Upsala J Med Sci 88: 4 3 4 9 , 1983 Irradiation Effects upon Ischemic Regenerating Rat Liver Cells Lillemor Lewan' , Ola Forsberg', Herman Amneus3, Kerstin Lundberg4 and Borje Larsson3 Department of Zoophysiology', University of Lund, Lund, and Department of Surgery2, Central Hospital, Vasterds, and Department of Physical Biology3, The Gustaf Werner Institute, University of Uppsala, Uppsala, and Department of Experimental Medicine4, Pharmacia A B , Uppsala, Sweden ABSTRACT S t a r c h p a r t i c l e s i n j e c t e d i n t o t h e a r t e r i a l and p o r t a l s y s t e m s of t h e l i v e r of t h e r a t c a u s e d a temporary b l o c k a g e of t h e l i v e r c i r c u l a t i o n and consequent hypoxia i n t h e l i v e r c e l l s . I n t h e r e g e n e r a t i n g l i v e r t h i s r e s u l t e d i n a 30-402 d e c r e a s e of thymidine i n c o r p o r a t i o n i n t o D N A , when a n a l y s e d 1.5 h o u r s a f t e r i n j e c t i o n . I r r a d i a t i o n - i n d u c e d c e l l damage, e v a l u a t e d by thymidine i n c o r - p o r a t i o n 1.5 h o u r s a f t e r i r r a d i a t i o n w i t h a s i n g l e d o s e of X-rays, was n o t a m e l i o r a t e d by t h e i s c h e m i c c o n d i t i o n . It i s s u g g e s t e d t h a t t h i s depends on a n i n h i b i t e d n u c l e o t i d e metabolism and DNA s y n t h e s i s l e a d i n g t o a n a d d i t i v e meta- b o l i c hypoxic e f f e c t of t h e s t a r c h p a r t i c l e s on r a d i a t i o n damage. An e q u a l l e v e l of thymidine i n c o r p o r a t i o n , however, w a s found i n an i s c h e m i c and a non- - i s c h e m i c group of a n i m a l s 1 6 h o u r s a f t e r i r r a d i a t i o n . I n t h i s c a s e t h e l i v e r c e l l s i n t h e i s c h e m i c group had overcome t h e a d d i t i o n a l i n h i b i t i o n of DNA s y n t h e s i s c a u s e d by temporary h y p o x i a . INTRODUCTION Low LET-radiation i n d u c e s DNA damage by f r e e r a d i c a l s . A n e x t r e m e l y low t i s s u e c o n c e n t r a t i o n of m o l e c u l a r oxygen r e d u c e s damage. The d e g r e e of damage can be s t u d i e d h i s t o l o g i c a l l y (5,6) o r , a t t h e m o l e c u l a r l e v e l , e f f e c t s on DNA s y n t h e s i s c a n b e s t u d i e d by t h e i n c o r p o r a t i o n of r a d i o a c t i v e l y l a b e l l e d p r e c u r - s o r s e . g . t h y m i d i n e ( 1 , 1 4 ) . R a p i d l y growing l i v e r c e l l s o f f e r a good o p p o r t u n i t y f o r such s t u d i e s . A f t e r p a r t i a l hepatectomy l i v e r c e l l s a r e f a i r l y w e l l s y n c h r o n i z e d when e n t e r i n g t h e S-phase of t h e c e l l c y c l e w i t h i n 12-30 h o u r s of r e g e n e r a t i o n . The v a r i o u s s t a g e s o f t h e c e l l c y c l e have b e e n t h o r o u g h l y i n v e s t i g a t e d and t h e w e l l known t i m e c o u r s e f o r t h e growth of t h e r e g e n e r a t i n g l i v e r ( 9 , l O ) a l l o w s s t u d i e s of r a d i a t i o n e f f e c t s a t d i f f e r e n t s t a g e s ( 1 , 2 , 8 ) . I n t h e p r e s e n t s t u d y we a n a l y s e d t h e e f f e c t s of i r r a d i a t i o n on DNA-synthesis i n r e g e n e r a t i n g i s c h e m i c l i v e r t i s s u e . Ischemia was t e m p o r a r i l y induced by means of i n t r a v a s c u l a r l y i n j e c t e d , d e g r a d a b l e s t a r c h m i c r o s p h e r e s ( 4 ) . The a n i m a l s 43 were w h o l e - b o d y - i r r a d i a t e d d u r i n g t h e p r e r e p l i c a t i v e o r t h e r e p l i c a t i v e phase of l i v e r growth. DNA-synthesis w a s e v a l u a t e d by thymidine i n c o r p o r a t i o n i n t o DNA d u r i n g t h e r e p l i c a t i v e p h a s e of growth. MATERIAL AND METHODS Animals Young a d u l t male Sprague-Dawley r a t s weighing 160-180 g ( S P F - q u a l i t y , Anti- cimex, Sweden) were u s e d . The a n i m a l s were k e p t under c o n s t a n t c o n d i t i o n s , and had f r e e a c c e s s t o s t a n d a r d l a b o r a t o r y d i e t b e f o r e and a f t e r t h e e x p e r i m e n t a l p r o c e d u r e s . S u r g i c a l p r o c e d u r e s The s u r g i c a l p r o c e d u r e s have been t h o r o u g h l y d e s c r i b e d e a r l i e r (11). The e x p e r i m e n t s were accomplished i n f o u r d i f f e r e n t s e r i e s . S e r i e s I and I11 com- p r i s e d sham-operated and p a r t i a l l y h e p a t e c t o m i z e d a n i m a l s . Series I1 and I V comprised p a r t i a l l y h e p a t e c t o m i z e d a n i m a l s w i t h one c a t h e t e r i n t h e g a s t r o d u o - d e n a l a r t e r y , one i n t h e i l e o - c o l i c v e i n and one c a t h e t e r emptying i n t o t h e p e r i t o n e a l c a v i t y . The c a t h e t e r s were i n s e r t e d i n t o t h e v e s s e l s o r f i x e d t o t h e p e r i t o n e a l w a l l i n c o n n e c t i o n w i t h p a r t i a l hepatectomy as d e s c r i b e d p r e v i o u s l y ( 1 1 ) . Each c a t h e t e r w a s f l u s h e d , f i l l e d w i t h F y s k o s a l (Pharmacia) + Heparin (Vitrum) 500 IE/100 m l and plugged. The c a t h e t e r s were k e p t open by i n t e r m i t t e n t f l u s h i n g . I n j e c t i o n of s t a r c h p a r t i c l e s i n t o t h e v a s c u l a r c a t h e t e r s was p e r f o r - med 8 o r 25 h o u r s a f t e r s u r g e r y . To a v o i d d i u r n a l v a r i a t i o n a l l o p e r a t i o n s were performed between 8 - 11 a . m . I r r a d i a t i o n The r a t s were exposed t o t o t a l d o s e s of 4 - 16 Gy whole body i r r a d i a t i o n . S e r i e s I, I1 and I V w e r e i r r a d i a t e d w i t h 8 MV X-rays from a l i n e a r a c c e l e r a t o r a t a dose r a t e of 4 Gy/min. S e r i e s 111 was i r r a d i a t e d i n a n i r r a d i a t i o n chamber w i t h y - r a y s from a 1 3 7 C e s o u r c e ( S c a n d i t r o n i x , B 349, S c a n d i t r o n i x i n s t r u m e n t s A B , Uppsala, Sweden) a t a d o s e r a t e o f 0 . 8 4 Gy/min. N o n - i r r a d i a t e d c o n t r o l g r o u p s were i n c l u d e d i n e a c h series. S t a r c h p a r t i c l e s D i g e s t i b l e s t a r c h p a r t i c l e s (Spherex 60 mg/ml, b a t c h 78 08 2 4 , Pharmacia) were a d m i n i s t e r e d i n t o t h e p o r t a l and a r t e r i a l c a t h e t e r s 3.5 m i n u t e s b e f o r e i r r a d i a t i o n ( s e r i e s I1 and IV). Mean d i a m e t e r of t h e s p h e r e s was 30 p. 200 p1 of Spherex s u s p e n s i o n was i n j e c t e d i n t o e a c h c a t h e t e r and t h e c a t h e t e r s were f l u s h e d w i t h 200 p 1 s a l i n e ( F y s k o s a l , Pharmacia) . 44 R a d i o n u c l i d e s Thymidine i n c o r p o r a t i o n i n t o DNA was a n a l y s e d on t h e second day a f t e r p a r t i a l hepatectomy and 16 o r 1 . 5 h o u r s a f t e r i r r a d i a t i o n . 100 ~1 c o n t a i n i n g 185 kBq ( 5 pCi) of ( m e t ~ l - ' ~ C ) thymidine (185 GBq, Amersham) o r 100 1.11 c o n t a i n i n g 3.7 MBq (100 VCi) of (metyl- H) thymidine (555-1110 G B q , Amersham) was adminis- t e r e d i . p . I n a n i m a l s w i t h p e r i t o n e a l c a t h e t e r s i n j e c t i o n w a s made by way of t h i s c a t h e t e r . Animals i r r a d i a t e d 1 . 5 h o u r s b e f o r e a n a l y s i s of DNA s y n t h e s i s were p r e l a b e l l e d w i t h 3H-thymidine one hour b e f o r e i r r a d i a t i o n t o g i v e t h e pre- t r e a t m e n t l e v e l of DNA s y n t h e s i s i n e a c h a n i m a l . I n t h e s e a n i m a l s I4C-thymidine w a s u s e d f o r a n a l y s i s of t h y m i d i n e i n c o r p o r a t i o n i n t o DNA 1 . 5 h o u r s a f t e r i r r a - d i a t i o n . The d o u b l e - l a b e l l i n g allowed a n a l y s i s of e f f e c t s of r a d i a t i o n i n e a c h a n i m a l (11). 3 I n t a c t Regen. Regen. Regen. A n a l y s i s of DNA DNA was e x t r a c t e d a c c o r d i n g t o Schmidt-Tannhluser a s m o d i f i e d by Munro & F l e c k ( 1 2 ) and q u a n t i f i e d a c c o r d i n g t o Burton ( 3 ) o r i s o l a t e d and q u a n t i f i e d a c c o r d i n g t o Kasche Amneus ( 7 ) . I n c o r p o r a t i o n of l a b e l l e d thymidine was a n a l y s e d by l i q u i d s c i n t i l l a t i o n (Mark I1 S c i n t i l l a t i o n C o u n t e r ) and t h e s p e c i f i c a c t i v i - t y of DNA w a s c a l c u l a t e d a s t h e amount of r a d i o a c t i v i t y p e r mg of DNA p e r i n j e c - t e d amount o f r a d i o a c t i v i t y . The e f f e c t of r a d i a t i o n d o s e w a s c a l c u l a t e d a s t h e r a t i o between t h e d i f f e r e n c e i n DNA s p e c i f i c a c t i v i t y i n i r r a d i a t e d and non- i r r a d i a t e d a n i m a l s and t h e r a d i a t i o n dose. - - 24 25 2 0.24 -f 0 . 0 8 - - - 24 25 4 2.30 1.12 4 8 24 25 3 0.86 z 0.16 0.4 8 8 24 25 4 0.62 0.19 3.2 RESULTS T a b l e 1. Thymidine i n c o r p o r a t i o n i n t o l i v e r DNA. S e r i e s I. E f f e c t of p a r t i a l hepatectomy and r a d i a t i o n d o s e . I r r a d i a t i o n performed 8 h o u r s a f t e r p a r t i a l hepatectomy. Mean s t a n d a r d d e v i a t i o n , r e l a t i v e v a l u e s . L i v e r I R a d i a t i o n I3H-thymidine / K i l l i n g \Number I3H-spec. / E f f e c t of s t a t u s 1- lhours a f t e r / h o u r s a f t e r If 1;;i;;ty i n I r a d i a t i o n d o s e x l o 3 p a r t .hep. p a r t .hep. a n i m a l s DNA 8 I 24 I 25 I 4 10.54 0.121 I p.1 Regen- I I Thymidine i n c o r p o r a t i o n i n c r e a s e d t e n f o l d i n l i v e r t i s s u e 24 h o u r s a f t e r p a r t i a l hepatectomy (Table 1). The v a r i a t i o n of t h y m i d i n e i n c o r p o r a t i o n b e t - ween d i f f e r e n t a n i m a l s w a s h i g h . I r r a d i a t i o n 8 h o u r s a f t e r s u r g e r y d e c r e a s e d thymidine i n c o r p o r a t i o n 24 h o u r s p o s t o p e r a t i v e l y . The e f f e c t of t h e r a d i a t i o n 45 d o s e w a s h i g h e s t a t 4 Gy. I n c r e a s e d r a d i a t i o n d o s e (Table 1) reduced t h y m i d i n e i n c o r p o r a t i o n . A n i n c r e a s e d t h y m i d i n e i n c o r p o r a t i o n i n t o l i v e r t i s s u e made i s c h e m i c d u r i n g i r r a d i a t i o n was n o t found (Table 2 ) . The c a t h e t e r s i n t h e a r t e r i a l and p o r t a l s y s t e m s , n e c e s s a r y f o r a d m i n i s t r a t i o n of t h e s t a r c h p a r t i c l e s , d e p r e s s e d t h y m i d i n e i n c o r p o r a t i o n ( T a b l e 2 ) a s h a s been n o t e d b e f o r e (11). T a b l e 2 . Thymidine i n c o r p o r a t i o n i n l i v e r DNA on t h e second day a f t e r p a r t i a l hepatectomy. S e r i e s 11. E f f e c t of d i g e s t i b l e s t a r c h p a r t i c l e s d u r i n g i r r a d i a t i o n 8 h o u r s a f t e r p a r t i a l hepatectomy. 3H-thymidine w a s a d m i n i s t e r e d i . p . 24 h o u r s a f t e r p a r t i a l hepatectomy, and t h e r a t s were k i l l e d 25 h o u r s a f t e r p a r t i a l hepatectomy. S t a r c h p a r t i c l e s R a d i a t i o n dose h o u r s a f t e r Gy p a r t . h e p . - - - - - 7 8 + 7 8 C a t h e t e r s i n a. g a s t r o d . v . i l e o - c o l i c a Number of 3H-spec. a c t i v i t y i n DNA anima 1 s M SD 4 2.43 0.61 4 1.67 1.13 7 0.62 _+ 0.26 1 ) 7 0.85 0.36’’ R a d i a t i o n 14C-thymidine h o u r s a f t e r p a r t . hep. dose h o u r s a f t e r Gy p a r t . h e p . - - 26.5 3 25 26.5 7 25 26.5 1) D i f f e r e n c e n o t s i g n i f i c a n t . Student’s t - t e s t . k i l l e d 14C-spec. E f f e c t of 1 4 c / 3 ~ i n h o u r s a c t i v i t y r a d i a t i o n l i v e r DNA a f t e r i n DNA d o s e x 1 0 3 M -+ SD p a r t . h e p . M -+ SD 27.5 1.2320.421’ ) 0.5620. 27.5 0 . 7 4 4 . 2 7 1 ) 0.16 . 0 .39&0.09 3 1 27.5 0.59*0.212) 0.09 ‘0.344to.104) The d e p r e s s i v e e f f e c t o f r a d i a t i o n on t h y m i d i n e i n c o r p o r a t i o n w a s less pro- nounced on t h e second day a f t e r p a r t i a l hepatectomy and o n e hour a f t e r i r r a d i a - 46 t i o n (Table 3 and 4) t h a n on t h e second day a f t e r p a r t i a l hepatectomy and 16 h o u r s a f t e r i r r a d i a t i o n ( T a b l e 1 and 2 ) . However, t h e d i f f e r e n c e between thymi- d i n e i n c o r p o r a t i o n i n i r r a d i a t e d and n o n i r r a d i a t e d a n i m a l s i n t h e one h o u r group w a s s i g n i f i c a n t b o t h a t 3 and 7 Gy ( T a b l e 3 ) . The s i g n i f i c a n c e was s l i g h t l y im- proved when t h e r a t i o between 3H-thymidine i n c o r p o r a t i o n b e f o r e i r r a d i a t i o n and t h e "C-thymidine i n c o r p o r a t i o n a f t e r i r r a d i a t i o n was c a l c u l a t e d f o r e a c h a n i m a l ( T a b l e 3 ) . The temporary e x c l u s i o n of blood by s t a r c h p a r t i c l e s d e c r e a s e d t h y m i d i n e i n - c o r p o r a t i o n i n t o DNA as s e e n from a n a l y s e s 1 . 5 h o u r s a f t e r i n j e c t i o n ( T a b l e 4 ) . The 1 4 C - s p e c i f i c a c t i v i t y of DNA d e c r e a s e d from 1.00 t o 0.70 o r 30% and t h e r a t i o l 4 C I 3 H i n e a c h a n i m a l from 0 . 9 4 t o 0.58 o r 39%. m i - la'' 6 8 6 9 8 9 T a b l e 4 . Thymidine i n c o r p o r a t i o n i n l i v e r DNA on t h e second day a f t e r p a r t i a l hepatectomy. S e r i e s I V . Immediate e f f e c t of r a d i a t i o n a n d / o r d i g e s t i b l e s t a r c h p a r t i c l e s . A l l a n i m a l s had c a t h e t e r s i n t h e g a s t r o d u o d e n a l a r t e r y and i l e o c o l i c v e i n and were p r e l a b e l l e d w i t h 3H-thymidine 24 h o u r s a f t e r p a r t i a l hepatectomy. DNA - M 2 SD 1.00+0.36 0.7020.31 0.8020.82 0.4020.21 0.89?0.53 0.31?0.22 t a r c h a r t i c - - - 3 3 7 d i n e p a r t .hep. 26.5 26.5 25 2 6 . 5 25 26.5 25 26.5 7 25 26.5 L i l l e d irs a f t e r iar t . hep . 27.5 27.5 2 7 . 5 27.5 27.5 27.5 lumber I14C-spec. 1 I 4 C l 3 H i n )f l a c t i v i t y i n DNA M + SD 40 0.36?0.16 89 0.45?0.14 31 0.34?0.18 % ._ 00 6 1 50 38 48 36 The d e c r e a s i n g e f f e c t on t h y m i d i n e i n c o r p o r a t i o n a s a r e s u l t of temporary i s c h e m i a w a s o b v i o u s a l s o i n i r r a d i a t e d a n i m a l s . The mean I 4 C - s p e c i f i c a c t i v i t y d e c l i n e d from 0.80 t o 0 . 4 0 , i . e . a n e x t r a 40% a t 3 Gy a n d from 0.89 t o 0 . 3 1 o r a n e x t r a 58% a t 7 Gy r e s p e c t i v e l y when s t a r c h p a r t i c l e s were p r e s e n t d u r i n g i r r a d i a t i o n . The c o r r e s p o n d i n g I 4 C l 3 H r a t i o s d e c l i n e d from 0.47 t o 0.36 and from 0.45 t o 0.34 a t 3 and 7 Gy r e s p e c t i v e l y . Thus, i m m e d i a t e l y a f t e r i r r a d i a t i o n t h e temporary i s c h e m i a a c t s s y n e r g i s t i c a l l y w i t h t h e i r r a d i a t i o n i n d e p r e s s i n g t h y m i d i n e i n c o r p o r a t i o n . The mean e f f e c t of t h e temporary i s c h e m i a c a u s e d by t h e s t a r c h p a r t i c l e s i n n o n - i r r a d i a t e d and i r r a d i a t e d r a t s was a 43% d e c r e a s e of thymidine i n c o r p o r a t i o n (Table 4 ) . DISCUSS I O N The r e s u l t s show n o p r o t e c t i v e a c t i o n i n terms of t h y m i d i n e i n c o r p o r a t i o n 47 from temporary i s c h e m i a d u r i n g t h e f i r s t h o u r s a f t e r i r r a d i a t i o n . On t h e c o n t r a r y , t h e e x c l u s i o n of blood seemed t o a c t s y n e r g i s t i c a l l y w i t h t h e r a d i a - t i o n i n d i s t u r b i n g c e l l u l a r DNA s y n t h e s i s . The d i s t u r b a n c e s s h o u l d , however, be of o p p o s i t e c h a r a c t e r . R a d i a t i o n l e a d s t o i n c r e a s e d amounts o f DNA damaging oxygen f r e e r a d i c a l s . Blood e x c l u s i o n s h o u l d l e a d t o a low oxygen t e n s i o n and d e c r e a s e d amounts o f f r e e r a d i c a l s . Both e f f e c t s seem t o push t h e l i v e r c e l l s o u t of o r d e l a y t h e i r e n t r a n c e i n t o t h e S-phase. It h a s been shown, however, t h a t t h e p r o t e c t i v e a c t i o n o f blood e x c l u s i o n and low oxygen t e n s i o n d u r i n g i r r a d i a t i o n i s q u i t e e v i d e n t a t l a t e r s t a g e s of r e c o v e r y ( 5 , 6 ) . Thus, c e l l metabolism i s n o t permanently damaged and t h e c e l l s s h o u l d r e t u r n t o t h e c e l l c y c l e and s y n t h e s i z e DNA. I n t h e p r e s e n t e x p e r i m e n t t h e c e l l s o f t h e 24 h o u r r e g e n e r a t i n g l i v e r s w e r e p r o b a b l y r e t u r n i n g t o t h e c e l l c y c l e and t h e S-phase a f t e r b e i n g i r r a d i a t e d d u r i n g i s c h e m i a 1 6 h o u r s e a r l i e r . The s i m i l a r v a l u e s of DNA s p e c i f i c a c t i v i t y i n p r o t e c t e d and n o n - p r o t e c t e d a n i m a l s i n d i c a t e d t h a t t h e c e l l s i n p r o t e c t e d l i v e r s had overcome t h e a d d i t i o n a l d e c r e a s e c a u s e d by t h e t e m p o r a r i l y low oxygen t e n s i o n . 3 P r e l a b e l l i n g o f DNA by H-thymidine and comparisons of 3H/14C-ratios i n DNA was p r e v i o u s l y shown t o improve t h e s i g n i f i c a n c e o f r e s u l t s ( 1 1 ) . The v a l u e of d o u b l e l a b e l l i n g w a s n o t s o c l e a r i n t h i s s t u d y . The p e r i o d between i n j e c t i o n of 3H-thymidine and 14C-thymidine was r a t h e r l o n g , i . e . 2.5 h o u r s and t h e i r r a d i a t i o n i n t h e meantime makes h a n d l i n g of t h e a n i m a l s n e c e s s a r y . Both may i n f l u e n c e t h y m i d i n e i n c o r p o r a t i o n i n t o DNA. The o b s e r v e d d e c r e a s e o f thymidine i n c o r p o r a t i o n i n t o DNA d u r i n g a temporary t o t a l b l o c k of l i v e r blood s u p p l y h a s i m p l i c a t i o n s i n t h e u s e o f s t a r c h p a r t i c - l e s f o r t h e l o c a l a c c u m u l a t i o n o f d r u g s ( 1 3 ) . Drugs t h a t are e n e r g y dependent and have t o b e m e t a b o l i z e d t o be e f f e c t i v e r e q u i r e u n i n t e r r u p t e d blood f l o w . T h i s would n o t be a problem when s t a r c h p a r t i c l e s are used t o a c c u m u l a t e d r u g s w i t h a f f i n i t y t o c e l l membranes and d e l a y e d c e l l u l a r u p t a k e . REFERENCES 1. 2 . 3. 4. 5. Altman, K . I . , G e r b e r , G . B . & Okada, S . : The L i v e r . I n R a d i a t i o n B i o c h e m i s t r y , Vol 11, Ed. Altman, 1.1. & G e r b e r , G . B . AP 1970, pp. 160-194. 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Biochem. 8:477-487, 1977. Lewan, L., Amneus, H . , F o r s b e r g , J.O. & Lundberg, K.: I n t r a a r t e r i a l and I n t r a p o r t a l i n v i v o c a t h e t e r i z a t i o n o f t h e r e g e n e r a t i n g r a t l i v e r . E f f e c t s upon body and l i v e r w e i g h t s and DNA s y n t h e s i s . Uppsala J Med S c i , 1983. Munro, H. & F l e c k , A . : Recent developments i n t h e measurement of n u c l e i c a c i d s i n b i o l o g i c a l m a t e r i a l . A n a l y s t 91:78-88, 1966. T u n a , R . , F o r s b e r g , J . O . & Agerup, B.: Enhanced u p t a k e of a c t i n o m y c i n D i n t h e dog k i d n e y by s i m u l t a n o u s l y i n j e c t i o n of d e g r a d a b l e s t a r c h m i c r o s p h e r e s i n t o t h e r e n a l a r t e r y . Cancer 5O:l-5, 1982. Walters, R . A . & Enger, M . D . : E f f e c t s of I o n i z i n g R a d i a t i o n on N u c l e i c Acid S y n t h e s i s i n Mammalian C e l l s . I n : Adv Rad B i o l Vol 6 , AE' 1976, pp. 1-48. Address f o r r e p r i n t s : L. Lewan, Ph.D. Department of Zoophysiology Helgonavagen 3b Sweden S-223 62 LUND 4-832859 49