15.9. 1977
1221
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be r e q u i r e d to i n d u c e t h e g l a n d u l a r d i f f e r e n t i a t i o n a n d t h e i n h i b i t i o n of t h e k e r a t i n i z a t i o n in t h e e c t o d e r m a l e n d - b u d s . T h e b a s a l m e m b r a n e d i s r u p t i o n a n d t h e presence of p i n o c y t i c vesicles c o u l d f a c i l i t a t e t h e t r a n s f e r of large molecules. H o w e v e r , t h e p r e s e n c e of d i r e c t i n t e r cellular c o n t a c t s b e t w e e n t h e i n t e r a c t i v e tissues w o u l d s u g g e s t a d i r e c t i n d u c t i v e s i g n a l t r a n s m i s s i o n f r o m one cell to a n o t h e r , r a t h e r t h a n a m a t r i x i n t e r a c t i o n , w h i c h has been demonstrated during the morphogenetic p e r i o d 16,17.
Thus, during the preen gland development, several intera c t i o n m e c h a n i s m s e x i s t a t d i f f e r e n t stages, in d i f f e r e n t a n a t o m i c a l sites, a n d t h e y i n i t i a t e a local d i f f e r e n t i a t i o n of t h e u r o p y g i a l t u b u l e s .
16 J. Bride and L. Gomot, C. r. Soc. Biol. 770, 575 (1976). 17 L. Gomot and J. Bride, C. r. Soc. Biol. 170, 579 (1976).
P~acental ~_ermeability of a~senate~gn during early !efmbryogenesis in the]h_aarnster1 D. P. H a n l o n a n d V. H. F e r m
Department of Microbiology and Biochemistry, College of Agriculture, University o/ Vermont, Burlington (Vermont 05401, USA)," and Departments o/Anatomy/Cytology and Pharmacology/Toxicology, Dartmouth Medical School, Hanover (New Hampshire 03755, USA), 4 February 1977 Summary. S o d i u m a r s e n a t e crosses t h e p l a c e n t a l b a r r i e r in p r e g n a n t S y r i a n h a m s t e r s following i n j e c t i o n in t e r a t o g e n i c or t r a c e r doses o n t h e 8 t h d a y of g e s t a t i o n . A r s e n a t e ion p r o d u c e s specific m a l f o r m a t i o n s in s u r v i v i n g o f f s p r i n g of h a m s t e r s if i n j e c t e d o n d a y 8 of g e s t a t i o n 2. T e r a t o g e n i c lesions c o u l d r e s u l t f r o m a n effect of a r s e n a t e on t h e m a t e r n a l s y s t e m , o n p l a c e n t a l p e r m e a b i l i t y , or d i r e c t l y o n t h e e m b r y o . O b v i o u s l y , a d i r e c t effect o n t h e d e v e l o p i n g e m b r y o r e q u i r e s p l a c e n t a l t r a n s p o r t of t h e t e r a t o g e n . W e h a v e e x a m i n e d t h i s p o i n t w i t h t h e aid of r a d i o i s o t o p i c arsenic (74As). O u r d a t a s h o w t h a t 74arsenate crosses t h e p l a c e n t a l b a r r i e r w h e n i n j e c t e d in a t e r a t o genic dose, or t r a c e a m o u n t s , d u r i n g t h e critical p h a s e of organogenesis. Materials and methods. P r o c e d u r e s g e n e r a l l y followed those described by Hanlon and Ferm a with regard to t i m e d m a t i n g s of f e m a l e h a m s t e r s , i n j e c t i o n of t e r a t o g e n , a n d collection a n d r a d i o a s s a y of tissue samples. H o w e v e r , a Beckman Biogamma D counter replaced the previously used r a d i a t i o n a n a l y z e r s y s t e m . T h e whole e n e r g y spect r u m c h a n n e l was e m p l o y e d . C o u n t i n g efficiency was 55 % . S u f f i c i e n t Na2H74AsO4 A m e r s h a m - S e a r l e ) was a d d e d to I"
E]9th day (24 h) D12th day(96 h)
300
250 200-
-6 =e 150..m 100-
50
01
Maternal blood
Maternal liver
Uterus
A~lantoic Yolksac Embryo placenta placenta
Tissue concentrations of arsenate in the pregnant Syrian hamster on day 9 and day 12 following injection of radiolabelled arsenate on the 8th day of gestation. Vertical bars represent standard errors of mean values. Experimental conditions are described in the text.
a n a q u e o u s s o l u t i o n c o n t a i n i n g 4.0 m g N a 2 H A s O 4 • H 2 0 / m l to yield a n i n i t i a l c a l i b r a t i o n of 10 txCi/ml. On t h e 8 t h d a y of g e s t a t i o n , 12 h a m s t e r s were i n j e c t e d (subl i n g u a l vein) w i t h 0.5 m l of s o l u t i o n / 1 0 0 g m a t e r n a l b . w t to give dose levels of 8.37 m g AsO4/kg. 24 h, a n d 96 h a f t e r injection, 6 a n i m a l s were killed b y c h l o r o f o r m a n e s t h e s i a a n d tissue s a m p l e s t a k e n . D u e to t h e d i f f i c u l t y of s e p a r a t i o n , 9 t h d a y p l a c e n t a l tissue i n c l u d e s y o l k sac and chorioallantoic placenta together with a small amount ( a b o u t 15 %) of m a t e r n a l decidua. 12-day-old c h o r i o a l l a n toic p l a c e n t a e c o u l d b e s e p a r a t e d f r o m y o l k sac p l a c e n t a e a n d t h e i r r a d i o a c t i v i t y m e a s u r e d i n d i v i d u a l l y . Correct i o n s for t h e loss of r a d i o a c t i v i t y in t h e s a m p l e s (74As h a s a half-life of 18d) were m a d e b y c o m p a r i n g s a m p l e c o u n t s w i t h t h a t of a s t a n d a r d labelled a r s e n a t e s o l u t i o n on t h e d a y of assay. A l i m i t e d s t u d y of t h e d i s t r i b u t i o n of radiolabelled a r s e n a t e following i n j e c t i o n of t r a c e doses w a s also carried out. 4 h a m s t e r s were i n j e c t e d o n d a y 8 of p r e g n a n c y w i t h a 0.85% s o d i u m chloride s o l u t i o n cont a i n i n g 12.8 ~xCi a n d 75 n g a r s e n a t e (AsO4) p e r ml. Doses were a d m i n i s t e r e d a t t h e level of 0.5 m l solution/100 g of a n i m a l , i.e., 375 ng/kg. 2 a n i m a l s were sacrificed on d a y 9 a n d 2 on d a y 12. T h e r e m a i n d e r of tile p r o c e d u r e followed m e t h o d s d e s c r i b e d a b o v e . Results and discussion. T h e d i s t r i b u t i o n o f 74arsenate i n m a t e r n a l a n d f e t a l tissues of t h e S y r i a n h a m s t e r following i n j e c t i o n of a t e r a t o g e n i c dose is s h o w n in t h e figure. 24 h p o s t i n j e c t i o n s i g n i f i c a n t a m o u n t s of 74As a p p e a r e d in e m b r y o n i c tissues, i n d i c a t i n g t h a t t h e p l a c e n t a e do n o t f u n c t i o n as c o m p l e t e l y effective b a r r i e r s to t h e p a s s a g e of a r s e n a t e (or some m e t a b o l i t e of a r s e n a t e ) i n t o t h e e m b r y o . A r s e n a t e c o n c e n t r a t i o n s u n d e r g o i d e n t i c a l decreases (84%) for m a t e r n a l blood, u t e r u s , a n d p l a c e n t a e b e t w e e n 24 h a n d 96 h. T h e figures for m a t e r n a l liver a n d e m b r y o s are 77% a n d 9 1 % . T h e s e d a t a are b e s t e x p l a i n e d b y a s t e a d y s t a t e c o n d i t i o n in w h i c h a r s e n a t e r a p i d l y equilib r a t e s b e t w e e n c o m p a r t m e n t s . T h e overall decrease in c o n c e n t r a t i o n is p r o b a b l y d u e p r i m a r i l y t o e x c r e t i o n v i a t h e k i d n e y s 4. 1 2 3 4
This work was supported by USPHS grant ES-00697. V.H. Ferm and S. J. Carpenter, J. Reprod. Fert. 17, 199 (1968). D.P. Hanlon and V. H. Ferm, J. Reprod. Fert. 44, 109 (1975). J.M. Ginsburg, Am. J. Physiol. 208, 832 (1965).
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O u r l i m i t e d s t u d y of t h e d i s t r i b u t i o n of a t r a c e dose (375 ng/kg) of a r s e n a t e r e v e a l e d t h e following: a) d a y 9 e m b r y o s c o n t a i n e d d e t e c t a b l e a m o u n t s of a r s e n a t e - app r o x i m a t e l y 3 p g / g t i s s u e ; b) t h e p a t t e r n of a r s e n a t e d i s t r i b u t i o n a n d t h e f r a c t i o n a l decrease in tissue a r s e n a t e b e t w e e n d a y 9 a n d d a y 12 were t h e s a m e as for t h e t e r a t o g e n i c dose. T h u s , a r s e n a t e is p a r t i t i o n e d in i d e n t i c a l f a s h i o n for doses of t e r a t o g e n differing b y m o r e t h a n 2100 times. C o n s t a n c y i n t h e p a t t e r n of a r s e n a t e d i s t r i b u t i o n o v e r a wide c o n c e n t r a t i o n r a n g e implies t h a t v e r y little t e r a t o g e n is b o u n d t o m a c r o m o l e c n l e s or o t h e r tissue c o m p o n e n t s , c o n t r a r y t o e x p e c t a t i o n s for h e a v y m e t a l ion teratogens. T h e b i o c h e m i c a l basis of a r s e n a t e - i n d u c e d t e r a t o g e n e s i s m a y d e p e n d o n t h e c h e m i c a l s i m i l a r i t y of a r s e n a t e a n d p h o s p h a t e . F o r e x a m p l e , a r s e n a t e c o m p e t e s w i t h inor-
EXPERII~NTIA 33/9
g a n i c p h o s p h a t e in a v a r i e t y of m e t a b o l i c r e a c t i o n s 5 a n d could i n t e r f e r e w i t h one or m o r e b i o c h e m i c a l s t e p s a t a critical stage of organogenesis. A l t e r n a t i v e l y , a r s e n a t e m i g h t b e r e d u c e d to arsenite, as G i n s b u r g h a s d e m o n s t r a t e d in r e n a l c l e a r a n c e studies 4, a n d f u r t h e r m e t a b o lized t o a n o r g a n o - a r s e n i c a l w i t h t e r a t o g e n i c p r o p e r t i e s . R e g a r d l e s s of t h e b i o c h e m i c a l m e c h a n i s m of a r s e n a t e induced teratogenesis, our data show that the teratogen does e n t e r e m b r y o n i c tissues d u r i n g organogenesis. T h e s e findings, t h e r e f o r e , offer s u p p o r t to F e r m ' s s u g g e s t i o n t h a t a r s e n a t e exercises a specific effect on e m b r y o n i c c e p h a l i c m e s e n c h y m e 6.
5 L.M. Klevay, Pharmac. Ther. , A, I, 189 (1976). 6 V.H. Ferm, Adv. Teratol. 5, 51 (1972).
H i s t o c h e m i c a l d e m o n s t r a t i o n of a d r e n e r g i c nerve fibres in the renal c a p s u l e of rats C. I n a g a k i a n d C. T a n a k a 1
Department o/Pharmacology, Faculty o[ Medicine, Kyoto University, Kyoto 606, and Department o/Pharmacology, Kobe University School o/ Medicine, Kobe 650 (Japan), 22 February 1977 Summary. A d r e n e r g i c i n n e r v a t i o n in r a t r e n a l capsule was d e m o n s t r a t e d u s i n g t h e h i s t o c h e m i c a l fluorescence m e t h o d w i t h glyoxylic acid. T h e fluorescence h i s t o c h e m i c a l m e t h o d of F a l c k a n d H i l l a r p 2 h a s r e v e a l e d a d r e n e r g i c i n n e r v a t i o n in t h e r e n a l b l o o d vessels 3-5 a n d t u b u l e s S ; h o w e v e r , l o c a l i z a t i o n of m o n o a m i n e s in t h e r e n a l c a p s u l e h a s n o t b e e n r e p o r t e d . A n e w h i s t o c h e m i c a l t e c h n i q u e u s i n g glyoxylic acid i n s t e a d of f o r m a l d e h y d e h a s b e e n d e v e l o p e d 7, s a n d good r e s u l t s are seen w i t h t h e m e t h o d u s i n g a q u e o u s s o l u t i o n a n d h e a t i n g in t h e s t r e t c h p r e p a r a t i o n of p e r i p h e r a l t h i n tissues ~. W e a t t e m p t e d to d e m o n s t r a t e a d r e n e r g i c i n n e r r a t i o n in t h e r e n a l c a p s u l e u s i n g t h i s glyoxylic acid method. Male W i s t a r r a t s w e i g h i n g i50• g were sacrificed u n d e r p e n t o b a r b i t a l a n e s t h e s i a . T h e k i d n e y s w i t h t h e capsules were r a p i d l y excised a n d i m m e r s e d in ice-cold ~ 2 % glyoxylic acid solution. G l y o x y l i c acid m o n o h y d r a t e was dissolved in 0.1 M p h o s p h a t e b u f t e r a d j u s t e d t o p H 7.0 w i t h N a O H . 10 rain later, t h e capsules were r e m o v e d a n d
s t r e t c h e d o n clean glass slides. T h e s p e c i m e n s were d r i e d w i t h a h a i r - d r y e r for 20 min, h e a t e d a t 100 ~ for 5-10 m i n and then mounted with the entellan-xylene mixture. For microscopic analyses, a Zeiss epifluorescence microscope was used w i t h a h i g h p r e s s u r e m e r c u r y l a m p as a l i g h t source. T h e e x c i t a t i o n filter used was a BG12 a n d t h e b a r r i e r filter was Zeiss '47' or '50'. T h e o u t e r surface of t h e k i d n e y is c o v e r e d w i t h a d e l i c a t e f i b r o u s c a p s u l e c o m p o s e d of c o l l a g e n o u s a n d elastic fibres a n d a few s m o o t h m u s c l e cells. O u t s i d e tile t u n i c a fibrosa is a f a t t y l a y e r called t h e t u n i c a adiposa. F l u o r e s c e n t n e r v e fibres w i t h v a r i c o s i t i e s were f o u n d t o i n n e r v a t e t h e hilus a r t e r i e s of t h e k i d n e y a n d t h e i r r a m i f i c a t i o n s to e n t e r t h e r e n a l capsule. T h e r e n a l c a p s u l a r a r t e r i e s were a c c o m p a n i e d b y 2 m a i n b u n d l e s in w h i c h r a n 2 or m o r e i n t e n s e l y f l u o r e s c e n t fibres a n d b o t h b u n d l e s were c o n n e c t e d b y a few fine varicose t e r m i n a l s {figure 1}. T h e r e were 2 t y p e s
Fig. 1. The renal capsule of the rat. The adrenergie nerve fibres run along the blood vessels, on to the fat cells (F) and freely on the renal capsule ( ~' ). Yellow fluorescent mast (:ells ( ~") were seen around the blood vessels and the fat cells. • 30.
Fig. 2. The tunica fibrosa of the rat kidney. A systenl of branching terminals arising from one preterminal axon ( j ' ) is visible irl the tunica fibrosa. • 30.
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be r e q u i r e d to i n d u c e t h e g l a n d u l a r d i f f e r e n t i a t i o n a n d t h e i n h i b i t i o n of t h e k e r a t i n i z a t i o n in t h e e c t o d e r m a l e n d - b u d s . T h e b a s a l m e m b r a n e d i s r u p t i o n a n d t h e presence of p i n o c y t i c vesicles c o u l d f a c i l i t a t e t h e t r a n s f e r of large molecules. H o w e v e r , t h e p r e s e n c e of d i r e c t i n t e r cellular c o n t a c t s b e t w e e n t h e i n t e r a c t i v e tissues w o u l d s u g g e s t a d i r e c t i n d u c t i v e s i g n a l t r a n s m i s s i o n f r o m one cell to a n o t h e r , r a t h e r t h a n a m a t r i x i n t e r a c t i o n , w h i c h has been demonstrated during the morphogenetic p e r i o d 16,17.
Thus, during the preen gland development, several intera c t i o n m e c h a n i s m s e x i s t a t d i f f e r e n t stages, in d i f f e r e n t a n a t o m i c a l sites, a n d t h e y i n i t i a t e a local d i f f e r e n t i a t i o n of t h e u r o p y g i a l t u b u l e s .
16 J. Bride and L. Gomot, C. r. Soc. Biol. 770, 575 (1976). 17 L. Gomot and J. Bride, C. r. Soc. Biol. 170, 579 (1976).
P~acental ~_ermeability of a~senate~gn during early !efmbryogenesis in the]h_aarnster1 D. P. H a n l o n a n d V. H. F e r m
Department of Microbiology and Biochemistry, College of Agriculture, University o/ Vermont, Burlington (Vermont 05401, USA)," and Departments o/Anatomy/Cytology and Pharmacology/Toxicology, Dartmouth Medical School, Hanover (New Hampshire 03755, USA), 4 February 1977 Summary. S o d i u m a r s e n a t e crosses t h e p l a c e n t a l b a r r i e r in p r e g n a n t S y r i a n h a m s t e r s following i n j e c t i o n in t e r a t o g e n i c or t r a c e r doses o n t h e 8 t h d a y of g e s t a t i o n . A r s e n a t e ion p r o d u c e s specific m a l f o r m a t i o n s in s u r v i v i n g o f f s p r i n g of h a m s t e r s if i n j e c t e d o n d a y 8 of g e s t a t i o n 2. T e r a t o g e n i c lesions c o u l d r e s u l t f r o m a n effect of a r s e n a t e on t h e m a t e r n a l s y s t e m , o n p l a c e n t a l p e r m e a b i l i t y , or d i r e c t l y o n t h e e m b r y o . O b v i o u s l y , a d i r e c t effect o n t h e d e v e l o p i n g e m b r y o r e q u i r e s p l a c e n t a l t r a n s p o r t of t h e t e r a t o g e n . W e h a v e e x a m i n e d t h i s p o i n t w i t h t h e aid of r a d i o i s o t o p i c arsenic (74As). O u r d a t a s h o w t h a t 74arsenate crosses t h e p l a c e n t a l b a r r i e r w h e n i n j e c t e d in a t e r a t o genic dose, or t r a c e a m o u n t s , d u r i n g t h e critical p h a s e of organogenesis. Materials and methods. P r o c e d u r e s g e n e r a l l y followed those described by Hanlon and Ferm a with regard to t i m e d m a t i n g s of f e m a l e h a m s t e r s , i n j e c t i o n of t e r a t o g e n , a n d collection a n d r a d i o a s s a y of tissue samples. H o w e v e r , a Beckman Biogamma D counter replaced the previously used r a d i a t i o n a n a l y z e r s y s t e m . T h e whole e n e r g y spect r u m c h a n n e l was e m p l o y e d . C o u n t i n g efficiency was 55 % . S u f f i c i e n t Na2H74AsO4 A m e r s h a m - S e a r l e ) was a d d e d to I"
E]9th day (24 h) D12th day(96 h)
300
250 200-
-6 =e 150..m 100-
50
01
Maternal blood
Maternal liver
Uterus
A~lantoic Yolksac Embryo placenta placenta
Tissue concentrations of arsenate in the pregnant Syrian hamster on day 9 and day 12 following injection of radiolabelled arsenate on the 8th day of gestation. Vertical bars represent standard errors of mean values. Experimental conditions are described in the text.
a n a q u e o u s s o l u t i o n c o n t a i n i n g 4.0 m g N a 2 H A s O 4 • H 2 0 / m l to yield a n i n i t i a l c a l i b r a t i o n of 10 txCi/ml. On t h e 8 t h d a y of g e s t a t i o n , 12 h a m s t e r s were i n j e c t e d (subl i n g u a l vein) w i t h 0.5 m l of s o l u t i o n / 1 0 0 g m a t e r n a l b . w t to give dose levels of 8.37 m g AsO4/kg. 24 h, a n d 96 h a f t e r injection, 6 a n i m a l s were killed b y c h l o r o f o r m a n e s t h e s i a a n d tissue s a m p l e s t a k e n . D u e to t h e d i f f i c u l t y of s e p a r a t i o n , 9 t h d a y p l a c e n t a l tissue i n c l u d e s y o l k sac and chorioallantoic placenta together with a small amount ( a b o u t 15 %) of m a t e r n a l decidua. 12-day-old c h o r i o a l l a n toic p l a c e n t a e c o u l d b e s e p a r a t e d f r o m y o l k sac p l a c e n t a e a n d t h e i r r a d i o a c t i v i t y m e a s u r e d i n d i v i d u a l l y . Correct i o n s for t h e loss of r a d i o a c t i v i t y in t h e s a m p l e s (74As h a s a half-life of 18d) were m a d e b y c o m p a r i n g s a m p l e c o u n t s w i t h t h a t of a s t a n d a r d labelled a r s e n a t e s o l u t i o n on t h e d a y of assay. A l i m i t e d s t u d y of t h e d i s t r i b u t i o n of radiolabelled a r s e n a t e following i n j e c t i o n of t r a c e doses w a s also carried out. 4 h a m s t e r s were i n j e c t e d o n d a y 8 of p r e g n a n c y w i t h a 0.85% s o d i u m chloride s o l u t i o n cont a i n i n g 12.8 ~xCi a n d 75 n g a r s e n a t e (AsO4) p e r ml. Doses were a d m i n i s t e r e d a t t h e level of 0.5 m l solution/100 g of a n i m a l , i.e., 375 ng/kg. 2 a n i m a l s were sacrificed on d a y 9 a n d 2 on d a y 12. T h e r e m a i n d e r of tile p r o c e d u r e followed m e t h o d s d e s c r i b e d a b o v e . Results and discussion. T h e d i s t r i b u t i o n o f 74arsenate i n m a t e r n a l a n d f e t a l tissues of t h e S y r i a n h a m s t e r following i n j e c t i o n of a t e r a t o g e n i c dose is s h o w n in t h e figure. 24 h p o s t i n j e c t i o n s i g n i f i c a n t a m o u n t s of 74As a p p e a r e d in e m b r y o n i c tissues, i n d i c a t i n g t h a t t h e p l a c e n t a e do n o t f u n c t i o n as c o m p l e t e l y effective b a r r i e r s to t h e p a s s a g e of a r s e n a t e (or some m e t a b o l i t e of a r s e n a t e ) i n t o t h e e m b r y o . A r s e n a t e c o n c e n t r a t i o n s u n d e r g o i d e n t i c a l decreases (84%) for m a t e r n a l blood, u t e r u s , a n d p l a c e n t a e b e t w e e n 24 h a n d 96 h. T h e figures for m a t e r n a l liver a n d e m b r y o s are 77% a n d 9 1 % . T h e s e d a t a are b e s t e x p l a i n e d b y a s t e a d y s t a t e c o n d i t i o n in w h i c h a r s e n a t e r a p i d l y equilib r a t e s b e t w e e n c o m p a r t m e n t s . T h e overall decrease in c o n c e n t r a t i o n is p r o b a b l y d u e p r i m a r i l y t o e x c r e t i o n v i a t h e k i d n e y s 4. 1 2 3 4
This work was supported by USPHS grant ES-00697. V.H. Ferm and S. J. Carpenter, J. Reprod. Fert. 17, 199 (1968). D.P. Hanlon and V. H. Ferm, J. Reprod. Fert. 44, 109 (1975). J.M. Ginsburg, Am. J. Physiol. 208, 832 (1965).
1222
Specialia
O u r l i m i t e d s t u d y of t h e d i s t r i b u t i o n of a t r a c e dose (375 ng/kg) of a r s e n a t e r e v e a l e d t h e following: a) d a y 9 e m b r y o s c o n t a i n e d d e t e c t a b l e a m o u n t s of a r s e n a t e - app r o x i m a t e l y 3 p g / g t i s s u e ; b) t h e p a t t e r n of a r s e n a t e d i s t r i b u t i o n a n d t h e f r a c t i o n a l decrease in tissue a r s e n a t e b e t w e e n d a y 9 a n d d a y 12 were t h e s a m e as for t h e t e r a t o g e n i c dose. T h u s , a r s e n a t e is p a r t i t i o n e d in i d e n t i c a l f a s h i o n for doses of t e r a t o g e n differing b y m o r e t h a n 2100 times. C o n s t a n c y i n t h e p a t t e r n of a r s e n a t e d i s t r i b u t i o n o v e r a wide c o n c e n t r a t i o n r a n g e implies t h a t v e r y little t e r a t o g e n is b o u n d t o m a c r o m o l e c n l e s or o t h e r tissue c o m p o n e n t s , c o n t r a r y t o e x p e c t a t i o n s for h e a v y m e t a l ion teratogens. T h e b i o c h e m i c a l basis of a r s e n a t e - i n d u c e d t e r a t o g e n e s i s m a y d e p e n d o n t h e c h e m i c a l s i m i l a r i t y of a r s e n a t e a n d p h o s p h a t e . F o r e x a m p l e , a r s e n a t e c o m p e t e s w i t h inor-
EXPERII~NTIA 33/9
g a n i c p h o s p h a t e in a v a r i e t y of m e t a b o l i c r e a c t i o n s 5 a n d could i n t e r f e r e w i t h one or m o r e b i o c h e m i c a l s t e p s a t a critical stage of organogenesis. A l t e r n a t i v e l y , a r s e n a t e m i g h t b e r e d u c e d to arsenite, as G i n s b u r g h a s d e m o n s t r a t e d in r e n a l c l e a r a n c e studies 4, a n d f u r t h e r m e t a b o lized t o a n o r g a n o - a r s e n i c a l w i t h t e r a t o g e n i c p r o p e r t i e s . R e g a r d l e s s of t h e b i o c h e m i c a l m e c h a n i s m of a r s e n a t e induced teratogenesis, our data show that the teratogen does e n t e r e m b r y o n i c tissues d u r i n g organogenesis. T h e s e findings, t h e r e f o r e , offer s u p p o r t to F e r m ' s s u g g e s t i o n t h a t a r s e n a t e exercises a specific effect on e m b r y o n i c c e p h a l i c m e s e n c h y m e 6.
5 L.M. Klevay, Pharmac. Ther. , A, I, 189 (1976). 6 V.H. Ferm, Adv. Teratol. 5, 51 (1972).
H i s t o c h e m i c a l d e m o n s t r a t i o n of a d r e n e r g i c nerve fibres in the renal c a p s u l e of rats C. I n a g a k i a n d C. T a n a k a 1
Department o/Pharmacology, Faculty o[ Medicine, Kyoto University, Kyoto 606, and Department o/Pharmacology, Kobe University School o/ Medicine, Kobe 650 (Japan), 22 February 1977 Summary. A d r e n e r g i c i n n e r v a t i o n in r a t r e n a l capsule was d e m o n s t r a t e d u s i n g t h e h i s t o c h e m i c a l fluorescence m e t h o d w i t h glyoxylic acid. T h e fluorescence h i s t o c h e m i c a l m e t h o d of F a l c k a n d H i l l a r p 2 h a s r e v e a l e d a d r e n e r g i c i n n e r v a t i o n in t h e r e n a l b l o o d vessels 3-5 a n d t u b u l e s S ; h o w e v e r , l o c a l i z a t i o n of m o n o a m i n e s in t h e r e n a l c a p s u l e h a s n o t b e e n r e p o r t e d . A n e w h i s t o c h e m i c a l t e c h n i q u e u s i n g glyoxylic acid i n s t e a d of f o r m a l d e h y d e h a s b e e n d e v e l o p e d 7, s a n d good r e s u l t s are seen w i t h t h e m e t h o d u s i n g a q u e o u s s o l u t i o n a n d h e a t i n g in t h e s t r e t c h p r e p a r a t i o n of p e r i p h e r a l t h i n tissues ~. W e a t t e m p t e d to d e m o n s t r a t e a d r e n e r g i c i n n e r r a t i o n in t h e r e n a l c a p s u l e u s i n g t h i s glyoxylic acid method. Male W i s t a r r a t s w e i g h i n g i50• g were sacrificed u n d e r p e n t o b a r b i t a l a n e s t h e s i a . T h e k i d n e y s w i t h t h e capsules were r a p i d l y excised a n d i m m e r s e d in ice-cold ~ 2 % glyoxylic acid solution. G l y o x y l i c acid m o n o h y d r a t e was dissolved in 0.1 M p h o s p h a t e b u f t e r a d j u s t e d t o p H 7.0 w i t h N a O H . 10 rain later, t h e capsules were r e m o v e d a n d
s t r e t c h e d o n clean glass slides. T h e s p e c i m e n s were d r i e d w i t h a h a i r - d r y e r for 20 min, h e a t e d a t 100 ~ for 5-10 m i n and then mounted with the entellan-xylene mixture. For microscopic analyses, a Zeiss epifluorescence microscope was used w i t h a h i g h p r e s s u r e m e r c u r y l a m p as a l i g h t source. T h e e x c i t a t i o n filter used was a BG12 a n d t h e b a r r i e r filter was Zeiss '47' or '50'. T h e o u t e r surface of t h e k i d n e y is c o v e r e d w i t h a d e l i c a t e f i b r o u s c a p s u l e c o m p o s e d of c o l l a g e n o u s a n d elastic fibres a n d a few s m o o t h m u s c l e cells. O u t s i d e tile t u n i c a fibrosa is a f a t t y l a y e r called t h e t u n i c a adiposa. F l u o r e s c e n t n e r v e fibres w i t h v a r i c o s i t i e s were f o u n d t o i n n e r v a t e t h e hilus a r t e r i e s of t h e k i d n e y a n d t h e i r r a m i f i c a t i o n s to e n t e r t h e r e n a l capsule. T h e r e n a l c a p s u l a r a r t e r i e s were a c c o m p a n i e d b y 2 m a i n b u n d l e s in w h i c h r a n 2 or m o r e i n t e n s e l y f l u o r e s c e n t fibres a n d b o t h b u n d l e s were c o n n e c t e d b y a few fine varicose t e r m i n a l s {figure 1}. T h e r e were 2 t y p e s
Fig. 1. The renal capsule of the rat. The adrenergie nerve fibres run along the blood vessels, on to the fat cells (F) and freely on the renal capsule ( ~' ). Yellow fluorescent mast (:ells ( ~") were seen around the blood vessels and the fat cells. • 30.
Fig. 2. The tunica fibrosa of the rat kidney. A systenl of branching terminals arising from one preterminal axon ( j ' ) is visible irl the tunica fibrosa. • 30.
