m o r r h a g e s i n the s c r o t u m , the p l e u r a l c a v i t y , the p e l v i c and p e r i r e n a l p e r i t o n e u m , and the v e n t r a l c e r v i c a l a r e a .

C o i n c i d e n t a l to, and b e g i n n i n g a l m o s t s i m u l t a n e o u s l y w i t h , the h e m o r r h a g i c d i s e a s e in the r a t s has been the o c c u r r e n c e of a s i m i l a r s y n d r o m e i n o u r c o l o n y o f s p e c i f i c - p a t h o g e n - f r e e m i c e . T h i s c o l o n y was i n i t i a t e d b y o b t a i n i n g l i t t e r s b y c e s a r e a n s e c t i o n and f o s t e r - n u r s i n g t h e m o n g e r m f r e e d a m s . The a n i m a l s u s e d to s t a r t t h i s c o l o n y c o n s i s t e d o f 26 f e m a l e s and 13 m a l e s .

W h e n these m i c e w e r e about 10 w e e k s of age, four of the m a l e s d i e d . W i t h i n ano the r 10 days the r e m a i n i n g n ine m a l e s d i e d . The s igns shown p r e v i o u s t o death i n c l u d e d d y s p n e a , d e p r e s s i o n , and g e n e r a l i z e d a n e m i a . The m o s t c o n s i s t e n t s i n g l e l e s i o n was a m a r k e d b i l a t e r a l h e m o t h o r a x .

A few days a f te r the l a s t of the o r i g i n a l m a l e s had d i ed , one of the f e m a l e s d i e d w i t h s i m i l a r s igns p r e c e d i n g dea th . A t n e c r o p s y the s i g n f i c a n t f i n d i n g was the p r e s e n c e o f e x t e n s i v e h e m o r r h a g e in to the u t e r u s , w h i c h had been g r a v i d .

S i n c e the dea th o f th i s f i r s t f e m a l e , a p p r o x i m a t e l y 25 o t h e r s have d i e d . In each i n s t a n c e the s i gns o f a n e m i a w e r e p r o m i n e n t be fo re dea th i f the a n i m a l was o b s e r v e d to be s i c k a t a l l , and in e a c h i n s t a n c e e x t e n s i v e h e m o r r h a g e in to the g r a v i d u t e r u s was p r e s e n t . No nonpregnan t f e m a l e has been i n v o l v e d .

M e i e r e t a l . ( 1 3 , 1 4 ) r e p o r t e d a h e m o r r h a g i c d i a t h e s i s i n m i c e , w h i c h they r e l a t e d t o the i n g e s t i o n o f g l y c o l s f o r m e d i n b e d d i n g s t e r i l i z e d w i t h e thy lene o x i d e . T h a t ou r p r o b l e m m i g h t be due to a s i m i l a r i n t o x i c a t i o n was s u p p o r t e d by the fac t that the o r i g i n a l p u r c h a s e d r a t s w e r e r a i s e d on a type o f b e d d i n g that a t one t i m e was s u p p l i e d p r e s t e r i l i z e d w i t h e thy lene o x i d e . S i m i l a r l y , the m i c e we u s e d as a n u c l e u s for our c o l o n y w e r e r a i s e d on the s a m e type of b e d d i n g . The m a n u f a c t u r e r of the b e d d i n g has a s s u r e d us , h o w e v e r , that none of our supp ly was t r e a t e d w i t h e thy lene ox ide and that i t i s v e r y u n l i k e l y that the bedd ing o f the b r e e d e r f r o m w h o m we p u r c h a s e d o u r r a t s was so t r e a t e d .

W e a r e p r e s e n t l y h a v i n g our bedd ing a n a l y z e d for g l y c o l s . S h o u l d i t be found g l y c o l f r ee , the w o r k o f M e i e r e t a l . w i l l need to be r e e v a l u a t e d . E v e n though they w e r e ab le t o r e p r o d u c e t h e i r s y n d r o m e e x p e r i m e n t a l l y by gavage w i t h g l y c o l s , they may" have been g r e a t l y s i m p l i f y i n g a c o m p l e x s y n d r o m e . T h e y d i d show, h o w e v e r , that i n the d i a t h e s i s they s t u d i e d the re w e r e p r o t h r o m b i n - c o m p l e x d e f i c i e n c i e s , s p e c i f i c a l l y d e f i c i e n c i e s o f f a c t o r s II, V I I , I X , and X .

O u r l a b o r a t o r y tes t s a l s o sugges t d e f i c i e n c i e s i n f a c t o r s II, V I I and X . Y e t our s y n d r o m e d i f f e r s f r o m that o f M e i e r e t a l . i n that o u r s e c o n d g e n e r a t i o n m i c e , b o r n and r a i s e d on bedd ing that has had no chance o f b e i n g c o n t a m i n a t e d w i t h g l y c o l s , a r e a l s o d y i n g . T h i s i s a l s o t rue o f our s e c o n d g e n e r a t i o n r a t s .

M a l h o t r a ( l 5 ) d e s c r i b e d a s i m i l a r h e m o r r h a g i c s y n d r o m e i n r a t s . He d e m o n s t r a t e d that a d m i n i s t r a t i o n of v i t a m i n K and m e t h i o n i n e to a f fec ted a n i m a l s p r e v e n t e d spontaneous h e m o r r h a g e , but he d i d not e s t a b l i s h the r e l a t i o n s h i p o f m e t h i o n i n e to p r o t h r o m b i n o r show how the s e v e r a l o the r e s s e n t i a l f a c t o r s m i g h t b e i n f l u e n c e d .

B a r n e s and F i a l a ( ^ ) r e p o r t e d the r a p i d d e v e l o p m e n t o f v i t a m i n K d e f i c i e n c y i n r a t s r e c e i v i n g v i t a m i n K - f r e e d ie t s when c o p r o p h a g y was p r e v e n t e d , and G u s t a f s s o n U ? ) d e m o n s t r a t e d the o c c u r r e n c e of a v i t a m i n K d e f i c i e n c y and subsequent d e v e l o p m e n t o f a h e m o r r h a g i c d i s e a s e i n g e r m -f ree r a t s . S i nc e bo th our a f fec ted r a t s and m i c e o r i g i n a t e d i n a g e r m f r e e e n v i r o n m e n t , the i n t e s t i n a l f l o r a o r , r a t h e r the l a c k o f i t m a y be an i m p o r tant p r e d i s p o s i n g f a c t o r . T h i s i s now b e i n g s t ud i ed .

V . P n e u m o n i a E p i z o o t i c i n M i c e

R o b e r t J . F l y n n , T h o m a s E . F r i t z , P a t r i c i a C . B r e n n a n , and C a l v i n M . P o o l e

D u r i n g the p e r i o d c o v e r e d b y th i s r e p o r t , a n u n u s u a l l y h i g h i n c i d e n c e o f p n e u m o n i a was o b s e r v e d i n one a n i m a l r o o m . T h i s r o o m c o n t a i n e d about 750 CF#1 m i c e , 10 to 12 m o n t h s o l d , u n t i l M a y 1963, w h e n a b r e e d i n g c o l o n y of about 800 AX m i c e up „to 3 j mon ths of age, w e r e m o v e d in w i t h t h e m . P n e u m o n i a had been p r e v i o u s l y o b s e r v e d i n the o l d e r CF#1 m i c e , but u n t i l t h i s t i m e , i t had not been noted i n the A X m i c e .

I n J u l y , two o f the A X m i c e , w h i c h then n u m b e r e d 1900, w e r e found, bo th g r o s s l y and h i s t o l o g i c a l l y , t o have p n e u m o n i a . A n o t h e r c a s e o c c u r r e d a r o u n d the m i d d l e o f A u g u s t . T h e r e a f t e r n u m e r o u s c a s e s w e r e o b s e r v e d and

33

both the A X and CF#1 g roups w e r e c u l l e d h e a v i l y . M a n y m i c e f r o m both s t r a i n s w e r e n e c r o p s i e d . A l l the CF#1 m i c e e x a m i n e d showed v a r y i n g d e g r e e s o f p n e u m o n i c i n v o l v e m e n t ; m o s t w e r e s e v e r e l y a f fec ted . T h e r e was a l s o a h i g h i n c i d e n c e i n the A X m i c e e x a m i n e d . N o s i g n i f i c a n t g r o s s l e s i o n s w e r e o b s e r v e d i n any o ther o rgans o r t i s s u e s i n e i t h e r g roup except for s e c o n d a r y i n v o l v e m e n t of the l y m p h nodes of the t h o r a c i c c a v i t y .

H i s t o l o g i c a l l y , the m o s t c o n s i s t e n t and c o n s p i c u o u s fea ture was the f o r m a t i o n o f e x t e n s i v e p e r i b r o n c h i o l a r l y m p h o i d a g g r e g a t e s . The extent o f i n v o l v e m e n t v a r i e d f r o m a n i m a l t o a n i m a l , but i n g e n e r a l the CF#1 m i c e had a m u c h g r e a t e r p o r t i o n of the lungs i n v o l v e d and the f ea tu res of the r e a c t i o n sugges t ed it to be of a l o n g e r d u r a t i o n .

A t t e m p t s w e r e made t o i s o l a t e the c a u s a t i v e agent. L u n g s f r o m 1 Z A X and 9 CF#1 m i c e w e r e c u l t u r e d i n d i v i d u a l l y and then p o o l e d and s t o r e d f r o z e n fo r fu ture s t u d i e s . P a s t e u r e l l a p n e u m o t r o p i c a was r e c o v e r e d f r o m m o s t o f the s p e c i m e n s . P l e u r o p n e u m o n i a - l i k e o r g a n i s m s ( P P L O ) w e r e not r e c o v e r e d i n i t i a l l y b e c a u s e o f t e c h n i c a l d i f f i c u l t i e s , but they w e r e l a t e r r e c o v e r e d f r o m one A X m o u s e .

M i c e k n o w n t o b e f ree o f a l l c o m m o n pathogens w e r e i n o c u l a t e d i n t r a -n a s a l l y w i t h b a c t e r i a - f r e e f i l t r a t e s of a h o m o g e n i z e d s u s p e n s i o n of the f r o z e n , p o o l e d l u n g s , but we w e r e unable to r e p r o d u c e the d i s e a s e .

S tud ie s a r e now under way to tes t the effects of i n o c u l a t i n g f i l t r a t e s o f h o m o g e n i z e d s u s p e n s i o n s o f f r e s h p o o l e d lungs a lone and in c o m b i n a t i o n w i t h P . p n e u m o t r o p i c a , P P L O , o r both .

V I . Roden t B r e e d i n g P r o g r a m

T h o m a s E . F r i t z , R o b e r t J . F l y n n , and M e r l i n H . D i p e r t

A m e t h o d for m a i n t e n a n c e o f b r e e d i n g r e c o r d s that u t i l i z e s the c o m p u t e r f a c i l i t i e s o f the D i v i s i o n i s b e i n g d e v e l o p e d . D a t a now b e i n g r e c o r d e d f r o m the D i v i s i o n ' s p r o d u c t i o n c o l o n i e s i n c l u d e d a i l y o b s e r v a t i o n s o n a l l changes in the h e a l t h s ta tus , b i r t h s , deaths , w e a n i n g s , w e a n i n g w e i g h t s , c u l l i n g s , and the i s s u a n c e o f a n i m a l s for e x p e r i m e n t a l use . T h e s e data a r e a n a l y z e d w e e k l y , and a s u m m a r y i s p r e p a r e d that l i s t s the n u m b e r o f l i t t e r s b o r n , the n u m b e r o f a n i m a l s b o r n , the a v e r a g e l i t t e r s i z e , the n u m b e r o f young a n i m a l s dead , s i c k , c u l l e d , o r m i s s i n g , the n u m b e r o f l i t t e r s weaned , the n u m b e r of a n i m a l s weaned , the sex d i s t r i b u t i o n of the a n i m a l s weaned , the a v e r a g e l i t t e r s i z e weaned , the t o t a l b r e e d i n g p a i r s p r e s e n t l y i n p r o d u c t i o n and the n u m b e r o f b r e e d i n g a n i m a l s s i c k , dead, c u l l e d , o r m i s s i n g .

It a l s o i n c l u d e s a l i s t of a n i m a l s that w i l l be of wean ing age d u r i n g the nex t week and thus r e l i e v e s the t e c h n i c a l a s s i s t a n t s of the job of c a l c u l a t i n g the age o f e a c h i n d i v i d u a l l i t t e r on each wean ing day.

T h i s p r o g r a m w i l l p r o v i d e a con t inuous c r i t i c a l a n a l y s i s o f the b r e e d i n g e f f i c i e n c y of the c o l o n i e s and w i l l a l s o p r o v i d e a con t inuous c h e c k on the h e a l t h s ta tus o f a n i m a l s i n t ended for e x p e r i m e n t a l use . I t i s b e l i e v e d that these p r o c e d u r e s a r e unique in the b r e e d i n g o f l a b o r a t o r y a n i m a l s and that they w i l l p r o v e to be of g r e a t use fu lness i n p r o v i d i n g e x p e r i m e n t e r s w i t h w e l l - d e f i n e d l a b o r a t o r y a n i m a l s .

V I I . D o g C a r e P r o g r a m

C a l v i n M . P o o l e and R o b e r t J . F l y n n

A p r o g r a m t o p r o v i d e p r o f e s s i o n a l v e t e r i n a r y c a r e for a l l the D i v i s i o n ' s dogs and c l i n i c a l suppor t for the i n v e s t i g a t o r s u s i n g these a n i m a l s was i n i t i a t e d d u r i n g the f a l l o f 1963. S p e c i f i c a l l y the p r o g r a m p r o v i d e s d a i l y o b s e r v a t i o n s of a l l dogs; can ine d i s t e m p e r - h e p a t i t i s v a c c i n a t i o n s a t 1Z w e e k s of age, 4 months of age, 1 y e a r of age, and a n n u a l l y t he rea f t e r ; r o u t i n e p h y s i c a l e x a m i n a t i o n s e v e r y 4 mon ths ; rou t ine den ta l e x a m i n a t i o n s a t 1 y e a r of age and e v e r y 4 months the rea f t e r ; p a r a s i t o l o g i c a l e x a m i n a t i o n s at 6 weeks of age, 10 weeks of age, and e v e r y 4 mon ths the rea f t e r ; e x a m i n a t i o n , d i a g n o s i s , and t r e a t m e n t o f a l l o b s e r v e d d i s e a s e s o r a b n o r m a l i t i e s ; p r o f e s s i o n a l s u r g i c a l a s s i s t a n c e and p o s t o p e r a t i v e c a r e ; rou t ine c a r e o f n e w b o r n pups . A t y p i c a l m o n t h ' s a c t i v i t y i s shown in T a b l e 6 .

T A B L E 6

D o g c a r e a c t i v i t i e s for a t y p i c a l m o n t h ( D e c e m b e r , 1963)

A n e s t h e t i c s g i v e n : 1Z1 Rou t ine den t a l e x a m i n a t i o n s : 109 Rou t ine p h y s i c a l e x a m i n a t i o n s : 135 D i s t e m p e r - h e p a t i t i s v a c c i n a t i o n s : 90 T r e a t m e n t s of eye c o n d i t i o n s : Z3 T r e a t m e n t s of ear c o n d i t i o n s : 11 I n d i v i d u a l t r e a t m e n t s for i n t e r n a l p a r a s i t e s : 145 P r e g n a n c y and p r e - p a r t u m e x a m i n a t i o n s : 19 M i s c e l l a n e o u s t r e a t m e n t s o r e x a m i n a t i o n s : 74 S u r g i c a l p r o c e d u r e s ( c e s a r e a n s ec t i ons ) : Z

35

In g e n e r a l the h e a l t h s tatus of the dog c o l o n y is e x t r e m e l y good. T h e r e have been n o con tag ious d i s e a s e s no r e x t e r n a l p a r a s i t e s o b s e r v e d d u r i n g th i s p e r i o d . T h e i n c i d e n c e o f i n t e r n a l p a r a s i t i s m has not ye t been c o m p l e t e l y d e t e r m i n e d ; h o w e v e r , as o f th i s date on ly c o c c i d i a and one s p e c i e s o f r o u n d w o r m , T o x a s c a r i s l e o n i n a , have been o b s e r v e d .

As a p a r t o f t h i s p r o g r a m , s tud ies a r e be ing made o f the r e l a t i o n sh ip of den ta l c a l c u l u s to p e r i o d o n t a l d i s e a s e and of f a c t o r s a f f ec t ing and m e t h o d s o f p r e v e n t i n g c a l c u l u s f o r m a t i o n .

V I I I . U s e o f an U l t r a s o n i c U n i t i n the D e n t a l C a r e o f R e s e a r c h Dogs


C l e a n i n g dogs ' teeth and r e m o v i n g den t a l c a l c u l u s b y hand i s l a b o r i ous and t i m e c o n s u m i n g . F o r th i s r e a s o n we have p r o c u r e d a D e n t s p l y -C a v i t r o n U n i t , M o d e l 3 0 C *

The un i t o p e r a t e s b y c o n v e r t i n g o r d i n a r y 5 0 o r 6 0 c y c l e A C c u r r e n t in to 25,000 c y c l e c u r r e n t . T h i s i n t u r n i s c o n v e r t e d in to 25,000 s m a l l (0.001 in . ) m e c h a n i c a l s t r o k e s p e r s econd . T h e s e s t r o k e s a r e t r a n s m i t t e d to the t ee th v i a a hand p i e c e f i t t ed w i t h a p r o p h y l a x i s t i p and a c c o m p a n i e d by a w a t e r s p r a y . In a d d i t i o n to the m e c h a n i c a l s t r o k e s , c a v i t a t i o n a l a c t i o n i s p r o d u c e d i n the w a t e r .

T h e dog to be t r e a t e d i s a n e s t h e t i z e d w i t h an u l t r a - s h o r t - a c t i n g b a r b i t u r a t e and the s c a l i n g i s a c c o m p l i s h e d by g u i d i n g the p r o p h y l a x i s t i p o v e r the e x p o s e d tee th and s u b g i n g i v a l a r e a s , w h e r e v e r c a l c u l u s and d e b r i s have a c c u m u l a t e d , in the s a m e m a n n e r as w r i t i n g w i t h a b a l l po in t pen . I t i s not n e c e s s a r y t o app ly any m o r e p r e s s u r e than one w o u l d i n w r i t i n g , s i n c e the v i b r a t i o n s and c a v i t a t i o n , r a t h e r than p r e s s u r e e x e r t e d b y the o p e r a t o r , a c c o m p l i s h the s c a l i n g . T h e r e i s m i n i m a l h e m o r r h a g e a s s o c i a t e d w i t h the use o f th i s i n s t r u m e n t and no a p p a r e n t soft t i s s u e d a m a g e . The w a t e r s p r a y p r e v e n t s o v e r h e a t i n g , s e r v e s t o a i d the c a v i t a t i o n a l a c t i o n , and f l u shes away b l o o d and d e b r i s .

The un i t not on ly g r e a t l y r e d u c e s the s c a l i n g t i m e and ef for t but g i v e s b e t t e r r e s u l t s than t r a d i t i o n a l hand s c a l i n g . The use o f th i s type o f i n s t r u m e n t i n h u m a n d e n t i s t r y has been i n c r e a s i n g i n p o p u l a r i t y . I t i s a l s o b e i n g u s e d o c c a s i o n a l l y i n v e t e r i n a r y m e d i c i n e . H o w e v e r , w e b e l i e v e th i s i s the f i r s t r e p o r t o f i t s use i n the c a r e o f r e s e a r c h dogs .

* C a v i t r o n U l t r a s o n i c s Inc . , L o n g I s l a n d C i t y 1 , N e w Y o r k .

I X . D o g B r e e d i n g P r o g r a m


The p r e s e n t b r e e d i n g c o l o n y c o n s i s t s o f b e a g l e s d e r i v e d f r o m s t o c k p r o c u r e d f r o m the U n i v e r s i t y o f U t a h , the U n i v e r s i t y o f C a l i f o r n i a ( D a v i s ) , C a s s O ' D a y K e n n e l s ( M u r r a y , Utah) , and D r . J a m e s W . C a d e n ( A l e x a n d r i a , A l a b a m a ) .

F o r c o n t r o l l e d , r e p r o d u c i b l e r e s e a r c h , the a n i m a l s u s e d s h o u l d have a m i n i m u m of v a r i a t i o n . T h e r e f o r e , the dogs s h o u l d be of a f i x e d s t r a i n , w i t h i n a s p e c i f i e d s i z e r ange , hea l thy , free o f a n a t o m i c a l o r p h y s i o l o g i c a l defec ts and d o c i l e i n t e m p e r a m e n t . The ob j ec t i ve o f the b r e e d i n g p r o g r a m i s to p r o d u c e b e a g l e s that mee t these q u a l i f i c a t i o n s and to p r o d u c e t h e m in n u m b e r s s u f f i c i e n t to s a t i s f y the e x p e r i m e n t a l needs of the D i v i s i o n .

A f t e r r e v i e w i n g the advan tages and d i s a d v a n t a g e s o f i n b r e e d i n g and o u t b r e e d i n g and c o m p a r i n g the e x p e c t e d r e s u l t s f r o m each , we f e e l that a m o d i f i e d i n b r e e d i n g p r o g r a m ( c l o s e d l i n e o r f a m i l y ) w o u l d m o r e n e a r l y a p p r o a c h the d e s i r e d g o a l . B e c a u s e the n u m b e r o f a v a i l a b l e i n b r e d a n i m a l s i s i n s u f f i c i e n t and b e c a u s e o f the u n c e r t a i n t i e s a s s o c i a t e d w i t h any i n b r e e d i n g p r o g r a m , s o m e o u t b r e d p r o d u c t i o n w i l l b e c o n t i n u e d .

The two e x i s t i n g i n b r e d l i n e s , the " B l o n d e s " l i n e and the " A l a b a m a " l i n e , w i l l b e c o n t i n u e d . H o w e v e r , s i b l i n g m a t i n g s w i l l b e l i m i t e d i n n u m b e r and the m a j o r i t y o f m a t i n g s w i l l b e m o r e d i s t a n t l y r e l a t e d , not c l o s e r than f i r s t c o u s i n s i f p o s s i b l e . I t i s r e a l i z e d that th i s type o f b r e e d i n g does not r a p i d l y i n c r e a s e - t h e i n b r e e d i n g c o e f f i c i e n t b u t . i t does d e c r e a s e the p o s s i b i l i t i e s o f e n c o u n t e r i n g d i f f i c u l t i e s i n m a i n t a i n i n g the l i n e s .

As o f D e c e m b e r 31, 1963, the b r e e d i n g c o l o n y c o n s i s t e d o f 28 out -b r e d f e m a l e s and 2 o u t b r e d m a l e s ; 20 i n b r e d f e m a l e s and 8 i n b r e d m a l e s o f the A l a b a m a l i n e ; and 5 i n b r e d f e m a l e s and 4 i n b r e d m a l e s o f the B l o n d e l i n e .

The dog p r o d u c t i o n f r o m J u l y 1 , 1963 to D e c e m b e r 31, 1963 c o n s i s t e d of 11 7 pups in 20 w h e l p i n g s , g i v i n g an a v e r a g e l i t t e r s i z e of 5.8 pups . T w e n t y - n i n e of the 117 pups d i e d . M o s t o f these deaths w e r e s t i l l b i r t h s , n e o n a t a l o r a c c i d e n t a l , ' a n d d i d not sugges t any s e r i o u s b r e e d i n g o r d i s e a s e p r o b l e m . I t is of i n t e r e s t to note, h o w e v e r , that of the 20 w h e l p i n g s o n l y 2 w e r e f r o m i n b r e d m a t i n g s . T h i s c a n b e a c c o u n t e d for i n m a n y ways and i s o no g r e a t s i g n i f i c a n c e a t t h i s t i m e . I t does i n d i c a t e , h o w e v e r , that the p r o d u c t i v i t y o f the i n b r e d a n i m a l s w i l l have t o b e c a r e f u l l y o b s e r v e d and e v a l u a ted in the fu tu re .

http://but.it

37

X . N e w S t r a i n s and S p e c i e s : Japanese Q u a i l ( C o t u r n i x co tu rn ix )

D u r w a r d D . B a n i s t e r , C a l v i n M . P o o l e , R o b e r t J . F l y n n , and T h o m a s E . F r i t z

The c o t u r n i x q u a i l i s a s m a l l game b i r d that o c c u r s n a t u r a l l y in m o s t pa r t s of the w o r l d outs ide the A m e r i c a s . I t r e s e m b l e s our na t ive q u a i l i n s i z e and g e n e r a l c h a r a c t e r i s t i c s . Padge t t and Ivey ( l8 ) a n d . Howes and I v e y ( 1 9 )

have sugges ted the use of the Japanese v a r i e t y in r e s e a r c h . B e c a u s e of i n t e r e s t e x p r e s s e d by some m e m b e r s o f th is D i v i s i o n , i t was d e c i d e d to obta in some and to study the i r c a r e and b r e e d i n g r e q u i r e m e n t s and p o s s i b l e use fu lness .

In M a y 1963, 100 m a l e s and 100 f ema le s were obta ined f r o m A u b u r n U n i v e r s i t y . These and t he i r subsequent p rogeny we re housed in s t anda rd (24 i n . x 36 i n . x 9 in . ) c h i c k e n b r o o d e r s . T h i s type of hous ing appea red to be quite s a t i s f a c t o r y for a l l but newly ha tched c h i c k s . The adul t f ema le s w e r e housed in g roups of 20 -25 , but we q u i c k l y found that the m a l e s had to be housed i n d i v i d u a l l y , except when they we re put w i th the f e m a l e s , to p r e vent t h e m f r o m k i l l i n g each o ther .

Japanese q u a i l m a t u r e quite r a p i d l y and s t a r t l a y i n g a t 4 5 - 5 0 days of age, w i th f u l l p r o d u c t i o n , about one egg e v e r y other day, by 70 days of age. We have incuba ted a p p r o x i m a t e l y 400 eggs at 1 0 0 ° F and 60% r e l a t i v e h u m i d i t y in a r e g u l a r au toma t i c i ncuba to r of the type used to ha t ch c h i c k e n eggs . B e c a u s e of the s m a l l s i z e of the q u a i l eggs, i t was n e c e s s a r y to put t h e m in the i ncuba to r in s p e c i a l w i r e tubes to keep t h e m and the newly ha tched c h i c k s f r o m f a l l i n g th rough the i ncuba to r t r a y openings . We found that eggs f r o m hens 70 days o ld or o lde r had a h a t c h a b i l i t y of 50-54% wh i l e those f r o m hens that w e r e 45 -50 days o ld had a h a t c h a b i l i t y of only 4-6%. We a l s o found a l o w e r e d h a t a c h a b i l i t y in eggs kept a t r o o m t e m p e r a t u r e for m o r e than f ive days . The i n c u b a t i o n p e r i o d v a r i e d f r o m 1 5 to 18 days , w i t h the m a j o r i t y ha t ch ing on the 17th day.

As the c h i c k s ha tched , they w e r e r e m o v e d f r o m the r e t a i n i n g tubes, p l a c e d in s m a l l p l a s t i c c o n t a i n e r s , and r e t u r n e d to the i ncuba to r for another 24 h o u r s . At the end of th is t i m e , they w e r e r e m o v e d f r o m the i ncuba to r and put in to i m p r o v i s e d b r o o d e r s c o n s i s t i n g o f s t anda rd A N L p l a s t i c r a t -cage bo t toms (23 i n . x 11 i n . x 6 y i n . ) heated w i t h l igh t bulbs or heat l a m p s . I n i t i a l l y 10-20 o n e - d a y - o l d c h i c k s we re p l a c e d i n each p l a s t i c cage bo t tom. P a p e r towe l s we re u sed as bedd ing , and a pint M a s o n j a r f i t t ed wi th a p l a s t i c s c r e w - o n wa te r t r ough was used for w a t e r i n g . The c h i c k s w e r e fed c h i c k e n s t a r t e r - g r o w e r m a s h f r o m a s h a l l o w p e t r i d i s h .

38

W h e n they w e r e 9 to 10 days o l d , they w e r e r a t h e r w e l l f e a t h e r e d and the heat s o u r c e was r e m o v e d . At about 14 days of age, c o v e r s had to be p l a c e d on the p l a s t i c cage bo t t oms to keep t h e m f r o m f l y i n g out. B e cause of t h e i r r a p i d g r o w t h ra t e , they had to be s e p a r a t e d r a t h e r often to a v o i d o v e r c r o w d i n g . A t 25 -30 days o f age they w e r e p l a c e d i n the r e g u l a r c h i c k e n b r o o d e r s , 20 p e r b r o o d e r (16 f e m a l e s to 4 m a l e s ) . T h e y w e r e c o n t i nued on c h i c k e n s t a r t e r - g r o w e r m a s h and a p p e a r e d t o do qui te w e l l .

Sex can be d e t e r m i n e d by s i z e and b r e a s t f ea the r c o l o r a t i o n . The f e m a l e u s u a l l y w e i g h s about 125-175 g and has a g r e y and b l a c k s p e c k l e d b r e a s t that i s l i g h t e r i n c o l o r than that o f the m a l e . The m a l e u s u a l l y w e i g h s about 105-135 g and has b r o w n o r r u s t - c o l o r e d b r e a s t f e a t h e r s , w i t h f e w e r s p e c k l e s than the f e m a l e .

C l i n i c a l e v i d e n c e o f d i s e a s e o ther than o c c a s i o n a l fea ther p i c k i n g was no t ab ly absent . O n l y one c a s e o f i l l n e s s was o b s e r v e d , an adu l t f e m a l e that e x h i b i t e d t o r t i c o l l i s , i n c o o r d i n a t i o n , l o s s o f e q u i l i b r i u m and r o l l i n g t o the r i g h t when r e c u m b e n t . C h r o n i c s ep t i c m e n i n g i t i s and o s t e i t i s w e r e ob s e r v e d a t n e c r o p s y .

T o d e t e r m i n e the p r e s e n c e o f c h r o n i c o r s u b c l i n i c a l d i s e a s e s i n the f l o c k , a p p r o x i m a t e l y 180 o f the o r i g i n a l adu l t b i r d s w e r e k i l l e d and n e c r o p -s i e d . N o - s i g n i f i c a n t g r o s s l e s i o n s w e r e o b s e r v e d , n o r w e r e any i n t e r n a l p a r a s i t e s found b y e i t h e r d i r e c t o r m i c r o s c o p i c e x a m i n a t i o n . S e r u m s a m p l e s f r o m f ive b i r d s w e r e t e s t e d fo r N e w c a s t l e d i s e a s e , p u l l o r u m and P P L O i n f e c t i o n . A l l w e r e n e g a t i v e .

I n s u m m a r y , the J a p a n e s e q u a i l i s r e l a t i v e l y ea sy t o r a i s e , a p p a r e n t l y c a n b e p r o d u c e d the y e a r r o u n d , ' m a t u r e s r a p i d l y , i s v e r y h a r d y , and r e q u i r e s m u c h l e s s space than the c h i c k e n o r o the r c o m m o n l y u s e d l a b o r a t o r y b i r d s .

R e f e r e n c e s

1 . H o r m a e c h e , E . , and R . S a l s a m e n d i . N o r m a l hog a s c a r r i e r o f S a l m o n e l l a . A r c h . U r u g u a y M e d . C i r . E s p e c i a l . ( M o n t e v i d e o ) 14, 375-387 ( 1 9 3 9 ) .

2 . H o r m a e c h e , E . , C . A . P.eluffo, and P . L . A l e p p o . L a s S a l m o n e l a s e n p a t o l o g r a i n f a n t i l . A r c h . U r u g u a y M e d . C i r . E s p e c i a l . ( M o n t e v i d e o ) 19, 125-142 (1941).

3 . Wol f f , A . H . , N . D . H e n d e r s o n , and G . L . M c C a l l u m . S a l m o n e l l a f r o m dogs and the p o s s i b l e r e l a t i o n s h i p t o s a l m o n e l l o s i s i n m a n . A m . J . P u b l i c H e a l t h 38, 4 0 3 - 4 0 8 (1948).

B r u n e r , D . W . , a n d A . B . M o r a n . S a l m o n e l l a in fec t ions o f d o m e s t i c a n i m a l s . C o r n e l l V e t . 39, 53-63 (1949).

E d w a r d s , P . R . , D . W . B r u n e r , and A . B . M o r a n . S a l m o n e l l a in fec t ions of f owl s . C o r n e l l V e t . 38, 247-256 (1948).

C a r l q u i s t , P . R . A b i o c h e m i c a l tes t for s epa ra t i ng p a r a c o l o n g roups . J . B a c t e r i o l . 71, 339 -341 (1956).

J a w e t z , E . A la tent p n e u m o t r o p i c P a s t e u r e l l a o f l a b o r a t o r y a n i m a l s . P r o c . Soc . E x p . B i o l . M e d . 68_, 46-48 (1948).

J a w e t z , E . A p n e u m o t r o p i c P a s t e u r e l l a o f l a b o r a t o r y a n i m a l s . I . B a c t e r i o l o g i c a l and s e r o l o g i c a l c h a r a c t e r i s t i c s o f the o r g a n i s m . J . Infect. Diseases_86, 172-183 (1950).

J a w e t z , E . , a n d W. H . B a k e r . A p n e u m o t r o p i c P a s t e u r e l l a o f l a b o r a t o r y a n i m a l s . I . P a t h o l o g i c a l and i m m u n o l o g i c a l s tudies w i th the o r g a n i s m . J . Infect. Diseases_86, 184-196 (1950).

H o a g , W . , P . W . W e t m o r e , J . R o g e r s , and H . M e i e r . A study o f la tent P a s t e u r e l l a i n f e c t i o n in a mouse co lony . J . Infect. D i s e a s e s 111, 135-140 (1962).

H e y l , J . G . A study o f P a s t e u r e l l a s t r a i n s f r o m a n i m a l s o u r c e s . A n t o n i e van Leeuwenhoek J . M i c r o b i o l . S e r o l . _29, 79-83 (1963).

N e l s o n , J . B . A s s o c i a t i o n o f a s p e c i a l s t r a i n o f p l e u r o p n e u m o n i a - l i k e o r g a n i s m s w i t h c o n j u n c t i v i t i s in a mouse c o l o n y . J . E x p t l . M e d . 9_1, 309-320 (1951).

M e i e r , H . , R . C . A l l e n , and W . G . H o a g . Spontaneous h e m o r r h a g i c d i a t h e s i s i n i n b r e d m i c e due to s ing le o r m u l t i p l e " p r o t h r o m b i n -c o m p l e x " d e f i c i e n c i e s . B l o o d 1_9, 501-514 (1962).

M e i e r , H . , R . C . A l l e n , and W . G . H o a g . E t h y l e n e g l y c o l p r o d u c e d b y e thy l ene oxide s t e r i l i z a t i o n and i t s effect on b l o o d - c l o t t i n g f ac to r s in an i n b r e d s t r a i n o f m i c e . N a t u r e 193, 387-388 (1962).

M a l h o t r a , O . P . H e m o r r h a g i c d i a t h e s i s i n r a t s : E f f e c t o f s t r a i n , e s t r o g e n , t e s to s t e rone , v i t a m i n K , and me th ion ine , P h . D . T h e s i s , U n i v e r s i t y o f I l l i n o i s (1962).

B a r n e s , R . H . , and G . F i a l a . E f f e c t s o f the p r e v e n t i o n o f coprophagy in the ra t . V I . V i t a m i n K. J . N u t r . 6_8, 603 (1959).

17. Gus tafsson , B . E . V i t a m i n K def ic iency i n ge rmf ree ra t s . A n n . N . Y . A c a d . S c i . 78, 166-174 (1959).

18. Padgett , C. A . , and W. D. Ivey. C o t u r n i x qua i l as a l abo ra to ry r e s e a r c h a n i m a l . Sc ience 129, 267-268 (1959).

19. Howes , J . R . , and W. D. Ivey. C o t u r n i x qua i l for v e t e r i n a r y r e s e a r c h . J . A m . Ve t . M e d . A s s o c . 140, 162-163 (1962).

T O X I C I T Y A N D M E T A B O L I S M O F R A D I O N U C L I D E S

W i l l i a m P . N o r r i s , L o u i s e S . L o m b a r d , C a r l E . R e h f e l d , and R o b e r t J . Toft

P a t h o l o g i c A l t e r a t i o n s i n B e a g l e Dogs In jec ted I n t r a v e n o u s l y w i t h C e r i u m - 1 4 4 a t the 50 j t ic /kg L e v e l *

E x p e r i m e n t 003

One dog (001Z) d i e d 955 days after i n j e c t i o n w i t h 50 f i c / k g of c e r i u m - 144.

G r o s s P a t h o l o g y

The a n i m a l w a s d e h y d r a t e d . D e c u b i t a l u l c e r s o c c u r r e d o v e r the l a t e r a l s k e l e t a l p r o m i n e n c e s . The m u s c u l a t u r e o f the p o s t e r i o r l i m b s was a t r o p h i c . A n o s seous t u m o r i n v o l v e d the body o f the second t h o r a c i c v e r t e b r a and ex tended in to the f i r s t v e r t e b r a ( F i g u r e 8). The v e r t e b r a l ends o f the f i r s t and second r i b s , r i g h t and lef t , w e r e i n c o r p o r a t e d w i t h i n the t u m o r m a s s fo r a d i s t ance of 1 cm or l e s s . Due to e x t e n s i o n of the t u m o r m a s s into the v e r t e b r a l c a n a l , t he re w a s c o n s t r i c t i o n and a t rophy o f the s p i n a l c o r d a t the l e v e l o f the f i r s t and second t h o r a c i c v e r t e b r a e . L o n g i t u d i n a l s e c t i o n i n g o f the long bones r e v e a l e d c o a r s e t r a b e c u l a t i o n .

Figure 8

Sagittal section of first four vertebrae of dog 0012. The osteogenic sarcoma arises from the second thoracic vertebra and extends into the vertebral canal .

2 CHI

H i s t o p a t h o l o g y

The t u m o r o f the t h o r a c i c v e r t e b r a e was an os t eogen ic s a r c o m a . The g r o w t h was m a d e up o f c a r t i l a g e , o s t e o i d , bone, and soft t i s s u e , in w h i c h r o u n d and sp ind l e c e l l s w e r e n u m e r o u s ( F i g u r e 9). The m a s s e s o f

* T h i s w o r k was p e r f o r m e d b y L o u i s e S . L o m b a r d .

bone and car t i lage were r u d i m e n t a r y and i r r e g u l a r . Radioautographs showed both be ta aggrega tes and a lpha t r a c k s in the a r e a of the t u m o r as w e l l a s in the bone m a r r o w and c o m p a c t bone o f the long bone, v e r t e b r a e , r i b s , and s t e r n u m .

Figure 9

Histologic appearance of tumor shown in F i g ure 8. There are areas of bone, osteoid and cart i lage interspersed among areas of spindle-shaped sarcoma ce l l s . (Giemsa)

P a t h o l o g i c A l t e r a t i o n s i n B e a g l e Dogs In jec ted I n t r a v e n o u s l y w i t h C e s i u m - 1 3 7 a t the LD50/30 L e v e l

E x p e r i m e n t 004

One dog (0441) d i ed 52 days after i n j e c t i o n of 2704 jUc /kg of c e s i u m - 1 3 7 , w h i l e another (0449) was eu than ized on the 38th day af ter i n j e c t i o n of 2650 juc /kg , as pa r t of a s a c r i f i c e s e r i e s .

G r o s s P a t h o l o g y

D o g 0441. The a n i m a l was m a r k e d l y d e h y d r a t e d . The lungs w e r e pa le p i n k and con ta ined m u l t i p l e p e t e c h i a l and e c c h y m o t i c h e m o r r h a g e s i n a l l l o b e s . The h e a r t b lood was pa le p i n k and w a t e r y . The p e r i c a r d i a l sac w a s t h i c k e n e d by edematous f l u i d and the m y o c a r d i u m w a s s t r e a k e d w i t h m u l t i p l e l i n e a r h e m o r r h a g e s . M u c o u s m e m b r a n e s , c o n j u n c t i v a e , and

v i s c e r a l o rgans w e r e b l a n c h e d . The c o r t e x o f the a d r e n a l g l a n d was pa le p i n k w i t h c r e a m - c o l o r e d s t r e a k s i n the m i d - c o r t i c a l zone; the r a t i o o f c o r t ex to m e d u l l a was 3 to 1. The c o r t e x of the k idney w a s s t r e a k e d w i t h c r e a m - c o l o r e d l i n e a r m a r k i n g s . P e t e c h i a e appea red th roughout the b r a i n . The bone m a r r o w of the long bones w a s fatty throughout the l eng th of the shaft and con ta ined s p a r s e s m a l l r e d f o c i ( F i g u r e 10). The sp l een w a s f i b r o t i c . L y m p h o i d t i s s u e o f the sp l een and l y m p h nodes w a s h y p o p l a s t i c .

Figure 10

Section of femur of dog 0441. The marrow is cream-colored and fatty throughout the cavi ty and contains scattered reddish-purple foci .

D o g 0449. H e m m o r r h a g e s o f v a r y i n g s i z e s o c c u r r e d w i t h i n the v e n t r a l c e r v i c a l m u s c l e s , i n the hea r t , s m a l l and l a r g e i n t e s t i n e , k i d n e y s , t h y m u s , g a l l b l a d d e r , g lans pen is and t e s t e s . The bone m a r r o w was a b r i g h t r e d d i s h - p u r p l e .

H i s t o p a t h o l o g y

D o g 0441. The bone m a r r o w was a p l a s t i c w i t h the e x c e p t i o n o f s m a l l a r e a s w h i c h con ta ined aggrega tes o f d i v i d i n g c e l l s . M y e l o p o i e t i c f o c i w e r e n u m e r o u s and e r y t h r o p o i e t i c f o c i c o m p a r a t i v e l y s p a r s e ( F i g u r e 11). C o n t r a s t r a d i o a u t o g r a p h s of the f e m u r i n d i c a t e d a c o n c e n t r a t i o n o f r a d i o a c t i v i t y a long the endos teum and i n a r e a s con ta in ing a c t i v e l y d i v i d i n g c e l l s . The a d r e n a l c o r t i c a l l i p i d was l i m i t e d t o pa tchy l i n e a r a r e a s w i t h i n the zona f a s c i c u l a t a . A l t e r a t i o n s w i t h i n s k e l e t a l m u s c l e c o n s i s t e d of a s l i g h t t h i c k e n i n g of the s a r c o l e m m a due to g l y c o g e n i n f i l t r a t i o n .

44

Figure 11

Histologic appearance of the bone marrow of dog 0441. A smal l nest of ce l l s , including plasma and re t iculum cel ls , osteoclasts, and degenerating megakaryocytes, ly ing along the endosteum. (Giemsa)

T h e r e was v a s c u l a r degene ra t i ve change and t h r o m b o t i c i n v o l v e men t o f the v e n u l e s and c a p i l l a r i e s o f the hea r t , l u n g s , s k e l e t a l m u s c l e , b r a i n and l i v e r . The t h r o m b i c o n s i s t e d o f f i b r i n , l i p i d and u n i d e n t i f i e d h y a l i n e - l i k e m a t e r i a l . These t h r o m b i w e r e e i the r f i b r i l l a r , h y a l i n e o r v a c u o l a t e d and s t a ined m e t a c h r o m a t i c a l l y w i t h G i e m s a , w e r e p o s i t i v e f o l l o w i n g p e r i o d i c a c i d - S c h i f f ( P A S ) t r e a t m e n t o f d i a s t a s e - d i g e s t e d m a t e r i a l , p o s i t i v e for the SH r a d i c a l ( d i h y d r o x y d i n a p h t h y l d i s u l p h i d e tes t ) , and s t a i n e d blue w i t h phospho tungs t i c a c i d h e m a t o x y l i n . The f i b r i l l a r t h r o m b i s t a ined blue w i t h W e i g e r t ' s f i b r i n and phospho tungs t i c a c i d h e m a t o x y l i n . The v a s c u l a r l e s i o n s c o n s i s t e d o f t h i c k e n i n g o f the v e s s e l w a l l w i t h a h y a l i n e l i k e m a t e r i a l and w i t h e x t r a v a s a t i o n o f e r y t h r o c y t e s w i t h i n and about the v e s s e l w a l l .

D o g 0449. The bone m a r r o w was h y p o p l a s t i c ( F i g u r e 12); a l l long bones and r i b s con ta ined f o c i o f g r a n u l o p o i e t i c and e r y t h r o p o i e t i c c e l l s . M i t o t i c f i g u r e s w e r e noted a long the endos t eum. The M a l p i g h i a n c o r p u s c l e s w i t h i n the s p l e e n and l y m p h nodes w e r e s m a l l and s p a r s e , and t h e r e w e r e n o m e g a k a r y o c y t e s . M i t o t i c f i g u r e s w e r e r a r e i n the s p l e e n and n u m e r o u s w i t h i n the l y m p h nodes w h e r e a b n o r m a l m i t o t i c f i g u r e s w e r e o b s e r v e d . A p p r o x i m a t e l y o n e - t h i r d o f each l y m p h node was r e p l a c e d b y ad ipose t i s sue . The s k e l e t a l m u s c l e s con ta ined i n c r e a s e d c o n c e n t r a t i o n s o f g l y c o g e n w i t h i n the s a r c o l e m m a ( F i g u r e 13).

The tes tes w e r e a t r o p h i c . O n l y r a r e tubules con ta ined p r i m a r y s p e r m a t o c y t e s , w h i l e s p e r m a t o z o a , S e r t o l i c e l l s and s p e r m a t o g o n i a w e r e

p r e s e n t i n m o s t t ubu l e s . M i t o t i c f i g u r e s w e r e r a r e l y o b s e r v e d . M u l t i n u c l e a t e d giant c e l l s a p p e a r e d f ree w i t h i n the l u m e n of the t ubu l e s .

Figure 12. Histologic appearance of the bone mar- Fig row of dog 0449. The marrow contains differentiated cells, necrotic cells and mitotic figures. (Giemsa)

ure 13. Cross section of the temporal muscle of dog 0449. The glycogen infiltration appears as a thick cuff between the sarcolemma and the muscle fiber. (Periodic acid-leucofuchsin-Schiff)

The venu le s throughout the body w e r e t h i c k e n e d by a s u b e n d o t h e l i a l h y a l i n e - l i k e m a t e r i a l . E x t r a v a s a t i o n s o f e r y t h r o c y t e s a p p e a r e d w i t h i n o r about the v e s s e l w a l l . The venu le s conta ined t h r o m b i s i m i l a r t o those de s c r i b e d for dog 0441.

C o n t r o l Dogs

Dogs o f d i f fe ren t ages w e r e s a c r i f i c e d for c o n t r o l t i s s u e s . The four dogs l i s t e d in the p r e s e n t r e p o r t w e r e : n u m b e r s 0049 (66 mon ths ) ; 0325 (31 mon ths ) ; 0405 (25 mon ths ) ; and 0463 (15 mon ths ) .

On g r o s s e x a m i n a t i o n i t was found that dog n u m b e r 0049 had c h r o n i c v a l v u l a r e n d o c a r d i t i s o f both a t r i o v e n t r i c u l a r v a l v e s . M i c r o s c o p i c f ind ings i n c l u d e d the o b s e r v a t i o n that g l y c o g e n i n f i l t r a t i o n o f the s a r c o l e m m a was p r e s e n t . T h i s change w a s s i m i l a r t o that o b s e r v e d in the e x p e r i m e n t a l dog 0449.

Dog 0325 r e c e i v e d 7 mc of t r i t i a t e d thymid ine two hours before being euthanized w i t h nembuta l . The t i s sues c o l l e c t e d f r o m the dog w e r e used for a study of c e l l r e n e w a l ra tes in a l l ma jo r o rgans , in add i t ion to being used for h i s t o l o g i c study. No g ross o r m i c r o s c o p i c a b n o r m a l i t i e s w e r e obse rved .

Dog 0405 was cons ide red n o r m a l on g r o s s and m i c r o s c o p i c examina t i on .

Dog 0463, p r e v i o u s l y used for K 4 0 t r a c e r s tudies , had c l i n i c a l l y obs e r v e d hear t m u r m u r s . On g ross examina t ion , a congeni ta l v e n t r i c u l a r sep ta l defect was found. The a o r t i c va lve leaf le ts had m i n e r a l i z e d annular th icken ings at the free m a r g i n s . The septa l defect o c c u r r e d beneath the a o r t i c v a l v e . The left v e n t r i c l e was d i l a t ed , and the a t r i o v e n t r i c u l a r v a l v e s had f i b rous , nodular th icken ings on the i r free m a r g i n s . The lungs conta ined m u l t i p l e f i b r o t i c a r eas i n a s s o c i a t i o n w i th b r o n c h i e c t a s i s .

Stock Dogs

The causes of death in the nonrad ioac t ive dogs on s tandard and semisyn the t i c diets are l i s t e d in Tab le 7 .

T A B L E 7

Causes of death in nonrad ioac t ive dogs on s tandard and s emi syn the t i c diets

N u m b e r of dogs

Cause of death Standard Semisyn the t i c diet diet

A d u l t

U r e m i a , u r e t h r a l obs t ruc t i on wi th c a l c u l i 1

Young

S e p t i c e m i a * 1 1 B r o n c h o p n e u m o n i a 2 1

N e w b o r n

P n e u m o n i a , h e m o l y t i c S t rep tococcus 2 B i r t h in ju ry 1 S e p t i c e m i a * 4 P e r i t o n i t i s , u m b i l i c a l in fec t ion 2

S t i l l b o r n 4

*In those a n i m a l s in w h i c h cu l tu res for b a c t e r i a l e x a m i n a t i o n w e r e p r e p a r e d a beta h e m o l y t i c S t rep tococcus was found.

P a t h o l o g i c A l t e r a t i o n s i n S p r a g u e - D a w l e y R a t s I n j e c t e d I n t r a v e n o u s l y w i t h C e r i u m - 144 a t the L D 1 0 0 / 3 0 L e v e l

P i l o t s t u d i e s w e r e u n d e r t a k e n w i t h c e r i u m - 1 4 4 i n r a t s t o d e v e l o p t e c h n i c s fo r e s t i m a t i n g the d e g r e e o f d a m a g e i n f l i c t e d u p o n the r e t i c u l o e n d o t h e l i a l s y s t e m b y h i g h dose l e v e l s o f c e r i u m - 1 4 4 . T a b l e 8 l i s t s the a n i m a l s b y n u m b e r , dose l e v e l , and days t o dea th .

T A B L E 8

The acute t o x i c i t y o f c e r i u m - 144 in Sprague D a w l e y r a t s

N u m b e r D L -

e th ion ine , g

C e 1 4 4

i n j e c t e d , lie

C e 1 4 4

dose l e v e l , M c / g

P r o f e r r i n i n j ec t ed ,

m g

S u r v i v a l a f ter i n j e c t i o n , days N u m b e r

D L -e th ion ine ,

g

C e 1 4 4

i n j e c t e d , lie

C e 1 4 4

dose l e v e l , M c / g

P r o f e r r i n i n j ec t ed ,

m g C e 1 4 4 P r o f e r r i n

1 ( c o n t r o l ) * 0 0 0 0 2 0 640 5 0 8 3 0 1000 5 0 9 4 0 715 4 0 11 5 0 663 4 0 12 6 0 715 4 0 12 7 0 663 4 0 1 1 8 0 715 4 0 1 1 9 0 848 4 14.2 8 3

10 0 904 4 15.1 5 11 0 840 4 0 3t 12 0 650 3 19.5 15** 2 h r s 13 0 0 0 17.0 2 h r s 14 t+ 1.5 0 0 0 15 1.5 884 3 0 5** 16 1.5 723 3 0 5** 17 0 845 3 0 5** 18 ( c o n t r o l ) * 0 0 0 0

* E u t h a n i z e d 12 days af ter i n i t i a t i o n of e x p e r i m e n t . * * E u t h a n i z e d w i t h n e m b u t a l .

^ D i e d f o l l o w i n g l i v e r b i o p s y . t f E u t h a n i z e d r a t 14 on 25th day af ter D L - e t h i o n i n e t r e a t m e n t was i n i t i a t e d

T h e r a t s w e r e i n j e c t e d v i a the l a t e r a l c a u d a l v e i n s w h i l e u n d e r n e m b u t a l s e d a t i o n . R a t s p r e t r e a t e d w i t h D L - e t h i o n i n e w e r e g i v e n a 0.5% s o l u t i o n o f D L - e t h i o n i n e i n t h e i r d r i n k i n g w a t e r f o r 2 0 days b e f o r e i n j e c t i o n w i t h c e r i u m - 1 4 4 . R a t s w h i c h r e c e i v e d i r o n d e x t r a n ( P r o f e r r i n ) * w e r e i n j e c t e d w i t h 6.7 m g P r o f e r r i n / l 0 0 g o f r a t . T i s s u e s w e r e t a k e n f o r h i s t o l o g i c a l s e c t i o n and p r e p a r e d fo r r a d i o a u t o g r a p h s a s d e s c r i b e d p r e v i o u s l y

* P r o f e r r i n ( s a c c h a r a t e d i r o n o x i d e ) c o n t a i n s the e q u i v a l e n t o f 2 0 m g o f a v a i l a b l e i r o n and i s m a n u f a c t u r e d b y S h a r p and D o h m e , P h i l a d e l p h i a , P e n n s y l v a n i a .

fo r can ine t i s s u e s . L i v e r b i o p s y was a t t empted i n one r a t (No. 12) u s i n g the M i i l l e r b i o p s y need le ; h o w e v e r , the a n i m a l s u c c u m b e d w i t h i n one h o u r f o l l o w i n g s u r g e r y .

P r i o r t o i n j e c t i o n , the c e r i u m - 1 4 4 s o l u t i o n was e x a m i n e d for d e g r e e o f u l t r a f i l t e r a b i l i t y u s i n g the t e c h n i c o f L i n d e n b a u m .

U) The i n j e c t i o n s o l u

t ions of c e r i u m - 1 4 4 w e r e found to be 90 to 94% n o n f i l t e r a b l e . G r o s s P a t h o l o g y

The r a t s w h i c h d i e d showed the f o l l o w i n g l e s i o n s . B l o o d y f l u i d was noted about the n a r e s and anus . H e m o r r h a g e s o c c u r r e d in the subcu t i s and

d e r m i s , i n the h e a r t m u s c l e , l u n g s , s t o m a c h and i n t e s t i n e s ; t h e r e was c o n g e s t i o n o f the bone m a r r o w , l i v e r and k i d n e y s . N e c r o t i c f o c i w e r e s c a t t e r e d th roughout the l i v e r .

H i s top atho logy

C e r i u m - 1 4 4 G r o u p

L i v e r . F o c a l a r e a s o f h e p a t i c c e l l n e c r o s i s o c c u r r e d i n the l i v e r o f a l l r a t s ( F i g u r e 14). B a c t e r i a l e m b o l i w e r e o b s e r v e d on ly i n those a n i m a l s that d i e d . E m b o l i w e r e a s s o c i a t e d w i t h l a r g e a r e a s o f h e p a t i c c e l l n e c r o s i s . I n such l e s i o n s , h e p a t i c c e l l c o r d s had unde rgone c o a g u l a t i o n n e c r o s i s w h i l e the r e t i c u l o e n d o t h e l i a l c e l l s r e m a i n e d in tac t . U p o n r a d i o -a u t o g r a p h i c e x a m i n a t i o n , i t was no ted that n u m e r o u s agg rega t e s of be ta r a d i o a c t i v i t y o c c u r r e d i n r e t i c u l o e n d o t h e l i a l c e l l s o f the l i v e r . D i f fu se r a d i o a c t i v i t y a p p e a r e d i n hepa t i c c e l l s about the c e n t r a l v e i n s .

Bone M a r r o w and B o n e . The bone m a r r o w was a p l a s t i c ( F i g u r e s 15 and 16). C e l l s r e m a i n i n g w i t h i n the

bone m a r r o w c o n s i s t e d o f fat c e l l s , m a t u r e e r y t h r o c y t e s , m a s t c e l l s and phagocy t e s . In the r a t s k i l l e d a t 5 d a y s , o c c a s i o n a l d i f f e r e n t i a t e d g r a n u l o cy tes w e r e seen . M a s t c e l l s w e r e c o n s i d e r a b l y m o r e n u m e r o u s i n the a n i m a l s r e c e i v i n g c e r i u m - 1 4 4 than i n the c o n t r o l r a t s . The m a s t c e l l g r a n u l e s v a r i e d c o n s i d e r a b l y i n s i z e and s t a i n i n g c h a r a c t e r i s t i c s . L a r g e ,

* '̂ ^» • HAP!.':

. 100 It

Figure 14. Liver of rat 4. Wedge-shaped areas of hepatic cell necrosis occur above and below the central vein. In the necrotic areas, many reticuloendothelial cells remain. (Hematoxylin and eosin)

r o u n d p h a g o c y t i c c e l l s w e r e p r o m i n e n t ; w i t h i n t h e i r c y t o p l a s m t h e r e w e r e as m a n y as 20 e r y t h r o c y t e s ( F i g u r e s 17 and 18). R a d i o a u t o g r a p h s i n d i c a t e d that be t a r a d i o a c t i v i t y w a s w i t h i n the p h a g o c y t e s i n bone m a r r o w , a l o n g the e n d o s t e u m o f t r a b e c u l a r and c o m p a c t bone , and i n b l o o d c a p i l l a r i e s w i t h i n the e p i p h y s e a l z o n e .

Figure 15. Femur of rat 6. The bone marrow above Figure 16. Higher magnification of the femur and below the epiphyseal line is aplastic. shown in Figure 15. The bone marrow (Giemsa) in the metaphysis consists mainly of

loose mesenchymal tissue. (Giemsa)

S p l e e n and L y m p h N o d e s . T h e l y m p h o i d t i s s u e s w e r e h y p o p l a s t i c o r a p l a s t i c . T h e r a d i o a c t i v i t y w i t h i n the s p l e e n w a s h e a v i e s t i n the p e r i f o l l i c u l a r r e g i o n , w h e r e a s i n the l y m p h nodes the a c t i v i t y w a s c o n c e n t r a t e d i n the s u b c a p s u l a r s i n u s e s .

K i d n e y , L u n g , G o n a d s , E n d o c r i n e O r g a n s , D u o d e n u m and C o l o n . D i f f u s e r a d i o a c t i v i t y w a s o b s e r v e d i n the e p i t h e l i a l c e l l s o f the c o n v o l u t e d t u b u l e s and i n the g l o m e r u l a r tufts o f the k i d n e y ; i n a d d i t i o n , a g g r e g a t e s o f r a d i o a c t i v i t y o c c u r r e d w i t h i n p h a g o c y t i c c e l l s i n the i n t e r s t i t i u m .

A g g r e g a t e s o f be ta r a d i o a c t i v i t y a p p e a r e d i n the a l v e o l a r l i n i n g c e l l s , i n p h a g o c y t i c c e l l s l y i n g f r ee w i t h i n a l v e o l i , and i n p h a g o c y t i c c e l l s w i t h i n the i n t e r s t i t i a l t i s s u e .

Figure 17. The femur of rat 5. The sinusoids of the shaft are distended with erythrocytes. Erythrophagocytes are numerous. (Giemsa)

Figure 18. Higher magnification of Figure 17. Mast cells (M) are interspersed within the mesenchymal tissue of the marrow. Phagocytes (P) contain large numbers of engulfed erythrocytes. (Giemsa)

A g g r e g a t e s o f beta r a d i o a c t i v i t y w e r e noted in the p h a g o c y t i c c e l l s of the gonads , the e n d o c r i n e o r g a n s , and the d i g e s t i v e t r a c t .

C e r i u m - 1 4 4 and D L - E t h i o n i n e G r o u p

The f ind ings w e r e s i m i l a r i n a l l o rgans t o those seen i n r a t s r e c e i v i n g c e r i u m - 1 4 4 a lone , w i t h the e x c e p t i o n that, i n a d d i t i o n , the l i v e r s of the D L - e t h i o n i n e - t r e a t e d r a t s con ta ined a few b i z a r r e m i t o t i c f i g u r e s and a reas of b i l e duct p r o l i f e r a t i o n ( F i g u r e 19).

C e r i u m - 1 4 4 and S a c c h a r a t e d I r o n O x i d e G r o u p

The d i s t r i b u t i o n o f s a c c h a r a t e d i r o n oxide was w i d e s p r e a d t h r o u g h out the r e t i c u l o e n d o t h e l i a l s y s t e m ; h o w e v e r , i t was m o s t abundant in the sp l een and l i v e r . C e l l s con ta in ing beta r a d i o a c t i v i t y a l so con ta ined s a c c h a r a t e d i r o n ox ide .

51

Figure 19

Radioautograph of the liver of rat 15. Diffuse and aggregated beta radioactivity appear in the liver. In the right field there are two areas of bile duct proliferation (BD). (Hematoxylin and eosin)

Iod ine and T h y r o i d M e t a b o l i s m *

T h e f o l l o w i n g i s a p r o g r e s s r e p o r t o f w o r k b e i n g c a r r i e d o n i n 131

w h i c h the e f fec t s o f I o n t h y r o i d f u n c t i o n w i l l b e s t u d i e d . T h i s r e p o r t c o v e r s the p e r i o d f r o m J u l y 1 to D e c e m b e r 3 1 , 1963, and r e p r e s e n t s i n i t i a l e x p l o r a t o r y e x p e r i m e n t s d e s i g n e d t o g i v e g e n e r a l i n f o r m a t i o n r e g a r d i n g t e c h n i q u e s w h i c h w i l l b e u s e f u l i n the l a t e r s tudy o f i r r a d i a t e d a n i m a l s .

T h e f i r s t o b j e c t i v e o f the n e w p r o g r a m w a s , and s t i l l i s , t o m e a s u r e c h a n g e s i n t h y r o i d f u n c t i o n i n dogs that have been g i v e n I 1 3 1 a lone o r w i t h a n o t h e r i s o t o p e , s u c h a s C s 1 3 7 o r C e 1 4 4 . I t i s p o s s i b l e w i t h e x i s t i n g m e t h o d s t o d e t e r m i n e e x t r e m e s t a t e s o f h y p e r - o r h y p o a c t i v i t y o f the t h y r o i d . B e t w e e n t h e s e e x t r e m e s t h e r e i s a c o n t i n u u m o f f u n c t i o n a l a c t i v i t y w h i c h m a y f l u c t u a t e i n r e s p o n s e t o m a n y v a r i e d p h y s i o l o g i c a l c h a n g e s . I t i s i n t h i s a r e a o f s u b t l e change that one e n c o u n t e r s the g r e a t e s t d i f f i c u l t y i s a s s e s s i n g t h y r o i d f u n c t i o n . H o w e v e r , i t i s p r e c i s e l y i n t h i s a r e a that the m o s t r e v e a l i n g i n f o r m a t i o n r e g a r d i n g the n a t u r e of t he se changes m u s t and w i l l b e g a i n e d . T h e f o l l o w i n g e x p l o r a t o r y e x p e r i m e n t s have b e e n p e r f o r m e d w i t h the hope that the i n s i g h t g a i n e d m i g h t l e a d to a r e f i n e m e n t o f t e c h n i q u e s f o r m e a s u r e m e n t and a s s e s s m e n t o f t h y r o i d f u n c t i o n . S e v e r a l o f the p r o c e d u r e s to be o u t l i n e d have been p e r f o r m e d on s i n g l e a n i m a l s fo r a n u m b e r o f r e a s o n s , not the l e a s t o f w h i c h i s the l i m i t e d s u p p l y o f a v a i l a b l e

* T h i s w o r k w a s p e r f o r m e d b y R o b e r t J . To f t .

dogs, and the data are n e c e s s a r i l y f ragmenta ry and not a lways e n t i r e l y r e v e a l i n g . They do, however , give ind ica t ions of the p robab le succes s or f a i l u r e of a p a r t i c u l a r technique or approach and for that r e a s o n a re va luab le .

131 S h o r t - t e r m Uptake of I by T h y r o i d

L a r s s o n and Jonnson(^) d e s c r i b e d a method for d e t e r m i n i n g t h y r o i d function based upon the uptake of I by the t h y r o i d w i t h i n the f i r s t 15-20 minu tes after in t ravenous in jec t ion . A fast r i s i n g rate of uptake was i nd i ca t i ve of h y p e r t h y r o i d i s m whi l e a g radua l d i m i n u t i o n in count rate ove r the t h y r o i d after an i n i t i a l r a p i d r i s e ind ica ted h y p o t h y r o i d i s m . A m o d i f i c a t i o n of th is technique was at tempted w i th dog number 0570. The dog was in jec ted in t r avenous ly w i th 1.4 ml (75 mg) of p r o m a z i n e h y d r o c h l o r i d e (Sparine) in the r ight cepha l ic v e i n . T h i s was fo l lowed by an i n j e c t i o n of 2.5 ml (125 mg) of sod ium pentothal . A f t e r being anes the t ized the dog was suspended in a s l i n g , cons t ruc ted of p l a s t i c and tape, w h i c h a l l o w e d the legs to hang in a n o r m a l standing p o s i t i o n w h i l e the body of the a n i m a l was supported. The dog was suspended over a table on w h i c h was p l aced a l — i n . x l - | - i n . sod ium iodide s c i n t i l l a t i o n c r y s t a l . The c r y s t a l was pos i t ioned under the a r e a of the t h y r o i d S c i n c h e s f r o m the sur face of the neck. A 400 channel R I D L ana lyze r p r o g r a m e d for sequent ia l counting w i t h an i n t e r v a l of 10 seconds per channel was at tached to the c r y s t a l -p h o t o m u l t i p l i e r - p r e a m p l i f i e r uni t .

The ana lyze r was s ta r ted and background counts w e r e s to r ed in s e v e r a l channels before the sy r inge conta in ing the I was brought into the a r ea . The in jec t ion was made in the left cepha l ic v e i n . A sequent ia l count was taken over the t h y r o i d a r e a for a p p r o x i m a t e l y 20 minu tes after the in jec t ion . The amount of I 1 3 1 in jec ted was d e t e r m i n e d by m e a s u r e m e n t of the sy r inge both before and after the in jec t ion w i th a 4 i n . x 8 i n . sod ium iodide c r y s t a l i n the i r o n r o o m .

The data f r o m the in j ec t ion so lu t ion and r e s idue ind ica t ed that dog 0570 r e c e i v e d 4.95 m i c r o c u r i e s of I 1 3 1 (0.45 juc/kg) . When the data f r o m the uptake we re plot ted, a pos i t i ve slope was noted. I t was , howeve r , v e r y s l igh t and would be c o n s i d e r e d to be in the eu thyro id range a c c o r d i n g to the c r i t e r i a of L a r s s o n and Jonnson.(3) T h e r e was noted a d i s t u r b i n g o s c i l l a t i o n in the counts f r o m channel to channel . These o s c i l l a t i o n s suggested that there might be some sys t ema t i c ma l func t ion in the m a c h i n e r y . When a pulse genera tor was coupled w i th the scanning dev ice , i t was found that the counts v a r i e d not at a l l f r o m channel to channel . The answer was then sought in the data i t se l f . A p r o g r a m for the I B M 1620 computer was d e v i s e d w h i c h would average s e v e r a l channels , d rop the f i r s t , add the next , and give a running average w h i c h would tend to smooth the cu rve and e l im ina t e some of the e x t r e m e s , thus enabl ing one to see better the nature of the o s c i l l a t i o n s i f they s t i l l ex i s ted . Af t e r some d i s c u s s i o n i t was

5 3

d e c i d e d that the o s c i l l a t i o n s no ted w e r e r a n d o m and w e r e c a u s e d by the s m a l l n u m b e r o f counts i n each c h a n n e l . D a t a w i t h o s c i l l a t i o n s s i m i l a r i n n a t u r e but d i f f e ren t in p e r i o d w e r e ob ta ined u s i n g a v a r i e t y o f s t a n d a r d s , p r o v i n g that the no ted event w a s not in any w a y connec ted w i t h the p e c u l i a r i t i e s o f the uptake of I 1 3 1 by the t h y r o i d .

B e c a u s e of the p r o b l e m of quan t i t a t ing the degree of the s lope w i t h any p a r t i c u l a r p h y s i o l o g i c a l func t ion and because the a r r a n g e m e n t fo r m e a s u r i n g the t h y r o i d was not ye t s t a n d a r d i z e d , i t was d e c i d e d to p o s t pone fu r the r e x c u r s i o n s into th i s m e t h o d u n t i l a l a t e r date. A s e r i e s of e x p e r i m e n t s i s now be ing p l anned w h i c h w i l l once a g a i n i n c o r p o r a t e th i s d e s i g n w i t h m a n y r e f i n e m e n t s and w i l l , w e hope, g ive m o r e i n s i g h t in to the r e l a t i o n s h i p be tween the speed of uptake and the p h y s i o l o g y of the t h y r o i d g l a n d .

A f t e r the i n i t i a l m e a s u r e m e n t o f uptake the dog w a s m e a s u r e d d a i l y in the i r o n r o o m u s i n g the s l i n g a r r a n g e m e n t and the 4 i n . x 8 i n . c r y s t a l w i t h the 4 0 0 - c h a n n e l a n a l y z e r i n pu l se he ight m o d e . B e c a u s e o f i n e x p e r i ence w i t h th i s type o f m e a s u r e m e n t the dog was not m e a s u r e d i m m e d i a t e l y af ter the i od ine i n j e c t i o n , and so no m e a s u r e of i n i t i a l a b s o r p t i o n was o b t a i n e d . T h i s has been c o r r e c t e d in subsequent i n j e c t i o n s w i t h a couple o f e x c e p t i o n s . T h e i n i t i a l w h o l e - b o d y m e a s u r e m e n t w h i c h was m a d e one day af ter i n j e c t i o n showed a b i o l o g i c a l r e t e n t i o n of 63% of the i n j ec t ed dose . T h i s f i g u r e r e p r e s e n t s the amount r e m a i n i n g after 24 h o u r s d u r i n g w h i c h t i m e a c o n s i d e r a b l e amount m a y have been e x c r e t e d in the u r i n e a s w i l l be no ted l a t e r . I t i s a l s o in e r r o r by the amount w h i c h the a b s o r p t i o n by the a n i m a l * s own t i s s u e s p r e v e n t s the r a d i a t i o n f r o m r e a c h i n g the c r y s t a l . L a t e r s tud ies have i n d i c a t e d that t h i s a b s o r p t i o n fac to r m a y r e p r e s e n t a 25-35% l o s s of coun t ing r a t e w i t h an i n t r a v e n o u s l y i n j e c t e d dose and as m u c h as a 50% l o s s i m m e d i a t e l y after o r a l i n g e s t i o n of a capsu le con t a in ing the I 1 3 1 . F u t u r e w o r k i n c l u d e s p lans for a c o m p l e t e s tudy of the g e o m e t r i c and abs o r p t i o n f a c t o r s . The a n a l y s i s of the data by the c o m p u t e r i s s u c h that these f a c t o r s m a y be i n c l u d e d at any t i m e and a r e c o m p u t a t i o n m a d e of the t rue b i o l o g i c a l r e t e n t i o n . I t s h o u l d be borne i n m i n d that a l l subsequent da ta to be p r e s e n t e d suffer f r o m the s ame l a c k of c o r r e c t i o n for a b s o r p t i o n . The abso lu t e v a l u e s to be p r e s e n t e d a r e , t h e r e f o r e , not c o r r e c t ; h o w e v e r , the r e l a t i v e c o m p a r i s o n s a r e v a l i d .

T h e r e was a de f in i t e b r e a k in the r e t e n t i o n c u r v e a t about the e ighth day w h e n the l o s s o f i od ide b e c a m e m u c h l e s s . A n i n c r e a s e i n r e t e n t i o n noted at day 20 can on ly be e x p l a i n e d by an e r r o r in the t echn ique of coun t ing w h e r e i n the g e o m e t r y of the coun t ing setup m u s t have changed .

W h o l e B o d y R e t e n t i o n - P r e l i m i n a r y M e a s u r e m e n t

F o l l o w i n g the w o r k w i t h 0570 th ree m o r e dogs w e r e g i v e n I 1 3 1 for a s tudy of w h o l e - b o d y r e t e n t i o n of the i so tope . The f i r s t two of these , 0078 and 0181 w e r e i n j e c t e d i n t r a v e n o u s l y w i t h I 1 3 1 a s was 0570. N u m b e r 0233

w a s g i v e n a c a p s u l e o r a l l y con t a in ing the i s o t o p e . D o g 0181 w a s m e a s u r e d at i n t e r v a l s for 1 week , w h i l e the r e t e n t i o n in the 2 o ther dogs was f o l l o w e d for 2 w e e k s . The four dogs a l l e x h i b i t e d s i m i l a r pa t t e rns o f r e t e n t i o n . T h e r e was an i n i t i a l r a p i d d e c r e a s e i n r e t e n t i o n w i t h a change i n the ra te o c c u r r i n g about the s i x t h to e igh th day after i n j e c t i o n or i n g e s t i o n of the i s o t o p e . The i n i t i a l l e v e l v a r i e d c o n s i d e r a b l y f r o m one dog t o ano the r . I n e a c h c a s e the l e v e l i n g off of the ra te of e x c r e t i o n c a m e a t about 6-8% r e t e n t i o n . I t i s unfor tuna te that dog 0181 was not m e a s u r e d fo r a l o n g e r p e r i o d o f t i m e . T h e r e i s ev idence that a t the t i m e o f the l a s t m e a s u r e m e n t w h i c h was m a d e at. 7 days that the c u r v e h a d begun to f l a t t en as in the o the r 2 dogs . One can on ly s p e c u l a t e as to w h e t h e r o r not i t w o u l d have a t t a ined the s a m e d e g re e of s lope as d i d the o the r dogs af ter e ight d a y s .

T h y r o i d R e t e n t i o n o f I A p p a r a t u s for M e a s u r e m e n t

B e c a u s e o f r e p o r t s in the l i t e r a t u r e that the ra te o f r e l e a s e o f i od ine f r o m the t h y r o i d was a s e n s i t i v e m e a s u r e o f t h y r o i d a c t i v i t y a r e newed i n t e r e s t was t a k e n i n t h y r o i d m e a s u r e m e n t s . A n a l u m i n u m c o l l a r was d e s i g n e d w h i c h w o u l d f i t o n t o the l e a d s h i e l d s u r r o u n d i n g the c r y s t a l u s e d for t h y r o i d m e a s u r e m e n t s . I t w a s so d e s i g n e d and c o n s t r u c t e d that the n e c k of the dog c o u l d be c e n t e r e d o v e r the c r y s t a l in a r e p r o d u c i b l e f a s h i o n . T h e r e a r e f ! w i n g s " p r o j e c t i n g u p w a r d f r o m the c o l l a r o n e i t h e r s i de o f the c r y s t a l w h i c h s e r v e t o p r e v e n t the dog f r o m e i t h e r t u r n i n g h i s head o r f r o m m o v i n g h i s n e c k f r o m s ide t o s i d e . T h e a n t e r i o r p o r t i o n o f the c o l l a r r e s t s j u s t p o s t e r i o r to the angle o f the j a w w h i l e the p o s t e r i o r end o f the c o l l a r i s j u s t in f ront o f the c o l l a r bone. In t h i s way the a n i m a l can be h e l d in a f i x e d p o s i t i o n fo r the f i v e - m i n u t e p e r i o d d u r i n g w h i c h a count i s b e i n g m a d e . I t i s n e c e s s a r y on ly for an a s s i s t a n t to h o l d h i s hand on the top o f the dog ' s head gen t ly t o r e s t r a i n h i m . T h e w h o l e c r y s t a l a s s e m b l y i s m o u n t e d o n a n u n i s t r u t f r a m e w h i c h can b e e l e v a t e d . The a s s e m b l y c a n be m o v e d back and f o r t h on the f r a m e so that any p o s i t i o n m a y b e a c h i e v e d w i t h the c r y s t a l . The c r y s t a l a s s e m b l y i s f u r t h e r m a d e t o r o tate on an a x i s p e r p e n d i c u l a r to the p lane of e l e v a t i o n . The w h o l e a p p a r a t u s i s m o u n t e d on a c a r t for ease in m o v i n g f r o m one l o c a t i o n to a n o t h e r . A dog is p l a c e d on the top of the c a r t and a l l o w e d e i t h e r to s i t or to s tand . T h e c r y s t a l a s s e m b l y w i t h the a l u m i n u m c o l l a r i s then e l e v a t e d , a d v a n c e d , and t i l t e d a s n e c e s s a r y t o f i t c o m f o r t a b l y i n p l a c e a t the dog ' s neck . R e pea ted m e a s u r e m e n t s on a s i n g l e dog s h o w e d that the r e p r o d u c i b i l i t y of the p o s i t i o n w a s s a t i s f a c t o r y . W i t h th i s v a s t l y i m p r o v e d coun t ing d e v i c e a new i n t e r e s t w a s t a k e n i n t h y r o i d m e a s u r e m e n t .

U p t a k e o f I 1 3 1 b y the T h y r o i d G l a n d

To o b t a i n i n f o r m a t i o n r e g a r d i n g the r e t e n t i o n o f i o d i d e by the t h y r o i d g l a n d and the w h o l e body , t h r e e dogs , 0499, 0500, and 0503, w e r e set a s i d e for i n g e s t i o n o f I 1 3 1 and subsequen t m e a s u r e m e n t and c o l l e c t i o n o f u r i n e and f e c e s . T h e y w e r e kept i n i n d i v i d u a l m e t a b o l i s m cages fo r s e v e r a l days

p r e c e d i n g the a d m i n i s t r a t i o n o f i o d i n e i n o r d e r t o a c c l i m a t e t h e m s e l v e s t o the new e n v i r o n m e n t . The I 1 3 1 was a d m i n i s t e r e d o r a l l y i n a g e l a t i n c a p s u l e . I m m e d i a t e l y af ter a d m i n i s t r a t i o n o f the i so tope the a n i m a l s w e r e fed t h e i r r e g u l a r d i e t . I n t h i s way the iod ine was i n c o m p a n y w i t h the n o r m a l d i e t a r y c o m p l e m e n t o f i o d i d e . U r i n e c o l l e c t i o n s w e r e begun a t the t i m e o f f eed ing . F o l l o w i n g i n g e s t i o n of the i so tope the a n i m a l s w e r e counted in the w h o l e -body coun t ing f a c i l i t y w i t h the 4 i n . x 8 i n . c r y s t a l . T h e y w e r e p l a c e d on the c a r t , and t h y r o i d m e a s u r e m e n t s w e r e m a d e at 3 , 6 , 10, 12, 24, and 48 h o u r s , and d a i l y t h e r e a f t e r for a w e e k . W h o l e - b o d y m e a s u r e m e n t s w e r e m a d e d a i l y fo r the s a m e p e r i o d . The v e r y r a p i d t h y r o i d uptake o f I 1 3 1 i s c l e a r l y d i s -c e r n a b l e . The peak o f uptake was r e a c h e d a t a p p r o x i m a t e l y two days in each o f the a n i m a l s . A f t e r th i s t i m e the m e a s u r e m e n t s w e r e m a d e w i t h l e s s r e g u l a r i t y for the r e m a i n d e r of the 12 d a y s . The m a x i m u m p e r cent uptake v a r i e d o v e r qu i te a r ange f r o m 8% to 15% at two d a y s . The s lopes of the subsequent l o s s o f i od ine f r o m the t h y r o i d w e r e v e r y s i m i l a r fo r the s e v e r a l dogs . W h o l e body r e t e n t i o n was s i m i l a r i n each o f the t h ree dogs . T h e y w e r e not m e a s u r e d fo r a long enough p e r i o d of t i m e to e s t a b l i s h the b r e a k noted in the e a r l i e r a n i m a l s a l though the r e t e n t i o n at 10 days i n d i c a t e d a b r e a k f r o m the p r e v i o u s s l o p e .

E x c r e t i o n o f I 1 3 1 i n U r i n e and F e c e s

U r i n e c o l l e c t i o n s w e r e made a t 12, 24, and 48 h o u r s af ter a d m i n i s t r a t i o n of the I . B l o o d s a m p l e s t a k e n by j u g u l a r punc tu re w e r e c o l l e c t e d at 2, 6, 10, and 24 h o u r s . B e c a u s e of d i f f i c u l t i e s w i t h the coun t ing equ ipmen t m a n y o f the data f r o m these c o l l e c t i o n s w e r e l o s t . R a d i o a c t i v e m e a s u r e m e n t s o f the u r i n e , b l o o d , and feces f r o m dog 0503 w e r e made and a r e p r e sen ted in T a b l e 9 .

T A B L E 9

I c o n c e n t r a t i o n in b lood

T i m e , h r P e r cent o f T i m e , h r in jec ted dose

2 9.9 6 11.2

10 10.6 24 8.6

E x c r e t i o n of I

T i m e , h r P e r cent o f in j ec ted dose

P e r cent o f r e m a i n i n g I 1 3 1

U r i n e 12 19.4 19.4 12-24 14.1 17.5 24-48 28.0 42.1

F e c e s 24 0.5 0.5

T o t a l 48 62.0 -

56

A l t h o u g h the I was a d m i n i s t e r e d o r a l l y to each dog w i t h the d a i l y d ie t , i t c an be no ted f r o m the tab le that by two h o u r s af ter i n g e s t i o n t h e r e was a s u b s t a n t i a l amount of the i so tope a l r e a d y in the b l o o d . The amount of I in the b l o o d d i d not change s i g n i f i c a n t l y d u r i n g the 2 4 - h o u r p e r i o d f o l l o w i n g i n g e s t i o n of the i so tope . T h e r e i s the s u g g e s t i o n of a d r o p in b l o o d i od ide by 24 h o u r s . I t i s r e g r e t t a b l e that t h i s was not f o l l o w e d up by s a m p l e s o f b l o o d t aken a t l a t e r t i m e s . The e x c r e t i o n da ta c l e a r l y show that the b u l k o f the i o d i d e i s e x c r e t e d in the u r i n e w i t h an i n s i g n i f i c a n t amoun t i n the f e c a l m a t e r i a l . T h e ra te o f e x c r e t i o n r e m a i n s r e l a t i v e l y cons tan t d u r i n g the 4 8 - h o u r p e r i o d d u r i n g w h i c h data a r e a v a i l a b l e . E v e n though the p e r cent o f the i n g e s t e d dose e x c r e t e d d u r i n g each of the t i m e p e r i o d s i n d i c a t e s that t h e r e i s a d e c r e a s e i n e x c r e t i o n r a t e , t h i s i n t e r p r e t a t i o n i s m i s l e a d i n g b e cause o f the r e l a t i v e l y l a r g e l o s s o f i so tope d u r i n g each c o l l e c t i o n p e r i o d . The c o r r e c t e d v a l u e b a s e d upon the pe r cent o f the dose r e m a i n i n g af ter e ach o f the c o l l e c t i o n p e r i o d s i s m u c h m o r e r e v e a l i n g and i n d i c a t e s that, the ra te i s not changing a p p r e c i a b l y . T h i s w o u l d be e x p e c t e d i f i o d i d e i s t r e a t e d n o n s e l e c t i v e l y by the k i d n e y o r i f t h e r e w e r e no m e c h a n i s m for i t s c o n s e r v a t i o n . W i t h no change in the p h y s i o l o g y o f the a n i m a l t h e r e w o u l d be l e s s than 5% of the i n j e c t e d dose r e m a i n i n g in the body at 8 d a y s , b a s e d upon the e x c r e t i o n f i g u r e s a lone . T h i s i s a c o m p l i c a t e d p i c t u r e b e c a u s e o f the uptake and subsequent r e l e a s e o f i o d i d e f r o m the v a r i o u s o r g a n s i n g e n e r a l , and the t h y r o i d , s t o m a c h , and in t egumen t i n p a r t i c u l a r . E a c h o f these o r g a n s c o n c e n t r a t e s and r e l e a s e s i o d i d e a t d i f f e r en t r a t e s . To e x p l o r e these r e l a t i o n s h i p s w o u l d r e q u i r e a n e x t e n s i v e s a c r i f i c e s e r i e s i n w h i c h v a r i o u s o r g a n s and t i s s u e s c o u l d be s a m p l e d a t v a r y i n g t i m e s af ter i n g e s t i o n o f I 1 3 1 . T h i s i s not p r a c t i c a l a t the p r e s e n t t i m e .

D i e t a r y Intake o f Iodide and R e l e a s e Ra t e f r o m T h y r o i d

To tes t the effect of i od ide c o n s u m p t i o n on subsequent uptake and r e l e a s e o f I 1 3 1 four dogs , 0174, 0272, 0275, and 0881, w e r e put on four s e p a r a t e d ie t s con t a in ing d i f f e r i n g amount s o f i n o r g a n i c i o d i d e . T h e a n i m a l s had a l l been r a i s e d o n the s e m i s y n t h e t i c d ie t o f K a s p a r . ( ^ ) T h e n o r m a l s e m i s y n t h e t i c d ie t con ta ins 165.7 fig of i od ide p e r 100 g of food . T h i s d ie t was g i v e n t o dog 0174. H i s d a i l y food c o n s u m p t i o n a v e r a g e d 322 g, g i v i n g a d a i l y in take of 534 / ig of i o d i d e . D o g 0281 w a s fed a d ie t c o n t a i n i n g \/b the n o r m a l amount o f i o d i d e . W i t h an a v e r a g e food c o n s u m p t i o n of 195 g / d a y th i s p r o v i d e d 65 / i g i od ide p e r day . D o g 0275 c o n s u m e d 265 g / d a y of a d ie t c o n t a i n i n g l / 5 0 the n o r m a l amount of i o d i d e (3.3 / i g / l 0 0 g) g i v i n g a d a i l y t o t a l of 8.7 / ig i o d i d e . D o g 0272 had no i o d i d e added to h i s d ie t and c o n s u m e d 227 g p e r day . The i o d i d e i n t ake was v i r t u a l l y z e r o . The a n i m a l s w e r e m a i n t a i n e d o n these d ie t s for 2 8 days p r i o r t o the i n g e s t i o n o f I 1 3 1 . T h e y w e r e m e a s u r e d w i t h the t h y r o i d c r y s t a l and who le body coun te r fo r a p e r i o d of 16 d a y s . At the end of t h i s p e r i o d they w e r e g i v e n a s e c o n d capsu l e c o n t a i n i n g I and m e a s u r e d for r a d i o a c t i v i t y fo r ano ther 18 d a y s . D u r i n g t h i s e n t i r e p e r i o d the a n i m a l s w e r e m a i n t a i n e d o n t h e i r v a r i o u s d i e t s . The da ta f r o m the t h y r o i d and w h o l e - b o d y counts have been c o m b i n e d w i t h those f r o m a l l o the r dogs m e a s u r e d t o date and a r e p r e s e n t e d in T a b l e s 10 and 11.

TABLE 10

Retention of 1*31 by the body as a whole

Dog no.

Time Biological retention, Dog no.



elapsed, fraction of original Dog no.



days dose

Dog no.

days dose

Dog no.

days dose

0078 0.0183 0.6399 0233 1.0175 0.2501 0281 13.7396 0.06873 0078 1.1829 0.3688 0233 2.0833 0.1239 0281 16.8125 0.06186 0078 2.2079 0.2244 0233 2.7993 0.09279 0281 17.7799 0.05540 0078 2.9302 0.1889 0233 3.8590 0.09126 0281 21.0250 0.05832 0078 3.9850 0.1468 0233 6.9868 0.07295 0499 0.0368 0.5216 0078 6.9940 0.09027 0233 7.8063 0.07137 0499 1.1125 0.5050 0078 8.1496 0.07404 0233 8.8590 0.06783 0499 2.0785 0.3239 0078 9.2142 0.06633 0233 9.7882 0.06454 0499 3.0250 0.1809 0078 9.9274 0.06141 0233 10.7542 0.06945 0499 8.0326 0.1079 0078 11.0149 0.06094 0233 13.7618 0.05980 0499 10.1792 0.1074 0078 14.1142 0.04186 0272 0.9403 0.3590 0500 0.0410 0.6725 0078 14.9350 0.05176 0272 2.9306 0.2926 0500 1.1306 0.5972 0174 0.9361 0.2475 0272 3.8292 0.2551 0500 2.0854 0.4707 0174 2.9250 0.1092 0272 6.7958 0.1838 0500 3.0361 0.2659 0174 3.8243 0.1319 0272 13.7375 0.1311 0500 8.0493 0.1024 0174 6.7917 0.1132 0272 16.7958 0.07443 0500 10.1889 0.08685 0174 13.7306 0.07513 0272 17.7764 0.08888 0503 0.0458 0.5949 0174 16.7910 0.08068 0272 21.0083 0.06791 0503 1.1389 0.4779 0174 17.7715 0.06442 0275 0.9493 0.3881 0503 2.0938 0.2750 0174 21.0007 0.06805 0275 2.9333 0.3045 0503 3.0458 0.1566 0181 0.0136 0.7540 0275 3.8389 0.2766 0503 8.0743 0.07069 0181 0.7351 0.6298 0275 6.8056 0.2380 0503 10.2000 0.05683 0181 1.1136 0.6039 0275 13.7396 0.1630 0570 1.0527 0.6315 0181 1.1198 0.6097 0275 16.8076 0.1367 0570 3.9221 0.2368 0181 1.7358 0.4338 0275 17.7792 0.1322 0570 7.0082 0.08879 0181 2.1198 0.3766 0275 21.0208 0.1120 0570 9.0742 0.06202 0181 4.0782 0.1088 0281 0.9569 0.2635 0570 10.9575 0.05054 0181 7.0754 0.05707 0281 2.9347 0.1613 0570 16.1895 0.04968 0233 0.0139 0.5084 0281 3.8431 0.1411 0570 21.1513 0.05645 0233 0.0972 0.5327 0281 6.8104 0.1162

TABLE 11

Retention of by the thyroid

Dog no.







days dose

Dog no.

days dose

Dog no.

days dose

0174 0.9403 0.07719 0272 16.7535 0.04118 0281 14.7882 0.03360 0174 1.0021 0.06623 0272 16.8882 0.05315 0281 15.7875 0.03635 0174 1.9326 0.08385 0272 17.9882 0.05245 0281 16.7639 0.03885 0174 1.9500 0.07864 0275 0.9451 0.1489 0281 16.8882 0.03233 0174 2.9625 0.07617 0275 1.0139 0.2425 0281 17.9896 0.03002 0174 2.9785 0.07001 0275 1.9368 0.2222 0499 0.1111 0.02826 0174 3.7778 0.07714 0275 1.9625 0.2653 0499 0.2340 0.04013 0174 3.9451 0.06652 0275 2.9736 0.2152 0499 0.4132 0.05431 0174 6.9500 0.06292 0275 2.9847 0.2221 0499 0.5000 0.05313 0174 7.9972 0.05607 0275 3.7826 0.1902 0499 1.2028 0.07764 0174 9.9618 0.05228 0275 3.9563 0.2235 0499 2.1069 0.08093 0174 11.0285 0.05199 0275 6.9632 0.1670 0499 3.0715 0.06294 0174 13.7861 0.05855 0275 8.0104 0.1166 0499 6.2840 0.03541 0174 13.7924 0.04707 0275 9.9750 0.1327 0499 8.0979 0.02638 0174 14.7861 0.05481 0275 11.0396 0.1084 0499 10.1007 0.02512 0174 15.7840 0.05841 0275 13.7910 0.1067 0500 0.1167 0.03537 0174 16.7493 0.04393 0275 13.8035 0.1032 0500 0.2410 0.05340 0174 16.8882 0.05546 0275 14.7896 0.1137 0500 0.4250 0.06345 0174 17.9847 0.04555 0275 15.7882 0.1182 0500 0.5056 0.07940 0272 0.9444 0.2005 0275 16.7611 0.09338 0500 1.2090 0.1286 0272 1.0063 0.1694 0275 16.8896 0.09361 0500 2.1125 0.1366 0272 1.9361 0.1878 0275 17.9896 0.09166 0500 3.0792 0.1355 0272 1.9542 0.2089 0281 0.9438 0.09432 0500 6.2903 0.09638 0272 2.9660 0.1825 0281 1.0167 0.08198 0500 8.1049 0.07454 0272 2.9819 0.1926 0281 1.9361 0.09298 0500 10.1097 0.05167 0272 3.7813 0.1682 0281 1.9653 0.1023 0503 0.1236 0.03482 0272 3.9493 0.1642 0281 2.9764 0.09367 0503 0.2479 0.05160 0272 6.9556 0.1012 0281 2.9840 0.1112 0503 0.4326 0.05510 0272 8.0014 0.1026 0281 3.9590 0.09073 0503 0.5125 0.08057 0272 9.9660 0.08424 0281 6.9688 0.06309 0503 1.2167 0.09853 0272 11.0319 0.04658 0281 8.0160 0.05722 0503 2.1194 0.1019 0272 13.7896 0.06717 0281 9.9778 0.05613 0503 3.0868 0.09604 0272 13.7958 0.04817 0281 11.0424 0.04913 0503 6.2972 0.05602 0272 14.7889 0.06826 0281 13.7896 0.03762 0503 8.1208 0.04991 0272 15.7875 0.06275 0281 13.8069 0.04415 0503 10.1201 0.04253

58

Thyro id and Whole Body Retention of I - Combined Data

W h i l e each o f the p r o c e d u r e s o u t l i n e d above s e r v e d s o m e p a r t i c u l a r p u r p o s e , each y i e l d e d s o m e i n f o r m a t i o n c o m m o n t o a l l m e t h o d s . T h i s was w i t h r e g a r d to the l o s s o f i od ide as i n d i c a t e d by I 1 3 1 f r o m e i t h e r the body as a w h o l e or f r o m the t h y r o i d in p a r t i c u l a r . I t i s p e r t i n e n t to point out that the data ob t a ined f r o m the a n a l y z e r a r e t r a n s f o r m e d into m i c r o c u r i e s and then into f r a c t i o n a l r e t e n t i o n by a p a r t i c u l a r p r o g r a m for the I B M 1620 c o m pu te r . The r e s u l t s ob t a ined f r o m the c o m p u t e r a r e i n t e r m s o f both b i o l o g i c a l and p h y s i c a l p r o p e r t i e s and cons t i tu te e f fec t ive r e t e n t i o n . T h e s e a r e not s u c h u s e f u l data for t r a c e r s tud ies of th i s type as for the a s s e s s m e n t of r a d i a t i o n dosage due to the p r e s e n c e of abso lu te amounts of r a d i o a c t i v e s u b s t ances . I t was d e s i r a b l e to know the b i o l o g i c a l r e t e n t i o n and so I d e v i s e d and w r o t e a s i m p l e p r o g r a m for the c o m p u t e r w h i c h took the data f r o m the e x i s t i n g p r o g r a m and c o n v e r t e d i t t o b i o l o g i c a l r e t e n t i o n by c o r r e c t i n g for the amount o f p h y s i c a l decay a t the t i m e of m e a s u r e m e n t . The b i o l o g i c a l r e t e n t i o n p r o g r a m was w r i t t e n in so that the output f r o m i t c o u l d be u s e d as input for the l e a s t - s q u a r e s p r o g r a m for the d e t e r m i n a t i o n o f the b e s t - l i n e f i t of the data . In th i s w a y the data w e r e s u b m i t t e d to f u r t h e r a n a l y s i s and c o u l d be g r o u p e d i n d i f f e r e n t w a y s to e x a m i n e the s lope of the b i o l o g i c a l d e c a y .

The f i r s t point to be noted r e g a r d i n g a l l the data f r o m the v a r i o u s a n i m a l s i s that the l o s s o f I 1 3 1 f r o m the t h y r o i d or f r o m the body as a w h o l e is n e i t h e r a l i n e a r func t ion nor is i t a s i n g l e e x p o n e n t i a l func t ion . A s i m p l e p lo t of the data on s e m i l o g paper r e v e a l s the change in s lope of the b e s t -l i n e f i t a s t i m e after a d m i n i s t r a t i o n p r o c e e d s . By g r o u p i n g the data i n s e v e r a l w a y s and t r y i n g each w i t h the l e a s t - s q u a r e s p r o g r a m , i t was ap pa ren t that the m o s t c o n s i s t e n t b r e a k in the s lope was at about 8 days af ter a d m i n i s t r a t i o n o f the i so tope whe the r w h o l e body o r t h y r o i d counts w e r e u s e d . On th i s a s s u m p t i o n the data w e r e a r r a n g e d as in T a b l e 12 and the

TABLE 12

Retention of I'

Dog no.

Interval, days

Biological half-life, days Dog no.

Interval, days

Biological half-life, days Dog no.

Interval, days

Thyroid Whole body

Dog no.

Interval, days Thyroid Whole body

0078 0-2 1.5 (0 0375 2-8 6.5 11.5 2-8 3.1 4.0 9-18 20.1 13.5 9-15 - 12.3 0-18 - 11.9 0-15 - 4.3 2-18 11.6 -

0174 2-8 14.0 118 0281 2-8 5.9 8.7 9-18 72.2 39.6 9-18 10.0 29.0 0-18 - 13.8 0-18 - 9.7 2-18 23.5 - 2-18 9.0 -

0181 0-2 - 2.1 0499 0-2 - 3.0 2-8 2.5 3.2 2-8 4.0 6.7 0-8 - 1.7 0-8 - 4.2

0233 0-2 - 1.0 0500 0-2 - 4.0 2-8 7.4 12.0 2-8 5.9 4.3 9-14 - 28.9 0-8 - 3.2 0-14 - 4.9

0503 0-2 - 1.9 0272 2-8 5.3 5.9 2-8 5.2 4.8

9-18 19.4 8.1 0-8 - 3.0

0-18 - 8.4 0570 1-7 _ 2.1

2-18 7.5 -0570

9-21 - 173 0-21 - 6.2

h a l f - t i m e o f each segment o f the decay c u r v e was c a l c u l a t e d . T h e r e i s an i n i t i a l r a p i d l o s s o f i o d i d e , w h i c h takes p l a c e i n the f i r s t two days, due t o e x c r e t i o n . T h i s m a y be noted i n the w h o l e - b o d y r e t e n t i o n c u r v e s . The b r e a k a t 2-3 days is l e s s d r a m a t i c than that a t 8 days but is p r o b a b l y s i g n i f i c a n t .

E x a m i n a t i o n o f the h a l f - p e r i o d s for t h y r o i d r e t e n t i o n r e v e a l s that t h e r e i s a g r e a t e r deg ree o f u n i f o r m i t y i n the l o s s o f I 1 3 1 f r o m t h e m d u r i n g the p e r i o d f r o m 2-8 days than the re i s a t any l a t e r t i m e . W i t h the e x c e p t i o n of dog N o . 0174, the a n i m a l s range f r o m 4.0 to 6.5 days fo r the h a l f -p e r i o d in that s egmen t be tween 2 and 8 days af ter a d m i n i s t r a t i o n of the i s o t o p e . T h e r e i s l e s s u n i f o r m i t y i n the s a m e p e r i o d w i t h r e g a r d t o w h o l e -body r e t e n t i o n a l though i n s e v e r a l a n i m a l s the h a l f - p e r i o d for w h o l e - b o d y r e t e n t i o n i s v e r y c l o s e to that o f the t h y r o i d . One w o u l d expec t t h i s b e -cause of the l a r g e s h a r e of the I w h i c h i s found in the t h y r o i d d u r i n g t h i s p e r i o d . T h e r e i s , h o w e v e r , n o s u c h n i c e c o r r e l a t i o n be tween w h o l e - b o d y and t h y r o i d a l h a l f - p e r i o d s in the t i m e s after 8 d a y s . A l t h o u g h a n u m b e r of i n t e r e s t i n g t r e n d s a r e t o be no ted in these f ind ings they a r e w i thou t r e a l s i g n i f i c a n c e because o f the v a r i e t y o f c o n d i t i o n s under w h i c h the v a r i o u s da ta w e r e c o l l e c t e d .

In p a r t i c u l a r a n i m a l s 0174, 0272, 0275, and 0281 a r e to be set a s i d e f r o m the o t h e r s because of the na tu re of the d ie t to w h i c h each, was e x p o s e d fo r a p r o l o n g e d p e r i o d . I t i s my f e e l i n g that dog 0174, w h i l e a c t i n g as a c o n t r o l fo r the d ie t e x p e r i m e n t , i s not n o r m a l because o f the d e v i a t i o n o f a l l i n f o r m a t i o n g a t h e r e d for h i m f r o m that t a k e n f r o m the o the r a n i m a l s , w h e t h e r i n t h i s e x p e r i m e n t o r not. The uptake w a s d e p r e s s e d , and the r e t e n t i o n w a s v e r y h i g h , i n d i c a t i n g a v e r y s l o w t u r n o v e r o f i o d i n e . I t i s r e g r e t t a b l e that m o r e w a s not k n o w n about t h i s a t the t i m e so that a be t te r a s se s smen t : o f t h i s dog and i t s c o n d i t i o n m i g h t have been m a d e . T h e uptake of i o d i n e by the o ther t h r e e dogs was as m i g h t be e x p e c t e d w h e r e t h e r e w a s i o d i n e d e p r i v a t i o n . I t i s c u r i o u s to note that dog 0275, w h i c h h a d only 8.7 / i g o f i o d i n e p e r day , was h i g h e r in uptake and r e t e n t i o n than dog 0272 w h i c h h a d no i o d i n e added to i t s food . B o t h 0272 and 0275 e x h i b i t e d g r e a t e r r e t e n t i o n o f i 1 3 1 th an d id e i t h e r 0174 or 0281. I t w o u l d appea r that the dogs w i t h the least, i o d i n e in t h e i r d ie t w e r e c o n s e r v i n g that, which, they had in a be t t e r f a s h i o n than w e r e the ones w i t h su f f i c i en t o r a t l e a s t g r e a t e r i od ine i n t a k e . T h a t t h i s c o n s e r v a t i o n , a s m e a s u r e d by the p e r cent r e t en t ion^ i s not g r e a t e r m a y be e x p l a i n e d by the fact that the t r a c e r a m o u n t e d to a m u c h l a r g e r f r a c t i o n o f the t o t a l d a i l y i o d i n e in take in those dogs d e p r i v e d o f the n o r m a l amount, than i t d i d i n the n o r m a l a n i m a l s .

T h e da ta have been t u r n e d o v e r i n s e v e r a l w a y s i n a n a t t empt t o a r r i v e a t s o m e m e a n i n g f u l c o m p a r a t i v e f i g u r e s for the amount o f i o d i n e in the t h y r o i d . I n a s m u c h as a supposed e q u i l i b r i u m e x i s t e d a t the t i m e of m e a s u r e m e n t be tween the d i e t a r y i n t ake and the s e c r e t i o n , i t w a s fe l t that s o m e a p p r o x i m a t i o n o f t h y r o i d a l i od ine content c o u l d be m a d e . C o n s i d e r i n g that the m a x i m u m per cent uptake r e f l e c t e d the d a i l y t u r n o v e r o f i o d i n e ,

and that the d a i l y i od ide in take was m e a s u r e d , and that the d a i l y loss f r o m the t h y r o i d c o u l d be m e a s u r e d f r o m the s lope of the r e l e a s e , a r a t i o w a s set up w h e r e b y the m a x i m u m p e r cent uptake by the t h y r o i d t i m e s the m i c r o g r a m s uptake d i v i d e d by the p e r cent r e l e a s e d p e r day w o u l d e q u a l the t o t a l m i c r o g r a m s i n the t h y r o i d . The f i g u r e s ob ta ined for the s e v e r a l a n i m a l s showed g r e a t v a r i a t i o n but i n d i c a t e d that dogs 0275 and 0281 w e r e d e f i c i e n t i n t h y r o i d a l i o d i n e . T h e r e a r e s o m e s e r i o u s e r r o r s i n s u c h r o u g h c a l c u l a t i o n s , but they m a y s e r v e to i n d i c a t e r e l a t i o n s h i p s not o t h e r w i s e seen .

P r o t e i n - b o u n d Iodine D e t e r m i n a t i o n s

A m i c r o a n a l y t i c a l k i t for the d e t e r m i n a t i o n of p r o t e i n - b o u n d iod ine ( P B I ) was p u r c h a s e d f r o m H y c e l , Inc . T h i s appa ra tus u t i l i z e s o n l y 0.3 m l o f s e r u m for a d e t e r m i n a t i o n . The t echn ique i n v o l v e d in the d e t e r m i n a t i o n s i s v e r y i n v o l v e d and i n c l u d e s p r e c i p i t a t i o n , b o i l i n g , and l a t e r d i s t i l l a t i o n o f the i o d i d e . A l l r eagen t s w e r e p u r c h a s e d w i t h the k i t i n o r d e r t o i n s u r e t h e i r pu r i t y .

M a n y s a m p l e s o f s e r u m and s t anda rds have been r u n t h r o u g h the p r o c e d u r e . As a r e s u l t i t i s p o s s i b l e to g ive a r ange o f P B I fo r the n o r m a l a n i m a l s o f about 3-8 jUg/lOO m l s e r u m . The i n o r g a n i c i o d i d e i n the s e r u m r a n g e s f r o m 0 .1 -5 .0 / i g . The t echn ique has not been a p p l i e d t o m a n y a n i m a l s , h o w e v e r , because the range w h i c h i s g i v e n above w i l l be found not on ly be tween a n i m a l s but i n d u p l i c a t e s a m p l e s t a k e n f r o m one a n i m a l o r f r o m p o o l e d s e r u m f r o m s e v e r a l a n i m a l s . T h e r e has been a g r e a t d e a l o f d i f f i c u l t y e n c o u n t e r e d in p e r f e c t i n g the t echn ique to the extent that r e p r o d u c i b i l i t y i s p o s s i b l e . The t echn ique r e q u i r e s that t i m e b e a v a i l a b l e fo r c o n t i n u a t i o n o f s a m p l e p r o c e s s i n g once i t has begun . B e c a u s e o f the l a c k o f t e c h n i c a l h e l p th i s has been i m p o s s i b l e w h e n an e x p e r i m e n t has been unde r w a y because of the demands of the- coun t ing p r o c e d u r e s .

R e f e r e n c e s

1 . N o r r i s , W . P . , L . S . L o m b a r d , C . E . ' R e h f e l d , T . W . S p e c k m a n , and H . M . S l a t i s . P r o g r e s s r e p o r t : T o x i c i t y and m e t a b o l i s m o f r a d i o n u c l i d e s . III. S e m i a n n u a l r e p o r t fo r p e r i o d ending M a y 31 , 1962. A r g o n n e N a t i o n a l L a b o r a t o r y B i o l o g i c a l and M e d i c a l R e s e a r c h D i v i s i o n S e m i annua l R e p o r t , J a n u a r y t h r o u g h June 1962. A N L - 6 7 2 3 , pp . 100- 137.

2 . L i n d e n b a u m , A . , and W . W e s t f a l l , 1963. C o l l o i d a l p r o p e r t i e s o f p l u t o n i u m i n d i l u t e aqueous s o l u t i o n . T o b e p u b l i s h e d .

3 . L a r s s o n , L a r s , and L a r s J o n s s o n . C o n t i n u o u s r e g i s t r a t i o n o f t h y r o i d uptake af ter i n t r a v e n o u s i n j e c t i o n o f r a d i o a c t i v e i o d i n e . A c t a R a d i o l . 43 , 81 -88 (1955).

4 . N o r r i s , W . P . , L . S . L o m b a r d , and C . E . R e h f e l d . T o x i c i t y and m e t a b o l i s m o f r a d i o n u c l i d e s . V . P r o g r e s s r e p o r t f o r p e r i o d end ing June 29, 1963. A r g o n n e N a t i o n a l L a b o r a t o r y B i o l o g i c a l and M e d i c a l R e s e a r c h D i v i s i o n S e m i a n n u a l R e p o r t , J a n u a r y t h r o u g h June 1963. A N L - 6 8 2 3 , pp. 4 4 - 7 5 .

61

D R Y E M U L S I O N A U T O R A D I O G R A P H Y

L l o y d J . R o t h , * A k e H a n n g r e n , * * and W i l l i a m P , N o r r i s

T h e r e s o l u t i o n o f an a u t o r a d i o g r a m depends in p a r t on the d i s t a n c e b e t w e e n the s o u r c e o f i r r a d i a t i o n and pho tog raph i c e m u l s i o n . C l o s e ap p r o x i m a t i o n o f e m u l s i o n t o t i s s u e i s u s u a l l y a c h i e v e d b y t echn iques u t i l i z i n g f l o t a t i o n on w a t e r o r l i q u i d e m u l s i o n . A s e r i o u s th rea t o f t r a n s l o c a t i o n i s thus i n t r o d u c e d i f the l a b e l e d subs tance i n v e s t i g a t e d i s w a t e r s o l u b l e . D r y a p p l i c a t i o n o f e m u l s i o n t o t i s s u e w i t h r e s o l u t i o n a t the c e l l u l a r l e v e l i s d e m o n s t r a t e d .

The technique o f a u t o r a d i o g r a p h y a p p l i e d to t i s s u e s c o n t a i n i n g w a t e r -s o l u b l e subs t ances has , i n g e n e r a l , been u n s u c c e s s f u l because the me thods o f t i s s u e p r o c e s s i n g and a p p l i c a t i o n o f e m u l s i o n i n t r o d u c e a h i g h p r o b a b i l i t y of l o s s or t r a n s l o c a t i o n of the r a d i o a c t i v e l y - l a b e l e d c o m p o u n d , A c o m p l e t e l y r e l i a b l e m e t h o d w o u l d b e one i n w h i c h a l l o p p o r t u n i t i e s fo r t r a n s l o c a t i o n d u r i n g the p r e p a r a t i o n o f the t i s s u e a r e e l i m i n a t e d . D e s c r i b e d h e r e i n i s a m e t h od for m e c h a n i c a l l y s u p e r i m p o s i n g a n u c l e a r e m u l s i o n upon an unwet ted t i s s u e s e c t i o n . T o d e t e r m i n e whe the r th is t echn ique w o u l d p r o v i d e adequate r e s o l u t i o n and r e r e g i s t r y a t the c e l l u l a r l e v e l , the m e t h o d was t e s t ed w i t h r a t i l e u m s e c t i o n s f r o m a n a n i m a l i n j e c t e d w i t h H 3 - t h y m i d i n e . S u c h s e c t i o n s w e r e u s e d b e c a u s e they p r o v i d e m a t e r i a l i n w h i c h the l o c a l i z a t i o n o f the l a b e l i n t i s s u e i s k n o w n .

M e t h o d

A g l a s s c o v e r s l i p 1 x 2~in0 was i m m e r s e d in a g e l a t i n - c h r o m a l u m s o l u t i o n and d r i e d . P i e c e s o f a u t o r a d i o g r a p h i c s t r i p p i n g e m u l s i o n ( K o d a k L i m i t e d ) 1 x Z i n , w e r e f loa ted on w a t e r u s i n g the technique d e s c r i b e d by D o n i a c h and P e l c , ( l ) but w i t h the e m u l s i o n s ide up 0 The f i l m was then p i c k e d up on the p r e p a r e d c o v e r s l i p l e a v i n g a 1 - c m long s e c t i o n a t one end f ree o f e m u l s i o n . The s l i p s w i t h e m u l s i o n w e r e d r i e d o v e r c a l c i u m c h l o r i d e i n a l i g h t - t i g h t b o x a t r o o m t e m p e r a t u r e .

T h e t i s s u e s e c t i o n was p r e p a r e d b y s t a n d a r d f i x a t i o n and p a r a f f i n i m b e d d i n g t echn iques and was m o u n t e d on the e t ched s ide of a m i c r o s c o p e s l i d e c e n t e r e d about - f i n , f r o m the une tched end. The s l i d e was d ipped t w i c e in x y l o l fo r 3 minu te s to r e m o v e the p a r a f f i n and the x y l o l was r e m o v e d b y e v a p o r a t i o n a t r o o m t e m p e r a t u r e .

* D e p a r t m e n t o f P h a r m a c o l o g y , The U n i v e r s i t y o f C h i c a g o . * R o y a l V e t e r i n a r y C o l l e g e , S t o c k h o l m , Sweden .

In a d a r k r o o m w i t h a W r a t t e n N o . 2 safe l i g h t , the f ree end of the c o v e r s l i p was g lued w i t h an epoxy r e s i n * to the e tched end o f the m i c r o scope s l i d e c a r r y i n g the t i s s u e s e c t i o n . The " d r y " e m u l s i o n and the t i s s u e s e c t i o n w e r e p r e s s e d together u s i n g a s econd s h o r t e n e d m i c r o s c o p e s l i d e ( l x l ^ i n . ) w h i c h was p l a c e d o v e r the c o v e r s l i p and c l a m p e d w i t h 6 pape r c l i p s * * ( three on e a c h s ide ) . T h e s h o r t s l i d e was u s e d t o a v o i d con tac t w i t h the r e g i o n of the epoxy bond. The a s s e m b l y was then p l a c e d in a l i g h t - t i g h t box c o n t a i n i n g d e s i c c a n t c a l c i u m c h l o r i d e , and a l l o w e d t o s tand i n a r e f r i g e r a t o r a t 5°C.

A f t e r e x p o s u r e fo r 2 weeks the c l a m p s w e r e r e m o v e d , and the f ree end o f the c o v e r s l i p c a r r y i n g the e m u l s i o n was s e p a r a t e d s l i g h t l y f r o m the s l i d e a t the ung lued end. A n i m p e r m e a b l e p l a s t i c bag was d r a w n o v e r the t i s s u e s l i d e w i t h s e p a r a t i o n m a i n t a i n e d by i n s e r t i o n o f a 2 mm r u b b e r s p a c e r a t the end . T h e e m u l s i o n was p r o c e s s e d a t 20°C by i m m e r s i n g the a s s e m b l y in K o d a k D 1 9 d e v e l o p e r fo r 5 m i n u t e s , i n w a t e r fo r 1 m i n u t e and in K o d a k a c i d f i x a t i v e fo r 8 m i n u t e s . The d e v e l o p e d e m u l s i o n was a l l o w e d to d r y a t r o o m t e m p e r a t u r e . The p l a s t i c bag was r e m o v e d f r o m the s l i d e and the c o v e r s l i p was s i m i l a r l y p r o t e c t e d w i t h a p l a s t i c bag . T h e t i s s u e was s t a i n e d w i t h C e l e s t i n e B l u e (1 gm d i s s o l v e d in 100 cc o f w a t e r c o n t a i n i n g « | cc H 2 S 0 4 and f i l t e r e d ) for 5 m i n u t e s and then w a s h e d in d i s t i l l e d w a t e r , 70% a l c o h o l , 95% a l c o h o l , and f i n a l l y abso lu t e a l c o h o l , e ach for 2 m i n u t e s . The p l a s t i c bag was r e m o v e d f r o m the c o v e r s l i p and the a s s e m b l y d r i e d , f i r s t i n r o o m a i r and f i n a l l y o v e r c a l c i u m c h l o r i d e ; w i t h the two p o r t i o n s s e p a r a t e d by the 2 mm s p a c e r a t the ung lued ends . T h e c o v e r s l i p and m i c r o s c o p e s l i d e w e r e r e a p p o s e d c a r e f u l l y and s e a l e d w i t h P e r m o u n t .

R e s u l t s and D i s c u s s i o n

C u r r e n t me thods o f c e l l u l a r a u t o r a d i o g r a p h y u t i l i z i n g l i q u i d e m u l s i o n s o r s t r i p p i n g f i l m t echn iques a r e unsu i t ab l e fo r w o r k w i t h w a t e r -s o l u b l e l a b e l e d compounds not f i r m l y bound in t issue.(2> 3 ) The m e c h a n i c a l a p p l i c a t i o n o f f i l m t o t i s s u e s e c t i o n s fo r a u t o r a d i o g r a p h y has b e e n p r e v i o u s ly d e s c r i b e d . ( 4 , 5 ) H o w e v e r , these l a t t e r me thods have not g a i n e d p o p u l a r i t y , p a r t i c u l a r l y i n w o r k r e q u i r i n g r e s o l u t i o n a t the c e l l u l a r l e v e l b e c a u s e o f d i f f i c u l t i e s e n c o u n t e r e d i n r e r e g i s t r a t i o n . E p o x y r e s i n s p r o v i d e a m e a n s o f f i r m l y bond ing g l a s s to g l a s s and thus the dange r o f m i s m a t c h i n g i n h e r e n t i n me thods that use m e c h a n i c a l c l a m p s o r e m u l s i o n s m o u n t e d o n p l a s t i c i s a v o i d e d .

C e l l s l a b e l e d w i t h H 3 - t h y m i d i n e p r o v i d e a tes t s y s t e m for e v a l u a t i n g r e s o l u t i o n and r e r e g i s t r y . F i g u r e 2 0 i s a n a u t o r a d i o g r a m o f r a t i l e u m p r e p a r e d by the m e t h o d d e s c r i b e d h e r e i n . I t i s c l e a r that the d e v e l o p e d s i l v e r g r a i n s c o i n c i d e c l o s e l y w i t h the e s t a b l i s h e d l o c a l i z a t i o n o f H 3 ~ t h y m i d i n e i n the c e l l n u c l e i .

* A r m s t r o n g A d h e s i v e C - 4 , A c t i v a t o r P . 4 / l m i x t u r e . * * L - E - B N o . 2 S m a l l , C u s h m a n and D e n n i s o n Mfg. , C o . , N e w Y o r k , U S A .

Figure 20. Autoradiogram of rat ileum prepared by the method described in the text

T h e p r o b l e m o f p r o c e s s i n g t i s s u e by methods that a v o i d t r a n s l o c a t i o n o f w a t e r - s o l u b l e subs tances p r i o r t o a p p l i c a t i o n o f d r y e m u l s i o n has not ye t been s o l v e d . The d e m o n s t r a t i o n that a u se fu l degree of r e s o l u t i o n a t the c e l l u l a r l e v e l c a n be ob ta ined w i t h " d r y " e m u l s i o n methods i s a f i r s t s tep t o w a r d p r o v i d i n g a m e t h o d for the a u t o r a d i o g r a p h i c study of w a t e r - s o l u b l e l a b e l e d subs t ances a t the c e l l u l a r l e v e l .

R e f e r e n c e s

1 . D o n i a c h , I . and S . R . P e l c . A u t o r a d i o g r a p h i c t echn ique . B r i t . J . R a d i o l . 23, 184-192 (1950).

2 . B e l a n g e r , L . F . , and C . P . L e b l o n d . A m e t h o d for l o c a t i n g r a d i o a c t i v e e l e m e n t s in t i s s u e s by covering h is to logica l sections with p h o t o g r a p h i c e m u l s i o n . E n d o c r i n o l o g y 39, 8- 13 (1946).

P e l c , S . R . , and A . H o w a r d . T e c h n i q u e s o f a u t o r a d i o g r a p h y and the ap p l i c a t i o n o f the s t r i p p i n g - f i l m m e t h o d to p r o b l e m s o f n u c l e a r m e t a b o l i s m . B r i t . M e d . B u l l . ^8, 132-135 (1952).

H o e c k e r , F . E . , and P . G . R o o f e . S t r u c t u r a l d i f f e r e n c e s i n bone m a t r i x a s s o c i a t e d w i t h m e t a b o l i z e d r a d i u m . R a d i o l o g y 5_2, 856-864 (1949).

W i l l i a m s , A , I . A m e t h o d for the p r e v e n t i o n o f l e a c h i n g and c h e m i c a l fogging in n u c l e a r t r a c k a u t o r a d i o g r a p h s , and the m a i n t e n a n c e o f h i g h s e n s i t i v i t y and la tent i m a g e s t a b i l i t y , U . S . A t o m i c E n e r g y C o m m i s s i o n R e p o r t , A E C U - 9 3 0 (Nov . 3 , 1950).

A R A P I D B L O O D C A T A L A S E S C R E E N I N G T E C H N I Q U E A D J U S T A B L E T O A N Y L E V E L O F A C T I V I T Y

R o b e r t N . F e i n s t e i n , Jud i th B e H o w a r d , L a r r y B , Ba l lono f f , * and John E . S e a h o l m

We w i s h e d to s c r e e n a l a rge number of m i c e in o rde r to detect a mutant exh ib i t i ng an a b n o r m a l l y low l e v e l of b lood ca ta lase Q S ince the de f in i t ion of " a b n o r m a l " w i l l depend on the n o r m a l l e v e l of b lood ca ta lase a c t i v i t y of a g iven s t r a i n of m i c e , i t was n e c e s s a r y to dev ise a technique w h i c h cou ld r e a d i l y be adjusted to indica te ' ' n o r m a l " or " s u b n o r m a l " at any d e s i r e d l e v e l of ca ta lase a c t i v i t y . U s i n g the p rocedure descr ibed, h e r e i n , one i n d i v i d u a l can p roces s perhaps 1Z5 m i c e per day, i n c l u d i n g d r a w i n g of b lood , p r e p a r a t i o n of so lu t ions , and wash ing g l a s s w a r e .

The technique is based on perbora te d e c o m p o s i t i o n by c a t a l a s e . 0 ) H o w e v e r , no t i t r a t i o n is p e r f o r m e d ; after incuba t ion of the b lood w i t h the subs t ra te m i x t u r e , enzyme a c t i v i t y i s ha l ted by the add i t ion of H 2 S 0 4 2 and a s m a l l amount of K M n 0 4 i s added as an i n d i c a t o r to test for excess N a B 0 3 , I f the b lood i s low in ca ta lase , r e s i d u a l perbora te d e c o l o r i z e s the K M n 0 4 . On the other hand, p e r s i s t e n c e of the K M n 0 4 c o l o r ind ica tes that suff ic ient ca ta lase i s p resen t to decompose a l l pe rbora te .

The s c r e e n i n g test i s adjusted to any d e s i r e d l e v e l of b lood ca ta lase a c t i v i t y by v a r y i n g the vo lume of b lood a s sayed . T h i s i s a c c o m p l i s h e d by d e t e r m i n i n g the length of b lood c o l l e c t i n g tube occup ied by a known vo lume of f l u i d , then e x p r e s s i n g vo lume in t e r m s of length. F i n a l l y , the s c r e e n i n g a s say is s t anda rd i zed by means of a s e r i e s of b lood samples of known ca ta lase ac t iv i ty*

E x p e r i m e n t a l and R e s u l t s

P r e p a r a t i o n of Buf fe red Subs t ra te

A so lu t ion of s o d i u m perbora te was p r e p a r e d by adding 2Q5 g N a B 0 3

4 H z O to 100 ml water and s t i r r i n g wi th a magnet ic m i x e r whi le adding c o n - , cen t ra t ed HC1 to pH 6.8. At this p H , so lu t ion i s n e a r l y comple te , and the s l i gh t i n so lub l e r e s idue i s r e m o v e d by f i l t r a t i o n , Nine vo lumes of this N a B O s so lu t ion m i x e d w i th one vo lume o f M / l 5 phosphate buffer , pH 6.8, cons t i tu tes the subs t ra te .

* A s s o c i a t e d C o l l e ges of the Tvlidwest S e m e s t e r Student f r o m C a r l e t o n C o l l e g e ,

Other Solut ions

Other so lu t ions n e c e s s a r y a re :

a) K M n 0 4 , a p p r o x i m a t e l y 0.05 The exact concen t r a t ion is not c r i t i c a l , s ince the K M n 0 4 s e rves m e r e l y as an i n d i c a t o r o f the p resence o r absence o f excess N a B 0 3 .

t>) 4 N H 2 S 0 4 conta in ing 0,2% M n C l 2 .

c) O c t y l a l c o h o l in a d ropper bot t le .

P r o c e d u r e

The r e a c t i o n i s c a r r i e d out in 5 0 - m l E r l e n m e y e r f l a s k s , to each of w h i c h 1 ml of d i s t i l l e d water i s i n i t i a l l y added. B l o o d i s c o l l e c t e d by the o r b i t a l s inus t e c h n i q u e ^ ] in h e p a r i n i z e d m i c r o c o l l e c t i n g tubes,* to the des i r e d point w h i c h i s p r e v i o u s l y m a r k e d on the tube. The b lood i s t r a n s f e r r e d to the water by b lowing and r i n s i n g a few t i m e s . The b lood is p robab ly c o m p l e t e l y l aked w i t h i n 30 sec or so, but i t is convenient to set up 10 or 15 such f l a sks at a t i m e , and there w i l l be no poss ib l e ques t ion of i ncomple t e l a k i n g . In front of each f l a sk is p l aced a stop watch , and the mouse whose b lood i s be ing a s sayed i s kept in a s m a l l i n d i v i d u a l cage b e h ind the f lask .

As the stop watch is s ta r ted , 20 ml of buffered subs t ra te is added to the f lask . F o r this purpose , an au tomat ic d i spens ing pipet i s convenient . The m i x t u r e i s b r i e f l y s w i r l e d to m i x . A f t e r 10 m i n a t r o o m t e m p e r a t u r e , 5 ml of the H 2 S 0 4 - M n C l 2 so lu t ion is added by means of another d i spens ing pipet . T h i s hal ts the enzyme ac t i v i t y and p rov ides the a c i d m i l i e u n e c e s s a ry for the N a B 0 3 - K M n 0 4 r e a c t i o n . The M n C l 2 s e rve s a s ca t a ly s t for r educ t ion o f the K M n 0 4 .

A d rop of o c t y l a l c o h o l is then added to des t roy the a c c u m u l a t e d foam, and f i n a l l y 2 ml of K M n 0 4 i s added w i t h a t h i r d d i spens ing pipet . The f l a sk is m i x e d by s w i r l i n g , and the c o l o r is noted. A blue c o l o r p e r s i s t i n g for at l eas t 15 or 30 sec ind ica tes ca ta lase a c t i v i t y above the l e v e l being tested; d e c o l o r a t i o n ind ica tes a l o w e r ca ta lase ac t iv i ty*

R e l a t i o n of Leng th to V o l u m e of B l o o d C o l l e c t i n g Tube

S ince our p r i m e i n t e r e s t was m a x i m a l speed and e f f i c i ency w i t h i n a s a t i s f ac to ry range of a c c u r a c y , we w i s h e d to o m i t such steps as t r a n s f e r r i n g b lood f r o m c o l l e c t i n g tube to m e a s u r i n g m i c r o p i p e t . In a p r e l i m i n a r y

* M i c r o b lood c o l l e c t i n g E v a n s t o n , I l l i n o i s .

tubes, Ca ta log B 3095-2, S c i e n t i f i c P r o d u c t s ,

67

e x p e r i m e n t we, therefore , m e a s u r e d the length of c o l l e c t i n g tube occup ied by 100 u 1 of wate r . The mean for 9 different tubes, 3 f r o m each of 3 separate packets , was 50.2 m m , w i th a s tandard dev ia t ion of 1.03 m m . We have, there fore , used the value of 1 mm length as equivalent to 2 / i l v o l u m e . A s l igh t e r r o r is in t roduced by the c o n s t r i c t e d tip of the tube, and if one were tes t ing for an e x t r e m e l y h igh b lood ca ta lase l e v e l ( i . e . , v e r y s m a l l amounts of b lood used), this migh t be an apprec iab le fac tor . Howeve r , in a s c r e e n f o r low ca ta lase ( i . e., l a rge amount of b lood used), this m i n o r factor may be neglec ted .

S t anda rd i za t i on of Sc reen ing A s s a y

A s e r i e s of 15 n o r m a l m i c e was k i l l e d by cut t ing the throat , and the b lood was caught on watch g lasses on wh ich a l i t t l e hepa r in so lu t ion had been d r i e d down. The b lood was b r i e f l y m i x e d to d i s s o l v e the hepa r in , then t r a n s f e r r e d to 5 - m l beakers conta in ing m i c r o m a g n e t i c s t i r r e r s and c o v e r e d wi th s m a l l watch g l a s se s . A f t e r gentle s t i r r i n g , samples were nea r ly s i m u l t a n e ous ly r e m o v e d by two i n d i v i d u a l s , one of whom p e r f o r m e d a quanti tat ive ca ta lase assay,(1) and the other of whom p e r f o r m e d the s c reen ing assay des c r i b e d above. In the la t te r case , ca ta lase ac t i v i t y was tested at 30 mm and at 3 5 m m of b lood in the c o l l e c t i n g tubes. Depending on the r e s u l t s , the test was then repeated on a graded s e r i e s of b lood lengths spaced at 1 -mm i n t e r v a l s . The b lood length a t w h i c h an in te rmed ia te c o l o r was obtained was c o n s i d e r e d the "equivalence length; " i f no in te rmedia te c o l o r was seen, the equiva lence length was c o n s i d e r e d to be halfway between those of the adjacent pos i t ive and negative tubes. Tab le 13 l i s t s for each mouse the "equivalence length ," the ca ta lase a c t i v i t y , and the i r product . S ince this product is a c o n stant, the mean product can be used to de te rmine the length of b lood sample n e c e s s a r y to s c r e e n for ca ta lase at any d e s i r e d b lood l e v e l .

T A B L E 1 3

R e l a t i o n s h i p o f q u a n t i t a t i v e b l o o d c a t a l a s e a c t i v i t y t o s c r e e n i n g t e s t " e q u i v a l e n c e l e n g t h " *

M o u s e Q u a n t i t a t i v e a s s a y

( P U / m l ) * *

S c r e e n i n g a s s a y

( e q u i v a l e n c e l e n g t h i n m m ) P r o d u c t

1 1 0 1 3 7 . 0 3 7 4 0

2 1 0 8 3 6 . 0 3 8 9 0

3 1 0 9 3 6 . 0 3 9 2 0 4 1 1 1 3 7 . 5 4 1 6 0 5 1 1 1 3 6 . 5 4 0 5 0

6 1 1 2 3 5 . 5 3 9 8 0 7 1 1 7 3 4 . 0 3 9 8 0 8 1 1 9 3 3 . 0 3 9 3 0

9 1 2 5 3 4 . 5 4 3 1 0 1 0 1 2 7 3 6 . 5 4 6 4 0 1 1 1 2 9 3 1 . 5 4 0 6 0 1 2 1 3 3 3 0 . 5 4 0 6 0 1 3 1 3 7 2 8 . 5 3 9 0 0 1 4 1 4 2 3 2 . 0 4 5 4 0 1 5 1 4 6 2 7 . 5 4 0 2 0

A v e r a g e : 4 0 8 0 ± 2 5 0

* T h e " s c r e e n i n g a s s a y e q u i v a l e n c e l e n g t h " i s t h a t l e n g t h o f b l o o d c o l u m n w h i c h g i v e s a r e

s u l t j u s t i n t e r m e d i a t e b e t w e e n a " p o s i t i v e " a n d a " n e g a t i v e " t e s t .

* * P e r b o r a t e u n i t s p e r m l .

A c c u r a c y o f S c r e e n i n g A s s a y

The a c c u r a c y of the s c r een ing p rocedure was tested on 30 m i c e having b lood ca ta lase l eve l s ranging f r o m 93 to 147 perbora te units (PU) pe r m l . (These l eve l s were not made known to the e x p e r i m e n t e r un t i l the test was completed . ) The s c r een ing value was set at 35 mm of b lood (equivalent to 117 P U / m l ) . Twen ty -e igh t of the 30 m i c e were p r o p e r l y c l a s s i f i e d by the s c r e e n i n g test, One mouse , b lood ca ta lase 108 P U / m l , was e r r o n e o u s l y c a l l e d h igh -ca t a l a se ( i . e . , g rea te r than 117 P U / m l ) , and one, b lood ca ta lase 126 P U / m l , was e r r o n e o u s l y c a l l e d l o w - c a t a l a s e ( i . e . , l e s s than 117 P U / m l ) .

No explana t ion is ava i l ab l e for the la t te r of these e r r o r s , but the f o r m e r i s p robab ly due to the de layed d e c o l o r a t i o n of K M n 0 4 , w h i c h i s o c c a s i o n a l l y seen even in the p resence of excess r educ ing agent. T h i s o c c u r r e d in an e a r l y exper imen t , before M n C l 2 was rou t ine ly added as a ca t a ly s t o f K M n 0 4 r educ t ion .

D i s c u s s i o n

T h i s s c r e e n i n g technique is not intended to se rve as a subst i tute for a quant i ta t ive assay . It has, however , three p a r t i c u l a r advantages as a s c r e e n i n g test: a) it is nondes t ruc t ive , b) it is quite r a p i d , and c) it is r e a d i l y adjustable to separate a popula t ion into groups w i th b lood ca ta lase h igher than, and l o w e r than, any d e s i r e d l e v e l .

I t is n e c e s s a r y to p repa re the subst ra te w i th somewhat m o r e ca re than in the quant i ta t ive perbora te a s say for c a t a l a s e . 0 ) In the l a t t e r test , two f l asks conta in ing no ca ta lase source a re incubated s i m u l t a n e o u s l y w i t h each set of e x p e r i m e n t a l f l a s k s . These c o n t r o l f l asks then se rve as a m e a s ure of the pe rbora te p resen t before ca ta lase ac t ion . H o w e v e r , there a re no c o n t r o l f l a sks in the s c r e e n i n g method d e s c r i b e d , hence the need for m o r e c a r e f u l p r e p a r a t i o n of the subs t ra te .

The only so lu t ions for w h i c h the concen t r a t i on or vo lume i s c r i t i c a l a re the subs t ra te so lu t ion and the added b lood . H 2 S 0 4 and M n C l 2 a re both p resen t i n c o n s i d e r a b l e exces s , The K M n 0 4 s e rve s m e r e l y a s a n i n d i c a t o r , and i ts exact concen t r a t i on or vo lume becomes of i m p o r t a n c e only i f the amount of b lood added is n e a r l y exac t ly that n e c e s s a r y to decompose the N a B 0 3 p resen t . The d e - f o a m e r o c t y l a l c o h o l i s innocuous i f added after the r e a c t i o n has been hal ted by the H 2 S 0 4 . In one e x p e r i m e n t i t was added about a minute before the H 2 S 0 4 , and the r e su l t s s eemed to show an i n h i b i t ion of the ca t a l a t i c a c t i v i t y .

The c o l o r mus t be o b s e r v e d f a i r l y sho r t l y after add i t ion of the K M n 0 4 , s ince a l m o s t a l l b loods w i l l s l o w l y d e c o l o r i z e a so lu t i on o f K M n 0 4

by a nonenzymat ic r eac t i on .

69

In the i n t e r e s t s of t i m e , we have t r i e d to e l i m i n a t e the p r e l i m i n a r y l a k i n g o f the b l o o d in w a t e r . H o w e v e r , the buf fe red p e r b o r a t e w h i c h s e r v e s as s ubs t r a t e a p p e a r s to be a p p r o x i m a t e l y i s o t o n i c , and we have found that u n l a k e d e r y t h r o c y t e s e x h i b i t s l i g h t l y l e s s than ha l f the c a t a l a s e a c t i v i t y o f l a k e d c e l l s , In a d d i t i o n , the b e h a v i o r o f s u c h an u n l a k e d p r e p a r a t i o n a p p e a r s to be s o m e w h a t m o r e e r r a t i c than that o f l a k e d b l o o d . We t r i e d to i n c o r p o r a t e in to the bu f f e r ed subs t r a t e s such l y s i n g agents as s apon in , de t e rgen t s , and d e o x y c h o l a t e . Of these , on ly the d e o x y c h o l a t e l y s e d the c e l l s s u f f i c i e n t l y r a p i d l y , but so m u c h deoxycho l a t e was n e c e s s a r y that the s o l u t i o n s tended to g e l and b e c a m e v e r y d i f f i c u l t to handle ,

R e f e r e n c e s

1 . F e i n s t e i n , R . N. P e r b o r a t e as subs t r a t e in a new a s s a y of c a t a l a s e . J . B i o l . C h e m , 180, 1197-1202 (1949).

2 . R i l e y , V . A d a p t a t i o n o f o r b i t a l b l e e d i n g technique t o r a p i d s e r i a l b l o o d s t u d i e s . P r o c . Soc . E x p t l . B i o l . M e d . 104, 751-754 ( i 9 6 0 ) .

70

E F F E C T O F P U R O M Y C I N O N B L O O D A N D L I V E R C A T A L A S E

R o b e r t N . F e i n s t e i n , J u d i t h B . H o w a r d , and J o h n E . S e a h o l m

P u r o m y c i n ( a m i n o n u c l e o s i d e ) has been r e p o r t e d t o i n h i b i t p r o t e i n s y n t h e s i s . (1) S i n c e we have been a t t emp t ing for a l ong t i m e to d e v e l o p t echn iques fo r r e d u c i n g the c a t a l a s e a c t i v i t y o f b l o o d and t i s s u e s , and fo r m a i n t a i n i n g i t a t a l o w l e v e l fo r p r o l o n g e d p e r i o d s o f t ime , (2 ) i t a p p e a r e d w o r t h w h i l e to see whe the r a g e n e r a l i n h i b i t o r o f p r o t e i n s y n t h e s i s , s u c h a s p u r o m y c i n , w o u l d a l s o have l o n g - t e r m effects on the c a t a l a s e l e v e l .

E x p e r i m e n t a l

M i c e w e r e C F N o . 1 / A n l m a l e s , about 6 weeks o f age. P u r o m y c i n ( N u t r i t i o n a l B i o c h e m i c a l s C o r p o r a t i o n ) was i n j e c t e d i n t r a p e r i t o n e a l l y a t 100 m g / k g , t w i c e d a i l y . In the f i r s t e x p e r i m e n t , the p u r o m y c i n was i n j e c t e d on days 1, Z, 3 , and 4, and b l o o d and l i v e r c a t a l a s e a s s a y s w e r e c o n d u c t e d on days Z, 3 , and 5. In the s econd e x p e r i m e n t , p u r o m y c i n was i n j e c t e d t w i c e on day 1 on ly , and a n i m a l s w e r e s a c r i f i c e d and a s s a y e d on days Z, 3 , and 5 . V a l u e s for three m i c e w e r e a v e r a g e d fo r each poin t , and in e a c h ca se c o n t r o l a n i m a l s w e r e i n j e c t e d w i t h s a l i n e and a s s a y e d i n the s a m e m a n n e r a s the p u r o m y c i n - t r e a t e d m i c e .

C a t a l a s e a s s a y s w e r e p e r f o r m e d by the p e r b o r a t e m e t h o d , (3) the l i v e r b e i n g h o m o g e n i z e d and d i l u t e d w i t h 1 % T r i t o n X - 1 0 0 fo r m a x i m u m e x p r e s s i o n of c a t a l a s e a c t i v i t y . (4)

R e s u l t s

T a b l e 14 shows the effect of e ight i n j e c t i o n s of p u r o m y c i n on b l o o d and l i v e r c a t a l a s e a c t i v i t y . I t i s ev iden t that, a l t hough e r y t h r o c y t e c a t a l a s e

T A B L E 14

Effect of repeated inject ions of p u r o m y c i n on blood and l i v e r cata lase

L i v e r catalase B l o o d cata lase

Day Diff e r ence, P u r o m y c i n Dif ference , Sal ine P u r o m y c i n % Sal ine P u r o m y c i n

%

2 960 895 -7 165 140 - 15 3 991 635 -36 153 165 +8 5 1003 441 -56 162 184 + 14

M i c e were injected in t r ape r i tonea l ly with 100 mg p u r o m y c i n per kg , or wi th sa l ine , on days 1, 2, 3, and 4. They were s a c r i f i c e d and assayed in groups of three, except for the last p u r o m y c i n datum, where only one s u r v i v o r was ava i l ab le . Cata lase ac t iv i t i e s are expressed as perborate units per g r a m of l i v e r or per ml of whole blood.(3)

i s unaf fec ted , the l e v e l o f c a t a l a s e a c t i v i t y i n the l i v e r p r o g r e s s i v e l y de c r e a s e s , u n t i l by the f i f th day af ter beg inn ing p u r o m y c i n t r e a t m e n t , c a t a l a s e a c t i v i t y i s l e s s than h a l f that o f c o n t r o l a n i m a l s . B y th i s t i m e , a n i m a l s have l o s t c o n s i d e r a b l e we igh t , have a s c ru f fy a p p e a r a n c e , and a r e o b v i o u s l y i n p r e c a r i o u s p h y s i c a l c o n d i t i o n . A c t u a l l y , some deaths had o c c u r r e d i n the t r e a t e d a n i m a l s by the t i m e o f l a s t a s s a y .

A n o t h e r set o f m i c e was i n j e c t e d tw ice on ly , on day one, and g roups o f th ree w e r e s a c r i f i c e d a t v a r i o u s t i m e s t he rea f t e r and a s s a y e d for l i v e r c a t a l a s e . ( B l o o d c a t a l a s e a s s a y s w e r e not i n c l u d e d , s i n c e e v e n the m o r e d r a s t i c t r e a t m e n t o f the f i r s t e x p e r i m e n t had shown no effects on b l o o d c a t a l a s e . ) T h e r e s u l t s a r e shown in T a b l e 15, w h i c h i n d i c a t e s a m a x i m u m of on ly 22% d e c l i n e in l i v e r c a t a l a s e by day 5 , and r e t u r n to n o r m a l by day 8 . In th i s t r e a t m e n t no d r u g t o x i c i t y was ev iden t , and no a n i m a l s d i e d f r o m the t r e a t m e n t .

T A B L E 1 5

E f f e c t o f one -day i n j e c t i o n o f p u r o m y c i n o n l i v e r c a t a l a s e

L i v e r c a t a l a s e D a y D a y

S a l i n e P u r o m y c i n D i f f e r e n c e , %

2 854 910 +7 3 870 850 -2 5 910 710 -22 8 not done 940 +7*

' B a s e d on a v e r a g e o f o the r c o n t r o l s .

M i c e w e r e i n j e c t e d i n t r a p e r i t o n e a l l y tw ice o n day one on ly , w i t h p u r o m y c i n , 100 m g / k g , o r w i t h s a l i n e . T h e y w e r e s a c r i f i c e d and a s s a y e d i n g r o u p s o f three,, C a t a l a s e a c t i v i t i e s a r e e x p r e s s e d a s p e r b o r a t e un i t s p e r g r a m o f l i v e r . ( 3 )

D i s c u s s i o n

T h e s e e x p e r i m e n t s a r e o b v i o u s l y on ly p r e l i m i n a r y , d e s i g n e d t o l e a r n s o m e t h i n g o f the t o x i c i t y o f p u r o m y c i n , and i t s p o s s i b l e use in l o n g - r a n g e i n h i b i t i o n o f p r o t e i n s y n t h e s i s . T h u s f a r the e x p e r i m e n t s sugges t a c o n c l u s i o n that m i g h t have been p r e d i c t e d , n a m e l y that i f p r o t e i n s y n t h e s i s i s s i g n i f i c a n t l y a f fec ted fo r a p r o l o n g e d t i m e , the effect on the a n i m a l i s l i k e l y to be d r a s t i c . F u r t h e r , s i nce i t i s a s i m p l e m a t t e r to r e d u c e the c a t a l a s e a c t i v i t y o f the l i v e r but not o f the e r y t h r o c y t e , ( 5 ) ou r p r i m e i n t e r e s t l i e s i n effects on the e r y t h r o c y t e , and h e r e we f ind that r e l a t i v e l y s h o r t - t e r m

expe r imen t s (e.g. , 8 days) are inadequate to demonst ra te ca ta lase d i m i n u t ion in the r e d c e l l . A g a i n this was a p red ic t ab le f inding , s ince i n h i b i t i o n of p r o t e i n synthes is would not be expected to affect p r o t e i n of c i r c u l a t i n g e ry th rocy te s

F u t u r e expe r imen t s w i l l be o r i en ted toward an at tempt to inh ib i t ca ta lase synthes is in the bone m a r r o w long enough to p e r m i t the l a t e r output of a wave of ca t a l a se - f r ee e r y t h r o c y t e s .

Refe rence s

1 . Y a r m o l i n s k y , M . B M and G . L . d e l a Haba , Inhib i t ion b y p u r o m y c i n o f amino a c i d i n c o r p o r a t i o n into p ro te in , P r o c , N a t l . A c a d . S c i . U . S . 45, 1721-1729 (1959).

2 . F e i n s t e i n , R . N , , and J , L . Dainko , Use o f 3 - a m i n o - 1 ,2 ,4 - t r i azo le to reduce body ca ta lase ac t i v i t y in cance r s tudies . C a n c e r R e s e a r c h 1 9, 612-617 (1959).

3 . F e i n s t e i n , R. N, P e r b o r a t e as subst ra te in a new assay of ca ta lase . J 0 B i o l . C h e m . J_80, 1197-1202 (1949).

4 . F e i n s t e i n , R . N , S o l u b i l i z a t i o n o f pa r t i cu la t e ca ta lase wi th nonionic detergent. A r c h , B i o c h e m . B i o p h y s . 2 9 , , 399-400 (1959),

5 . F e i n s t e i n , R . N . , S . B e r l i n e r and F . O . G r e e n , M e c h a n i s m o f i n h i b i t ion o f ca ta lase by 3 - a m i n o - 1 , 2 , 4 - t r i a z o l e . A r c h . B i o c h e m . B i o p h y s . 76, 32-44 (1958),

L A C K O F E F F E C T O F E X O G E N O U S C A T A L A S E A D M I N I S T E R E D D U R I N G P R E G N A N C Y O N C A T A L A S E C O N T E N T O F P R O G E N Y

R o b e r t N„ F e i n s t e i n , John E 0 Seaho lm, and Jud i th B . H o w a r d

We have been in t e r e s t ed in techniques for obta ining a n i m a l s low in to ta l body c a t a l a s e . 0 ) A suggest ion was made* that perhaps the i n t r o d u c t ion of s ign i f i can t amounts of exogenous ca ta lase du r ing pregnancy migh t produce l i t t e r s of a n i m a l s of low body ca ta lase . We have now tested this sugges t ion and have found that the added ca ta lase is without effect on the l i v e r or b lood ca ta lase of the progeny.

M a t e r i a l s and Methods

M i c e were CF N o . 1 m a l e s and females , a p p r o x i m a t e l y 8 weeks of age* E a c h of the 5 m a l e s was housed wi th 6 f emales . In each such group, 3 f emales we re m a r k e d by ear c l i p to r e c e i v e ca ta lase , and 3 were m a r k e d to r e c e i v e s a l i ne . Count ing f r o m the day of housing the a n i m a l s together , f emales r e c e i v e d ca ta lase (or sal ine) on days 3, 5, 7, 10, 12, 14, and 17. F o r f i na l a s s a y s , matched l i t t e r s (one ca ta lase , one sal ine) were se lec ted w h i c h were c h a r a c t e r i z e d by (a) having the same s i r e , and (b) having as n e a r l y as pos s ib l e the same b i r t h date. M o r t a l i t y was heavy, and i t was f i n a l l y pos s ib l e to use only two matched p a i r s , t o t a l l i ng 10 c a t a l a s e - t r e a t e d m i c e and 11 c o n t r o l s .

Ca ta l a se used was Cape ra se ,** a s table , d ry b e e f - l i v e r ca ta lase w h i c h was r e a d i l y recons t i tu ted by the addi t ion of s a l ine . The amount of each i n t r a p e r i t o n e a l in j ec t ion was suff ic ient to add 35% to the to ta l body ca ta lase of the r ec ip i en t , as d e t e r m i n e d by whole body ca ta lase a s says of other m i c e of the same o r i g i n a l shipment .

B l o o d was d r a w n f r o m the o r b i t a l sinus,(2) and the a n i m a l was then k i l l e d by c e r v i c a l d i s l o c a t i o n . The l i v e r was r e m o v e d and f r o z e n in a d ry i ce -ace tone bath and was ma in ta ined on d ry i ce un t i l a s say , w h i c h was done in 1% T r i t o n X - 1 0 0 for m a x i m u m e x p r e s s i o n of ca ta lase a c t i v i t y 0 ( 3 ) A l l ca ta lase a s says were p e r f o r m e d by the perbora te method 0 (4)

R e s u l t s

R e s u l t s are p resen ted in Table 16, w h i c h shows no s ign i f i can t d i f f e r ence in b lood o r l i v e r ca ta lase a c t i v i t y between the m i c e whose d a m r e c e i v e d ca ta lase du r ing pregnancy and those whose dam r e c e i v e d sa l ine .

*We w i s h to thank D r 0 P . K . Lala for this i n t e r e s t i n g sugges t ion . **We w i s h to thank D r . P . P u i g M u s e t , L a b o r a t o r i o P E V Y A , B a r c e l o n a ,

Spa in , for generous gifts of C a p e r a s e .

T A B L E 1 6

B l o o d and l i v e r c a t a l a s e a c t i v i t y o f p rogeny o f f e m a l e m i c e r e c e i v i n g r epea t ed c a t a l a s e o r s a l i n e i n j e c t i o n s d u r i n g p r e g n a n c y

T r e a t m e n t of d a m s

S a l i n e C a t a l a s e % D i f f e r e n c e

N u m b e r o f m i c e a s s a y e d 11 10 _

A v e r a g e l i v e r c a t a l a s e , P U / g* 990 958 - 10 A v e r a g e b l o o d c a t a l a s e / P U / / m l * 186 176 -9

^ P e r b o r a t e uni ts p e r g r a m l i v e r o r per m l who le b l o o d .

D i s c u s s i o n and C o n c l u s i o n s

We have shown e a r l i e r ( 5 ) that i n t r a p e r i t o n e a l l y i n j e c t e d c a t a l a s e r a p i d l y a p p e a r s i n the b l o o d s t r e a m but not i n the l i v e r . We have no i n f o r m a t i o n as to whe the r or not the c a t a l a s e m o l e c u l e c a n pass the p l a c e n t a , o r whe the r o r not a h i g h c a t a l a s e l e v e l i n the fe t a l c i r c u l a t i o n w i l l d i m i n i s h the s y n t h e s i s o f new c a t a l a s e . The p r e s e n t data i n d i c a t e on ly that r e p e a t e d i n j e c t i o n s o f exogenous, c a t a l a s e , p r e s u m a b l y g i v i n g r i s e t o r e p e a t e d h i g h m a t e r n a l b l o o d l e v e l s , i s wi thou t u l t i m a t e effect o n the c a t a l a s e l e v e l i n b l o o d o r l i v e r o f p r o g e n y .

R e f e r e n c e s

1 . F e i n s t e i n , R , N . , and J . D a i n k o . U s e o f 3 - a m i n o - 1 , 2 , 4 - t r i a z o l e t o r e duce body c a t a l a s e a c t i v i t y in c a n c e r studies, . C a n c e r R e s e a r c h 19> 612-617 (1959).

2 . R i l e y , V . A d a p t a t i o n o f o r b i t a l b l e e d i n g technique t o r a p i d s e r i a l b lood s t u d i e s . P r o c . Soc . E x p t l . B i o l . M e d . 104, 751-754 ( i 9 6 0 ) .

3 . F e i n s t e i n , R . N . S o l u b i l i z a t i o n o f p a r t i c u l a t e c a t a l a s e w i t h n o n i o n i c dete rgen t . A r c h , B i o c h e m . B i o p h y s , 79, 399-400 (1959) =

4 . F e i n s t e i n , R . N . P e r b o r a t e a s subs t r a t e i n a new a s s a y o f c a t a l a s e . J , B i o l , C h e m . JJ30, 1197-1202 (1949).

5 . F e i n s t e i n , R . N . A t t e m p t s a t p r o t e c t i o n o f m i c e aga ins t i o n i z i n g r a d i a t i o n by exogenous c a t a l a s e . A t o m p r a x i s _6_, 205-207 ( I960) ,

S E A R C H F O R A N A C A T A L A S E M I C M O U S E

R o b e r t N . F e i n s t e i n , J o h n E . S e a h o l m , and J u d i t h B 0 H o w a r d

I f a c a t a l a s e - f r e e s t r a i n o f a n i m a l w e r e a v a i l a b l e , i t w o u l d p r o v i d e a m u c h needed r e s e a r c h t o o l w i t h w h i c h t o tes t c e r t a i n t h e o r i e s o f r a d i a t i o n effects and o n c o l o g y . T h e h u m a n s tud ies o f T a k a h a r a ( l ) and. l a t e r o f A e b i ( 2 ) d e m o n s t r a t e d c l e a r l y that i t i s p o s s i b l e for a n a c a t a l a s e m i c m a m m a l i a n o r g a n i s m to be p e r f e c t l y v i a b l e , and so a p r o j e c t was c o n c e i v e d and begun h e r e to s e a r c h for a m o u s e g e n e t i c a l l y a b e r r a n t w i t h r e s p e c t to c a t a l a s e a c t i v i t y .

A l t h o u g h A e b i(2) s e r e ened h i s h u m a n c a s e s w i t h a s i m p l e a l l - o r -none t echn ique , i t was d e c i d e d h e r e to d e t e r m i n e the n o r m a l l e v e l and r ange o f b l o o d c a t a l a s e , and then s c r e e n m i c e w i t h a s e m i q u a n t i t a t i v e c a t a l a s e a s say w h i c h w o u l d d i s t i n g u i s h n o r m a l f r o m s u b n o r m a l l e v e l s . S u c h a n a s s a y i s d e s c r i b e d e l s e w h e r e i n th is r epor t . (3 ) T h i s d e v i c e , though e n t a i l i n g c o n s i d e r a b l y m o r e e f for t than A e b f s a l l - o r - n o n e s c r e e n i n g , has the b a s i c a d vantage that i t w i l l de tec t h e t e r o z y g o t e s , w h e r e a s the o ther t echn ique w i l l s c r e e n out on ly h o m o z y g o t e s , a m u c h m o r e r a r e c o n d i t i o n . In a d d i t i o n , i t was d e c i d e d to use not a n o r m a l m o u s e popu l a t i on , but a p o p u l a t i o n c o n s i s t i n g o f p r o g e n y o f i r r a d i a t e d s i r e s . T h i s shou ld have the advantage o f v e r y c o n s i d e r a b l y e n h a n c i n g the p o s s i b i l i t y of f i n d i n g a h e t e r o z y g o t e , a l t hough one w o u l d not expec t a h o m o z y g o t e to be p r o d u c e d as a r e s u l t of p a r e n t a l i r r a d i a t i o n .

A l t h o u g h we had f i r s t p l anned to do the i r r a d i a t i o n s h e r e o u r s e l v e s , i t was pointed, out t o u s that D r . W i l l i a m L . R u s s e l l , O a k R i d g e N a t i o n a l L a b o r a t o r y , was engaged i n a m a s s i v e p r o g r a m o f i r r a d i a t i n g s i r e s and e x a m i n i n g p r o g e n y fo r c e r t a i n s p e c i f i c v i s i b l e m u t a t i o n s . A f t e r s u c h e x a m i n a t i o n , these a n i m a l s a r e o r d i n a r i l y d i s c a r d e d . D r . R u s s e l l v e r y k i n d l y c o n s e n t e d to save these a n i m a l s and send t h e m on to us fo r b i o c h e m i c a l t e s t i n g for l e v e l o f b l o o d c a t a l a s e . The s i r e s o f the m i c e t e s t ed r e c e i v e d a t o t a l of 600 r of X - r a y s to the l o w e r t h i r d of the body, in a d i v i d e d dose of 500 r , then 100 r , or 100 r , then 500 r , s p a c e d at 24 h o u r i n t e r v a l s .

One h u n d r e d and ten n o r m a l m i c e , e x a c t l y c o m p a r a b l e t o the m i c e to be s c r e e n e d e x c e p t that the s i r e s had not been i r r a d i a t e d , w e r e a s s a y e d quan t i t a t ive ly(4) fo r b l o o d c a t a l a s e 9 The m e a n and s t a n d a r d d e v i a t i o n f r o m the m e a n w e r e 149 ± 1 4 p e r b o r a t e un i t s ( P U ) pe r m l whole b lood . The range of 113-185 P u / m l w i l l e n c o m p a s s 99% of the n o r m a l a n i m a l s . The s c r e e n i n g tes t l e v e l fo r s u b n o r m a l i t y was t h e r e f o r e set a t a p p r o x i m a t e l y 1 10 P u / m l .

As of the end of 1963, a p p r o x i m a t e l y 5600 m i c e have been examined . F r o m these, the s c r e e n i n g test separa ted 19 m i c e as poss ib l e low ca ta lase a n i m a l s . A l l 19 were quant i ta t ive ly assayed , and five were found to have b lood ca ta lase of above 1Z0 and so were d i s c a r d e d . The r e m a i n i n g 14 may be grouped as f o l l o w s :

111-120 P U / m l 3 101-110 6 91-100 4 be low 91 1.

The mouse l i s t e d as "be low 91 M is a female whose ac tua l b lood ca ta lase is 40 P U / m l , She is undoubtedly a he te rozygote . C o n s i d e r a b l e genet ic data w i l l have to be accumula t ed before i t can be d e t e r m i n e d whether the other an ima l s l i s t e d above a re ac tua l mutants or m e r e l y "'low n o r m a l s / 5

The female of 40 P U / m l was pregnant when r e c e i v e d , She has s ince p roduced a l i t t e r of four m a l e s , the b lood ca ta lase a c t i v i t y of w h i c h is 75, 141, 150, and 123 P u / m l . It is hoped that the b a c k c r o s s of the mother by her low ca ta lase son w i l l produce a homozygote .

We w i s h to emphas ize one p a r t i c u l a r point, Not only do we fee l f a i r l y confident that we w i l l eventual ly obtain a s t r a i n of a c a t a l a s e m i c m i c e , but we fee l that we have demons t ra t ed the f e a s i b i l i t y of obta in ing, by c o m parab le techniques , other mutants sui table for tools in the study of v a r i o u s i n b o r n e r r o r s of m e t a b o l i s m . The r equ i r emen t s are (a) a supply of progeny of i r r a d i a t e d parents , and (b) a r a p i d assay capable m e r e l y of d i s t i n g u i s h i n g between n o r m a l and a b n o r m a l l eve l s of some b lood const i tuent . T h i s need not be an enzyme . In fact, such d i s o r d e r s as pheny lke tonur ia d i s p l a y the i r enzyme defects in the l i v e r . In the p a r t i c u l a r case of pheny lke tonur ia , however , i t seems probable f r o m the w o r k of Hsia(5) that i f the m i c e we re s i m p l y kept on diets h igh in phenyla lan ine , the he terozygote would show a h igh b lood phenyla lan ine , Other d i s o r d e r s , e .g. , g a l a c t o s e m i a ^ ) ev ident ly r ep re sen t e ry th rocy t e defects , and a s t r a i g h t f o r w a r d whole b lood s c r e e n for the enzyme should se rve to detect the he te rozygote .

S u m m a r y

By means of a r a p i d , semiquant i ta t ive assay , a p p r o x i m a t e l y 5600 m i c e have thus far been examined for l ow b lood ca ta lase . Of these, 14 have been set as ide as s u b n o r m a l in b lood ca ta lase a c t i v i t y . Of the 14;, one is a l m o s t su r e ly a he te rozygote , and some of the others may be. The f e a s i b i l i t y of this approach in obta ining spec i f i c mutants for the study of i n b o r n e r r o r s o f m e t a b o l i s m i s discussed, ,

We w i s h to acknowledge, w i th grat i tude, helpful d i s c u s s i o n s w i t h D r s . H e r m a n S la t i s (now a t M i c h i g a n State U n i v e r s i t y ) and M a x R . Z e l l e , A r g o n n e N a t i o n a l L a b o r a t o r y ; D r s . John B . S t o r e r and Thomas R o d e r i c k o f J a c k s o n M e m o r i a l L a b o r a t o r y ; and D r . W i l l i a m L . R u s s e l l , Oak R idge N a t i o n a l L a b o r a t o r y . To D r . R u s s e l l goes a l so our s i n c e r e thanks for h i s cont inuing c o r d i a l coopera t ion in p r o v i d i n g us w i th the m i c e used in this study.

Re fe r ence s

1. T a k a h a r a , S. P r o g r e s s i v e o r a l gangrene probably due to l a c k of c a t a lase in the b lood (aca ta lasemia ) . L a n c e t 263, 1 101-1104 (1952).

2 . A e b i , H . , J , P . H e i n i g e r , R . B u t l e r , and A , H a s s i g . Two cases o f aca t a -l a s i a in S w i t z e r l a n d . E x p e r i e n t i a J/7, 466 (1961).

3 . F e i n s t e i n , R . N . , J . B , H o w a r d , L . B . Bal lonof f , and J . E . Seaho lm. A r a p i d b lood ca ta lase s c r e e n i n g technique adjustable to any l e v e l of a c t i v i t y . T h i s r epor t , p . 65.

4 . F e i n s t e i n , R. N. P e r b o r a t e as subst ra te in a new assay of ca ta lase . J . B i o l . C h e m . 180, 119 7-1202 (1949).

5 . H s i a , D . Y . Y . , K . W . D r i s c o l , W . T r o l l , and W . E , K n o x . De tec t ion b y phenyla lan ine to le rance tests of he te rozygous c a r r i e r s of pheny lke tonur i a . Na tu re 178, 12 39-1240 (1956).

6. R o b i n s o n , A. The assay of ga lac tok inase and ga lac tose - 1 -phosphate u r i d y l t r ans fe rase a c t i v i t y in human e r y t h r o c y t e s . A p r e s u m e d test for he te rozygous c a r r i e r s o f the g a l a c t o s e m i c defect. J . E x p t l , M e d , 118, 359-370 (1963),

A T T E M P T S A T R A D I A T I O N P R O T E C T I O N B Y C O M P O U N D S R E L A T E D T O 3 - A M I N O - 1 , 2 , 4 - T R I A Z O L E

R o b e r t N . F e i n s t e i n and John E . S e a h o l m

We r e p o r t e d e a r l i e r that 3 - a m i n o - 1 ,2 ,4 - t r i azo le (AT) had a s m a l l but s ign i f i can t p ro tec t ive effect when a d m i n i s t e r e d to m i c e before whole body X - i r r a d i a t i o n . ( 1 ) We have r ecen t ly been so fortunate as to have p l aced a t our d i s p o s a l a wide v a r i e t y of c h e m i c a l agents r e l a t ed to A T . * S ince many of these are unusual compounds, not ava i l ab l e e l s e w h e r e , i t s eemed d e s i r a b l e to make m a x i m u m use of them, and one facet of i n t e r e s t was the i r pos s ib l e r ad iop ro t ec t i ve a b i l i t y . Some of these agents a re c h a r a c t e r i z e d by the c h e m i c a l groupings gene ra l l y a s s o c i a t e d w i th many r a d i o p ro tec t ive agents, namely , a free m e r c a p t o group in p r o x i m i t y to an a m i n o group.

Of the many compounds ava i l ab l e (for comple te l i s t , see R e f e r e n c e 2), we se lec ted s e v e r a l agents m o r e or l e s s r ep re sen ta t i ve of spec i f i c c h e m i c a l g roups . To a c e r t a i n extent, a l so , our cho ice was d ic ta ted by the amount of compound s t i l l ava i l ab l e after our p r i m a r y tes t ing had been completed . (^)

E x p e r i m e n t a l

M i c e used were CF N o . 1 f ema les , about three months of age. X - i r r a d i a t i o n d e l i v e r e d was 700 r to the whole body, at a rate of 36-37 r per m i n . X - r a y fac tors we re : 250 K V , 15 m a , 0.5 mm copper and 3 mm B a k e -l i t e f i l t r a t i o n . The ha l f -va lue l a y e r was 1.72 mm of copper .

D r u g s were a d m i n i s t e r e d i n t r a p e r i t o n e a l l y w i t h i n ten minu tes before the s ta r t of i r r a d i a t i o n . Dosages are l i s t e d w i th the i n d i v i d u a l e x p e r i m e n t s . In some cases , because of l i m i t e d s o l u b i l i t y , i t was n e c e s s a r y to in jec t the agent as a suspens ion in 5% gum a r a b i c .

R e s u l t s

R e s u l t s are shown i n Tab le 17. It w i l l be seen that of the seven agents tes ted, s i x p e r m i t t e d g rea t e r s u r v i v a l , and s l i gh t ly g r ea t e r m e a n s u r v i v a l t ime , than the i r r e s p e c t i v e c o n t r o l s . The excep t ion , 2 - a m i n o t h i a z o l e appeared to be d e t r i m e n t a l in that the mean s u r v i v a l t ime was v e r y g r ea t l y shor tened. In this l a t te r group, f i r s t deaths d id not o c c u r e a r l i e r than in other groups ; r a the r , a l l a n i m a l s succumbed w i t h i n an unusua l ly s h a r p l y c i r c u m s c r i b e d p e r i o d .

*We w i s h to exp re s s our w a r m e s t thanks to D r . C a m e r o n A i n s w o r t h , o f E l i L i l l y and C o . R e s e a r c h L a b o r a t o r i e s , for h is c o r d i a l c o o p e r a t i o n in supp ly ing us wi th these compounds .

79

T A B L E 17

R a d i o p r o t e c t i v e a b i l i t y o f c e r t a i n c o m p o u n d s r e l a t e d t o a m i n o t r i a z o l e

E x p t . C o m p o u n d t e s t e d D o s e ,

m g / k g

N o .

m i c e

N o .

s u r v i v i n g

% s u r v i v a l

M e a n s u r v i v a l

t i m e , d a y s

1 S a l i n e 1 6 0 0 1 2 . 7

4 - a m i n o - 3 - m e r c a p t o -

1 , 2 , 4 - t r i a z o l e 5 0 0 1 6 3 1 9 1 4 . 0

2 - a m i n o t h i a z o l e 2 5 0 * 1 6 0 0 7 . 4

5 - m e r c a p t o - 1 , 2 , 3 , 4 - t e t r a z o l e 1 0 0 0 * 1 6 5 3 1 1 2 . 8

2 S a l i n e 1 6 4 2 5 1 3 . 3

1 - m e t h y l - 5 - m e r c a p t o -

1 , 2 , 4 - t r i a z o l e 1 5 0 1 6 9 5 6 1 3 . 4

2 - m e t h y l - 1 , 3 , 4 , 4 H - o x a d i a z o l i n e -

5 - t h i o n e 6 0 0 1 0 4 4 0 1 4 . 7

3 S a l i n e 1 6 1 6 1 2 . 3

1 - i s o p r o p y l - 5 - m e r c a p t o -

1 , 2 , 3 , 4 - t e t r a z o l e 1 0 0 0 * 9 4 4 4 1 5 . 0

1 - t e r t - b u t y l - 5 - m e r c a p t o -

1 , 2 , 3 , 4 - t e t r a z o l e 5 0 0 * 1 1 2 1 8 1 4 . 1

* I n d i c a t e s s u s p e n s i o n i n 5 % g u m a r a b i c .

D i s c u s s i o n

T h e s e compounds w e r e o r i g i n a l l y tes ted a s i n h i b i t o r s o f c a t a l a s e i n v i v o and i n v i t r o .

(2) S ince i t s c a t a l a se i n h i b i t o r y power was the o r i g i n a l

r e a s o n for t e s t ing the r a d i o p r o t e c t i v e a b i l i t y o f A T , i t i s o f i n t e r e s t to c o m p a r e the c a t a l a s e i n h i b i t o r y p r o p e r t y of these agents(2) w i t h the p r e s ent tes ts of r a d i o p r o t e c t i o n . The r e s u l t s i nd i ca t e c l e a r l y that there i s no d i r e c t c o r r e l a t i o n . T h u s , for e x a m p l e , 5 - m e r c a p t o - 1,2, 3 , 4 - t e t r a z o l e i s the m o s t ef fec t ive c a t a l a s e i n h i b i t o r yet deve loped , and the 1 -me thy l , 1 - i s o p r o p y l , and 1 - t e r t - b u t y l ana logs t he reo f a re to t a l ly d e v o i d o f c a t a l a s e i n h i b i t o r y a b i l i t y , yet there i s l i t t l e d i s t i n c t i o n be tween them, so far a s r a d i o p r o t e c t i o n i s c o n c e r n e d . A l t h o u g h the r a d i o p r o t e c t i v e a b i l i t y o f these agents m a y s t i l l be in some way r e l a t e d to the e n z y m e c a t a l a s e , the r e l a t i o n s h i p i s not i m m e d i a t e l y apparen t .

W o r k on the r a d i o p r o t e c t i v e a b i l i t y o f these agents has been d i s con t inued for s e v e r a l r e a s o n s : (a) u n a v a i l a b i l i t y of fu r the r supp l i e s of s o m e of the agents ; (b) l ow l e v e l of r a d i o p r o t e c t i o n ob ta ined ; and (c) our u n d e r s t a n d i n g that some of these compounds have a l s o been s u b m i t t e d to the m a s t e r s c r e e n i n g p r o g r a m for r a d i a t i o n p r o t e c t o r s a t W a l t e r R e e d A r m y H o s p i t a l .

Refe rences

F e i n s t e i n , R . N . , and S . B e r l i n e r . P r o t e c t i o n agains t X - i r r a d i a t i o n 3 - a m i n o - 1 ,2 ,4 - t r i azo le . Sc ience 125, 936 (1957).

F e i n s t e i n , R . N . , J . E . Seaho lm, and L . B . Ba l lonof f . Ef fec t o f c o m pounds r e l a t ed to a m i n o t r i a z o l e on ca ta lase in v i vo and in v i t r o , E n z y m o l o g i a 17, 30-40 (1964).

O C C A S I O N A L R E S I S T A N C E O F M I C E T O G L U C O S E O X I D A S E I N J E C T I O N

R o b e r t N . F e i n s t e i n and J o h n E . S e a h o l m

In a r e c e n t p u b l i c a t i o n , ( 1 ) we r e p o r t e d the d r a s t i c effects of i n t r a p e r i t o n e a l i n j e c t i o n in to m i c e o f the e n z y m e g l u c o s e o x i d a s e , a m o l d -d e r i v e d p r o d u c t c a t a l y z i n g the r e a c t i o n : g l u c o s e + o x y g e n h y d r o g e n p e r o x i d e + g l u c o n i c a c i d . T h e s e e f fec t s , w h i c h depend q u a l i t a t i v e l y and q u a n t i t a t i v e l y upon the dose a d m i n i s t e r e d , i n c l u d e e r y t h r o c y t e h e m o l y s i s , m e t h e m o g l o b i n f o r m a t i o n , s t r i k i n g changes i n h e m a t o c r i t , and dea th .

D u r i n g the c o u r s e o f that w o r k i t was o b s e r v e d that o c c a s i o n a l a n i m a l s - p e r h a p s f ive pe r cent , a l t hough exac t r e c o r d s w e r e not kep t -a p p e a r e d to be t o t a l l y unaf fec ted by the e n z y m e . I n j e c t i o n of as m u c h as 100 l e t h a l dose s c a u s e d no b l o o d changes o r o ther s igns o f t o x i c i t y in these m i c e .

We d e c i d e d to i n v e s t i g a t e th i s u n u s u a l phenomenon , and we c o n s i d e r e d two p o s s i b l e e x p l a n a t i o n s a s m o s t p r o b a b l e : (a) s o m e s o r t o f i m mune r e a c t i o n , * o r ( b ) a gene t i c phenomenon . In b r i e f s u m m a r y , n e i t h e r o f these p o s s i b l e m e c h a n i s m s has p r o v i d e d a s a t i s f a c t o r y e x p l a n a t i o n .

E x p e r i m e n t a l and R e s u l t s

I m m u n o l o g i c a l T e s t i n g

T h r e e s o r t s o f i m m u n o l o g i c a l t e s t i n g w e r e p e r f o r m e d . I n the f i r s t , we a t t e m p t e d to f i n d a n t i b o d i e s to g l u c o s e o x i d a s e in the p l a s m a o f m i c e k n o w n to have r e c e i v e d , and s u r v i v e d , l e t h a l doses o f the e n z y m e , and in the p l a s m a o f n o r m a l m i c e . B l o o d was w i t h d r a w n f r o m a l l m i c e b y the o r b i t a l s i n u s t echnique , (2) and the p l a s m a was s e p a r a t e d by c e n t r i f u g a t i o n . T h e p l a s m a was p l a c e d i n s m a l l tubes , and s o l u t i o n s o f g l u c o s e o x i d a s e o f v a r y i n g c o n c e n t r a t i o n ( l . 0 - 0 , 0 1 m g / m l ) w e r e c a r e f u l l y l a y e r e d o v e r the p l a s m a . T h e i n t e r f a c e was e x a m i n e d a t i n t e r v a l s fo r s igns o f an a n t i g e n -an t ibody p r e c i p i t a t e . The r e s u l t s o f th is e x p e r i m e n t w e r e n e g a t i v e . A l though s l i g h t t u r b i d i t i e s w e r e o c c a s i o n a l l y seen a t the i n t e r f a c e , these w e r e no ted i n bo th " r e s i s t a n t " and " n o r m a l " p l a s m a s , and a t n o t i m e was a de f in i t e p r e c i p i t a t e o b s e r v e d .

T h e s e c o n d i m m u n o l o g i c a l e x p e r i m e n t was a n a t t empt t o i m m u n i z e n o r m a l m i c e a g a i n s t g l u c o s e o x i d a s e , to see w h e t h e r t h e i r r e s p o n s e to a l a t e r c h a l l e n g i n g dose o f g l u c o s e o x i d a s e r e s e m b l e d the r e s p o n s e o f the

*We w i s h t o thank D r 0 B e r n a r d J a r o s l o w , A r g o n n e N a t i o n a l L a b o r a t o r y , fo r h i s h e l p i n the i m m u n o l o g i c a l i n v e s t i g a t i o n s ,

o c c a s i o n a l t r u l y r e s i s t a n t m o u s e . F o r th i s p u r p o s e , 1 0 m i c e ( C F N o . 1 f e m a l e s , 10 weeks of age) w e r e i n j e c t e d subcu t aneous ly 6 t i m e s in the c o u r s e of 12 d a y s , e a c h t i m e w i t h a p p r o x i m a t e l y -|- of a l e t h a l dose of g l u c o s e o x i d a s e m i x e d w i t h a n e q u a l v o l u m e o f F r e u n d ' s adjuvant . N i n e t e e n days af te r the l a s t i m m u n i z i n g dose , these 10 m i c e , p lus 10 s i m i l a r but u n t r e a t e d m i c e , w e r e c h a l l e n g e d w i t h a n i n t r a p e r i t o n e a l dose o f a p p r o x i m a t e l y 4 l e t h a l doses o f g l u c o s e o x i d a s e . A l l 10 of the u n t r e a t e d m i c e w e r e dead w i t h i n 6 h r ; w h e r e a s on ly 3 of the t r e a t e d m i c e w e r e ; the o the r 6 t r e a t e d m i c e , h o w e v e r , a p p e a r e d to be qui te i l l , and, in fact , 3 m o r e d i e d w i t h i n the next 20 hr„ I t thus a p p e a r s that a p a r t i a l i m m u n i z a t i o n was a c h i e v e d , but i t i s c o n s i d e r e d that t h i s b e a r s l i t t l e r e s e m b l a n c e to the • • res i s tance phenomenon b e i n g i n v e s t i g a t e d , fo r r e a s o n s d i s c u s s e d l a t e r .

T h e t h i r d tes t o f the g l u c o s e o x i d a s e r e s i s t a n c e a s an i m m u n e p h e n o m e n o n was a n a t t empt t o t r a n s f e r ' ' p a s s i v e r e s i s t a n c e " f r o m i n h e r e n t l y r e s i s t a n t ( 4 m i c e ) , p a r t i a l l y i m m u n i z e d ( 4 m i c e , a s above) , o r n o r m a l m i c e (8 m i c e ) t o o ther p r e s u m e d n o r m a l m i c e . T h i s was done by m i x i n g 200 m i c r o l i t e r s of the p l a s m a in q u e s t i o n w i t h 50 [A 1 of g l u c o s e o x i d a s e s o l u t i o n , and i n j e c t i n g the e n t i r e 2 50 fll in to the t a i l v e i n of a n o r m a l r e c i p i e n t . T h e dose o f g l u c o s e o x i d a s e was s u c h that e a c h a n i m a l r e c e i v e d s l i g h t l y m o r e than one l e t h a l dose . As a r e s u l t o f these i n j e c t i o n s , on ly one dea th o c c u r r e d , and that was i n the g r o u p r e c e i v i n g g l u c o s e o x i d a s e p lus p l a sma , f r o m a 5 1 p a r t i a l l y i m m u n i z e d 7 ' m o u s e ,

G e n e t i c T e s t i n g

B e c a u s e o f the i n c o n c l u s i v e , but g e n e r a l l y nega t i ve , r e s u l t s o f the i m m u n o l o g i c a l t e s t i n g , and because of the p o s s i b i l i t y that a gene t i c s u b s t r a i n o f CF N o , 1 m i c e i s r e s i s t a n t t o g l u c o s e o x i d a s e , we i n v e s t i g a t e d the q u e s t i o n f r o m th is po in t o f v i e w a l s o , The f o l l o w i n g m i c e w e r e c h a l l enged w i t h 5 l e t h a l doses of g l u c o s e o x i d a s e :

(a) T e n r e p r e s e n t a t i v e s of e a c h of 11 g e n e t i c a l l y i n b r e d s t r a i n s ;

(b) T e n n o r m a l , p r e v i o u s l y u n t r e a t e d C F N o , 1 m i c e ; and

(c) E i g h t e e n CF N o . 1 m i c e , the o f f s p r i n g o f p a r e n t s bo th o f w h o m had p r o v e n r e s i s t a n t to i n j e c t i o n s of a t l e a s t 20 l e t h a l d o s e s ,

F r o m r e s u l t s , g i v e n i n T a b l e 18, two g e n e r a l c o n c l u s i o n s c a n b e d r a w n :

(a) None of the i n b r e d s t r a i n s i s p a r t i c u l a r l y r e s i s t a n t to g l u c o s e o x i d a s e ; and

(b) The p r o g e n y of two r e s i s t a n t m i c e a r e not t h e m s e l v e s r e s i s t a n t

T A B L E 1 8

G l u c o s e oxidase chal lenge of va r i ous mouse s t r a ins and groups

Sex A g e , weeks No . cha l l enged No, s u r v i v i n g

" R e s i s t a n t " * M 7-10 10 0 " R e s i s t a n t " * F 7-10 8 1 C F N o . 1 M 15-17 10 0 M A / j / A n l M 7-10 4 1 M A / j / A n l F 7 6 0 C 5 7 B r F c a . 35 10 0 R F F ca , 35 10 0 S W R F ca , 35 10 0 C 5 7 B 1 / 6 F ca . 20 10 2 L P / j F ca . 20 10 0 S J L F c a . 20 10 1 P L / J F c a . 16 10 1 129 / J F c a . 20 10 0 A / d F c a . 20-24 10 0 B A L B F c a . 20 10 1 C B A F c a . 20 10 0

" R e s i s t a n t " m i c e a re f i r s t genera t ion progeny of CF No . 1 paren ts , both of w h o m had s u r v i v e d chal lenge wi th about 20 l e tha l doses of g l u cose ox idase . Cha l l enge in the present e x p e r i m e n t was w i th 5 l e t h a l doses .

D i s c u s s ion

It appears that no explana t ion has yet been found for the phenomenon of e r r a t i c r e s i s t a n c e to g lucose ox idase . The genetic explana t ion seems r u l e d out a l m o s t e n t i r e l y , s ince the progeny of two r e s i s t an t m i c e a re not t hemse lves r e s i s t an t . A s l igh t r e m a i n i n g p o s s i b i l i t y is that the parents showed r e s i s t a n c e because of genetic v a r i a n c e at d i f ferent l o c i , hence the progeny w i l l s t i l l not be homozygous for this t r a i t . We intend to test this p o s s i b i l i t y when, and if, the p rope r m i c e become a v a i l a b l e , namely , a parent and o f f sp r ing of the opposite sex, to p e r m i t b a c k c r o s s i n g . At p r e s ent we do not have enough s u r v i v o r s for this test.

At the same t i m e , the i m m u n o l o g i c a l explanat ion a l so seems i n adequate, for the fo l lowing reasons , based l a r g e l y on the second of the i m m u n o l o g i c a l expe r imen t s d e s c r i b e d above:

(a) Unt rea ted m i c e give an a l l - o r - n o n e response to g lucose o x i dase; a n i m a l s r e s i s t an t to a g iven dose of g lucose oxidase gene ra l l y show

few or no toxic s y m p t o m s . The " i m m u n i z e d " m i c e in the presen t e x p e r i ments were a l l in e x t r e m e l y poor p h y s i c a l condi t ion , even though some eventua l ly s u r v i v e d .

(b) The s u r v i v a l cu rve for n o r m a l m i c e g iven g lucose oxidase i s u s u a l l y sharp and c l o s e l y demarcated. ( -0 The " i m m u n i z e d " m i c e suc cumbed over a range of 12 or m o r e h r .

(c) S ince the 6 - in jec t ion s e r i e s p roduced only p a r t i a l i m m u n i z a t ion , i t seems e x t r e m e l y u n l i k e l y that a s ingle mouse , in a cageful , wou ld o c c a s i o n a l l y develop fu l l - f l edged i m m u n i t y .

(d) The negative r e su l t s of the f i r s t and t h i r d of the i m m u n o l o g i c a l expe r imen t s fur ther b o l s t e r the c o n c l u s i o n that r e s i s t a n c e to g lucose oxidase i s not an i m m u n o l o g i c a l phenomenon.

S u m m a r y

The o c c a s i o n a l s t r i k i n g r e s i s t ance to in jec t ion of g lucose oxidase seen in apparent ly n o r m a l m i c e cannot be exp la ined e i ther as an i m m u n o l o c a l or as a genetic phenomenon.

Re fe rences

1 . F e i n s t e i n , R , N . , M . Cou lon , and J . E . Seaho lm. Ef fec t s o f g lucose oxidase in m i c e . B i o c h e m . P h a r m a c o l . I3j 105-113 (1964).

2. R i l e y , V„ Adap ta t ion of o r b i t a l b leeding technique to r a p i d s e r i a l b lood s tudies . P r o c , Soc. E x p t l . B i o l . M e d . 104, 751-754 (1960).

M U T A T I O N I N D U C E D W I T H C A F F E I N E , U L T R A V I O L E T L I G H T , O R 2 - A M I N O P U R I N E

H e r b e r t E , K u b i t s c h e k and H a r o l d E , B e n d i g k e i t

T h e d e p e n d e n c e o f m u t a t i o n r a t e u p o n g r o w t h r a t e , that i s , the m u t a t i o n a l r e s p o n s e , has b e e n d e t e r m i n e d f o r s e v e r a l k i n d s o f m u t a g e n i n s t r a i n s o f E s c h e r i c h i a c o l i B i n c o n t i n u o u s ( c h e m o s t a t ) c u l t u r e s l i m i t e d w i t h a v a r i e t y o f g r o w t h f a c t o i s i n m i n i m a l s a l t s m e d i a . B o t h the m a g n i tude o f m u t a t i o n r a t e a n d i t s dependence upon g r o w t h r a t e d e p e n d upon the f a c t o r u s e d t o l i m i t g r o w t h w h e n ca f f e ine i s the m u t a g e n . W h e n t r y p t o p h a n -r e q u i r i n g c u l t u r e s w e r e l i m i t e d w i t h t r y p t o p h a n , m u t a t i o n r a t e s d i d not d e p e n d upon g r o w t h r a t e but w e r e c o n s t a n t . W h e n c u l t u r e s w e r e l i m i t e d w i t h g l u c o s e , l a c t a t e , s u c c i n a t e , o r p h o s p h o r u s , m u t a t i o n r a t e s w e r e d i r e c t l y p r o p o r t i o n a l t o g r o w t h r a t e . P a r a l l e l r e s p o n s e s w e r e o b s e r v e d f o r s p o n taneous m u t a t i o n . O n l y one e x c e p t i o n was found to these two k i n d s o f r e s p o n s e ; a m o r e c o m p l e x dependence w a s found f o r n i t r o g e n - l i m i t e d c u l t u r e s . I t i s thought that t h i s c o m p l e x i t y m i g h t be due to s e l e c t i o n , s i n c e two d i f f e r e n t s t r a i n s gave d i f f e r e n t r e s p o n s e s w h e n n i t r o g e n w a s u s e d t o l i m i t g r o w t h , W i t h a l l o t h e r g r o w t h f a c t o r s , m u t a t i o n r a t e s w e r e the s a m e f o r d i f f e r e n t s t r a i n s , F o r t h i s r e a s o n , i t i s thought that t h e r e a r e b a s i c a l l y o n l y two k i n d s o f m u t a t i o n a l r e s p o n s e , and that i n n i t r o g e n - l i m i t e d c u l t u r e s the m u t a t i o n a l r e s p o n s e i s m o d i f i e d b y s e l e c t i o n o r b y s o m e o t h e r k i n d o f s e c o n d a r y p r o c e s s .

T h e m o s t d i r e c t i n t e r p r e t a t i o n o f the two k i n d s o f r e s p o n s e i s that the m u t a g e n a f fec t s the gene e i t h e r d u r i n g o r a f t e r i t s s y n t h e s i s , I f the m u t a g e n ac t s o n l y d u r i n g the s y n t h e s i s o f D N A , then the m u t a t i o n r a t e s h o u l d be p r o p o r t i o n a l t o the r e p l i c a t i o n r a t e , a n d hence to g r o w t h r a t e , a s p r e d i c t e d b y the c o p y - e r r o r h y p o t h e s i s . A l t e r n a t i v e l y , i f m u t a t i o n o c c u r s a f t e r s y n t h e s i s then m u t a t i o n s h o u l d o c c u r a t a n e s s e n t i a l l y c o n s t a n t r a t e , s i n c e D N A i s p r e s e n t i n r e l a t i v e l y c o n s t a n t a m o u n t . T h i s i n t e r p r e t a t i o n i s s u p p o r t e d b y the r e s p o n s e s w i t h two o t h e r m u t a g e n s , u l t r a v i o l e t l i g h t a n d Z - a m i n o p u r i n e , T h e r e i s e v i d e n c e that the f i r s t o f t he se , u l t r a v i o l e t l i g h t , a c t s upon D N A r a t h e r t han i t s p r e c u r s o r s , I f so , m u t a t i o n r a t e s h o u l d b e i n d e p e n d e n t o f g r o w t h r a t e f o r c u l t u r e s l i m i t e d e i t h e r w i t h g l u c o s e o r w i t h t r y p t o p h a n . T h i s c o n s t a n c y i s found . F o r the s e c o n d , 2 - a m i n o p u r i n e , m u t a t i o n i s e x p e c t e d t o r e s u l t f r o m the i n c o r p o r a t i o n o f t h i s b a s e a n a l o g i n t o D N A , H e n c e m u t a t i o n r a t e i s e x p e c t e d t o b e p r o p o r t i o n a l t o g r o w t h r a t e f o r e i t h e r l i m i t i n g f a c t o r , T h i s p r o p o r t i o n a l i t y i s a l s o o b s e r v e d ,

A c c o r d i n g t o t h i s i n t e r p r e t a t i o n , then , the o b s e r v a t i o n o f two d i f f e r e n t c a f f e i n e - i n d u c e d m u t a t i o n a l r e s p o n s e s that depend upon the f a c t o r u s e d t o l i m i t g r o w t h i s a r e s u l t o f the o c c u r r e n c e o f d i f f e r e n t p r o c e s s e s

of mutat ion; The constant muta t ion rate obse rved wi th t r y p t o p h a n - l i m i t e d cu l tu re s is due to muta t ion o c c u r r i n g after synthes i s of a gene, and the p r o p o r t i o n a l i t y to g rowth rate for g l u c o s e - l i m i t e d cu l tu re s is due to m u t a t ion o c c u r r i n g du r ing syn thes i s . The nature of the changes in c e l l u l a r p r o c e s s e s leading to these different muta t iona l responses is s t i l l unknown.

S ince there has been no evidence that caffeine reac ts c h e m i c a l l y w i th D N A , we have been e s p e c i a l l y conce rned about an exp lana t ion for the manner i n w h i c h caffeine affects muta t ion rates i n t r y p t o p h a n - l i m i t e d c u l tu res . A poss ib le explanat ion for this effect of caffeine comes f r o m the obse rva t i on of T s ' o O ) that caffeine reduces the t h e r m a l denatura t ion t e m pera tu re of D N A . T h i s obse rva t ion leads to the fo l lowing ' l o c a l i z e d m e l t i ng" m o d e l : the p resence of caffeine i n c r e a s e s the spontaneous rate of muta t ion by i n c r e a s i n g the frequency of spontaneous d i s s o c i a t i o n of duplex D N A along l o c a l i z e d areas of the m o l e c u l e . B o t h ca f fe ine - induced and spontaneous muta t ion in t r y p t o p h a n - l i m i t e d cu l tu res a re thought to o c c u r p r i m a r i l y in s ingle separa ted s t rands o f D N A . The frequency of s epa ra t i on i n c r e a s e s when caffeine is p resen t because the denatura t ion t empera tu re is then l o w e r e d , A v e r y s i m i l a r a l t e rna t ive m o d e l that cannot be r u l e d out is that caffeine s i m p l y adsorbs to s ingle s t rands as they a re f o r m e d and p r e vents r e f o r m a t i o n of the duplex m o l e c u l e .

The e x p e r i m e n t a l support for this k i n d of m o d e l comes f r o m an ex p e r i m e n t w i th the dye a c r i d i n e orange.(2) A l though a c r i d i n e orange was found to be an exce l l en t photodynamic mutagen, the o b s e r v a t i o n that the p resence of a c r i d i n e orange r a i s e d the m e l t i n g t empera tu re of

DNA(3) l ed to the p r e d i c t i o n that this dye would be a good ant imutagen to caffeine in the absence of l igh t . T h i s r e s u l t was found.

Refe rences

1. Ts o, P. O- P . , Go K. H e l m k a m p , and C. Sander , In t e rac t ion of nuc l eos ides and r e l a t ed compounds w i th nuc l e i c ac ids as i nd ica t ed by the change o f h e l i x - c o i l t r a n s i t i o n t empera tu re . P r o c , Nat, A c a d , S c i . U S , , 48, 686-698 (1962).

2 . Webb, R . B . , and H, E . Kub i t s chek . Mutagen ic and ant imutagenic effects o f a c r i d i n e orange i n E s c h e r i c h i a c o l i , B i o c h e m , B i o p h y s . R e s . C o m m u n , 13, 90-94 (1963),

3 . F r i e f e l d e r , D . , P . F , D a v i s o n , and E . P . Ge iduschek . Damage b y v i s i b l e l igh t to the a c r i d i n e o r a n g e - D N A complex , B i o p h y s , J , 389-400 (1961)

C O L L O I D A L P R O P E R T I E S O F P L U T O N I U M I N D I L U T E A Q U E O U S S O L U T I O N

A r t h u r L i n d e n b a u m and W i l l i a m M . W e s t f a l l

The c o l l o i d a l p rope r t i e s o f P u ( l V ) in solut ions sui table for i n j e c t i o n have been inves t iga ted as a funct ion of p H , t ime after pH adjustment, and r e l a t i v e concen t ra t ions of Pu and c o m p l e x i n g c i t r a t e ions . Techniques have been d e s c r i b e d for the p r e p a r a t i o n of Pu(IV) c i t r a t e solut ions of m i n i m a l c o l l o i d a l i t y and for r a p i d e s t i m a t i o n of c o l l o i d a l f o r m a t i o n by an u l t r a f i l t r a t i o n procedure . (1) P o l y m e r i z a t i o n i s i n c r e a s e d w i th i n c r e a s i n g pH and d e p r e s s e d by a l a rge m o l a r excess of c i t r a t e . D i f fe rences in the ra tes of p o l y m e r i z a t i o n and d e p o l y m e r i z a t i o n are d e m o n s t ra ted . The rate of d e p o l y m e r i z a t i o n is d i r e c t l y p r o p o r t i o n a l to c i t r a t e concen t r a t ion , t ime , and a c i d i t y . The p o l y d i s p e r s e nature o f Pu(IV) c o l lo ids is shown by k i n e t i c d i a l y s i s studies suggest ing the p resence of s e v e r a l d i f fus ib le spec i e s . A p p a r e n t d i s c r e p a n c i e s between values for the f r a c t i o n of d i f fus ib le Pu obtained by the u l t r a f i l t r a t i o n and d i a l y s i s methods a re r e l a t e d to r e v e r s i b l e p o l y m e r i z a t i o n .

Refe rence

1 . L i n d e n b a u m , A . , W . M . W e s t f a l l , M . W . Rosen tha l , and J . F . M a r k l e y . P r o g r e s s R e p o r t : P l u t o n i u m r e m o v a l . I X . A n u l t r a f i l t r a t i o n technique for e s t i m a t i n g the c o l l o i d a l p rope r t i e s of p lu ton ium so lu t ions . A r g o n n e N a t i o n a l L a b o r a t o r y B i o l o g i c a l and M e d i c a l R e s e a r c h D i v i s i o n S e m i annual R e p o r t , J anua ry through June 1963, A r g o n n e N a t i o n a l L a b o r a t o r y . A N L - 6 8 2 3 , pp. 111-113.

P R O G R E S S R E P O R T : P L U T O N I U M R E M O V A L . X . D I S T R I B U T I O N A N D R E M O V A L O F M O N O M E R I C A N D P O L Y M E R I C

P L U T O N I U M I N T H E R A T

M a r c i a W . R o s e n t h a l , J o a n F . M a r k l e y and A r t h u r L i n d e n b a u m

The d i s t r i b u t i o n and r e s p o n s e to c h e l a t i o n t h e r a p y o f both m o n o m e r i c and p o l y m e r i c f o r m s o f p l u t o n i u m i n the r a t have been d e t e r m i n e d s o that c o m p a r i s o n m a y b e m a d e w i t h e a r l i e r e x p e r i m e n t s i n the m o u s e . ( ^ ^ ) The r e s u l t s s u m m a r i z e d h e r e w i l l be p u b l i s h e d i n f u l l e l s e w h e r e . ^ ) The d i s t r i b u t i o n of the two f o r m s of p l u t o n i u m in ra t t i s s u e s 3 days af ter i n t r a venous i n j e c t i o n a g a i n d e m o n s t r a t e s that the p a r t i t i o n i n g of a h y d r o l y z a b l e r a d i o e l e m e n t depends on i t s deg ree o f p o l y m e r i z a t i o n . The p o l y m e r i c f o r m i s d e p o s i t e d m o r e h e a v i l y i n the l i v e r than i s the m o n o m e r i c , and the p o l y m e r i c f o r m i s a l w a y s d e p o s i t e d t o a g r e a t e r extent i n l i v e r than i n bone . The m a j o r d i f f e r e n c e be tween the r a t and the m o u s e i s that the s k e l e t a l uptake o f both f o r m s o f p l u t o n i u m i s t w i c e a s h i g h in the r a t a s in the m o u s e - 12% v e r s u s 6% of i n j ec t ed p o l y m e r i c p l u t o n i u m and about 60% v e r s u s 24% o f the i n j e c t e d m o n o m e r i c f o r m . The o v e r a l l r e s u l t i s that the i n i t i a l t o t a l - b o d y b u r d e n i s h i g h e r i n the r a t , s i nce the d e p o s i t i o n i n l i v e r i s about the s ame for the two a n i m a l s p e c i e s (3 /4 o f the i n j e c t e d p o l y m e r i c and about l / 3 o f the m o n o m e r i c f o r m ) .

T h e r e t e n t i o n and l o s s o f i n i t i a l p l u t o n i u m b u r d e n s i n the t i s s u e s o f r a t s i n g e n e r a l f o l l o w e d the s a m e pa t t e rn s d e s c r i b e d i n m i c e o v e r the f i r s t 12 days : no change in bone b u r d e n s , r e l a t i v e l y r a p i d l o s s of about 2 /3 of the d e p o s i t e d m o n o m e r i c p l u t o n i u m f r o m the l i v e r , and l i t t l e i f any l o s s o f p o l y m e r i c p l u t o n i u m f r o m l i v e r . O v e r a 9 0 - d a y p e r i o d the r a t l i v e r l o s t l / 2 , and the m o u s e l i v e r only l / 4 o f the d e p o s i t e d p o l y m e r i c p l u t o n i u m .

T h e r a p y w i t h the c h e l a t i n g agent d i e t h y l e n e t r i a m i n e p e n t a a c e t i c a c i d ( D T P A ) (300 m g / k g i n j e c t e d i n t r a p e r i t o n e a l l y once d a i l y f r o m day 3 t h r o u g h day 11) i n r a t s i n j e c t e d w i t h e i t h e r m o n o m e r i c o r p o l y m e r i c p l u t o n i u m h a d a n effect s i m i l a r t o that found after D T P A t h e r a p y i n m i c e . A b o u t l / 2 o f both f o r m s o f p l u t o n i u m d e p o s i t e d i n r a t bone and a l m o s t a l l o f the m o n o m e r i c p l u t o n i u m d e p o s i t e d i n l i v e r w e r e r e m o v e d . H o w e v e r , the t h e r a p y d i d not r e m o v e any p o l y m e r i c p l u t o n i u m f r o m the r a t l i v e r , w h e r e a s i t h a d r e m o v e d about l / 3 o f that d e p o s i t e d i n the m o u s e l i v e r .

R e f e r e n c e s

S c h u b e r t , J . , J . F . F r i e d , M . W . R o s e n t h a l , and A . L i n d e n b a u m . T i s s u e d i s t r i b u t i o n o f m o n o m e r i c and p o l y m e r i c p l u t o n i u m as m o d i f i e d by a c h e l a t i n g agent . R a d i a t i o n R e s . 15^ 220-226 (1961).

R o s e n t h a l , M . W . , J . F . M a r k l e y , and A . L i n d e n b a u m . P r o g r e s s r e p o r t : P l u t o n i u m r e m o v a l , t u m o r i n c i d e n c e s t u d i e s . I V . A r g o n n e N a t i o n a l L a b o r a t o r y B i o l o g i c a l and M e d i c a l R e s e a r c h D i v i s i o n S e m i a n n u a l R e p o r t , J u l y t h r o u g h D e c e m b e r 1962. A N L - 6 7 9 0 , pp. 8 7 - 9 1 .

M a r k l e y , J . F . , M . W . R o s e n t h a l , and A . L i n d e n b a u m . D i s t r i b u t i o n and r e m o v a l o f m o n o m e r i c and p o l y m e r i c p l u t o n i u m i n r a t s and m i c e . S u b m i t t e d t o I n t e r n . J . R a d i a t i o n B i o l .

P R O G R E S S R E P O R T : P L U T O N I U M R E M O V A L . X I . R E M O V A L O F P O L Y M E R I C P L U T O N I U M F R O M M I C E B Y C O M B I N E D

T H E R A P Y W I T H T H E C A L C I U M C H E L A T E A N D P E N T A E T H Y L E S T E R O F D T P A

J o a n F . M a r k l e y

D i e t h y l e n e t r i a m i n e p e n t a a c e t i c a c i d ( D T P A ) , one o f the m o s t e f f ec t i v e agents fo r r e m o v a l o f p l u t o n i u m d e p o s i t e d i n the body , p e n e t r a t e s c e l l u l a r m e m b r a n e s t o a v e r y l i m i t e d extent; i t s a c t i o n i s e s s e n t i a l l y l i m i t e d t o e x t r a c e l l u l a r p l u t o n i u m . A c e r t a i n p r o p o r t i o n o f p l u t o n i u m i n the t i s s u e s i s i n t r a c e l l u l a r , and t h i s p r o p o r t i o n i s h i g h e r af ter a d m i n i s t r a t i o n of a c o l l o i d a l f o r m of p l u t o n i u m . T h u s , a change in the c h e l a t i n g agent t o i n c r e a s e i t s c e l l u l a r p e r m e a b i l i t y i s o f i n t e r e s t . S i nce an e s t e r f o r m o f D T P A w o u l d b e e x p e c t e d t o b e m o r e l i p i d s o l u b l e and , t h e r e f o r e , be t t e r ab le t o pene t ra t e c e l l m e m b r a n e s , the p e n t a e t h y l e s t e r o f D T P A w a s p r e p a r e d b y D r . N o r m a n F r i g e r i o o f t h i s D i v i s i o n . T h i s r e p o r t s u m m a r i z e s the r e s u l t s o f a n e x p e r i m e n t , p u b l i s h e d i n d e t a i l e l s e w h e r e , ( - 0 to c o m p a r e the e f f ec t i venes s of the pen t ae thy l e s t e r and the c a l c i u m sa l t o f D T P A i n the r e m o v a l o f a p o l y m e r i c f o r m o f p l u t o n i u m f r o m m i c e .

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 for 11 days of 75 m g / k g of the e s t e r , e i t h e r a lone o r i m m e d i a t e l y p r e c e d e d b y C a - D T P A a t 300 m g / k g , o r o f 300 m g / k g C a - D T P A a lone , w e r e i n i t i a t e d in. g r o u p s o f 6 m i c e 3 days af ter i n t r a v e n o u s i n j e c t i o n o f 0.092 / ic P u 2 3 9 i n a h i g h l y p o l y m e r i z e d f o r m (16% u l t r a f i l t e r a b l e ) . The m i c e w e r e k i l l e d e i t he r 3 days o r 14 days af ter p l u t o n i u m i n j e c t i o n . L i v e r s , s p l e e n s , and both f e m u r s w e r e r e m o v e d fo r p l u t o n i u m a n a l y s i s .

The d i s t r i b u t i o n o f p l u t o n i u m a t t h r e e d a y s , w h e n t r e a t m e n t w a s begun , w a s t y p i c a l o f that ob t a ined af ter i n j e c t i o n of a p o l y m e r i c s o l u t i o n : d e p o s i t i o n w a s h i g h in. l i v e r and s p l e e n and l o w i n the s k e l e t o n ( F i g u r e 21). T r e a t m e n t w i t h e i t he r C a - D T P A o r the D T P A e s t e r g i v e n a lone r e d u c e d the p l u t o n i u m content o f the l i v e r by about 28% in each c a s e . C o m b i n e d t r e a t m e n t w i t h both compounds gave a t o t a l r e d u c t i o n of 59%, w h i c h i s the s u m of e i t h e r a lone . The p l u t o n i u m content o f the s p l e e n w a s i n c r e a s e d by t r e a t m e n t w i t h e i t he r agent a lone , i n d i c a t i n g r e d e p o s i t i o n o f s o m e o f the p l u t o n i u m r e m o v e d f r o m the l i v e r , H o w e v e r , w h e n c o m b i n e d t r e a t m e n t w a s g i v e n , t h i s i n c r e a s e w a s p r e v e n t e d and the p l u t o n i u m content o f the s p l e e n w a s r e d u c e d 60% b e l o w the l e v e l o f the c o n t r o l s . The p l u t o n i u m content o f bone w a s r e d u c e d b y t r e a t m e n t w i t h C a - D T P A a l o n e , but w a s una f fec ted b y the D T P A e s t e r , and the effect o f c o m b i n e d t r e a t m e n t on bone w a s the s a m e a s that o f C a - D T P A a lone ( F i g u r e 21). S ince the a c t i o n o f D T P A i s e s s e n t i a l l y e x t r a c e l l u l a r i t i s r e a s o n a b l e to a s s u m e that the a d d i t i v e effect o f the D T P A e s t e r o n the l i v e r i s due t o r e m o v a l o f i n t r a c e l l u l a r p l u t o n i u m . A u t o r a d i o g r a p h i c s tud ies shou ld be o f h e l p i n c o n f i r m i n g t h i s po in t .

91

100 -

90 -

80 -

70 -

I

L I V E R

O Pu only, or Pu + Saline

[H Pu + DTPA Ester ( 7 5 mg/kg)

H Pu + Co-DTPA (350 m g / k g )

• Pu+ Co-DTPA ( 3 0 0 m g / k g ) + DTPA Ester (75mg/kg^

T Standard Error of the Mean

S P L E E N BONE

3 days 14 days 3 days

DAYS AFTER

14 days 3 days

Pu INJECTION

14 days

Figure 21. T h e effect of treatment with C a - D T P A and D T P A ester, s ingly and in combina t i on , on the distr ibution of p o l y m e r i c p l u t o n i u m . Trea tment was g iven once a day, b e g inn ing at 3 days after the in jec t ion of p lu ton ium and cont inuing for 11 days. A l l values are the means from three pairs of m i c e . Bone content is obtained by m u l t i p ly ing the mean femur content by 20.

R e f e r e n c e

1 . M a r k l e y , J . F . R e m o v a l o f p o l y m e r i c p l u t o n i u m f r o m m i c e b y c o m b i n e d t h e r a p y w i t h the c a l c i u m c h e l a t e and p e n t a e t h y l e s t e r o f D T P A . I n t e r n . J . R a d i a t i o n B i o l . 7_(4), 4 0 5 - 4 0 7 ( O c t o b e r 1963).

R A D I O R E S T O R A T I O N O F A M O E B A E A F T E R I N J E C T I O N O F U N I R R A D I A T E D P R O T O P L A S M

E d w a r d W . D a n i e l s and E v e l y n P . B r e y e r

S u p r a l e t h a l l y X - i r r a d i a t e d (30 k r ) P e l o m y x a i l l i n o i s e n s i s a m o e b a e d ie w i t h i n a w e e k af te r e x p o s u r e , but death c a n be p r e v e n t e d or d e l a y e d in 85 to 100% of the amoebae by t r a n s f e r of u n i r r a d i a t e d p r o t o p l a s m (cy to p l a s m + n u c l e i ) o r c y t o p l a s m a lone , b y d i r e c t m i c r o f u s i o n . W e have m a d e m a n y u n s u c c e s s f u l a t t empts t o p r e v e n t death i n these amoebae b y m i c r o -p ipe t te i n j e c t i o n o f homogena t e s o f u n i r r a d i a t e d amoebae a t 1 -4°C. To sepa ra t e the c a u s e s of f a i l u r e of th i s p r o c e d u r e , we a t t e m p t e d to t r a n s f e r u n i r r a d i a t e d p r o t o p l a s m in to i r r a d i a t e d a m o e b a e b y m i c r o p i p e t t e r a t h e r than m i c r o f u s i o n a t 1 -4°C. T h i r t y - s i x s u p r a l e t h a l l y i r r a d i a t e d a m o e b a e w e r e i n j e c t e d w i t h u n i r r a d i a t e d p r o t o p l a s m , i s o l a t e d , and c u l t u r e d , t o ge ther w i t h 47 u n t r e a t e d i r r a d i a t e d c o n t r o l s . S e v e n of the 36 t r e a t e d a m o e b a e r e p r o d u c e d to f o r m m a s s c u l t u r e s that had an a v e r a g e o f 33 c e l l s p e r c l o n e w i t h i n a m o n t h . T h e r e w e r e no d i v i s i o n s a m o n g the i r r a d i a t e d c o n t r o l s . T h e r e m a i n i n g a m o e b a e i n the t r e a t e d g roup l i v e d s i g n i f i c a n t l y l o n g e r than t h e i r c o n t r o l s (16. 0 ± 2.4 days v e r s u s 4.6 ± 0. 14 days ; T = 6.03).

T H E M E T A B O L I S M O F M I T O C H O N D R I A L P R O T E I N S . I . T H E I S O Z Y M E S O F G L U T A M I C - P Y R U V I C T R A N S A M I N A S E

R o b e r t W . S w i c k , P a u l a L . B a r n s t e i n , * and J o h n L . Stange

We have been a t t emp t ing to m e a s u r e i n an u n e q u i v o c a l f a s h i o n the m e t a b o l i c t u r n o v e r o f v a r i o u s p r o t e i n cons t i tuen t s o f the l i v e r ; in teres t , r e c e n t l y has f o c u s e d on the m i t o c h o n d r i a l p r o t e i n s . E l u c i d a t i o n o f the p a t t e r n o f r e p l a c e m e n t o f the i n d i v i d u a l e n z y m e s and s t r u c t u r a l m e m b r a n e s m a y l e a d to a be t te r u n d e r s t a n d i n g of the n o r m a l d y n a m i c s of t h i s p a r t i c l e , i t s f o r m a t i o n o r a s s e m b l y , " l i f e - s p a n , " and d i s a p p e a r a n c e . The f i r s t e x p e r i m e n t s , u s i n g i s o t o p e s , showed s i m i l a r t u r n o v e r r a t e s for the p u r i f i e d s t r u c t u r a l p r o t e i n and s e v e r a l c rude f r a c t i o n s o f the r e m a i n i n g p r o t e i n s . T h i s has sugges ted that the m i t o c h o n d r i o n m a y be r e p l a c e d as a un i t .

The a r r a y o f e n z y m e s i n th i s p a r t i c l e has u s u a l l y been r e g a r d e d a s qui te cons tan t ; e .g . , c y t o c h r o m e o x i d a s e i s f r equen t ly u s e d as an i n d e x o f the m i t o c h o n d r i a l p o p u l a t i o n . On the o ther hand , the a c t i v i t i e s of a n u m b e r o f e n z y m e s in the c e l l have been shown to be subjec t to m a r k e d a l t e r a t i o n as a r e s u l t o f d i e t a r y o r h o r m o n a l m a n i p u l a t i o n . M o s t o f the s tud ie s have been c o n c e r n e d w i t h changes i n a c t i v i t y i n the w h o l e c e l l , a s m e a s u r e d i n a h o m o g e n a t e , a l though s o m e of these e n z y m e s o c c u r as i s o z y m e s , each o f w h i c h m a y be found on ly in c e r t a i n s p e c i f i c l o c a t i o n s w i t h i n the c e l l . A c o n s i d e r a t i o n o f the effects o f v a r i o u s t r e a t m e n t s on the i n d i v i d u a l i s o z y m e s w o u l d be o f s i g n i f i c a n c e . A l s o , i f the a c t i v i t y o f a m i t o c h o n d r i a l i s o z y m e i s m o d i f i a b l e by t r e a t m e n t o f the hos t , then the r a t e o f change in i t s a c t i v i t y shou ld be s i m i l a r to the ra te o f s y n t h e s i s o f new m i t o c h o n d r i a a s m e a s u r e d by the i n c o r p o r a t i o n o f i so tope in to i t s p r o t e i n . T h i s w o u l d g i v e an independent m e a s u r e o f the t u r n o v e r ra te o f th i s s t r u c t u r e .

T h i s r e p o r t i s c o n c e r n e d w i t h e s t a b l i s h m e n t of the e x i s t e n c e of a m i t o c h o n d r i a l i s o z y m e o f g l u t a m i c - p y r u v i c t r a n s a m i n a s e ( G P T ) , w i t h m o d i f i c a t i o n o f i t s a c t i v i t y b y v a r i o u s t r e a t m e n t s , and w i t h s o m e c h e m i c a l c h a r a c t e r i s t i c s that d i s t i n g u i s h i t f r o m the s o l u b l e i s o z y m e .

M e t h o d s

Y o u n g adul t f e m a l e r a t s w e r e fed d ie t s o f v a r y i n g p r o t e i n c o m p o s i t i o n f o r p e r i o d s o f 5-10 d a y s , o r w e r e m a d e d i a b e t i c b y i n j e c t i o n w i t h a l l o x a n , o r w e r e i n j e c t e d w i t h g l u c o n e o g e n i c c o r t i c o s t e r o i d s ( p r e d n i s o l o n e and c o r t i s o n e ) fo r 2-4 d a y s . C o n t r o l r a t s w e r e m a i n t a i n e d on a 12% c a s e i n d ie t , a s w e r e a l l o t h e r s excep t those o n s p e c i a l p r o t e i n d i e t s . The a n i m a l s

* A C M S e m e s t e r Student f r o m L a w r e n c e College.

w e r e s a c r i f i c e d by decapi ta t ion , and the l i v e r was q u i c k l y r e m o v e d and c h i l l e d to 0°. A 2% homogenate of the l i v e r was p r e p a r e d in wate r for e s t i m a t i o n of the to ta l G P T ac t i v i t y . A 10% homogenate in 0.25 M s u c r o s e was used for en r i chmen t of the pa r t i cu l a t e f rac t ions by cen t r i fuga t ion in the u s u a l way or by grad ien t cen t r i fuga t ion . Techniques such as f reeze- thawing son ica t ion , and incuba t ion w i th detergents are f requent ly used to i n c r e a s e the a c c e s s i b i l i t y of m i t o c h o n d r i a l enzymes to the i r subs t ra tes . Such t r e a t ments d id not enhance G P T ac t iv i ty in m i t o c h o n d r i a ; hence, the m i t o c h o n d r i a w e r e suspended in buffer and a s sayed i m m e d i a t e l y .

G P T ca ta lyzes the fo l lowing r eac t ion :

C O O " coo" I I

C O O " C=0 C O O " H - C - N H 2

I I I I H - C - N H 2 + C H 2 C=0 + C H 2

I I I I C C H 3 C H 2 C H 3 C H 2

C O O " C O O "

alanine a -ke tog lu ta ra te pyruva te glutamate A c t i v i t y of the enzyme was m e a s u r e d by incubat ing L - a l a n i n e and a -ke tog lu ta ra te w i th the sample at 30°. A l i q u o t s w e r e pipet ted at i n t e r v a l s into 20% t r i c h l o r o a c e t i c a c i d and the p r o t e i n was r e m o v e d by c e n t r i fuga t ion . An al iquot (now conta in ing pyruvate) and suff ic ient phosphate buffer, pH 7.2, to b r i n g the f ina l vo lume to 3.0 ml w e r e p ipet ted into a cuvette. c t - D i h y d r © n i c o t i n a m i d e adenine d inuc leo t ide ( N A D H ) was added and the o p t i c a l dens i ty was r ead in a B e c k m a n DU spec t ropho tomete r a t 340 and r e c o r d e d on a G i l f o r d M u l t i p l e Sample A b s o r b a n c e R e c o r d e r . L a c t i c dehydrogenase was added and the change in o p t i c a l dens i ty was r e c o r d e d .

C O O " C O O "

C ^ O + N A D H + H + - H C O H + N A D +

I I C H 3 C H 3

pyruva te lacta te

Unde r the condi t ions of the assay a dec rease in o p t i c a l dens i ty of 2.07 uni t is equivalent to the ox ida t ion of 1 / imole of N A D H , and the re fo re , to the re duc t ion of 1 jumole of py ruva te . It was n e c e s s a r y to use an i n d i r e c t a s say method because the a c t i v i t y was low in the pa r t i cu l a t e f r a c t i o n s , and the p r e sence of other enzymes and endogenous subs t ra tes gave r i s e to l a r g e b lank v a l u e s .

The r e v e r s e r e a c t i o n was a l so m e a s u r e d i n d i r e c t l y u s i n g pyruva te and glu tamate as subs t ra tes and m e a s u r i n g the a -ke tog lu ta ra t e f o r m e d

w i t h g l u t a m i c d e h y d r o g e n a s e and N A D H . C o n c e n t r a t i o n s o f the v a r i o u s s u b s t r a t e s w h i c h w o u l d g ive a z e r o - o r d e r r e a c t i o n r a t e w e r e d e t e r m i n e d by v a r y i n g the amount o f each subs t r a t e independen t ly u n t i l o p t i m u m c o n d i t i o n s ob t a ined .

P r o t e i n c o n c e n t r a t i o n was m e a s u r e d b y the b i u r e t r e a c t i o n , and s p e c i f i c a c t i v i t y was e x p r e s s e d a s m i c r o m o l e s o f p r o d u c t f o r m e d pe r m i n ute p e r mg o f p r o t e i n .


C y t o l o g i c a l D i s t r i b u t i o n

E n z y m e a s s a y s c a r r i e d out on the f ive f r a c t i o n s ob ta ined by d i f f e r e n t i a l c e n t r i f u g a t i o n showed a s m a l l amount o f G P T a c t i v i t y i n the n u c l e a r (2.2% of the to ta l ) , m i c r o s o m a l ( l . 2 % ) , and "poor ly s e d i m e n t a b l e " (0.3%) f r a c t i o n s . A c t i v i t y i n the m i t o c h o n d r i a was h i g h e r (4.7%) than in the o ther p a r t i c u l a t e s , but m o s t o f the G P T a c t i v i t y was i n the superna tan t f r a c t i o n (89%). S i m i l a r d i s t r i b u t i o n s have been r e p o r t e d by o t h e r s , ^ ~3) a l though S e g a l e t a l .

(4) found no a c t i v i t y in the p a r t i c u l a t e f r a c t i o n s .

B e c a u s e g r a d i e n t c e n t r i f u g a t i o n a f fords a c l e a n e r f r a c t i o n a t i o n of c e l l u l a r c o m p o n e n t s , th i s t echn ique was u s e d to show m o r e c l e a r l y the p r e s e n c e o f G P T i n the m i t o c h o n d r i a . * S u c c i n i c d e h y d r o g e n a s e , a m i t o c h o n d r i a l e n z y m e , was u s e d as a r e f e r e n c e to show the p r e c i s e l o c a t i o n of peak m i t o c h o n d r i a l a c t i v i t y . F i g u r e 22 shows the d i s t r i b u t i o n o f G P T and s u c c i n i c d e h y d r o g e n a s e a c t i v i t y i n f r a c t i o n s o f a who le homogena te ; F i g u r e 23 shows t h e i r d i s t r i b u t i o n in a m i t o c h o n d r i a l f r a c t i o n i s o l a t e d by d i f f e r e n t i a l c e n t r i f u g a t i o n . In the l a t t e r the peak o f G P T a c t i v i t y c o i n c i d e s w i t h that o f s u c c i n i c d e h y d r o g e n a s e , i n d i c a t i n g that G P T a c t i v i t y i s d e f i n i t e l y p r e s e n t i n the m i t o c h o n d r i a .

Figure 22

Distribution of GPT (A-A) and succinic dehydrogenase (0"0) a f t e r gradient centrifugation of liver homogenate

*We a r e g r a t e f u l t o D r . J o h n F . T h o m s o n for p e r f o r m i n g the g r a d i e n t c e n t r i f u g a t i o n s and the a n a l y s i s o f s u c c i n i c d e h y d r o g e n a s e .

i i 1 1 1 1 r

Figure 23

Distribution of GPT (A - A) and succinic dehydrogenase (O - O) after gradient centrifugation of the mitochondrial fraction of a l iver homogenate

M e t a b o l i c S t u d i e s

A s t u d y o f t h e e f f e c t o f v a r i o u s d i e t a r y a n d h o r m o n a l t r e a t m e n t s o n G P T a c t i v i t y i n t h e s u p e r n a t a n t a n d m i t o c h o n d r i a l f r a c t i o n s o f r a t l i v e r w a s m a d e t o c o m p a r e t h e r e s p o n s e s o f t h e t r a n s a m i n a s e s i n t h e t w o f r a c t i o n s . T h e i s o z y m e s r e a c t e d s i m i l a r l y t o t r e a t m e n t w i t h g l u c o n e o g e n i c h o r m o n e s , b u t f a i l e d t o d o s o i n a n i m a l s e i t h e r k e p t o n d i e t s c o n t a i n i n g d i f f e r e n t a m o u n t s o f p r o t e i n o r m a d e d i a b e t i c ( T a b l e 1 9 a n d F i g u r e 2 4 ) . I n a l l c o n d i t i o n s t h a t c a u s e d t h e b r e a k d o w n o f p r o t e i n , w h e t h e r o f e n d o g e n o u s o r e x o g e n o u s o r i g i n , t h e a c t i v i t y o f t h e s o l u b l e e n z y m e w a s c o n s i s t e n t l y e l e v a t e d . C o n v e r s e l y , i n c o n d i t i o n s t h a t c a u s e d a r e d u c e d c a t a b o l i s m o f p r o t e i n , G P T a c t i v i t y i n t h e s u p e r n a t a n t f r a c t i o n w a s l o w e r e d . T h e s e r e s u l t s a r e c o n s i s t e n t w i t h t h o s e r e p o r t e d b y o t h e r s . ( ^ - 9 ) Q n t h e o t h e r h a n d , t h e m i t o c h o n d r i a l e n z y m e d i d n o t r e s p o n d i n t h i s m a n n e r i n a l l c a s e s , s u g g e s t i n g t h a t t h e i s o z y m e s a r e d i f f e r e n t p r o t e i n s a n d o n l y p a r t l y u n d e r t h e s a m e c o n t r o l .

TABLE 19

Metabolic studies of the isozymes of GPT

Treatment No. of

animals used

Change in body weight,

9

Mitochondria Supernatant Homogenate M + S**

Treatment No. of

animals used

Change in body weight,

9

Protein, mg/g

Specific activity, umoles/min/mg

Total units, Mmoles/min






Control: 12% Casein 8 -6 50.9 0.014 ± 0.002+ 0.64 ± 0.07 0.248 ± 0.017 20 ± 1.0 0.081 ± 0.006 20 ± 1 20.6

2% Casein 6 -14 36.9 0.013 ± 0.001 0.48 ± 0.037 0.153 ± 0.021 11 ± 1.7 0.056 ± 0.008 12.2 ± 1.4 11.5

40% Casein 4 +8 63.9 0.027 ± 0.002 1.7 ± 0.137 0.366 ± 0.063 34.2 ± 5.83 0.115 ± 0.018 33.6 ± 3.4 35.9

Fasted 6 -40 50.1 0.015 ± 0.002 0.731 ± 0.123 0.369 ± 0.039 32.3 ± 3.07 0.147 ± 0.013 34.9 ± 2.89 33.0

Essential amino acids 4 -9 33.0 0.0053 ± 0.002 0.19 ± 0.09 0.068 ± 0.021 5.95 ± 0.81 0.032 ± 0.007 6.1 ± 1.55 6.1

Essential amino 6.9 acids plus alanine 4 -3 34.0 0.0059 ± 0.003 0.21 ± 0.11 0.077 ± 0.022 6.68 ± 1.8 0.035 ± 0.007 7.24 1 1.56 6.9

Prednisolone, 4 -58 46.5 0.057 ± 0.008 2.6 ± 0.360 0.955 ± 0.041 71.3 ± 6.96 0.353 ± 0.021 71.5 ± 3.9 73.9

2 mg/rat/day

Cortisone, 4 -26 49.9 0.033 ± 0.003 1.7 ± 0.16 0.619 ± 0.125 50.6 ± 6.43 0.229 ± 0.015 50.1 ± 5.1 52.3

13 mg/rat/day

DOCA? 4 -12 45.9 0.011 ± 0.003 0.52 ± 0.238 0.172 ± 0.019 14.6 ± 1.79 0.066 ± 0.011 13.7 ± 1.23 15.1

10 mg/rat/day

Alloxan, 175 mg/kg 4 -44 43.9 0.019 ± 0.001 0.818 ± 0.069 0.402 ± 0.052 32.4 ± 2.13 0.159 ± 0.020 33.4 ± 3.14 33.2

^Deoxycorticosterone. "Mitochondria plus supernatant. +Standard error of the mean.

Mitochondria • Mitochondria Specific Activity Protein Q .Q| .02 .03 .04 .05 .06 .07 .08 .09 .10

Figure 24

Change in act ivi ty of mitochondria l and soluble isozymes in response to diets c o n taining different amounts of protein, to treatment with hormones, to diabetes, and to fasting

6 0 2 0 0 0.1 02 0.3 0.4 0-5 0.6 0,7 0-8 0.9 1.0 mg/qm liv. • Supernatant Specific Activity

In a n i m a l s fed the d i f f e ren t l e v e l s of p r o t e i n , the changes in the m i t o c h o n d r i a l p r o t e i n ( e x p r e s s e d a s t o t a l p r o t e i n p e r g r a m o f f r e s h l i v e r ) r o u g h l y p a r a l l e l e d the change in a c t i v i t y o f the m i t o c h o n d r i a l i s o z y m e (Tab le 19 and F i g u r e 24). On the o ther hand , m i t o c h o n d r i a l p r o t e i n l e v e l s i n a n i m a l s t r e a t e d w i t h h o r m o n e s w e r e u n a l t e r e d a l though the G P T a c t i v i t y changed m a r k e d l y . I t w o u l d appear that the m i t o c h o n d r i a l p o p u l a t i o n m a y be m o d i f i e d by p r o t e i n l e v e l of the die t but not by changes in p r o t e i n m e t a b o l i s m a s s o c i a t e d w i t h g l u c o n e o g e n e s i s .

C h e m i c a l C h a r a c t e r i s t i c s

F u r t h e r ev idence fo r the e x i s t e n c e o f two G P T p r o t e i n s was ob ta ined f r o m the e x a m i n a t i o n o f the c h e m i c a l c h a r a c t e r i s t i c s o f the i s o z y m e s p r e p a r e d f r o m r a t s i n j ec t ed w i t h p r e d n i s o l o n e . The superna tan t o r so lub le e n z y m e was e n r i c h e d a c c o r d i n g to p u r i f i c a t i o n p r o c e d u r e I I of Sega l e t al.(^) t h r o u g h the D E A E step. The f i r s t p r o b l e m encoun te red i n the i s o l a t i o n o f the m i t o c h o n d r i a l i s o z y m e was that of s o l u b i l i z a t i o n of the e n z y m e . F r e e z e -t hawing a t e i t h e r - 195° or - 1 0 ° , s o n i c a t i o n , and t r e a t m e n t w i t h de te rgen ts a l l f a i l e d t o y i e l d a n a p p r e c i a b l e amount o f s o l u b i l i z e d e n z y m e . H o w e v e r , w h e n the m i t o c h o n d r i a w e r e f i r s t suspended in c o l d , 50% gly c e r o l - 0 . 0 0 1 M a l a n i n e , s e d i m e n t e d , and r e s u s p e n d e d in 0.05 M phosphate bu f f e r -0 .025 M a l a n i n e , an a c t i v e , so lub le p r e p a r a t i o n of the e n z y m e c o u l d be ob ta ined .

F u r t h e r e n r i c h m e n t has so far been f r u s t r a t e d because o f the m a r k e d i n s t a b i l i t y of the e n z y m e . W h e r e a s the so lub le i s o z y m e a t a l l s tages of p u r i f i c a t i o n was s tab le for weeks when s t o r e d a t 0°, the m i t o c h o n d r i a l f o r m l o s t m o s t o f i t s a c t i v i t y in a few h o u r s . Hea t , pH changes away f r o m n e u t r a l i t y , f r a c t i o n a t i o n w i t h a m m o n i u m sul fa te , o r d i a l y s i s l e d t o r a p i d l o s s of a c t i v i t y . T h i s l o s s w a s not the consequence of d i s s o c i a t i o n of the c o e n z y m e f r o m the p r o t e i n , s i nce a d d i t i o n of, o r i n c u b a t i o n w i t h , p y r i d o x a l phosphate d i d not r e s t o r e a c t i v i t y . The i n a c t i v a t i o n c o u l d be s l o w e d s o m e what by s to rage of the e n z y m e in a 50% g l y c e r o l s o l u t i o n or in 0.05 to 0.1 M a l a n i n e . I t was n e c e s s a r y , t h e r e f o r e , to m a k e p r e l i m i n a r y e s t i m a t e s o f the p H o p t i m a and apparen t M i c h a e l i s cons tants ( K m ) o n the c rude s o l u b i l i z e d e n z y m e .

Assays for the reverse reaction were carried out on both e n z y m e s and the r a t i o s o f the s p e c i f i c a c t i v i t i e s w e r e c o m p a r e d (Tab le ZO). E x a m i n a t i o n o f the pH c u r v e s o f o p t i m u m a c t i v i t y for both i s o z y m e s i n d i c a t e d that the two m a y have d i f fe ren t pH o p t i m a ( F i g u r e 25). The s o l u b l e e n z y m e shows a b r o a d peak of a c t i v i t y c e n t e r i n g about pH 7.2; the m i t o c h o n d r i a l e n z y m e shows a peak a t about pH 8.0. T h e s e r e s u l t s a r e in c o n t r a s t to those of K a t u n u m a et a l .

(3)

who r e p o r t e d o p t i m u m a c t i v i t y a t pH 9.7 for the

T A B L E 2 0 C o m p a r i s o n of f o r w a r d and r e v e r s e r e a c t i o n s o f the two i s o z y m e s

F o r w a r d r e a c t i o n , p imoles p y r u v a t e /

m i n / m g

R e v e r s e r e a c t i o n , jUmoles a - k e t o g l u t a r a t e /

m i n / m g

R a t i o : f o r w a r d / r e v e r s e

M i t o c h o n d r i a *

F r o m f r a c t i o n a l cen t r i fuga t ion 0.11 0.04 2.75

S o l u b i l i z e d e n z y m e 0.013 0.016 0.81

So lub le

F r o m f r a c t i o n a l cen t r i fuga t ion 0.3Z 0.24 1.33

P u r i f i e d enzyme 12.4 9.49 1.30

L i t e r a t u r e ' 1 0 )

So lub le (homogenate) 0.74

* T h e s e s a m p l e s w e r e not f r o m the same a n i m a l and w e r e a s s a y e d a t d i f fe rent t i m e s .

99

s o l u b l e e n z y m e and p H 8.5 for the m i t o c h o n d r i a l i s o z y m e . S e g a l e t a l . W ob t a ined a b r o a d peak for the s o l u b l e (homogenate) e n z y m e r a n g i n g f r o m pH 7 . 2 - 8 . 5 . A b s o l u t e a c t i v i t y o f the so lub le e n z y m e in the d i f f e ren t buf fe rs v a r i e d w i d e l y , w h i l e that o f the m i t o c h o n d r i a l e n z y m e i n T r i s and phospha te buf fe rs w a s n e a r l y the s a m e .

K i n e t i c cons tan t s ob t a ined fo r the two e n z y m e s a r e shown in T a b l e 21 . The appa ren t M i c h a e l i s cons tan t s fo r cc-ketoglutarate a r e s i m i l a r fo r the i s o z y m e s , but those for a l a n i n e a r e qui te d i f f e r en t . The abso lu te K m fo r a - k e t o g l u t a r a t e f o r the s o l u b l e e n z y m e has been c a l c u l a t e d to be 1.1 and that fo r a l an ine t o b e 3 4 . ' ^ ' The M i c h a e l i s cons tan ts fo r a s p a r t i c a c i d fo r the i s o z y m e s of g l u t a m i c - o x a l a c e t a t e t r a n s a m i n a s e ^ ^> ^ ) a l s o d i f fe r g r e a t l y .

T A B L E 2 1

M i c h a e l i s cons tan t s

E n z y m e a - k e t o g l u t a r a t e , m m o l e s

L - a l a n i n e , m m o l e s

S o l u b l e 0.81 34.0 M i t o c h o n d r i a 0.68 5.0

W h e n c o n d i t i o n s for s t a b i l i z a t i o n o f the m i t o c h o n d r i a l G P T a r e found, i t m a y be p o s s i b l e to e n r i c h the e n z y m e so that the k i n e t i c and c h e m i c a l p r o p e r t i e s o f t h i s i s o z y m e m a y b e s tud i ed m o r e d e f i n i t i v e l y .

S u m m a r y

M e a s u r e m e n t o f e n z y m e a c t i v i t y u n d e r v a r i o u s c o n d i t i o n s o f p r o t e i n in t ake and c o r t i c o s t e r o i d a d m i n i s t r a t i o n showed that the e n z y m e s do not a l w a y s r e a c t i n an i d e n t i c a l m a n n e r t o t r e a t m e n t o f the hos t . D i f f e r ences i n the s t a b i l i t y , m e t h o d s o f e n r i c h m e n t , p H o p t i m a , and k i n e t i c c o n stants s t r o n g l y sugges t that these a r e , i n d e e d , i s o z y m e s o f g l u t a m i c -p y r u v i c t r a n s a m i n a s e .

A new t echn ique fo r s o l u b i l i z i n g m i t o c h o n d r i a l e n z y m e s w a s d e v e l o p e d ; the t echn ique i n v o l v e s o s m o t i c shock after p a r t i a l r e p l a c e m e n t o f w a t e r i n the m i t o c h o n d r i a w i t h g l y c e r o l .

100

R e f e r e n c e s

1 . K a f e r , E . , and J . K . P o l l a k . A m i n o a c i d m e t a b o l i s m o f g r o w i n g t i s sues , II. A l a n i n e - g l u t a m i c a c i d t r a n s a m i n a s e a c t i v i t y o f e m b r y o n i c ra t l i v e r . E x p t l . C e l l . R e s . _22, 120- 136 (1961).

2 . R o w s e l l , E . V . S u b c e l l u l a r d i s t r i b u t i o n o f a - o x o g l u t a r a t e and py ruva t e t r a n s a m i n a s e s i n ra t l i v e r . B i o c h e m . J . 89, 65P (1963).

3 . K a t u n u m a , N . , K . M i k u m o , M . M a k o t o , and M . O k a d a . D i f f e r e n c e s be tween the t r a n s a m i n a s e s i n m i t o c h o n d r i a and so lub le f r a c t i o n , I . G l u t a m i c - p y r u v i c t r a n s a m i n a s e . J . V i t a m i n o l o g y _8, 68-73 (1962).

4 . Sega l , H . L . , D . S . B e a t t i e , and S . H o p p e r . P u r i f i c a t i o n and p r o p e r t i e s o f l i v e r g l u t a m i c - a l a n i n e t r a n s a m i n a s e f r o m n o r m a l and c o r t i c o i d - t r e a t e d r a t s . J . B i o l . C h e m . 237, 1914-1920 (1962).

5 . R o s e n , F . , N . R . R o b e r t s , L . E . B u d n i c k , a n d . C . A . N i c h o l . C o r t i c o -s t e r i o d s and t r a n s a m i n a s e a c t i v i t y : the s p e c i f i c i t y of the g l u t a m i c -p y r u v i c t r a n s a m i n a s e r e s p o n s e . E n d o c r ino logy 65, 256-264 (1959).

6 . G a v o s t o , F . , A . P i l e r i , and A . B r u s c a . I n c r e a s e d t r a n s a m i n a s e a c t i v i t y i n the l i v e r after a d m i n i s t r a t i o n o f c o r t i s o n e . B i o c h e m . B i o p h y s . A c t a 24, 250-254 (1957).

7 . W a l d o r f , M . A . , M . C . K i r k , H . L i n k s w i l e r , and A . E . H a r p e r . M e t a b o l i c adaptat ions i n h ighe r a n i m a l s . V I I . R e s p o n s e s o f g l u t a m a t e -oxa lace ta t e and g l u t a m a t e - p y r u v a t e t r a n s a m i n a s e s to d ie t . P r o c . Soc . E x p t l . B i o l . M e d . 112, 764-768 (1963).

8 . M u r a m a t s u , K . , and K . A s h i d a . E f f ec t o f d i e t a r y p r o t e i n l e v e l o n g r o w t h and l i v e r enzyme a c t i v i t i e s of r a t s . J . N u t r i t i o n 7_6, 143-150 (1962).

9 . S c h i m k e , R . T . A d a p t i v e c h a r a c t e r i s t i c s o f u r e a c y c l e e n z y m e s in the ra t . J . B i o l . C h e m . 237, 459-468 (1961).

10. H o p p e r , S., and H . L . Sega l . K i n e t i c s tudies o f ra t l i v e r g l u t a m i c -a lan ine t r a n s a m i n a s e . J . B i o l . . C h e m . 237, 3189-3195 (1962).

11. F l e i s h e r , G . A . , C . S . P o t t e r , and K . G . W a k i n a . S e p a r a t i o n o f 2 g l u t a m i c - o x a l a c e t i c t r a n s a m i n a s e s by paper e l e c t r o p h o r e s i s . P r o c . Soc . E x p t l . B i o l . M e d . 103, 229-231 (1961).

12. B o y d , J . W . The i n t r a c e l l u l a r d i s t r i b u t i o n , l a t ency , and e l e c t r o -p h o r e t i c m o b i l i t y o f 1 -g lu tamate -oxa lace ta t e t r a n s a m i n a s e f r o m r a t l i v e r . B i o c h e m . J . 81 , 434-441 (1961).

101

E F F E C T O F I S O T O P E E X C H A N G E O N T H E P R O T O N A T I O N O F D N A

L y l e G . B u n v i l l e

In an i n v e s t i g a t i o n of the i n t r a m o l e c u l a r i n t e r a c t i o n of the ba se s in D N A , h y d r o g e n - i o n b i n d i n g c u r v e s have been d e t e r m i n e d i n the so lven t s H z O and D z O , each a t an i o n i c s t r eng th o f 0.10 i n N a C l , 2 5 ° C . I n the l a t t e r s y s t e m the t i t r a t i o n c u r v e a t l o w d e g r e e s o f b i n d i n g i s sh i f t ed t o w a r d s h i g h e r pH v a l u e s by an amount ( A p H •= 0.42) c o n s i s t e n t w i t h that e x p e c t e d f r o m the e q u i l i b r i u m i so tope effect on the d i s s o c i a t i o n cons tan t s o f l o w m o l e c u l a r we igh t a c i d s . A s the deg ree o f b i n d i n g i n c r e a s e s , A p H i n c r e a s e s to a cons tan t v a l u e of 0.52 th roughout the r e g i o n of the h e l i x - c o i l t r a n s i t i o n . U p o n c o m p l e t i o n o f the t r a n s i t i o n , A p H a g a i n i n c r e a s e s w i t h the d e g r e e of b i n d i n g . In each s y s t e m the end po in t of the t r a n s i t i o n o c c u r s a t the s a m e deg ree of b i n d i n g , i n d i c a t i n g that no s i g n i f i c a n t change of s t a b i l i t y r e s u l t s f r o m the exchange o f h y d r o g e n for d e u t e r i u m i n the double h e l i x , o r that the s t a b i l i t y o f both h e l i x and c o i l a r e a l t e r e d to the s ame extent . The i n c r e a s i n g d i s p l a c e m e n t o f the two b i n d i n g c u r v e s w i t h i n c r e a s i n g deg ree o f p r o t o n a t i o n i s i n d i c a t i v e o f enhanced b i n d i n g b y D N A i n D z O , e i t h e r by p e r t u r b a t i o n o f the i n t r i n s i c d i s s o c i a t i o n cons tan t s o f the b a s e s , o r b y a l t e r a t i o n o f m o l e c u l a r d i m e n s i o n s .

N U C L E A R C H A N G E S I N R A T L I V E R C E L L S D U R I N G T H I O A C E T A M I D E A D M I N I S T R A T I O N

A . D . B a r t o n and E l i z a b e t h A . C e r n y *

R a t h e r ^ ) has shown that the a d m i n i s t r a t i o n o f t h i o a c e t a m i d e to r a t s p r o d u c e s a r e m a r k a b l e i n c r e a s e i n the v o l u m e o f p a r e n c h y m a l c e l l n u c l e i i n the l i v e r and a s t r i k i n g i n c r e a s e i n the s i z e o f the l i v e r c e l l n u c l e o l i . L a i r d ( ^ ) has shown that i n s h o r t - t e r m a d m i n i s t r a t i o n o f the d r u g , the i n c r e a s e i n n u c l e a r v o l u m e i s not due t o an i n c r e a s e i n p l o i d y , s i n c e the a v e rage d e o x y r i b o n u c l e i c a c i d ( D N A ) content p e r c e l l does not i n c r e a s e . L a i r d ( ^ ) and o thers (3 ,4 ) have d e m o n s t r a t e d a n i n c r e a s e i n the r i b o n u c l e i c a c i d ( R N A ) and p r o t e i n o f the e n l a r g e d n u c l e i . The p r e s e n t s tudy w a s u n d e r t a k e n t o l e a r n m o r e about these changes i n R N A and p r o t e i n , u s i n g a c o n s i d e r a b l y i m p r o v e d m e t h o d o f i s o l a t i n g n u c l e i that w a s not a v a i l a b l e w h e n the e a r l y s tud ies w e r e m a d e .

M e t h o d s

The a n i m a l s u s e d w e r e adul t m a l e S p r a g u e - D a w l e y a l b i n o r a t s , body w e i g h t 300-425 g ; Wayne " L a b - b l o x " ( A l l i e d M i l l s ) and w a t e r w e r e a v a i l a b l e a d l i b . The a n i m a l s i n the t r e a t e d g roups r e c e i v e d t h i o a c e t a m i d e d a i l y , (10 mg as a 1% s o l u t i o n in i s o t o n i c s a l i n e - 0 . 1 8 mM C a C l 2 ) i n j e c t e d subcu taneous ly in the s c a p u l a r r e g i o n . The t r e a t m e n t p e r i o d s w e r e 4 , 7 , 11, and 14 d a y s . At e ach t i m e poin t , a t o t a l of t h r e e t r e a t e d g r o u p s and one c o n t r o l g roup that had r e c e i v e d c o r r e s p o n d i n g i n j e c t i o n s o f the sa l t s o l u t i o n w i t h the t h i o a c e t a m i d e o m i t t e d , w e r e s a c r i f i c e d for s tudy. A z e r o -t i m e c o n t r o l g roup r e c e i v e d n o i n j e c t i o n s . F o r e a c h g r o u p , four r a t s w e r e a n e s t h e t i z e d w i t h e ther , and the l i v e r s w e r e r e m o v e d and t r a n s f e r r e d to a c o l d r o o m m a i n t a i n e d a t 2 - 4 ° C ; p e r f u s i o n o f the l i v e r s w i t h i s o t o n i c s a l i n e -0.18 m M C a C l 2 and a l l subsequent o p e r a t i o n s w e r e c a r r i e d out a t c o l d r o o m t e m p e r a t u r e .

F r o m the p o o l e d l i v e r s , one r e p r e s e n t a t i v e s a m p l e w a s t a k e n t o p r e p a r e a 10% homogena te in 30% s u c r o s e , and f r o m th i s h o m o g e n a t e , a l i quot s w e r e t a k e n for n u c l e a r counts and for a n a l y s i s t o d e t e r m i n e R N A and D N A by S c h n e i d e r ' s m e t h o d . ( 5 ) A C o o k e i m a g e - s p l i t t i n g e y e p i e c e w a s u s e d t o m e a s u r e n u c l e a r d i a m e t e r s ; a t o t a l o f 36 -60 n u c l e i w e r e m e a s u r e d a t e ach t i m e po in t .

A s econd r e p r e s e n t a t i v e s a m p l e w a s t a k e n f r o m the p o o l e d l i v e r s t o p r e p a r e a 10% homogena te i n 75% s u c r o s e - 0 . 1 8 m M C a C l 2 , f r o m w h i c h the l i v e r c e l l n u c l e i w e r e i s o l a t e d by a m o d i f i c a t i o n o f the m e t h o d o f C h a v e a u e t a l . (^) a s d e s c r i b e d p r e v i o u s l y . ( ? )

* A s s o c i a t e d C o l l e g e s o f the M i d w e s t S e m e s t e r Student f r o m C o e C o l l e g e .

103

The " so lub le" nuc lea r p ro te ins were obtained by ex t rac t ing the i s o la ted n u c l e i w i th 0.14 M N a C l - 0 . 1 8 mM C a C l 2 and cent r i fuging the ex t rac ted n u c l e i . T h e R N A and D N A contents o f the nuc lea r sediment we re de te r m i n e d after ex t r ac t i on wi th hot t r i c h l o r o a c e t i c acid.(^) The res idue was washed twice w i th 95% ethanol and twice w i th ether; i t was then d r i e d to constant weight and the " r e s idue" p ro t e in was de t e rmined g r a v i m e t r i c a l l y .

Re suits

The r e s u l t s of the de te rmina t ions made on the v a r i o u s p repa ra t ions are g iven in Table 22. The re was l i t t l e change in c e l l s i ze (as ind ica ted by the number of n u c l e i per g r a m of l i v e r ) or in the amount of D N A per nucleus or in the amount of R N A per c e l l o r per g r a m of t i s sue . The i n c rease in nuc lea r vo lume was s t a t i s t i c a l l y s igni f icant (P = 0.01) by the e leventh day of th ioace tamide a d m i n i s t r a t i o n .

TABLE 22

Changes in rat liver cells with daily administration ot thioacetamide

Preparation Control n* = 5

4 days n =3

7 days n = 3

11 days n = 3

14 days n = 3

Whole Tissue

Nuclei/g liver (x 10" 6) DNA (mg/g liver) DNA/cell (g x 10"11) RNA (mg/g liver) RNA/cell (gx l O ' l l )

131 ± 4** 1.97 ± 0.06 1.51 ± 0.06 5.14 ±0.15 3.94 ± 0.09

138 ± 5 1.99 ± 0.03 1.45 ± 0.07 5.84 ± 0.24 4.24 ± 0.05

140 ±7 1.82 ± 0.04 1.30 ± 0.05 5.10 ± 0.15 3.65 ± 0.12

120 ± 3 1.69 ± 0.01 1.41 ±0.10 4.71 ± 0.10 3.92 ± 0.02

122 ± 3 1.57 ± 0.08 5.30 ± 0.08 5.30 ± 0.08 4.34 ± 0.04

Nuclei

RNA/DNA "Residue" protein/DNA "Soluble" protein/DNA Nuclear volume (u^)

0.194 ±0.016 3.30 ±0.12 1.19 ±0.08 1043 ± 70

0.278 ± 0.021 3.52 ± 0.29 1.07 ±0.15 1625 ± 158

0.395 ± 0.005 4.78 ± 0.02 1.15 ±0.07 1359 ± 162

0.431 ± 0.020 4.66 ± 0.15 1.07 ± 0.08 f

1945 ± 212

0.407 ± 0.026 4.38 ± 0.22 1.21 ± 0.10 1933 ± 200

*Number of preparations on which determinations were made. **Mean ± standard error . t n = 2.

In the i so l a t ed n u c l e i , the ra t io of R N A to D N A i n c r e a s e d by m o r e than 100%, the i nc rease being s igni f icant at the 0.01 l e v e l by the seventh day of t rea tment . The r a t i o of " r e s idue" p ro t e in to D N A i n c r e a s e d by n e a r l y 50%, and again the i n c r e a s e was s t a t i s t i c a l l y s igni f icant by the seventh day of t rea tment . There was no s igni f icant change in the r a t io of " so lub l e " p r o t e i n to D N A throughout the course of th ioace tamide t rea tment

P i s cus s ion

The i n c r e a s e s in nuc lea r v o l u m e s , and the l ack of change in c e l l s i z e , or in the amount of D N A per nuc leus , or in the amount of R N A per c e l l o r per g r a m of l i v e r , are a l l in agreement wi th the r e su l t s o f e a r l i e r s tudies v 2 ' ^ The i n c r e a s e s i n nuc lea r R N A and p ro t e in r epor t ed p r e -v i o u s l y t 2 * 4 ) are con f i rmed by the present r e su l t s wi th nuc lea r p repara t ions containing cons ide rab ly l e s s contaminat ing (non-nuclear) m a t e r i a l . ^ 6 " 8 )

By studying the " so lub l e " ( sa l ine -ex t r ac t ab le ) and " r e s i d u e " p r o te in sepa ra te ly , i t has been poss ib l e to show that the i n c r e a s e in p r o t e i n is e n t i r e l y in the " r e s i d u e " p r o t e i n f r ac t i on . O u r p r ev ious s tudies showed that the s a l i n e - e x t r a c t a b l e p ro te ins are r e t a ined in the nucleus by ion b ind ing , p r e s u m a b l y to n u c l e i c a c i d , s ince they are bas i c pro te ins . (9 ,10) In the presen t e x p e r i m e n t s , there was no i n c r e a s e in s a l i n e - e x t r a c t a b l e p r o t e i n despi te the l a rge i n c r e a s e in nuc l ea r R N A ; this r e s u l t , together w i t h the low ra t io of R N A to D N A in the i s o l a t e d n u c l e i i nd ica te s that these s a l i n e - e x t r a c t a b l e p ro te ins are p robab ly a s s o c i a t e d w i t h the D N A , r a the r than R N A in the nuc leus , i . e . , w i t h the c h r o m o s o m e s . A l though the t h i o ace tamide t rea tment l ed to cons ide rab le i n c r e a s e s in both p r o t e i n and R N A in the l i v e r n u c l e i , i t d id not produce any quant i ta t ive change in the capac i ty of the c h r o m o s o m e s to b ind these soluble p r o t e i n s .

Re fe r ences

1 . R a t h e r , L . J . E x p e r i m e n t a l a l t e r a t i on o f n u c l e a r and c y t o p l a s m i c components of the l i v e r c e l l w i t h t h ioace t amide . I . E a r l y onset and r e v e r s i b i l i t y of vo lume changes of the nuc l eo lu s , nuc l eus , and cy to p l a s m . B u l l . Johns H o p k i n s H o s p . 88, 38-58 (1951).

2 . L a i r d , A . K . N u c l e a r changes induced i n ra t l i v e r c e l l s b y t h i o ace tamide . A r c h . B i o c h e m . B i o p h y s . 46, 119-127 (1953).

3 . T h o m s o n , R . Y . , F . C . Heagy , W . C . H u t c h i n s o n , and J . N . D a v i d s o n . The d e o x y r i b o n u c l e i c a c i d content of the ra t c e l l nuc leus and i t s use in e x p r e s s i n g the r e s u l t s o f t i s sue a n a l y s i s , w i t h p a r t i c u l a r r e f e rence to the c o m p o s i t i o n of l i v e r t i s s u e . B i o c h e m . J . 53, 460-474 (1953).

4 . A d a m s , H . R . , and H . B u s c h . Ef fec t s o f th ioace tamide o n i n c o r p o r a t i on o f o r o t i c a c i d - 2 - C 1 4 into R N A f rac t ions i n l i v e r . C a n c e r R e s . 23, 576-582 (1963).

5 . Schne ide r , W. C . Phosphorus compounds in a n i m a l t i s s u e s . I . E x t r a c t i o n and e s t i m a t i o n of desoxypentose n u c l e i c a c i d and of pentose n u c l e i c a c i d . J . B i o l . C h e m . 16T, 293-303 (1945).

6 . Chaveau , J . , Y . M o u l e , and C h . R o u i l l e r . I so l a t i on o f pure and u n a l t e r e d l i v e r n u c l e i . M o r p h o l o g i c a l and b i o c h e m i c a l c o m p o s i t i o n . E x p t l . C e l l R e s . U , 317-321 (1956).

7 . B a r t o n , A . D . P r o g r e s s R e p o r t . Soluble p ro te ins f r o m c e l l n u c l e i . A r g o n n e N a t i o n a l L a b o r a t o r y B i o l o g i c a l and M e d i c a l R e s e a r c h D i v i s i o n Semiannua l R e p o r t , J u l y through D e c e m b e r , 1962. A N L - 6 7 9 0 , pp. 138-144.

M a g g i o , R . , P . S i ekev i t z , and G . E . Pa l ade . Studies o n i so l a t ed n u c l e i . I . I so la t ion and c h e m i c a l c h a r a c t e r i z a t i o n of a nuc lea r f r a c t ion f r o m guinea pig l i v e r . J . C e l l B i o l . 18, 267-291 (1963).

B a r t o n , A . D . Soluble pro te ins o f the c e l l nuc leus . The C e l l N u c l e u s , ed. J . S . M i t c h e l l . A c a d e m i c P r e s s , New Y o r k , I960, pp. 142-143.

B a r t o n , A . D . Soluble prote ins f r o m l i v e r c e l l n u c l e i . F e d e r a t i o n P r o c . 21, 143 (1962).

N U C L E A R C H A N G E S I N R A T K I D N E Y C E L L S A F T E R C A S T R A T I O N A N D A D M I N I S T R A T I O N

O F T E S T O S T E R O N E

A . D . B a r t o n and K a t h l e e n M . T r a c y *

R a b i n o v i t c h and V a l e r i , ^ ) s tudy ing h i s t o l o g i c a l s e c t i o n s , have r e p o r t e d that c a s t r a t i o n of m i c e l eads to a d e c r e a s e in the v o l u m e of the n u c l e i in the c e l l s o f the k i d n e y , and that the n u c l e a r v o l u m e i n c r e a s e s w h e n t e s t o s t e r o n e i s a d m i n i s t e r e d . B e r n and A l f e r t , ^ 2 ^ u s i n g quan t i t a t i ve m i c r o s p e c t r o p h o t o m e t r y on s t a ined h i s t o l o g i c a l s e c t i o n s , showed that the i n c r e a s e i n n u c l e a r v o l u m e was not due t o a n i n c r e a s e i n p l o i d y . T h e i r r e s u l t s i n d i c a t e d an i n c r e a s e in the nonh i s tone p r o t e i n content o f the e n l a r g e d n u c l e i . I n the p r e s e n t s tudy, k i d n e y c e l l n u c l e i have been i s o l a t e d f r o m a n i m a l s i n w h i c h the t e s t o s t e r o n e ba l ance was a l t e r e d i n v a r i o u s w a y s . The changes i n n u c l e a r cons t i tuen t s that a c c o m p a n y changes i n the l e v e l o f t e s t o s t e r o n e a c t i v i t y have been f o l l o w e d b y d i r e c t m e a s u r e m e n t s on the i s o l a t e d n u c l e i .

The a n i m a l s u s e d w e r e adul t , m a l e , S p r a g u e - D a w l e y a l b i n o r a t s ; age 3-4 m o n t h s ; we igh t 250-300 g . Wayne " L a b - b l o x " ( A l l i e d M i l l s ) and w a t e r w e r e a v a i l a b l e a d l i b . I n a d d i t i o n t o c o n t r o l s , a n i m a l s w e r e e x a m i n e d i n four e x p e r i m e n t a l c o n d i t i o n s : c a s t r a t e ; c a s t r a t e t r e a t e d w i t h t e s t o s t e r o n e for 4 days ; same t r e a t e d for 8 days ; and n o r m a l t r e a t e d w i t h t e s t o s t e r o n e for 6 days . C a s t r a t i o n was p e r f o r m e d unde r e ther a n e s t h e s i a . ^ The c a s t r a t e s w e r e k i l l e d for study 17-30 days after the o p e r a t i o n , and the c o r r e s p o n d i n g c o n t r o l s w e r e s a c r i f i c e d as c l o s e to the s a m e age as the schedu le o f e x p e r i m e n t s w o u l d p e r m i t . The h o r m o n e t r e a t m e n t c o n s i s t e d of a d a i l y subcutaneous i n j e c t i o n of 1 mg t e s t o s t e r o n e ( L i l l y ) .

T h e a n i m a l s w e r e s a c r i f i c e d b y c e r v i c a l d i s l o c a t i o n unde r e ther a n e s t h e s i a . The k i d n e y s w e r e r e m o v e d and p e r f u s e d w i t h c o l d i s o t o n i c s a l i n e i n a c o l d r o o m a t 2 - 4 ° C , w h e r e a l l subsequent o p e r a t i o n s w e r e c a r r i e d out; the k i d n e y s f r o m 8 a n i m a l s w e r e p o o l e d for e ach p r e p a r a t i o n .

F r o m the p o o l e d k i d n e y s , a r e p r e s e n t a t i v e s a m p l e w a s t a k e n t o p r e p a r e a 10% homogena te in 30% s u c r o s e , and f r o m th i s homogena te a l i q u o t s w e r e t a k e n for the d e t e r m i n a t i o n o f R N A and D N A b y S c h n e i d e r ' s m e t h o d / 4 ) F o r n u c l e a r counts and m e a s u r e m e n t o f n u c l e a r d i a m e t e r s i n the C o o k e i m a g e - s p l i t t i n g e y e p i e c e , a l i q u o t s o f the homogena te w e r e d i l u t e d 1:60 w i t h 2 % a c e t i c a c i d c o n t a i n i n g gen t i an v i o l e t ; d i a m e t e r s o f 30 -40 n u c l e i w e r e m e a s u r e d for each e x p e r i m e n t a l c o n d i t i o n .

A second r e p r e s e n t a t i v e s a m p l e w a s t a k e n f r o m the p o o l e d k i d n e y s t o p r e p a r e a 10% homogena te i n 75% s u c r o s e - 0 . 1 8 m M C a C l 2 , f r o m w h i c h the k i d n e y c e l l n u c l e i w e r e i s o l a t e d by the m o d i f i c a t i o n o f the m e t h o d o f C h a v e a u e t a l . (^) u s e d p r e v i o u s l y for the i so l a t ion , o f l i v e r c e l l n u c l e i . ^ '

* Associated Colleges of the Midwest Semester Student from C o r n e l l C o l l e g e .

107

T h e " s o l u b l e " ( s a l i n e - e x t r a c t a b l e ) n u c l e a r p r o t e i n s w e r e o b t a i n e d b y e x t r a c t i n g the i s o l a t e d n u c l e i w i t h 0.14 M N a C l - 0 . 1 8 m M C a C l 2 and c e n t r i f u g i n g the e x t r a c t e d n u c l e i . ( 6 ) T h e R N A and D N A con ten t s o f the n u c l e a r s e d i m e n t w e r e d e t e r m i n e d a f te r e x t r a c t i o n w i t h hot t r i c h l o r o a c e t i c a c i d . T h e r e s i d u e w a s w a s h e d w i t h 95% e t h a n o l and t w i c e w i t h e t h e r , and the " r e s i d u e " p r o t e i n w a s d e t e r m i n e d g r a v i m e t r i c a l l y .

T h e w e i g h t o f the s e m i n a l v e s i c l e s d r o p p e d s h a r p l y a f te r c a s t r a t i o n and i n c r e a s e d m a r k e d l y w i t h a d m i n i s t r a t i o n o f t e s t o s t e r o n e ( T a b l e 23); m o s t o f the c h a n g e s a r e s i g n i f i c a n t a t the 0.001 l e v e l . T h r o u g h o u t the v a r i o u s g r o u p s , i n c r e a s e d l e v e l s o f t e s t o s t e r o n e p r o d u c e d a c o n s i s t e n t t r e n d t o w a r d s i n c r e a s e d k i d n e y w e i g h t and a l o w e r c o n c e n t r a t i o n o f D N A p e r g r a m o f k i d n e y , but e v e n b e t w e e n the e x t r e m e c o n d i t i o n s the c h a n g e s w e r e not l a r g e and w e r e b a r e l y s i g n i f i c a n t a t the 0.05 l e v e l . T h e p r o d u c t o f t h e s e t w o m e a s u r e s , w h i c h g i v e s the t o t a l D N A p e r k i d n e y , s h o w e d n o c o n s i s t e n t c h a n g e s . I n a g r e e m e n t w i t h a p r e v i o u s r e p o r t ,

(7) t h e r e w a s n o s i g n i f i c a n t

change i n the a m o u n t o f R N A p e r g r a m o f k i d n e y , w h i c h i m p l i e s that the t o t a l R N A p e r k i d n e y i n c r e a s e s w i t h t e s t o s t e r o n e a d m i n i s t r a t i o n .

TABLE 23

Changes in rat seminal vesicles and kidney cells in response to castration and administration of testosterone

Normal Castrate n • 5

Castrate + Testosterone Normal + Testosterone

n* = 3 Castrate

n • 5 4 days n = 3

8 days n =3

6 days n • 2

Seminal Vesicles

weight, g 1.40 ± 0.12** 0.13 ± 0.02 0.58 ± 0.08 1.72 ± 0.22 2.20 ± 0.06

Kidneys

Whole Tissue weight, g DNA, mg/g kidney RNA, mg/g kidney

2.95 ± 0.10 3.94 ± 0 . 1 2 3.70 ± 0 . 1 3

2.72 ± 0.07 4.46 ± 0.20 3.77 ± 0.21

2.92 ± 0.07 3.58 ± 0.30 3.27 ± 0.24

3.07 ± 0.04 3.77 ± 0.23 3.89 ± 0.31

3.21 ± 0.18 3.64 ± 0.14 3.72 ± 0.08

Nuclei

Nuclear volume, RNA/DNA "Residue" protein/DNA "Soluble" protein/DNA

575 ± 30 0.092 ± 0.010

2.32 ± 0.08 0.492 ± 0.004 +

472 ± 19 0.094 ± 0.006

2.72 ± 0.15 0.603 ± 0.058

504 ± 2 9 0.084 ± 0.006

2.34 ± 0.08 0.570 ± 0.016

462 ± 30 0.089 ± 0.005

2.17 ± 0 . 4 2 0.472 ± 0.011

705 ± 51 0.094 ± 0.008

2.10 ± 0.16 0.414 ± 0.023

•Number of preparations on which determinations were made; kidneys from 8 animals pooled for each preparation. ' * M e a n ± standard e r ro r . t n - 2 .

C a s t r a t i o n r e d u c e d the n u c l e a r v o l u m e i n the k i d n e y c e l l s and t e s t o s t e r o n e t r e a t m e n t i n c r e a s e d i t . T h e n u c l e a r v o l u m e i n the c a s t r a t e s w a s s i g n i f i c a n t l y d i f f e r e n t a t the 0.01 l e v e l f r o m that i n the n o r m a l s o r i n the t e s t o s t e r o n e - t r e a t e d n o r m a l s . T h e r e w a s n o s i g n i f i c a n t change i n the r a t i o o f R N A t o D N A i n the k i d n e y c e l l n u c l e i . I n c r e a s i n g l e v e l s o f t e s t o s t e r o n e w e r e a c c o m p a n i e d by a c o n s i s t e n t t r e n d t o w a r d s a d e c r e a s e in the r a t i o o f b o t h " r e s i d u e " p r o t e i n t o D N A and " s o l u b l e " p r o t e i n t o D N A i n the i s o l a t e d n u c l e i . R e g r e s s i o n a n a l y s i s i n d i c a t e d that t h i s t r e n d i s s i g n i f i c a n t a t the 0.05 l e v e l i n the c a s e o f the " r e s i d u e " p r o t e i n and i s not s i g n i f i c a n t i n the c a s e o f the " s o l u b l e " p r o t e i n .

The d r a s t i c changes i n the we igh t o f the s e m i n a l v e s i c l e s i n d i c a t e that the v a r i o u s t r e a t m e n t s w e r e a l t e r i n g the t e s t o s t e r o n e b a l a n c e e f f e c t i v e l y . The t r e n d t o w a r d i n c r e a s e d k i d n e y w e i g h t and n u c l e a r v o l u m e w i t h i n c r e a s i n g l e v e l s o f t e s t o s t e r o n e c o n f i r m e d p r e v i o u s r e p o r t s . ^ »^»^) T h e s e r e s u l t s , t oge the r w i t h the d e c r e a s e i n D N A c o n c e n t r a t i o n wi thou t change i n the t o t a l amount o f D N A , sugges t that the effect o f t e s t o s t e r o n e i s to i n c r e a s e the s i z e o f the k i d n e y c e l l s and t h e i r n u c l e i , w i thou t chang ing the t o t a l n u m b e r o f c e l l s p r e s e n t .

The w o r k o f B e r n and A l f e r t ( ^ ) i n d i c a t e d a d e c r e a s e in the c o n c e n t r a t i o n o f h i s tone upon a d m i n i s t r a t i o n o f t e s t o s t e r o n e , due to d i l u t i o n f r o m e n l a r g e m e n t of the n u c l e i ; the c o n c e n t r a t i o n of nonh i s tone p r o t e i n in the n u c l e u s r e m a i n e d the s ame and, t h e r e f o r e , the t o t a l amount was a s s u m e d to have i n c r e a s e d , s i nce the n u c l e a r v o l u m e i n c r e a s e d . In the p r e s e n t e x p e r i m e n t s , the t o t a l amount o f p r o t e i n i n the i s o l a t e d n u c l e i was h i g h e r i n the absence of the h o r m o n e , and d e c r e a s e d w i t h t e s t o s t e r o n e , desp i t e the fact that n u c l e a r v o l u m e i n c r e a s e d . The d i f f e r e n c e i n r e s u l t s i s p r o b a b l y r e l a t e d t o the d i f f e ren t m e t h o d s u s e d . B e r n and A l f e r t m e a s u r e d p r o t e i n i n f i x e d t i s s u e s e c t i o n s , w h i c h p r e s u m a b l y i n c l u d e d a l l o f the p r o t e i n p r e s e n t i n the n u c l e i . ^ O u r i s o l a t e d n u c l e i con ta ined on ly those p r o t e i n s that w e r e bound f i r m l y enough to be r e t a i n e d d u r i n g the i s o l a t i o n p r o c e d u r e . ( 9 ) T h u s , w h i l e the i n c r e a s e i n n u c l e a r v o l u m e p r o d u c e d b y t e s t o s t e r o n e a d m i n i s t r a t i o n w a s a c c o m p a n i e d b y a n i n c r e a s e i n the p r o t e i n s that w e r e bound v e r y l o o s e l y , i f a t a l l , i n the n u c l e u s , our r e s u l t s i n d i c a t e d that t h e r e w a s a s l i g h t d e c r e a s e i n the p r o t e i n s that a r e m o r e f i r m l y bound, and l i t t l e , i f any, change in the s a l i n e - e x t r a c t a b l e p r o t e i n o f the n u c l e i .

C o m p a r i n g the n u c l e a r e n l a r g e m e n t p r o d u c e d b y t h i o a c e t a m i d e a d m i n i s t r a t i o n ^ ^ ) w i t h that p r o d u c e d b y i n c r e a s e d l e v e l s o f t e s t o s t e r o n e a c t i v i t y , w e f ind s o m e s i m i l a r i t y and s o m e d i s t i n c t d i f f e r e n c e s . W i t h t h i o a c e t a m i d e a d m i n i s t r a t i o n , t he re i s a p r o m i n e n t i n c r e a s e in the s i z e o f the n u c l e o l i , and t h e r e i s a l a r g e i n c r e a s e i n the amount o f n u c l e a r R N A and " r e s i d u e " p r o t e i n . I n the n u c l e a r e n l a r g e m e n t p r o d u c e d by t e s t o s t e r o n e a d m i n i s t r a t i o n , the re i s no obv ious change i n n u c l e o l a r s i z e , no i n c r e a s e i n n u c l e a r R N A , and , i f any th ing , a s l i gh t d e c r e a s e i n " r e s i d u e " p r o t e i n . N e i t h e r t r e a t m e n t p r o d u c e d any quan t i t a t ive change in the c a p a c i t y o f the c h r o m o s o m e s to b i n d the s a l i n e - e x t r a c t a b l e p r o t e i n s .

109

R e f e r e n c e s

1 . R a b i n o v i t c h , M . , and V ; V a l e r i . E f f ec t s o f c a s t r a t i o n and o f t e s t o s te rone p rop iona te a d m i n i s t r a t i o n on n u c l e a r v o l u m e s of the k idney c o r t e x c e l l s o f the m o u s e . R e v . B r a s i l . B i o l . J_2(4), 417-420 (1952).

2 . B e r n , H . A . , and M . A l f e r t . H o r m o n a l inf luence o n n u c l e a r s y n t h e s i s . II. V o l u m e , d e s o x y r i b o n u c l e i c a c i d and p r o t e i n content o f r e n a l n u c l e i i n c a s t r a t e d and t e s tos te rone t r ea t ed m i c e . R e v . B r a s i l . B i o l . 14 ( l ) , 25-29 (1954).

3 . D ' A m o u r , F . E . , and F . R . B l o o d . M a n u a l for L a b o r a t o r y W o r k i n P h y s i o l o g y . The U n i v e r s i t y o f C h i c a g o P r e s s , C h i c a g o , 1948.

4 . S c h n e i d e r , W. C . E x t r a c t i o n and e s t i m a t i o n o f desoxypentose n u c l e i c a c i d and of pentose n u c l e i c a c i d . J . B i o l . C h e m . 161, 293-303 (1945).

5 . C h a v e a u , J . , Y . M o u l e , and C h . R o u i l l e r . I s o l a t i o n o f pure and u n a l t e r e d l i v e r n u c l e i . M o r p h o l o g i c a l and b i o c h e m i c a l c h a r a c t e r i z a t i o n . E x p t l . C e l l R e s . 1 1, 3 17-321 (1956).

6 . B a r t o n , A . D . P r o g r e s s r e p o r t : Soluble p ro t e in s f r o m c e l l n u c l e i . A r g o n n e N a t i o n a l L a b o r a t o r y B i o l o g i c a l and M e d i c a l R e s e a r c h D i v i s i o n S e m i a n n u a l R e p o r t , J u l y th rough D e c e m b e r 1962. A N L - 6 7 9 0 , pp. 138-144.

7 . K a s s e n a a r , A . , A . K o u w e n h o v e n , and A . Q u e r i d o . O n the m e t a b o l i c a c t i o n of t e s tos t e rone and r e l a t e d compounds . V. The effect o f t e s tos t e rone on the n u c l e i c a c i d c o m p o s i t i o n o f s e m i n a l v e s i c l e s and k idneys of m i c e . A c t a E n d o c r i n o l . 39 (2), 223-233 (1962).

8 . P o l l i s t e r , A . W . , and C . L e u c h t e n b e r g e r . The n u c l e o p r o t e i n content o f who le n u c l e i . P r o c . N a t l . A c a d . S c i . ( U . S . ) 3J3, 66-71 (1949).

9 . B a r t o n , A . D . Soluble p ro t e in s o f the c e l l nuc l eus . The C e l l N u c l e u s , ed. J . S . M i t c h e l l . A c a d e m i c P r e s s , N e w Y o r k , I960, pp. 142- 143.

0 . B a r t o n , A . D . , and E . A . C e r n y . N u c l e a r changes i n r a t l i v e r c e l l s d u r i n g t h i o a c e t a m i d e a d m i n i s t r a t i o n . T h i s r e p o r t , p . 102.

E F F E C T O F X - R A D I A T I O N O N O X I D A T I V E P H O S P H O R Y L A T I O N I N R A T L I V E R M I T O C H O N D R I A . II.

J o h n F . T h o m s o n and S h a r r o n L . N a n c e

The p r e v i o u s r e p o r t i n t h i s s e r i e s was c o n c e r n e d w i t h s tud ies o n the c a p a c i t y o f m i t o c h o n d r i a l f r a c t i o n s i s o l a t e d f r o m l i v e r s o f i r r a d i a t e d r a t s t o e s t e r i f y i n o r g a n i c phospha te , u s i n g the ene rgy d e r i v e d f r o m the o x i d a t i o n o f fumara t e o r a - k e t o g l u t a r a t e . \ l ) The data i n d i c a t e d no c o n s i s t en t effect o f X - i r r a d i a t i o n on e i t he r p h o s p h o r y l a t i v e c a p a c i t y o r p h o s p h o r y l a t i v e e f f i c i e n c y , i n c o n t r a s t t o the e a r l i e r o b s e r v a t i o n s o f H a l l e t al.(^)

T h i s r e p o r t i s c o n c e r n e d w i t h a d d i t i o n a l s tud ies c a r r i e d out w i t h m a l e r a t s ( ins tead o f the f e m a l e s u s e d in m o s t o f t h i s w o r k ) , w i t h the effect o f f l u o r i d e on a - k e t o g l u t a r a t e o x i d a t i o n , and w i t h 3 - h y d r o x y b u t y r a t e a s the s u b s t r a t e .

M e t h o d s

I n most, e x p e r i m e n t s , B a d g e r R e s e a r c h S p r a g u e - D a w l e y f e m a l e r a t s , Z to 4 mon ths o l d , w e r e u sed ; one s e r i e s of e x p e r i m e n t s was c a r r i e d out w i t h m a l e r a t s o f the s a m e s t r a i n , 11 mon ths o l d , I r r a d i a t e d r a t s r e c e i v e d 1000 r t o t a l - b o d y X - r a d i a t i o n at a dose ra te of 200 r / m i n for f e m a l e s and 100 r / m i n for m a l e s ; they w e r e k i l l e d 3y t o 4 h r af ter e x p o s u r e .

The p r o c e d u r e fo r i s o l a t i o n o f m i t o c h o n d r i a l f r a c t i o n s was the s a m e a s that d e s c r i b e d p r e v i o u s l y . ^ ) F o u r m i t o c h o n d r i a l f r a c t i o n s w e r e ob t a ined f r o m each l i v e r .


T a b l e 24 is a s u m m a r y of data , ob ta ined under a v a r i e t y of e x p e r i m e n t a l c o n d i t i o n s w i t h f e m a l e r a t s , o n o x y g e n c o n s u m p t i o n , phosphate e s t e r i f i c a t i o n , P / o r a t i o s , and t o t a l n i t r o g e n i n d i f f e ren t m i t o c h o n d r i a l f r a c t i o n s . The data f r o m the p r e v i o u s r e p o r t a r e i n c l u d e d i n t h i s t a b l e . The s a m e m e a s u r e m e n t s , c a r r i e d out o n p r e p a r a t i o n s f r o m m a l e r a t s w i t h a - k e t o g l u t a r a t e a s s u b s t r a t e , a r e shown in T a b l e 25.

A m o n g the 72 c o m p a r i s o n s m a d e in these two t a b l e s , on ly two s i g n i f i c a n t d i f f e r e n c e s w e r e o b s e r v e d , both w i t h r e s p e c t t o o x y g e n c o n s u m p t i o n w i t h a - k e t o g l u t a r a t e a s the subs t r a t e : Zone B , f e m a l e r a t s , f l u o r i d e p r e s ent; and Z o n e A , m a l e r a t s . A s far a s p h o s p h o r y l a t i v e e f f i c i e n c y ( P / O ) and c a p a c i t y ( P / N ) w e r e c o n c e r n e d , t he re was not even a t r e n d to show an effect o f i r r a d i a t i o n ; i n fact , i n e l e v e n o f the 2 1 c o m p a r i s o n s , both the P / O and P / N r a t i o s w e r e s l i g h t l y h i g h e r after i r r a d i a t i o n .

I l l

TABLE 24

Oxidative phosphorylation in mitochondria prepared from livers of control and irradiated female rats

Zone* A B C D l D 2

Size range (|J) 0.37-0.55 0.55-0.69 0.69-0.82 0.82-0.94 >0.82

N Av. a p N Av. a p N Av. a p N Av. o p N Av. a p

a-ketoglutarate + mafonate, F" present

P/N** Control Irradiated

0/N +

Control Irradiated

P/O t f

Control Irradiated

11 18.2 0.86 11 17.6 1.46

0.7

11 5.60 0.256 1 10 5.93 0.521 J

12 3.26 0.133 1 10 3.01 0.245 J u ' 4

11 27.9 11 26.2

0.68 0.61

0.1

11 11

12 11

7.46 6.69

3.67 3.86

0.2001 0.234 J u , u '

0.089 0.098

0.2

11 29.2 0.76 11 28.8 0.85

0.7

11 8.26 0.374 1 n„ 11 7.58 0.310 J U m £

12 3.65 0.077 \ n

11 3.82 0.090 J •

7 30.2 1.14 6 28.7 1.99

6 7.48 0.278 6 7.67 0.500

7 3.94 0.139 5 3.85 0.076

0.5

0.7

0.6

33.1 28.5

2.03 3.23

8.52 0.7031 7.58 0.578 J

3.91 0.1671 3.65 0.093 J

0.2

0.3

0.2

a-ketoglutarate + malonate, F" omitted

P/N Control Irradiated

O/N Control Irradiated

P/O Control Irradiated

3 10.8 1.33 5 11.4 1.39

0.8

4 6.28 0.5401 n . 5 5.71 0.508 J U - 4

3 1.81 0.2421 n, 5 1.96 0.071 J U , t )

26.8 25.2

8.24 8.41

3.13 3.02

1.45 1.40

0.5

0.247 1 0.364 J u > /

0.191 0.159

0.7

4 28.2 0.97 5 29.7 1.12

4 8.95 0.414 1 5 8.91 0.467 J

4 3.16 0.1701 5 3.35 0.106 J

0.3

0.9

0.4

4 21.9 5 24.1

0.35 2.89

2.93 0.179 2.97 0.153

0.5

4 7.57 0.3831 5 8.13 0.480 J

0.9

Fumarate, F" present

P/N Control 7 12.2 0.80 1 Irradiated 7 13.1 0.50 J

O/N Control 8 4.97 0.488 1 Irradiated 5 5.51 0.567 J

P/O Control 6 2.44 0.1281 Irradiated 5 2.50 0.179 J

0.4

0.4

0.8

15.8 16.8

5.59 6.12

2.78 2.87

0.86 1 1.41 J u , : ?

0.357 0.531

0.127 0.149

0.3

0.7

16.6 17.9

1.71 0.81

0.5

8 6.12 0.375 1 5 6.12 0.342 J '

7 2.78 0.060 1 n, 5 2.97 0.101 J 0 , 1

7 13.5 7 15.9

1.11 1.05

7 5.33 0.345 5 6.09 0.576

2.57 0.118 1.18 0.194

0.15

0.3

0.6

(3-hydroxybutyrate, F" present

P/N Control Irradiated

O/N Control Irradiated

P/O Control Irradiated

14.1 13.3

0.54 0.81

6.23 0.260 5.30 0.344

2.26 2.54

0.026 0.202

0.4

0.1

0.2

12.6 13.3

5.48 5.72

2.31 2.34

0.48 0.94

0.239 0.663

0.080 0.099

0.5

0.7

0.8

5 12.2 4 13.2

0.84 1.01

5 5.35 0.308 4 5.88 0.481

2.29 0.210 2.32 0.066

0.4

0.3

0.8

5 10.1 4 11.1

0.84 0.50

5 2.32 0.182 4 2.31 0.157

0.3

5 4.35 0.098 \ n -4 4.92 0.614 J *

0.9

Nitrogen conten t (composite of above experiments )

mg N/g liver Control Irradiated

29 0.89 0.032 1 27 0.90 0.031 J

• 0.9 29 1.38 0.039 1 27 1.34 0.053 J

, 0 , 29 1.47 0.046 1 27 1.38 0.033 J

[ 0.1 15 0.82 0.043 1 14 0.74 0.152 J

' 0.2 14 2.98 0.2181 13 2.78 0.187 J

\ 0.4

*Dj: nuclei not removed before layering, sediment not included in bottom layer. D2: nuclei removed before layering, sediment included in bottom layer. "Micromoles P esterified/mg N/20 min.

+Microatoms Oconsumed/mg N/20 min. ^Micromoles P esterified/microatoms O consumed.

O u r w o r k o n the e f fec t s o f r a d i a t i o n o n the p h o s p h o r y l a t i v e e f f i c i e n c y o f r a t s p l e e n m i t o c h o n d r i a l ) i n d i c a t e d that a r a d i a t i o n - i n d u c e d d e p r e s s i o n o f the P / o r a t i o c o u l d b e o b s e r v e d o n l y w h e n e x p e r i m e n t a l c o n d i t i o n s w e r e s u c h that c o n t r o l p r e p a r a t i o n s w e r e u n a b l e t o e s t e r i f y p h o s p h a t e e f f i c i e n t l y .

H o w e v e r , i n t h e s e e x p e r i m e n t s w i t h l i v e r , l o w P / O r a t i o s w e r e o b s e r v e d i n c o n t r o l r a t s u n d e r a v a r i e t y o f c o n d i t i o n s - t h r o u g h o u t the s e r i e s

w i t h jS-hydroxybutyrate, in Zone A with a l l substrates, and in a l l zones with a - k e t o g l u t a r a t e w h e n f l u o r i d e w a s o m i t t e d - but no f u r t h e r d e p r e s s i o n of the P / O r a t i o was noted after t o t a l - b o d y i r r a d i a t i o n . H e n c e the e x p l a n a t i o n o f f e r e d t o accoun t for the o c c a s i o n a l d e c r e a s e i n P / o r a t i o s o f i r r a d i a t e d s p l e e n m i t o c h o n d r i a , i . e . , a r e l a t i v e i n c r e a s e i n m i t o c h o n d r i a l c o n t a m i n a n t s w i t h h i g h phospha tase a c t i v i t y , (3) w o u l d s e e m not to app ly in these s t u d i e s .

T A B L E 2 5

O x i d a t i v e p h o s p h o r y l a t i o n i n m i t o c h o n d r i a p r e p a r e d f r o m l i v e r s o f

c o n t r o l a n d i r r a d i a t e d m a l e r a t s *

Z o n e

S i z e r a n g e ( / i ) 0 . 3 7 - 0 . 5 5 0 . 5 5 - 0 . 6 9 0 . 6 9 - 0 . 8 2 > 0 . 8 2

A v . A v . A v . A v .

P / N

C o n t r o l

I r r a d i a t e d

O / N C o n t r o l

I r r a d i a t e d

p/o C o n t r o l

I r r a d i a t e d

m g N / g l i v e r

C o n t r o l

I r r a d i a t e d

1 0 . 0 1 . 3 3

8 . 9

1 . 3 3 "1

1 . 7 5 J

4 . 4 1 0 . 3 0 1

2 . 9 9 0 . 3 4 3

2 . 2 8 0 . 2 7 1

2 . 9 2 0 . 3 9 5

0 . 9 1 0 . 0 6 0

0 . 8 3 0 . 0 3 2 }

0 . 6

0 . 0 2

0 . 2

0 . 2

1 9 . 9 0 . 7 9

2 0 . 1

3 . 7 9 \

1 . 0 1 J

2 1 . 2 0 . 7 8

2 1 . 0

3 . 7 8 "I

1 . 2 8 J 0 . 9

1 4 . 3

1 4 . 4

2 . 2 7 \

1 . 3 4 J

6 . 0 9 0 . 2 5 2

5 . 7 4 0 . 3 4 5

3 . 2 7 0 . 0 1 8

3 . 5 6 0 . 1 2 4

1 . 6 3 0 . 1 6 9

1 . 4 7 0 . 0 9 1 }

0 . 4

0 . 1

0 . 4

5 . 7 1 0 . 1 8 0

5 . 8 9 0 . 3 9 1

3 . 7 0 0 . 0 6 2

3 . 5 9 0 . 1 4 1

1 . 6 0 0 . 1 2 3

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* a - k e t o g l u t a r a t e a s s u b s t r a t e . E a c h v a l u e i s t h e a v e r a g e o f 5 r a t s ; s e e T a b l e 2 4 f o r u n i t s .

The d i s c r e p a n c y be tween our r e s u l t s and those o f H a l l e t a l . (2) i s s t i l l u n r e s o l v e d . S ince w e have o b s e r v e d n o r a d i a t i o n effects w i t h m i t o c h o n d r i a i s o l a t e d f r o m S p r a g u e - D a w l e y o r H o l t z m a n r a t s , m a l e s o r f e m a l e s , a s s a y e d w i t h d i f fe ren t s u b s t r a t e s w i t h o r wi thou t f l u o r i d e , the on ly p l a u s i b l e e x p l a n a t i o n r e m a i n i n g i s a n u n r e c o g n i z e d v a r i a t i o n i n e x p e r i m e n t a l t e chn ique .

R e f e r e n c e s

1 . T h o m s o n , J . F . , and L . F . B o r d n e r . E f f e c t o f X - r a d i a t i o n o n o x i d a t i v e p h o s p h o r y l a t i o n i n r a t l i v e r m i t o c h o n d r i a . A r g o n n e N a t i o n a l L a b o r a t o r y B i o l o g i c a l and M e d i c a l R e s e a r c h D i v i s i o n S e m i a n n u a l R e p o r t , J a n u a r y t h r o u g h June , 1963. A N L - 6 8 2 3 , pp. 157-160 .

2 . H a l l , J . C , A . L . G o l d s t e i n , and B . P . S o n n e n b l i c k . R e c o v e r y o f o x i d a t i v e p h o s p h o r y l a t i o n i n r a t l i v e r m i t o c h o n d r i a af ter w h o l e body i r r a d i a t i o n . J . B i o l . C h e m . 238, 1137- 1140 (1963).

3 . T h o m s o n , J . F . E f f e c t s o f t o t a l - b o d y X - i r r a d i a t i o n o n phosphate e s t e r i f i c a t i o n and h y d r o l y s i s i n m i t o c h o n d r i a l p r e p a r a t i o n s o f r a t s p l e e n . R a d i a t i o n R e s . 2J_, 46 -60 (1964).

113

P H O S P H O L I P A S E C , L O W p H , A N D T E M P E R A T U R E E F F E C T S O N A C I D P H O S P H A T A S E R E L E A S E F R O M R A T L I V E R , S P L E E N A N D T H Y M U S

Y , E . R a h m a n

In t roduct ion

The fact that ' ' l y s o s o m e s a r e a s p e c i a l group of p a r t i c l e s , m o r p h o l o g i c a l l y and func t iona l ly d i s t i nc t f r o m other s u b c e l l u l a r p a r t i c l e s , has been ind i spu tab ly e s t ab l i shed du r ing the course of the past few y e a r s , H o w e v e r , s tudies on l y s o s o m e s have been l i m i t e d m a i n l y to ra t l i v e r t i s sue , and v e r y l i t t l e is known about the func t iona l and p h y s i o l o g i c a l c h a r a c t e r i s t i c s of l y s o s o m e s in other t i s sue s , a l though the i r ex is tence in a number of other t i s sues has been demonstrated,,

A study on the effect of X - i r r a d i a t i o n on the f r a g i l i t y of r a t sp leen l y s o s o m e s was r e p o r t e d e a r l i e r „ ( 1 »2) p resen t r e p o r t i s conce rned w i t h a c i d phosphatase r e l e a s e f r o m ra t l i v e r , sp leen , and thymus l y s o s o m e s under the effects of phosphol ipase C, low p H , and di f ferent t empe ra tu r e s .

Methods

T i s s u e p r e p a r a t i o n and a c i d phosphatase a s say are e s s e n t i a l l y the same as d e s c r i b e d previously,(^>3) except that the 0,25 _M s u c r o s e used for homogen i za t i on conta ined 0,05 M t r i s - m a l e a t e buffer, at pH 7,0 or pH 5,2 as i nd ica t ed for each expe r imen t , The f i l t e r e d t i ssue homogenate was then incubated at 0°, 25°, or 37°C, and a l iquots of the homogenate w e r e taken for a s say of free a c i d phosphatase a c t i v i t y at 10, 30, 60, 120, and 180 m i n u t e s . At the end of each e x p e r i m e n t , another a l iquot of the t i s sue homogenate was taken for a s say of to ta l a c t i v i t y , us ing 0,1% Igepal -630, a n o n - i o n i c de te r gent, w h i c h , a t this concen t ra t ion , causes r e l ea se of a l l the a c i d phosphatase bound in the l y s o s o m e s . A l l f ree a c t i v i t i e s a re c a l c u l a t e d as per cent of the tota l enzyme a c t i v i t y ,

P h o s p h o l i p a s e C was p u r c h a s e d f r o m S i g m a Chemica l . C o . ; the amount used for each e x p e r i m e n t was d i s s o l v e d in 0,25 M s u c r o s e before add i t ion to the t i s sue homogenate ,

Resu l t s

Ef fec t o f L o w pH and T e m p e r a t u r e on A c i d Phospha tase R e l e a s e

F i g u r e 26 shows the r e l e a s e of a c i d phosphatase f r o m l i v e r l y s o s omes incubated at 0°C and 37°C for t imes up to 3 hr . At 0°C, a c i d

114

phosphatase release d i d not change up to 3 hr, but at 37°C, th i s r e l e a s e is shown to i n c r e a s e w i t h t i m e , and a p p r o a c h e d 100% after 3 h r .

h- in — o

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100

80

60

40

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pH 5.2

20 < i—6 -

Figure 26

Effect of temperature on t ime-course of ac id phosphatase release from l iver lyso somes

0 10 30 60 120

T IME (minutes)

180

F i g u r e s 27 and 28 show the r e s u l t of the s a m e e x p e r i m e n t on s p l e e n and t h y m u s ; h e r e , c o n t r a r y t o the r e l e a s e o f a c i d phospha tase f r o m l i v e r , e v e n a t 37°C the amount o f r e l e a s e d e n z y m e i s p r a c t i c a l l y unchanged f o r m o r e than 3 h r .

Figure 27

Effect of temperature on t ime-course of ac id phosphatase release from spleen lysosomes

t S

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1 1 1 l I 0 10 30 60 120

TIME (minutes)

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Figure 28

Effect of temperature on t ime-course of ac id phosphatase release from thymus lysosomes

120

TIME (minu tes)

115

E f f e c t o f L o w p H , T e m p e r a t u r e and P h o s p h o l i p a s e C o n A c i d P h o s p h a t a s e R e l e a s e

F i g u r e s 29~31 show that the a d d i t i o n of p h o s p h o l i p a s e C at 25°C and p H 7.0 d i d not i n c r e a s e the r e l e a s e o f a c i d phosphatase f r o m l i v e r l y s o s o m e s fo r 2 hr or f r o m s p l e e n and thymus l y s o s o m e s for 3 h r . When the pH was l o w e r e d to 5.2, h o w e v e r , the r e l e a s e o f a c i d phosphatase i n c r e a s e d w i t h t i m e . T h i s r e l e a s e r e a c h e d 100% at about 2 hr fo r the l i v e r and the thymus but not m o r e than 80% fo r the s p l e e n , under the same c o n d i t i o n s .

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of 25 *C

-o pH 7.0

-x pH 5.2

0 10 30 6 0 120

TIME (minutes)

180

Figure 29

Effect of pH on time-course of ac id phosphatase release from phospholipase C-treated l iver lysosomes at 2 5 ° C

Figure 30

Effect of pH on time-course of ac id phosphatase release from phospholipase C-treated spleen lysosomes at 2 5 ° C

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at 25 °C

_L 0 10 30 6 0 120

TIME (minutes) 180

20

0 10

T h y m u s

30 60

P. lipase C 0.1 m g / 100 mg Thymus

at 25°C

—x

o o pH 7.0

x x pH 5.2

120

TIME (minutes)

180

Figure 31

Effect of pH on time-course of ac id phosphatase release from phospholipase C-treated thymus lysosomes at 2 5 ° C

F i g u r e s 32 and 33 show that i n bo th l i v e r and s p l e e n at p H 5.2 and t r e a t e d w i t h p h o s p h o l i p a s e C , but i ncuba ted a t 3 7 ° C , a c i d phospha tase r e l e a s e i s m o r e r a p i d than a t 2 5 ° C . H e r e aga in , the s p l e e n f a i l e d to show c o m p l e t e r e l e a s e a t th is low p H , w h e r e a s a t n e u t r a l p H , the a c i d p h o s phatase r e l e a s e is 100% at about 2 h r .

_L

P. l ipase C 0.1 m g / 1 0 0 mg Liver

o t 3 7 # C

o PH 7.0 x pH 5.2

0 10 30 6 0 120 TIME (m inu tes )

180

Figure 32

Effect of pH on t ime-course of ac id phosphatase release from phospholipase C-treated l iver lysosomes at 3 7 ° C

Figure 33

Effect of pH on t ime-course of ac id phosphatase release from phospholipase C-treated spleen lysosomes at 37°C

>- ~

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100

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-x pH 5.2

L 0 10 30 60 120

TIME (minutes)

180

D i s c u s s i o n

P r e v i o u s s tud ies on a c t i v a t i o n o f l y s o s o m a l e n z y m e s have been done m o s t l y w i t h r a t l i v e r , and f r o m the r e s u l t s thus ob ta ined , de Duve s p e c u l a t e d that a p o s s i b l e m e c h a n i s m of l y s o s o m e r u p t u r e in in tac t c e l l s w a s the l o w e r i n g of the i n t r a c e l l u l a r pH. (^ ) In v i e w of the r e s u l t s r e p o r t e d he re on s p l e e n and t h y m u s , we a r e not j u s t i f i e d in ex tend ing this s p e c u l a t i o n to t i s s u e s o ther than l i v e r .

The r e s u l t s ob ta ined w i t h p h o s p h o l i p a s e C s e e m r a t h e r too c o m p l i c a t e d to p e r m i t a c o n c l u s i o n a t th is m o m e n t ; m o r e e x p e r i m e n t s a r e needed before we w i l l be able to u n d e r s t a n d the m e c h a n i s m s of l y s o s o m e r u p t u r e i n d i f fe ren t m a m m a l i a n t i s s u e s .

R e f er ence s

R a h m a n , Y . E . F r a g i l i t y s tudies o f r a t sp l een l y s o s o m e s after X -i r r a d i a t i o n . A r g o n n e N a t i o n a l L a b o r a t o r y B i o l o g i c a l and M e d i c a l R e s e a r c h D i v i s i o n S e m i a n n u a l R e p o r t , J u l y th rough D e c e m b e r 1962. A N L - 6 7 9 0 . pp. 150- 155.

R a h m a n , Y, E . The effect o f X - i r r a d i a t i o n on the f r a g i l i t y o f ra t sp l een l y s o s o m e s . R a d i a t i o n R e s . _20, 741-750 (1963).

R a h m a n , Y. E . A c i d phosphatase and /3~ g l u c u r o n i d a s e a c t i v i t i e s of thymus and sp leen o f ra t s after w h o l e - b o d y X - i r r a d i a t i o n . P r o c . Soc. E x p t l . B i o l . M e d . 109, 378-381 (1962).

de D u v e , C. L y s o s o m e s , a new group of c y t o p l a s m i c p a r t i c l e s . Subc e l l u l a r P a r t i c l e s . A m . P h y s i o l . Soc. P u b l i c a t i o n , Wash ing ton , D . C . 1959, pp. 128-159.

F I N E S T R U C T U R E O F T H E L A C U N A E A N D C A N A L I C U L I I N B O N E

F . W a s s e r m a n n and J a m e s A . Y a e g e r

F r o m 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 o b s e r v a t i o n s and h i s t o c h e m i c a l s t a i n i n g , i t i s known that oste ocyte s s i t ua t ed in t h e i r l acunae a r e s u r r o u n d e d by a l a y e r of u n m i n e r a l i z e d m a t e r i a l , the s o - c a l l e d c a p s u l e of the o s t e o cyte s. (^ >^' ^) In a p r e v i o u s r epor t (^ ) the f ine s t r u c t u r e of th is c a p s u l e was d e s c r i b e d a s i t a p p e a r e d i n the e l e c t r o n m i c r o s c o p e af ter o s m i u m f i x a t i o n and d e m i n e r a l i z a t i o n . I t c o n s i s t e d of a r a t h e r s t r u c t u r e l e s s , s o m e t i m e s f l o c c u l e n t m a t e r i a l , o c c u p y i n g a space o f v a r y i n g w i d t h be tween the su r f ace of the os t eocy te and the m a t r i x of the bone. I t was s ep a r a t e d f r o m the m a t r i x by an o s m i o p h i l i c l a y e r about 3 0 m^ in d i a m e t e r . T h i s l a y e r w a s not v i s i b l e i n s ec t ions o f u n d e m i n e r a l i z e d bone, w h i c h i n d i c a t e d that i t was o c c u p i e d by bone c r y s t a l s , c a l l e d " c o a s t a l c r y s t a l s . "

T h e r e r e m a i n e d , h o w e v e r , some doubt as to whe the r the s o m e t i m e s f l o c c u l e n t m a t e r i a l o f the capsu le r e p r e s e n t e d , as w a s sugges ted , c o n n e c t ive t i s s u e g r o u n d subs tance and, be ing cont inuous w i t h the g r o u n d subs tance w i t h i n the H a v e r s i a n c a n a l s , c o u l d be c o n s i d e r e d the m a i n route o f t r a n s p o r t be tween the b l o o d and the bone m a t r i x . S ince o s m i u m t e t r o x i d e f i x a t i o n d i d not p r e s e r v e s u f f i c i e n t l y the f ine s t r u c t u r e of the os teocy te in d e m i n e r a l i z e d bone, and i n p a r t i c u l a r d i d not p r e s e r v e i ts p l a s m a m e m b r a n e , one m i g h t suspec t the f l o c c u l e n t m a t e r i a l a r o u n d the os t eocy te of a r i s i n g f r o m d i s i n t e g r a t i o n of the p e r i p h e r y of the c e l l . Thus , we thought i t d e s i r a ble to app ly to bone o ther f i x i n g agents in a d d i t i o n to o s m i c a c i d in an a t t empt to c o n f i r m and i m p r o v e the p r e v i o u s r e s u l t s . In the case of bone, i t has t u rned out that the use of d i f f e ren t f i x a t i v e s is not on ly d e s i r a b l e but n e c e s s a r y , s ince no s ing l e f i x a t i v e p r e s e r v e s a l l the s t r u c t u r a l d e t a i l s e q u a l l y w e l l .

M a t e r i a l s and M e t h o d s

A l l o f the bone w a s c h i p p e d f r o m the m i d - s h a f t o f the f e m u r . Bone f r o m m a t u r e dogs , gu inea p igs and r a t s w a s f i x e d i n bu f fe red o s m i u m t e t rox ide s o l u t i o n and d e c a l c i f i e d i n d i s o d i u m e t h y l e n e d i a m i n e t e t r a a c e t a t e ( E D T A ) , e i t h e r w i t h o r wi thou t bu f f e r i ng t o p H 7.3. Bone f r o m m i c e about th ree w e e k s o l d was f i x e d i n L u f f s p o t a s s i u m p e r m a n g a n a t e so lu t ion , (5) T a h m i s i a n ' s p e r m a n g a n a t e - o s m i u m f i x a t i v e , ( 6 / o r i n g lu t a r a ldehyde (^ ) f o l l o w e d b y p e r m a n g a n a t e - o s m i u m . T h i s m a t e r i a l was not d e m i n e r a l i z e d . F i n a l l y , bone f r o m young m i c e and m a t u r e r a t s was f i x e d i n g l u t a r a l d e h y d e , d e m i n e r a l i z e d i n E D T A a t p H 7.3, and p o s t f i x e d i n p o t a s s i u m p e r m a n g a n a t e -o s m i c a c i d . A l l o f the m a t e r i a l was embedded i n E p o n and s e c t i o n e d o n the P o r t e r - B l u m m i c r o t o m e w i t h a g l a s s k n i f e .

119

O b s e r v a t i o n s

F i g u r e 34 r e p r e s e n t s a s e c t i o n of m a t u r e r a t bone f i x e d in bu f fe red o s m i u m t e t r o x i d e and d e m i n e r a l i z e d i n E D T A . I t shows a n os teocy te i n i t s l a c u n a s u r r o u n d e d by the p e r i c e l l u l a r space , and the o s m i o p h i l i c l a y e r a d j a c e n t to the bone m a t r i x , w h i c h i s c h a r a c t e r i z e d by the l a m i n a e of c r o s s -banded c o l l a g e n f i b r i l s . C o m p a r e d w i t h the c e l l i n F i g u r e 37, th is o s t e o cyte i s not w e l l f i x e d . S ince the m e m b r a n e i s not c l e a r l y def ined , the few s t r u c t u r e s i n the p e r i c e l l u l a r space c o u l d be p a r t i c l e s o f c e l l u l a r o r i g i n .

Figure 34. Bone from femur of adult rat fixed in buffered osmium tetroxide, deminera l ized in E D T A . Section stained with uranyl acetate, a. osteocyte cytoplasm; b. per icel lular space; c. osmiophi l ic layer.

The m a t e r i a l o f F i g u r e 35 was p r e p a r e d a l s o f r o m the f e m u r of m a t u r e r a t and w a s f i x e d and s t a ined in the same w a y as that o f F i g u r e 34; i t p r e s e n t s a g a i n the c o n s p i c u o u s o s m i o p h i l i c l a y e r , w h i c h a l w a y s a p p e a r s i n th is w a y af ter o s m i u m f i x a t i o n . F i g u r e 3 5 shows m o r e c l e a r l y than F i g u r e 34 a zone of v e r y l oose t ex tu re be tween the o s m i o p h i l i c l a y e r and the r e s t of the d e c a l c i f i e d m a t r i x . In the m a t r i x the c r o s s - b a n d e d f i b r i l s a r e t h i c k e r and m o r e d e n s e l y p a c k e d than in the l o o s e - t e x t u r e d zone . In the space be tween the os teocy te and the o s m i o p h i l i c zone a few f i b r i l s m a y be seen .

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Figure 35. Bone from femur of adult rat, prepared as in Figure 34. Arrows indicate col lagen fibrils in per ice l lu lar space.

The os teocy te in F i g u r e 36 d e m o n s t r a t e s the s u p e r i o r i t y of p o t a s s i um pe rmangana t e f i x a t i o n for r e v e a l i n g the m e m b r a n e s t r u c t u r e s o f the c e l l , i n c l u d i n g the p l a s m a m e m b r a n e , a l though the n u c l e i c a c i d s s e e m to be w a s h e d out (no r i b o s o m e s or o ther dense m a t e r i a l s a r e seen in the n u c l e u s ) . The o s m i o p h i l i c l a y e r and the l o o s e l y s t r u c t u r e d zone beyond i t a r e not v i s i b l e , s i nce the bone was not d e m i n e r a l i z e d . The edges of bo th c e l l and m a t r i x a r e so w e l l de f ined that the r e a l i t y of the space a r o u n d the c e l l ( w h i c h is c e r t a i n l y not sh runken) cannot be doubted. The sugges t i on , m a d e r e p e a t e d l y in the past , that the os teocy te f i l l s the l a c u n a c o m p l e t e l y in l i v i n g bone i s no l o n g e r tenable i n v i e w o f these e l e c t r o n m i c r o s c o p i c f i n d i n g s . P o t a s s i u m pe rmangana te f i x a t i o n has a l s o p r e s e r v e d n u m e r o u s f i b r i l s w i t h i n the p e r i c e l l u l a r space ; these a re not v i s i b l e i n the l i g h t m i c r o s c o p e . They a re of v a r y i n g d i a m e t e r s , the t h i c k e r ones m e a s u r i n g about 250 A in w i d t h , and they a r e c l e a r l y c r o s s - b a n d e d . The l a r g e body and w e l l - d e v e l o p e d c y t o p l a s m i c s t r u c t u r e s ind ica te that th is os t eocy te was r e c e n t l y e n c a s e d and can be c o n s i d e r e d a "young" o s t eocy t e . C y t o p l a s m i c a c t i v i t y , sugges t ed by the s i z e and s t r u c t u r e of the c e l l , s e e m s to be i n d i c a t e d a l s o by the s m a l l b lebs w h i c h a re seen in m a n y p l a c e s on the s u r f a c e .

Figure 36. Bone from femur of two-week-o ld mouse fixed in permanganate. Section stained with lead citrate. Arrows indicate col lagen fibrils in per icel lu lar space.

Judged by the n u c l e a r - c y t o p l a s m i c r a t i o , the os teocy te of F i g u r e 37 is a l s o " y o u n g . " The s e c t i o n i s f r o m bone of a young m o u s e f i x e d in g l u t a r a l d e h y d e , d e m i n e r a l i z e d , and p o s t f i x e d i n p e r m a n g a n a t e - o s m i u m . T h i s f i x a t i v e p r e s e r v e s the n u c l e a r and c y t o p l a s m i c s t r u c t u r e s and the c e l l m e m b r a n e e x c e l l e n t l y , but the co l l agenous m a t e r i a l i n both the m a t r i x and the capsu l e of the os teocy te is not as c l e a r l y def ined as in the o s m i u m -f i x e d s p e c i m e n s . I t i s of s p e c i a l i n t e r e s t that the d a r k l i ne that s tands out i n the o s m i u m - f i x e d m a t e r i a l i s r e p r e s e n t e d i n F i g u r e 37 (and a l s o i n F i g u r e 38) by a c o r r e s p o n d i n g l a y e r whose e l e c t r o n dens i t y i s somewha t h i g h e r than that of the m a t r i x and the c a p s u l e . The r e a l i t y of the l a y e r that con ta ins the " c o a s t a l c r y s t a l s " has thus been d e m o n s t r a t e d by two d i f f e ren t f i x i n g m e t h o d s .

The s p e c i m e n in F i g u r e 38 shows a g a i n the c h a r a c t e r i s t i c s o f g l u t a r a l d e h y d e and p e r m a n g a n a t e - o s m i u m f i x a t i o n . T h e r e a r e , h o w e v e r , s i g n i f i c a n t d i f f e r e n c e s be tween th is case and that of F i g u r e 37: the c y t o p l a s m i c body o f the os t eocy te i s r e l a t i v e l y s m a l l and the c e l l su r f ace i s s m o o t h , w h i c h i n d i c a t e s that th is c e l l i s not as a c t i v e as that in F i g u r e 37

Figure 37. Bone from femur of two-week-old mouse fixed in glutaraldehyde, demineralized in EDTA, postfixed in permanganate-osmium. Section stained with lead citrate. Arrows indicate osmiophilic layer. The pericellular space is inside this layer.

Figure 38. Bone from femur of adult rat prepared as in Figure 37. Arrows indicate pericellular space.

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the p e r i c e l l u l a r space i s v e r y n a r r o w and does not c o n t a i n f i b r i l s ; and, f i n a l l y , there is a b r o a d zone be tween the out l ine of the p e r i c e l l u l a r space and the m a t r i x p r o p e r . T h i s zone c o r r e s p o n d s to the l o o s e l y s t r u c t u r e d zone in the o s m i u m - f i x e d s p e c i m e n s , w h e r e i t i s m o r e c o n s p i c u o u s but p r o b a b l y not a s w e l l f i x e d as in the g l u t a r a l d e h y d e p r e p a r a t i o n . S ince th is l a y e r has now been shown by two f i x i n g m e t h o d s , i t s r e a l i t y s e e m s c e r t a i n . S ince the os t eocy te in F i g u r e 35 i s f r o m the f e m u r of an adul t r a t we f e e l j u s t i f i e d in a s s u m i n g that the d i f f e r e n c e s be tween the o s t eocy t e s and t h e i r s u r r o u n d i n g s t r u c t u r e s in F i g u r e s 37 and 38 a re due to a d i f f e r ence in the ages of the c e l l s .

In o r d e r to show the v a r i a b i l i t y one encoun te r s in s a m p l i n g o s t e o cy tes f r o m d i f f e ren t m a t e r i a l s , the o s m i c - a c i d f i x e d c e l l i n F i g u r e 3 9 i s p r e s e n t e d . In th i s case the space a r o u n d the c e l l i s f i l l e d w i t h an u n u s u a l l y l a r g e n u m b e r o f r e l a t i v e l y t h i c k f i b e r s . The s i t u a t i o n sugges ts that under c e r t a i n , as ye t unknown, cond i t i ons the os teocy te m a y func t i on as a f i b r o b l a s t . That the f i b r i l s of the capsu le a r e a t some p l a c e s cont inuous w i t h those of the m a t r i x and that the l o o s e l y s t r u c t u r e d zone adjacent to the o s m i o p h i l i c l a y e r i s m i s s i n g m a y be r e l a t e d to the abundant f i b e r f o r m a t i o n .

Figure 39. Bone from femur of adult rat prepared as in Figure 34. a. large amount of col lagen in per ice l lu lar space; b. c a n a l i c u l i

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In F i g u r e 39 > two p r o c e s s e s of an osteocyte ( ind ica ted by a r r o w s ) are seen in the i r c a n a l i c u l i . We want to emphas ize that in no case have f i b r i l s been o b s e r v e d in the c a n a l i c u l i , a l though the other s t ruc tu re s des c r i b e d in the lacunae w e r e present . I t i s a l so impor t an t that in a l l c a n a l i c u l i obse rved , inc lud ing those l e s s than 1 jd in d i ame te r , c e l l p r o c e s s e s cou ld be ident i f ied .

Conc lus ions

1. It has been shown that m o r e complete i n f o r m a t i o n about the fine s t ruc tu re of bone can be obtained by combin ing the r e su l t s of s e v e r a l f i x i n g agents. In this r espec t , the exper i ence gained in the p re sen t study m a y be va luable for e l e c t r o n m i c r o s c o p i c i nves t iga t ion of any b i o l o g i c a l s t r u c t u r e .

2. The demons t r a t i on of a d i s t i nc t i ve l a y e r of g rea t e r e l e c t r o n dens i ty than e i ther the bone m a t r i x or the capsule of the osteocyte in g l u t a r a l d e h y d e - f i x e d bone shows that the o s m i o p h i l i c l a y e r p r e v i o u s l y d e s c r i b e d ^ ) i s not a r t i f i c i a l l y p roduced by depos i t ion of o s m i c a c i d a t the edge of the m a t r i x but r ep resen t s a l a m i n a e x i s t i n g in v i v o that is a pa r t of the t r anspo r t s y s t e m between the b lood and the bone m a t r i x .

3. It was a l so c o n f i r m e d that the space su r round ing the os teocyte co r r e sponds to the i n t e r c e l l u l a r m a t e r i a l of connect ive t i s sue , and that i t is not c o r r e c t to c h a r a c t e r i z e i t as t i s sue f l u i d , as has been done in the past, Th i s has been shown d e f i n i t i v e l y by the d e m o n s t r a t i o n of col lagenous f i b r i l s w i t h i n the space. I t i s , therefore , c e r t a i n that the i n t e r f i b r i l l a r m a t e r i a l of the capsule of the osteocyte r ep resen t s in bone, as in any other connect ive t i s sue , the m e d i u m of t r anspo r t between the b lood and both the c e l l s and the m a t r i x .

4. Since the s m a l l e s t c a n a l i c u l i we re found to conta in c y t o p l a s m i c p r o c e s s e s of the os teocytes , it is not c o r r e c t to a s sume that, as a r u l e , the osteocyte r e t r a c t s i ts p r o c e s s e s .

5. The f o r m a t i o n of f i b r i l s w i t h i n the lacunae, w h i c h can be so abundant as to amount to a k i n d of f i b r o s i s , shows that the os teocyte , after i ts enc lo su re in the l acuna , re ta ins at l eas t in pa r t the a c t i v i t y of the os teoblas t ,

6 . The zone outside the o s m i o p h i l i c l a m i n a , w h i c h d i f fe rs in s t ruc ture f r o m the r e s t of the m a t r i x , m a y w e l l c o n s i s t of bone that was l a i d down s e c o n d a r i l y after the enc lo su re of the os teoblas t . T h i s point needs fur ther inves t iga t ion . I f such secondary m i n e r a l i z a t i o n a c t u a l l y o c c u r s , one m u s t conclude that the d i s t i n c t i o n between os teob las t and osteocyte i s , f r o m the func t iona l point of v i e w , a r a the r a r b i t r a r y one; fo r the change in funct ion seems to be g radua l and p o s s i b l y r e v e r s i b l e .

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R e f e r e n c e s

1 . B a u d , C . A . M o r p h o l o g i e e t s t r u c t u r e i n f r a m i c r o s c o p i q u e des o s t e o -c y t e s , A c t a A n a t . J51, 209-225 (1962).

2 . B a u d , C . A , , and P . W . M o r g e n t h a l e r , S t r u c t u r e s u b m i c r o s c o p i q u e d u r e b o r d l a c u n o - c a n a l i c u l a i r e o s s e u x . M o r p h . J a h r b , 104, 476-486 (1963).

3 . L i p p , W. N e u u n t e r s u c h u n g e n des K n o c h e n g e w e b e s . A c t a A n a t . 20, 162-200 (1954).

4 . W a s s e r m a n n , F . 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 the w a l l o f the l acunae and c a n a l i c u l i i n bone. A r g o n n e N a t i o n a l L a b o r a t o r y B i o l o g i c a l and M e d i c a l R e s e a r c h D i v i s i o n S e m i a n n u a l R e p o r t , J u l y t h r o u g h D e c e m b e r 1961. A N L - 6 5 3 5 , pp. 129-138 (1962),

5 . Lu f t , J . H , P e r m a n g a n a t e , a new f i x a t i v e f o r e l e c t r o n m i c r o s c o p y . J . B i o p h y s . B i o c h e m , C y t o l . _2, 799-801 (1956).

6 . T a h m i s i a n , T . N. U s e of the f r e e z i n g po in t m e t h o d to adjust the t o n i c i t y of f i x i n g s o l u t i o n s , J . U l t r a s t r u c t . R e s . 10, 182- 188 (1964).

7 . S a b a t i n i , D , D . , K . B e n s c h , and R . J . B a r r n e t t . C y t o c h e m i s t r y and e l e c t r o n m i c r o s c o p y . The p r e s e r v a t i o n o f c e l l u l a r u l t r a s t r u c t u r e and e n z y m a t i c a c t i v i t y by a ldehyde f i x a t i o n . J . C e l l . B i o l . _17, 19 (1963).

V A R I O U S A S P E C T S O F F I X A T I V E S F O R U L T R A T H I N S E C T I O N S A N D R E S O L U T I O N I N E L E C T R O N M I C R O S C O P Y

Theodore N . T a h m i s i a n , C a r o l J . C h r i s t i a n s e n , R o s e m a r i e L . Dev ine , and Bet ty Jean W r i g h t

There are two ma jo r r equ i r emen t s for a f ixa t ive used in e l e c t r o n m i c r o s c o p y : f i r s t , i t m u s t impregnate the t i ssue w i t h a heavy e lement w h i c h w i l l abso rb e l ec t rons under the beam in p r o p o r t i o n to its depos i t ion ; second, i t mus t produce m i n i m u m d i s t o r t i o n of the t i s sue . O s m i u m , pe r manganate, d i ch roma te , and c e r t a i n a ldehydes , such as f o r m a l i n and g lu ta ra ldehyde , are a t p resent used for fine f ixa t ion . The f i r s t three are o x i d i z i n g agents that a re reduced by the t i s sue . T h e i r m e t a l l i c e lements f o r m complexes w i t h the unsatura ted bonds of s u b c e l l u l a r reductants . These heavy e lements are able to absorb e lec t rons m o r e e f f i c ien t ly than the t i ssue i t s e l f and thus p rov ide con t ras t for obse rv ing and photographing the spec imen . A d d i t i o n a l con t ras t m a y be gained by fur ther s t a in ing . The aldehydes act as p ro t e in -dena tu r ing compounds, but s ince o rgan ic c o m pounds are not by themse lves e l e c t r o n a b s o r b e r s , the a ldehyde - f ixed t i s sue m u s t l a t e r be s ta ined w i t h a heavy e l emen t -con ta in ing compound such as u r a n y l acetate, o s m i u m te t rox ide , l ead c i t r a t e , p o t a s s i u m permanganate , go ld ch lo r ide or phosphotungst ic a c i d ,

In o rde r to obtain fine f ixa t ion , we have found that the ton ic i ty of the f i x ing agent should be equivalent to i so ton ic s a l i n e , S o m e of the m e t a l l i c compounds are r e l a t i v e l y in so lub le , so that the i r i on ic s t rength in the concent ra t ions used is l ower than that of n o r m a l s a l ine . The a ldehydes , w h i c h do not i on i ze , cannot be used at h igh concent ra t ions because denatura t ion of the p ro t e in p roceeds too r ap id ly and t i s sue d i s t o r t ion r e s u l t s . On the other hand, at low concent ra t ions (5 or 10 per cent) they are hypotonic , We have used the f r e e z i n g - p o i n t - d e p r e s s i o n me thod( 1 ) to de te rmine ion ic s trengths of the v a r i o u s f ixa t ives and then have added r e q u i r e d amounts of sal ts to a r r i v e at the p r o p e r ton ic i ty ,

The ma te r i a l , used throughout to test f i xa t i on was g ra s shoppe r t e s t i s . F o r this t i s sue , the ion ic s t rength of the f i x ing so lu t ion is equ iva lent to 0,9% N a C L Such a so lu t ion w i l l hereaf ter be r e f e r r e d to as n o r m a l sa l ine equivalent (NSE) . However , n o r m a l sa l ine for other t i s sues m a y differ in sa l t concent ra t ion , The ion ic s t rength of n o r m a l sa l ine for a g iven t i ssue m u s t be e s t ab l i shed and the f i x ing so lu t ion adjusted a c c o r d i n g l y .

We have inves t iga ted the effects upon the t i s sue of v a r i o u s supp l e me n ta ry sal ts such a s N a C L K C L M g C l 2 and C a C l 2 , used s ing ly a s w e l l as in ba lanced [e.g. , B e l a r ( 3 ) ] sa l ine so lu t ions , The f i x i n g agents used in this phase o f the inves t iga t ion were O s 0 4 and K M n 0 4 . When N a C l was used as the supp lementa ry sal t , the t i s sue m a t r i x was f ine ly g r a n u l a r , Howeve r ,

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under n o r m a l p h y s i o l o g i c a l condi t ions , p o t a s s i u m , ra ther than s o d i u m , i s found in c y t o p l a s m . When K C 1 was used in the f ixa t ive the c e l l u l a r contents appeared c o a r s e l y g r a n u l a r . Th i s m a y or m a y not be n o r m a l ; in any case the effect is dec ided ly dif ferent f r o m that p roduced by the N a C l - s u p p l e m e n t e d so lu t ion . M g C . l 2 and C a C l 2 enhanced the d e m a r c a t i o n of m e m b r a n e s . On the other hand, a ba lanced sa l ine supplement p roduced a smooth c e l l outl ine w i t h r e l a t i v e l y d i s t i n c t m e m b r a n e s . We have conc luded that enhancement or r e p r e s s i o n of s t ruc tu re s w i t h i n the c e l l can be a funct ion of the sa l t used in the f ixa t ive and that this should be c o n s i d e r e d v e r y c a r e f u l l y in the e v a l u a t ion o f e l e c t r o n m i c r o g r a p h s ,

Unbuffered f i xa t i ve s adjusted to n o r m a l sa l ine ton ic i ty u s u a l l y have an a c i d p H , The c o m m o n l y - u s e d o s m i u m f ixa t ive i s buffered to pH 7,4 w i t h v e r o n a l - s o d i u m acetate, a buffer never found n o r m a l l y in c e l l s . Luft(4) r e c e n t l y r e p o r t e d that unna tura l buffers such as v e r o n a l and arsenate r e move a c o n s i d e r a b l e amount of p r o t e i n f r o m the t i s sues du r ing f i xa t i on ; this was shown by l a b e l i n g p ro te ins w i t h C and demons t r a t ing that some of the l a b e l e d m a t e r i a l was l eached out du r ing f i xa t i on . He found that f i xa t i ve s buffered w i t h phosphate, a n o r m a l component of c e l l s , ex t r ac t ed the l eas t p r o t e i n d u r i n g f i xa t i o n . In our e x p e r i m e n t s N S E f ixa t ives we re buffered w i t h phosphate a t pH 5.8 to 7,3, F r o m p r e l i m i n a r y w o r k , i t appears that as the i s o e l e c t r i c point of p ro te ins is approached m o r e g r a n u l a r i t y is seen throughout the c e l l . Since the i s o e l e c t r i c points of v a r i o u s p ro te ins d i f fe r , i t w i l l be e s s e n t i a l in th£ future to use f i x i n g so lu t ions of d i f ferent pH v a l ues to obta in s p e c i f i c i n f o r m a t i o n , We have noted the b reakdown of the nebenkern or the d i sappearance of c e n t r i o l e s in s p e r m a t i d m a t e r i a l when pH was a l t e r e d . I t i s a l s o known that m i t o c h o n d r i a tend to d i sappea r in a c i d f ixa t ives , (5 ) We expect to pursue the p r o b l e m of pH s p e c i f i c i t y in g rea t e r d e t a i l ,

F i g u r e s 40 and 41 a re e l e c t r o n m i c r o g r a p h s of g r a s s h o p p e r s p e r m a tocytes f i x e d in K M n 0 4 a t two di f ferent p H ! s , Note that in the t i s sue f i x e d at pH 6 ( F i g u r e 40) the nucleus has d i s t i n c t a reas of g r a n u l a t i o n w h i c h p r o v ide g rea t e r c h r o m a t i n d e t a i l . At pH 7,3 ( F i g u r e 41) the nucleus i s a l m o s t e n t i r e l y g r a n u l a r w i t h l i t t l e , i f any, c h r o m a t i n d i s t i n c t i o n . The G o l g i appara tus appears d i s t i n c t l y di f ferent a t the two p H ! s . At pH 7,3 the v a c u o l a r s t r u c t u r e a s s o c i a t e d w i t h the apparatus i s no t i ceab le , whe reas a t pH 6 the l a m e l l a r p o r t i o n i s w e l l ou t l ined and the v a c u o l a r p o r t i o n i s l e s s d i s t inc t . D i f f e r e n c e s a re a l s o seen in the e n d o p l a s m i c r e t i c u l u m , w h i c h has a d i s t i n c t m e m b r a n e and an absence of r i b p s o m e s at pH 6, w h i l e at pH 7.3 the m e m b r a n e i s l e s s c l e a r and r i b o s o m e s are v i s i b l e . M i t o c h o n d r i a l , m e m b r a n e s appear s ingle at pH 7.3 but a re def in i te ly double at pH 6, The dens i ty of s p e c i f i c s t r u c t u r e s in the i n t e r c e l l u l a r m a t e r i a l d i f fe rs a t the two p H ' s , The best r e s u l t s to date on g ra s shoppe r tes t i s m a t e r i a l have

Figure 40. Grasshopper spermatocyte fixed in NSE, K M n O ^ , phosphate buffer pH 6.0. Lead citrate stained. Within the nucleus the chromatin material is distinct and granules of two sizes can be seen in the karyoplasm. The membranous components of the ce l l are distinct and are enhanced by the fine textured matrix of the cytoplasm. The endoplasmic reticulum appears smooth and does not show ribosome attachment. The Golgi apparatus appears to be mainly lamellar with a few indistinct vacuoles.

Figure 41. Grasshopper spermatocyte fixed in NSE, KMnO^, phosphate buffer, pH 7.3. Uranyl acetate stained. Within the nucleus the chromatin masses are almost indistinguishable from the remainder of the karyoplasm. The cytoplasmic matrix is granular and the membranous structures appear coarse, a l though ribosomes are visible along the endoplasmic reticulum. The Golgi apparatus appears to be mainly vacuolar with only a slight indistinct membranous portion.

IS - Intercellular space M - Mitochondrion NU -Nucleus CH - Chromosome GO - Golgi apparatus ER - Endoplasmic reticulum

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been obtained w i t h f ixa t ive of the fo l lowing c o m p o s i t i o n :

K M n 0 4 : 0.13 g

Buffer (pH 5,9): 1.20 ml

M / l 5 K H 2 P 0 4 , 9 m l ;

M / l 5 N a 2 H P 0 4 , 1 m l ;

9% N a C L 10 ml

H 2 0 , t o make 10.0 m l

F i n a l p H : 6

We w i l l next at tempt to use manganese and a l u m i n u m sal ts i n the f ix ing so lu t ion , because these sa l ts a re ca ta lys t s for c e r t a i n enzymes app l i ed to t i s sue sec t ions in c y t o c h e m i c a l s tudies .

We fee l i t m a y someday be pos s ib l e to define the f i xa t i on capac i ty of a g iven so lu t ion by v i r t ue of a l l of i ts consti tuents and the i r concen t ra t ions . Such s p e c i f i c i t y of f i x i n g and s ta in ing p rocedures wou ld g rea t ly i n c r e a s e the usefulness o f e l e c t r o n m i c r o s c o p y for b i o l o g i c a l m a t e r i a l s .

Refe rences

1. T a h m i s i a n , T, N. Use of the f r e e z i n g point method to adjust the ton ic i ty of f i x i n g so lu t ions , J . U l t r a s t r u c t u r e Res , 10, 182-188 (1964).

2 . T a h m i s i a n , T. N. A method for obtaining f i x i n g solut ions o s m o t i c a l l y equivalent t o - p h y s i o l o g i c a l sa l ine so lu t ion , Argonne Na t iona l L a b o r a t o r y B i o l o g i c a l and M e d i c a l R e s e a r c h D i v i s i o n , Semiannua l Repor t , J u l y through D e c e m b e r , 1961. A N L - 6 5 3 5 , pp. 127-128.

3 . B e l a r , K. B e i t r a g e z u r K a u s a l a n a l y s e der m i t o s e . II, Unte rsuchungen an den s p e r m a t o c y t e n von Chor th ippus (Stenopothrus) l ineatus panz. A r c h . E n t w i c k l n g s m e c h n . Organ . 1 18, 359-484 (1929).

4 . Luf t , J , H . , and R, L, Wood, The e x t r a c t i o n of t i ssue p r o t e i n dur ing and after f i x a t i o n w i t h o s m i u m te t roxide i n v a r i o u s buffer s y s t e m s . J . C e l l B i o l . J19, 46a (1963).

5 . R o u i l l e r , C . P h y s i o l o g i c a l and pa tho log i ca l changes in m i t o c h o n d r i a l m o r p h o l o g y . Intern. Rev , C y t o l . 9, 227 ( i960) .

130

P U B L I C A T I O N S

J u l y 1 , 1963 t h r o u g h D e c e m b e r 31 , 1963

B a s e r g a , R 0 J a n d W . E . K i s i e l e s k i . A u t o b i o g r a p h i e s o f C e l l s , S c i 0 A m . 209(2 ) . 1 0 3 - 1 1 0 ( A u g u s t 1963)

B a s e r g a , R U J a n d W . E , K i s i e l e s k i . E f f e c t s o f H i s t o l o g i c and H i s t o c h e m i -c a l P r o c e d u r e s o n the I n t e n s i t y o f the L a b e l i n R a d i o a u t o -g r a p h s o f C e l l s L a b e l e d w i t h T r i t i a t e d C o m p o u n d s * J « Lab . . I n v e s t , 1̂ 2, 6 4 8 - 6 5 6 (1963) .

B a s e r g a , R „ , S „ A . T y l e r , a n d W „ E „ K i s i e l e s k i . T h e K i n e t i c s o f G r o w t h o f the E h r l i c h T u m o r , A r c h . P a t h o l . 76, 9 - 1 3 (1963 ) .

B l a c k , C . C , C , A . F e w s o n , M , G i b b s , a n d S . A , G o r d o n , S t u d i e s o n P h o t o -s y n t h e t i c P r o c e s s e s . III . F u r t h e r S t u d i e s o n A c t i o n S p e c t r a a n d Q u a n t u m R e q u i r e m e n t s f o r A d e n o s i n e T r i p h o s p h a t e R e d u c t i o n a n d the F o r m a t i o n o f T r i p h o s p h o p y r i d i n e N u c l e o t i d e b y S p i n a c h C h l o r o p l a s t s . J , B i o l . C h e m , 2_38;. 3 8 0 2 - 3 8 0 5 ( N o v e m b e r 1963) .

B r u e s , A . M . P h y s i c a l A g e n t s a s C a u s a t i v e F a c t o r s . . S y m p o s i u m o f the A m e r i c a n C a n c e r S o c i e t y , N e w Y o r k , O c t o b e r 2 2 - 2 3 , 1962 . A m . C a n c e r S o c , I n c . , N e w Y o r k , 1963 , p p . 2 6 - 3 0 .

D a n i e l s , E . W 0 ) a n d E e P . ' B r o w n . C o m p l e x P a t t e r n s i n the M i t o c h o n d r i a o f a R a d i o s e n s t i v i e G i a n t A m o e b a . J . P r o t o z o o L 10 s 1 5 ( A u g u s t 1 963) 0 A b s t r a c t .

D a n i e l s , E . W . , E 0 P . B r o w n , a n d Y , E . R a h m a n . S t r u c t u r a l a n d T o x i c i t y E f f e c t s o f V a r i o u s H o m o g e n a t e M e d i a o n S u b c e l l u l a r C o m p o n e n t s of A m o e b a e . J . C e l l B i o l . ^ 9 ( 2 ) , 8 5 A ( 1 9 6 3 ) . A b s t r a c t ,

D a n i e l s , E . W. , a n d N . R e e d . G o l g i F i n e S t r u c t u r e i n a R a d i o s e n s i t i v e G i a n t A m o e b a . P r o c . 16th I n t e r n . C o n g r . Z o o l o g y , W a s h i n g t o n , D . C , A u g u s t 2 0 - 2 7 , 1963 . V o l . 2 , p . 191 . A b s t r a c t ,

E h r e t , C . F . , a n d G . D e H a l l e r . O r i g i n , D e v e l o p m e n t a n d M a t u r a t i o n o f O r g a n e l l e s a n d O r g a n e l l e S y s t e m s o f the C e l l S u r f a c e i n P a r a m e c i u m , J e U l t r a s t r u c t u r e R e s . S u p p l , 6 , 3 - 4 2 ( O c t o b e r 1963) .

F e i n s t e i n , R . N . , a n d J , E . S e a h o l m . E f f e c t o f a S e r o t o n i n A n t a g o n i s t o n R a d i a t i o n L e t h a l i t y , P r o c . S o c . E x p t l , B i o L M e d . 1 14(1), 2 4 7 - 2 4 8 ( O c t o b e r 1963) .

131

P U B L I C A T I O N S (Contd.)

F l y n n , R . J . The D i a g n o s i s o f Some F o r m s o f E c t o p a r a s i t i s m o f M i c e L a b . A n i m a l C a r e 1_3_(2), 111-125 ( A p r i l 1963).

F l y n n , R . J . The D i a g n o s i s and C o n t r o l o f E c t r o m e l i a Infec t ion o f M i c e . L a b . A n i m a l C a r e 13(2), 130-136 ( A p r i l Supp l . 1963).

F l y n n , R . J . The D i a g n o s i s o f P s e u d o m o n a s a e r u g i n o s a Infect ion o f M i c e . L a b . A n i m a l C a r e 13(2), 126- 129 ( A p r i l Supp l . 1963).

F l y n n , R , J . , I . L . G r e c o , and P . B . J i n k i n s . A D i s e a s e o f M i c e C h a r a c t e r i z e d by L u n g C o n g e s t i o n . L a b . A n i m a l C a r e 13(4), P a r t 1 , 499-501 (Augus t 1963).

F r i g e r i o , N . A . , and R . F 0 C o l e y . C o m p l e x e s o f U r a n y l Ion w i t h D i m e t h y l -f o r m a m i d e and S e m i c a r b a z i d e . J . I n o r g . N u c l . C h e m . 25(9), 1111-1113 (September 1963).

F r i g e r i o , N . A . The S c i n t i l l a t i o n Count ing o f I o d o f o r m and Other A l k y l H a l i d e s . In te rn . J , A p p l . R a d i a t i o n Isotopes 14(6), 3 30-331 (Augus t 1963) . Note .

F r y , R . J . M . , S . L e s h e r , A . R . S a l l e s e , and E . F . Staffeldt . The G e n e r a t ion C y c l e o f Duodena l C r y p t C e l l s o f M i c e E x p o s e d t o 220 Roentgens of C o b a l t - 6 0 G a m m a I r r a d i a t i o n per Day . R a d i a t i o n R e s . 1_9, 628-635 (August 1963).

G r a h n , D . and J . K r a t c h m a n . V a r i a t i o n i n Neona t a l Dea th Rate and. B i r t h Weigh t i n the U n i t e d States and P o s s i b l e R e l a t i o n s to E n v i r o n m e n t a l R a d i a t i o n , Geo logy and A l t i t u d e , A m . J . H u m a n Genet . 1_5(4), 329-352 ( D e c e m b e r 1963)..

G u s t a f s o n , P . F , and S . S . B r a r . E x t e r n a l G a m m a - R a y Dose f r o m S h o r t -L i v e d F i s s i o n P r o d u c t s f r o m N u c l e a r Weapon T e s t s . H e a l t h Phys ._9 , 629-634 (June 1963).

H u m p h r e y , R . M . , and W . K . S i n c l a i r , The R e l a t i v e B i o l o g i c a l E f f e c t i v e n e s s o f 2 2 - M e v p X - R a y s , C o b a l t - 6 0 G a m m a R a y s and 200 K v c p X - R a y s , VII I . D e t e r m i n e d by the G r o w t h C a p a c i t y o f T u m o r C e l l s i n V i t r o . R a d i a t i o n Re s._20, 593-599 ( D e c e m b e r 1963).

J a r o s l o w , B . N , , T . G Baf f e s , and F . H . K e t o l a . A t t e m p t s t o E s t a b l i s h Immune T o l e r a n c e b y M a s s i v e O r g a n T r a n s p l a n t a t i o n . T r a n s , A m . Soc , A r t i f i c i a l I n t e rna l Organs 9 , 135-138 (1963).


K a s p a r , L . V . , a n d L , S . L o m b a r d , N u t r i t i o n a l Myodegene ra t ion i n a L i t ter o f Beag le s . J . A m . Vet . M e d , A s s o c , 143, Z84-288 (August 1, 1963).

Kub i t s chek , H . E , D N A . Argonne Na t iona l L a b o r a t o r y Rev iews 1(1), 3-9 (September 1963).

Kuet tner , K . E . , and R. W, Swick . M u c o p r o t e i n E x t r a c t s o£ Oss i fy ing C a r t i lage. F e d e r a t i o n P r o c . ZZ[Z): 647 ( M a r c h - A p r i l 1963). A b s t r a c t

L e s h e r , S., R . J , M , F r y , W , E . K i s i e l e s k i , and G . A . Sacher . C e l l P r o l i f e r a t ion i n the S m a l l Intestine: A n E m p i r i c i s t ' s A p p r o a c h t o a B i o l o g i c a l P r o b l e m . P r o c . Guinness Symp. on C e l l P r o l i f e ra t ion , Dub l in , I re land , Augus t 14-15, 1962, ed. L . F . L a m e r t o n and R . J . M . F r y . B l a c k w e l l S c i . P u b h , L t d . , Oxfo rd , Eng land , 1963. pp. 213-233.

L indenbaum, A . M u c o p o l y s a c c h a r i d e s and Mucopro t e in s o f O s s i f y i n g C a r t i lage. F e d e r a t i o n P r o c . 22(2), 647 ( M a r c h - A p r i l 1963). A b s t r a c t

M o t a , I . B i o l o g i c a l C h a r a c t e r i z a t i o n o f " M a s t C e l l Sens i t i z i ng ' ' A n t i b o d i e s . L i f e Sc iences No . 7, 465-474 (1963).

Nebe l , B . R . , E , M , Coulon , and D . J . M c W h i n n i e . Some Effects o f C h r o n i c G a m m a I r r a d i a t i o n on Mouse T e s t i s . C y t o l o g i a 28, 81-95 (1963).

Pat t , H. M', Quanti tat ive Aspec t s of Rad ia t i on Effec ts at the T i s s u e and T u m o r L e v e l . A m . J . Roentgenol . 90, 928-937 (November 1963).

Pat t , H . M . , and H . Quas t l e r . Rad ia t i on Effects o n C e l l R e n e w a l and R e l a t e d Sys t ems , P h y s i o l , Rev . 43, 357-396 (July 1963).

Rahman, Y . E . Effec t o f X - I r r ad ia t ion on the F r a g i l i t y o f Rat Spleen L y s o s o m e s . R a d i a t i o n R e s . ^20, 741-750 (December 1963).

Sacher , G. A. Effects o f X - R a y s on the S u r v i v a l o f D r o s o p h i l a Imagos. P h y s i o l . Z o o l . 36(4), 295-311 (October 1963).

Sacher , G . A . R e c o v e r y f r o m R a d i a t i o n Damage. E n c y c l o p e d i a o f X - R a y s and G a m m a R a y s , ed. G . L , C l a r k . R e i n h o l d P u b l i s h i n g C o m pany, New Y o r k , 1963. pp. 931-934,

133


S i n c l a i r , W . K , A b s o r b e d D o s e i n B i o l o g i c a l S p e c i m e n s I r r a d i a t e d E x t e r n a l l y w i t h C o b a l t - 6 0 G a m m a R a d i a t i o n . R a d i a t i o n R e s . Z0, 288-297 (Oc tobe r 1963).

S i n c l a i r , W . K . , and R . A . M o r t o n . V a r i a t i o n s i n X - R a y R e s p o n s e d u r i n g the D i v i s i o n C y c l e o f P a r t i a l l y S y n c h r o n i z e d C h i n e s e H a m s t e r C e l l s in C u l t u r e . N a t u r e 199, 1 158-1 160 (Sep tember 21, 1963).

S l a t i s , H . M . , and A . J . F i n k e l . The Study o f N o r m a l V a r i a t i o n i n M a n . I . I n t e r r e l a t i o n s o f A d i p o s i t y , A n c e s t r y and B l o o d T y p e . A m . J . H u m a n Genet . 1 5 (4), 398-407 ( D e c e m b e r 1963).

S t e a m e r , S . P . , and S , A . T y l e r . R a d i a t i o n M o r t a l i t y i n the M o u s e : M o d e l o f the K i n e t i c s o f In jury A c c u m u l a t i o n . I . P r o t r a c t e d D o s e s in the 3 0 - D a y L e t h a l R a n g e . R a d i a t i o n R e s . Z0, 619-630 ( D e c e m b e r 1963).

S t r a u b e , R . L . E x a m i n a t i o n o f D i u r n a l V a r i a t i o n i n L e t h a l l y I r r a d i a t e d R a t s . S c i e n c e 142, 1062 ( N o v e m b e r 22, 1963).

S u r r e y , K . A z o - C o u p l i n g R e a c t i o n s o f P r o t e i n - B o u n d F r e e A m i n o G r o u p s i n P l a n t T i s s u e s . A n n . H i s t o c h i m . jB, 149-156 (1963).

S u r r e y , K . A z o - C o u p l i n g R e a c t i o n o f P r o t e i n - B o u n d I m i n o G r o u p s i n P l a n t T i s s u e s . A n n . H i s t o c h i m . _8, 157-164 (1963).

S u r r e y , K . S p e c t r a l R e s p o n s e and I n t e r a c t i o n o f V a r i o u s Wave l eng th s o n the Phospha t e M e t a b o l i s m o f G e r m i n a t i n g L e t t u c e Seeds . P r o c . In t e rn . S y m p . o n P h y s i o l o g y , E c o l o g y and B i o c h e m i s t r y o f G e r m i n a t i o n , G r e i i s w a l d , E a s t G e r m a n y , S e p t e m b e r 8 -14, 1963. I t e m A - V I I - d 7 . A b s t r a c t .

T a l i a f e r r o , W . H . C e l l u l a r and H u m o r a l F a c t o r s i n I m m u n i t y t o P r o t o z o a , I m m u n i t y t o P r o t o z o a , ed . T . C . C . G a r n h a m , A . E , P i e r c e and I . R o i t t . B l a c k w e l l S c i , P u b l r s . , O x f o r d , 1963. pp. 22-38.

T a l i a f e r r o , W . H . , and L . G . T a l i a f e r r o . The E f f e c t o f A n t i g e n Dosage o n the F o r s s m a n H e m o l y s i n R e s p o n s e i n R a b b i t s . J . Infect , D i s e a s e s 1J_3, 155-169 ( N o v e m b e r - D e c e m b e r 1963),

T a l l e n t i r e , A . , and E . L . P o w e r s . M o d i f i c a t i o n o f S e n s i t i v i t y t o X - i r r a d i a t i o n by W a t e r i n B a c i l l u s m e g a t e r i u m . R a d i a t i o n R e s . 20. 270 -287 (Oc tobe r 1963). ~

P U B L I C A T I O N S (Contd . )

T r u c c o , E , O n the F o k k e r - P l a n c k E q u a t i o n i n the S t o c h a s t i c T h e o r y o f M o r t a l i t y , B u l l , M a t h . B i o p h y s . 25(3), 303-323 ( S e p t e m b e r 1963),

T r u c c o , E . O n the F o k k e r - P l a n c k E q u a t i o n i n the S t o c h a s t i c T h e o r y o f M o r t a l i t y . II. B u l l . M a t h . B i o p h y s . 25(4), 343- 366 (1963).

V o g e l , H . H , , J r . , and D , L . J o r d a n , F r a c t i o n a t e d I r r a d i a t i o n o f M i c e w i t h F i s s i o n N e u t r o n s and C o G a m m a R a y s , I n t e r n . A t o m i c E n e r g y A g e n c y S y m p . o n B i o l o g i c a l E f f e c t s o f N e u t r o n I r r a d i a t i o n , B r o o k h a v e n N a t i o n a l L a b o r a t o r y , N e w Y o r k , O c t o b e r 7 - 1 1 , 1963. A b s t r a c t s o f P a p e r s . S M - 4 4 / l 6 .

Webb , R . B . , a n d H . E . K u b i t s c h e k . M u t a g e n i c and A n t i m u t a g e n i c E f f e c t s o f A c r i d i n e O r a n g e i n E s c h e r i c h i a c o l i . B i o c h e m . B i o p h y s . R e s . C o m m u n . 13(2), 90 -94 (1963).

Y u , C . K . E v a l u a t i o n and N a t u r e o f the N e a r - D i p l o i d C e l l s o f C h i n e s e H a m s t e r ( C r i c e t u l u s g r i s e u s ) i n v i t r o . C a n . J . Gene t . C y t o l . 5(3), 307-317 ( S e p t e m b e r 1963).

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