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Effect of zinc on the teratogenic action and tissue distribution of cadmium in the rat Garcia, Miren Lizaso de 1980

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EFFECT OF ZINC ON THE TERATOGENIC ACTION AND TISSUE DISTRIBUTION OF CADMIUM IN THE RAT B.Sc. U n i v e r s i d a d C e n t r a l de Venezuela, 1976 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES D i v i s i o n of Human N u t r i t i o n School of Home Economics We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA by MIREN LIZASO DE ARCIA February 1980 (c) Miren L i z a s o de G a r c i a , 1980 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f fM/?*/ t)(flR\HGti - SCtfOOL GF- Hm £ Q £ 0 ^  M l c The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 w e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 D a t e JfvJt, M (11 tt D E - 6 B P 75-51 1 E ABSTRACT The i n t e r a c t i o n o f z i n c and cadmium was s t u d i e d i n pregnant r a t s and t h e i r f e t u s e s . Four e x p e r i mental groups of animals were used, with each group being given one of the f o l l o w i n g treatments by i n t r a p e r i t o n e a l i n j e c t i o n : (1) cadmium c h l o r i d e (16uM/kg body weight) with 115m-cadmium, (2) cadmium c h l o r i d e (16uM/kg body weight) p l u s z i n c c h l o r i d e (16uM/kg body weight) with 115m-cadmium, (3) z i n c c h l o r i d e (16uM/kg body weight) with 6 5-zinc, o r (4) z i n c c h l o r i d e (16uH/kg body weight) plus cadmium c h l o r i d e (16uM/kg body weight) with 65-zinc._ In the f i r s t experiment the chemicals were.administered on day 9 of pregnancy and the animals were s a c r i f i c e d on day 20 of pregnancy. Fetuses were examined f o r gross d e f e c t s and maternal t i s s u e s and f e t u s e s were counted f o r r a d i o a c t i v i t y i n order to determine the d i s t r i b u t i o n of cadmium and z i n c . . Gross a b n o r m a l i t i e s were seen i n group 1 (cadmium), where 28.8 percent of the f e t u s e s were malformed. Defec t s i n c l u d e d absent t a i l ( 7 . 7 % ) , d y s p l a s t i c t a i l (10.3%), t h i n abdominal w a l l (6.4%), d y s p l a s t i c neck (5.8%), d y s p l a s t i c eyes (3.2%), exencephaly (1.9%), d y s p l a s t i c ears (1.3%), d y s p l a s t i c limbs (0.6%), and stunted body (0.6%). In group 3 ( z i n c ) , 2 malformed f e t u s e s both with exencephaly were found i n one l i t t e r * No malformations were found i n groups 2 and 4 (cadmium plus z i n c ) . In a d d i t i o n , f e t a l weight was s i g n i f i c a n t l y decreased i n group 1 (cadmium) when compared with groups 2, 3, and 4. There were no d i f f e r e n c e s i n numbers of r e s o r p t i o n s i t e s , numbers of dead f e t u s e s , o r l i t t e r s i z e among the f o u r groups;. There were no d i f f e r e n c e s i n the maternal t i s s u e d i s t r i b u t i o n of cadmium between group 1 (cadmium) and group 2 (cadmium-zinc). On the other hand, z i n c accumulation was s i g n i f i c a n t l y i n c r e a s e d i n group 4 (zinc-cadmium) compared with group 3 ( z i n c ) , i n l i v e r , kidney, a d r e n a l * h e a r t , lung s p l e e n , p l a c e n t a , f e t u s , f a t , uterus, muscle, and plasma. In the second experiment pregnant Wistar r a t s were given treatments 1 (cadmium) and 2 (cadmium-zinc) on day 9, and t i s s u e s were sampled on day 10, 11, and 12 of g e s t a t i o n * . When animals give cadmium were compared with those given cadmium plus z i n c , a s i g n i f i c a n t l y g r e a t e r amount of r a d i o a c t i v i t y (115m-cadmium) was found i n the uter u s , uterus p l u s f e t u s , f e t u s , f a t , and plasma on day 10 i n the cadmium group. On day 11 the only d i f f e r e n c e s found were i n c r e a s e d amounts of r a d i o a c t i v i t y i n the a d r e n a l s and s m a l l i n t e s t i n e * On day 12 r a d i o a c t i v i t y was i n c r e a s e d o n l y i n the femur* When the 4 d i f f e r e n t sampling days (10, 11, 12, and 20) were compared, i t was found t h a t i n most t i s s u e s the amount o f cadmium d e p o s i t e d decreased with time except f o r the kidney, where cadmium i n c r e a s e d , and the l i v e r and spleen where: th e r e was no change. The data suggest t h a t cadmium reaches the f e t u s i n the e a r l y hours a f t e r i n j e c t i o n and i s excreted t h e r e a f t e r . Simultaneous a d m i n i s t r a t i o n of z i n c d i d not change cadmium accumulation i n maternal t i s s u e s but s i g n i f i c a n t l y reduced the amount of cadmium reachin g the f e t u s 24 hours p o s t - i n j e c t i o n . On the other hand, simultaneous a d m i n i s t r a t i o n of cadmium i n c r e a s e d the accumulation of z i n c i n most of the organs i n v e s t i g a t e d . i v TABLE OF CONTENTS Abstra c t i i Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i v L i s t o f Tables v i L i s t of F i g u r e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v i i i Acknowledgement x Chapter I 1 I n t r o d u c t i o n 1 Chapter I I 5 Beview o f L i t e r a t u r e 5 Ter a t o g e n i c A c t i o n : General C o n s i d e r a t i o n s ............ 7 Cadmium as a Teratogen 11 Cadmium and Zinc D i s t r i b u t i o n i n T i s s u e s .............. 18 Z i n c as a Teratogen 24 Cadmium and Zinc I n t e r a c t i o n s 26 Cadmium i n Human Health ...............................33 Chapter I I I .39 M a t e r i a l s and Methods • 39 Experiment 1 • 39 Treatment of Experimental Animals 39 Examination and C o l l e c t i o n of T i s s u e s 41 Experiment 2 43 Treatment of Experimental Animals . . . 4 3 Examination and C o l l e c t i o n of T i s s u e s ..,...,.,......44 Counting o f R a d i o i s o t o p e s ............................... 44 S t a t i s t i c a l A n a l y s i s 4 5 V Chapter IV 47 R e s u l t s 47 Chapter V 90 D i s c u s s i o n .............................................. 90 B i b l i o g r a p h y • • 100 v i LIST OF TABLES Table I E f f e c t of Zinc on Cadmium-Induced F e t a l M alformations, and Cadmium on Zinc-Induced F e t a l Malformations ..........................................43 Table I I E f f e c t of Z i n c on Cadmium and Cadmium on Z i n c -Induced C h a r a c t e r i s t i c s of Fetu s e s ...................... 49 Table I I I E f f e c t of Zin c and Cadmium on the Hematocrit of Wistar Bats ............................................50 Table IV E f f e c t o f Zinc on Cadmium D i s t r i b u t i o n i n Organs of Wistar Rats Sampled on Day 20 of G e s t a t i o n ... 5 2 Table V E f f e c t of Zinc on Cadmium D i s t r i b u t i o n i n Organs of Wistar Rats Sampled on Day 20 of G e s t a t i o n ............. 53 Table VI E f f e c t o f Cadmium on Zinc D i s t r i b u t i o n i n Organs o f Wistar Rats Sampled on Day 20 of G e s t a t i o n ... 54 Table VII E f f e c t o f Cadmium on Zinc D i s t r i b u t i o n i n Organs of Wistar Rats Sampled on Day 20 of G e s t a t i o n ... 56 Table V I I I E f f e c t of Zinc on Cadmium D i s t r i b u t i o n i n Organs of Wistar Rats Sampled on Day 10 of G e s t a t i o n *..,58 Table IX E f f e c t of Z i n c on Cadmium D i s t r i b u t i o n i n Organs of Wistar Rats Sampled on Day 10 of G e s t a t i o n ... 59 Table X E f f e c t of Zinc on Cadmium D i s t r i b u t i o n i n Organs of Wistar Rats Sampled on Day 11 of G e s t a t i o n .............60 Table XI E f f e c t o f Z i n c on Cadmium D i s t r i b u t i o n i n Organs of Wistar Rats Sampled on Day 11 of G e s t a t i o n . . . 6 1 Table XII E f f e c t o f Z i n c on Cadmium D i s t r i b u t i o n i n v i i Organs of Wistar Rats Sampled on Day 12 of G e s t a t i o n ... 62 Table XIII E f f e c t of Zinc on Cadmium D i s t r i b u t i o n i n Organs of Wistar Rats Sampled on Day 12 of G e s t a t i o n ... 63 Table XIV Cadmium D i s t r i b u t i o n i n Organs of Wistar Rats Sampled on Day 10, 11, and 12 of G e s t a t i o n .....64 Table XV Cadmium D i s t r i b u t i o n i n Organs of Wistar Rats Sampled on Day 10, 11, and 12 of G e s t a t i o n ............. 65 Table XVI E f f e c t o f Zinc on Cadmium D i s t r i b u t i o n i n Organs of Wistar Rats Sampled on Day 10, 11, and 12 of G e s t a t i o n ..............................................67 Tabl e XVII E f f e c t of Zinc on Cadmium D i s t r i b u t i o n i n Organs of Wistar Rats Sampled on Day 10, 11, and 12 of G e s t a t i o n .................................. Table XVIII E f f e c t o f Zin c and Cadmium on the Maternal Hematocrit, L i t t e r S i z e , and F e t a l Weight of Wistar Rats 6 9 v i i i LIST OF FIGURES F i g u r e I Accumulation of 115m-Cadmium i n L i v e r of Animals Given Cadmium or Cadmium plus Zinc .....................70 F i g u r e I I Accumulation of 115m-Cadmium i n Kidneys of Animals Given Cadmium or Cadmium Plus Zinc .............71 Fi g u r e I I I Accumulation of 115m-Cadmium i n Brains of Animals Given Cadmium or Cadmium plus Z i n c .............72 F i g u r e IV Accumulation of 115m-Cadraium i n Adrenals of Animals Given Cadmium or Cadmium plus Zinc .............73 Figu r e V Accumulation of 115m-Cadmium i n Hearts of Animals Given Cadmium or Cadmium plus Zinc ..................... 75 Fi g u r e VI Accumulation of 115m-Cadmium i n Lungs o f Animals Given Cadmium or Cadmium plus Z i n c .....................76 F i g u r e VII Accumulation of 115m-Cadmium i n Spleens of Animals Given Cadmium or Cadmium p l u s Z i n c ............. .77 Fi g u r e V I I I Accumulation of 115m-Cadmium i n Femurs of Animals Given Cadmium or Cadmium plus Z i n c .,,.,....,.,.78 Fi g u r e IX Accumulation of 115m-Cadmium i n Uterus of Animals Given Cadmium or Cadmium plus Z i n c .....................79 Fi g u r e X Accumulation of 115m-Cadmium i n Uterus plu s Fetus o f Animals Given Cadmium or Cadmium p l u s Z i n c .....81 Fi g u r e XI Accumulation of 115m-Cadmiura i n Fetuses of Animals Given Cadmium or Cadmium plus Z i n c ......,82 Fi g u r e XII Accumulation of 115m-Cadmium i n Blood of Animals Given Cadmium or Cadmium plus Zinc ..................... 83 i x F i g u r e X I I I Accumulation of 115m-Cadmium i n I n t e s t i n e of Animals Given Cadmium or Cadmium plus Zinc ............. 84 Fi g u r e XIV Accumulation of 115m-Cadmium i n Fat o f Animals . Given Cadmium or Cadmium plus Zinc ..................... 85 F i g u r e XV Accumulation of 115m-Cadmium i n Muscles of Animals Given Cadmium or Cadmium plus Z i n c 86 Figu r e XVI Accumulation of 115m-Cadmium i n Plasma of Animals Given Cadmium or Cadmium plus Zinc .............88 Fi g u r e XVII E f f e c t o f Cadmium or Cadmium plus Zinc i n the Maternal Hematocrit of Wistar Rats ..................... 89 X ACKNOWLEDGEMENT I would l i k e t o thank my s u p e r v i s o r Dr. Melvin Lee f o r h i s advice and encouragement throughout the whole of t h i s work..I am a l s o g r a t e f u l t o Dr. Kenneth Chan f o r h i s i n t e r e s t i n the p r o j e c t and f o r the many u s e f u l d i s c u s s i o n s we had together* , A l s o acknowledged are Dr. P a t r i c i a G a l l o and Dr. John Vanderstoep f o r t h e i r h e l p f u l s u g g e s t i o n s and comments, as well, as Mrs. V i r g i n i a Green f o r her help with the s t a t i s t i c a l a n a l y s i s of the data and Miss C h r i s t i n a Duke f o r her c o l l a b o r a t i o n i n the production o f t h i s manuscrit.. Thanks are a l s o due to my husband, F r a n c i s c o G a r c i a f o r h i s sug g e s t i o n s , p a t i e n c e , and understanding. F i n a n c i a l support from the Venezuelan Government through FGMA (Fundacion Gran M a r i s c a l de Ayacucho), from the N a t i o n a l Research C o u n c i l of Canada Grant A4692, and a grant from the U n i v e r s i t y of B r i t i s h Columbia Committee on Research, i s g r a t e f u l l y a p p r e c i a t e d . 1 CHAPTER I INTRODUCTION In r e c e n t years the presence of cadmium as an environmental contaminant has aroused concern because of i t s marked t o x i c i t y f o r humans ( F l i c k et a l . , 1971), with both acute and c h r o n i c p o i s o n i n g being d e s c r i b e d . Furthermore, cadmium i s of concern because the sources are so widespread i n nature;_ Cadmium i s c l o s e l y r e l a t e d , c h e m i c a l l y , t o z i n c and i s found wherever z i n c occurs i n nature, although the r e l a t i v e amounts of the two minerals vary from place to p l a c e . I n most mineral d e p o s i t s and s o i l s the r a t i o of cadmium to z i n c i s between 1:100 and 1:1000. Since z i n c i s an e s s e n t i a l n u t r i e n t f o r most forms of l i f e ( Onderwood, 1962; Bowen, 1966; Schroader e t a l . , 1967; and Yamagata and Shigematsu, 1970), the c l o s e a s s o c i a t i o n between cadmium and z i n c suggests t h a t probably no n a t u r a l l y o c c u r r i n g m a t e r i a l w i l l be completely f r e e of cadmium. Furthermore, cadmium occurs as an environmental p o l l u t a n t , because i t i s a byproduct of some commercial processes. For example, cadmium i s obtained as a byproduct i n the r e f i n i n g o f z i n c and other metals. Since i t i s d i f f i c u l t t o separate cadmium and z i n c , the former w i l l o f t e n be found i n s m a l l amounts i n commercially a v a i l a b l e z i n c compounds (Schroeder e t a l . , 1967). Environmental cadmium p o l l u t i o n probably has a very l o n g h i s t o r y , as copper, l e a d , and z i n c s m e l t i n g are o l d proc e s s e s . However, the concern with cadmium p o l l u t i o n and t o x i c i t y has become more urgent because of the i n c r e a s i n g use of cadmium compounds by i n d u s t r y i n t h i s c e n t u r y . Not on l y i s cadmium emitted i n t o the atmosphere by mining and smelting ( p a r t i c u l a r l y 2 l e a d , copper, and z i n c s m e l t i n g ) , but cadmium i s widely used i n b a t t e r i e s , a l l o y s , p a i n t s , and p l a s t i c s , as w e l l as i n the p l a t i n g i n d u s t r y and i n c e r t a i n ceramic f i n i s h e s . The burning o f o i l and wastes, and the treatment of scrap metals a l s o c o n t r i b u t e cadmium t o the environment, as does the use o f cadmium-containing p e s t i c i d e s . F i n a l l y the i n c r e a s i n g use of sewage sludge, which may c o n t a i n a c o n s i d e r a b l e amount of cadmium, f o r the f e r t i l i z a t i o n of farm land can i n t r o d u c e cadmium d i r e c t l y i n t o the food supply ( F r i b e r g e t a l . , 1974). . The growing r e c o g n i t i o n of the importance of heavy metal p o l l u t i o n of the environment, i n c l u d i n g cadmium p o l l u t i o n , has focussed a t t e n t i o n on the i m p l i c a t i o n s f o r human h e a l t h . The e f f e c t of cadmium on mammalian r e p r o d u c t i o n and f e t a l development has been o f p a r t i c u l a r i n t e r e s t s Although the p o s s i b l e r o l e of cadmium i n human t e r a t o g e n e s i s remains unresolved, i t has been recognized f o r some time t h a t the o r a l a d m i n i s t r a t i o n of cadmium s a l t s t o pregnant mice causes an i n c r e a s e i n f e t a l m o r t a l i t y , a r e t a r d a t i o n of f e t a l growth, and an i n c r e a s e i n malformations (Schroeder and Hitchener, 197 1). Cadmium s u l f a t e , given i n t r a v e n o u s l y , i s t e r a t o g e n i c i n hamsters, mainly producing malformations of the f a c e ( M u l v i h i l l et a l . , 1970). Given i n t r a p e r i t o n e a l l y , i t i s t e r a t o g e n i c i n r a t s (Barr, 1972), although the spectrum of malformations i s q u i t e d i f f e r e n t from t h a t seen i n hamsters. Cadmium as the c h l o r i d e , a c e t a t e , or l a c t a t e , was shown to produce p l a c e n t a l d e s t r u c t i o n and f e t a l m o r t a l i t y when administered subcutaneously to Wistar r a t s (Parizek, 1964) . However, there appear to be some d i f f e r e n c e s i n s u s c e p t i b i l i t y among d i f f e r e n t s t o c k s of the same 3 s t r a i n of r a t s , as Barr (1973) demonstrated t h a t the i n c i d e n c e and v a r i e t y of f e t a l malformations d i f f e r e d i n two s t o c k s o f Wistar r a t s given cadmium. In a d d i t i o n t o i t s t e r a t o g e n i c a c t i o n , cadmium has been shown t o evoke a number of other p a t h o l o g i c a l responses, ( F r i b e r g et a l . , 1974), s t i m u l a t i n g i n t e r e s t i n the metabolism and t i s s u e d i s t r i b u t i o n of the metal. Cadmium has been found t o be p r i m a r i l y concentrated i n the l i v e r and kidneys, although other t i s s u e s take up s m a l l e r amounts (Kotsonis and Klaassen, 1977). The p a t h o l o g i c a l consequences are t i s s u e and organ s p e c i f i c , i n c l u d i n g t e s t i c u l a r atrophy (Parizek and Lahor, 1956), r e n a l d y s f u n c t i o n ( P i s c a t o r , 1962), hypertension (Perry and E r l a n g e r , 1974), h e p a t i c damage ( F r i b e r g , 1955), and anemia ( F r i b e r g , 1950). However, the manner i n which cadmium e x e r t s i t s t o x i c e f f e c t s i s not c l e a r l y understood. There i s some evidence t h a t z i n c e x e r t s a p r o t e c t i v e a c t i o n a g a i n s t p a t h o l o g i c a l t e r a t o g e n i c i t y of cadmium. Ferm and Carpenter (1967, 1968) demonstrated t h a t when z i n c was a dministered t o pregnant r a t s s i m u l taneously with t e r a t o g e n i c amounts of cadmium, the i n c i d e n c e of f e t a l malformations was reduced, and Webb (1972) and Sarkar and Hondal (1973) r e p o r t e d t h a t z i n c prevents cadmium-induced t e s t i c u l a r damage i n r a t s , mice, and pigeons. Ferm and Carpenter (1967, 1968) suggested t h a t the t o x i c a c t i v i t y o f cadmium may be a consequence of i t s replacement of z i n c i n metalloenzymes. As z i n c has been shown t o be e s s e n t i a l f o r normal r e p r o d u c t i o n and e m b r y o l o g i c a l development ( V a l l e e , 1959; Forbes, 1967), i t s replacement by cadmium may be expected to have d e l e t e r i o u s consequences.. 4 T h e r e f o r e , the r e s e a r c h p r o j e c t d e s c r i b e d here was designed to i n v e s t i g a t e the r e l a t i o n s h i p between z i n c and cadmium i n r e p r o d u c t i o n and development i n the r a t . . Two a s p e c t s of t h i s r e l a t i o n s h i p were studied._ The t e r a t o g e n i c i t y of cadmium and z i n c and t h e i n t e r a c t i o n of the two minerals was examined by a d m i n i s t e r i n g them i n d i v i d u a l l y or together t o pregnant r a t s . Then the e f f e c t of simultaneous a d m i n i s t r a t i o n o f z i n c on the d i s t r i b u t i o n of cadmium i n the maternal t i s s u e s and f e t u s and o f cadmium on the d i s t r i b u t i o n of z i n c i n maternal t i s s u e s and f e t u s was examined, using 115m-cadmium and 65-zinc. 5 CHAPTER I I REVIEW OF LITERATURE Although i t has been known f o r more than a century t h a t cadmium i s an extremely t o x i c element and may c o n s t i t u t e a source o f damage t o human h e a l t h , i t was not u n t i l the outbreak of the endemic " I t a i - I t a i " d i s e a s e (chronic cadmium poisoning) among the i n h a b i t a n t s of Toyama, Japan t h a t the e f f e c t s o f cadmium on h e a l t h have a t t r a c t e d s p e c i a l a t t e n t i o n i n both environmental and medical p r o f e s s i o n s ( I s h i z a k i , 1971). Since World War I I , cadmium i n m e t a l l i c , i n o r g a n i c , and o r g a n i c forms has been widely used i n m e t a l l u r g i c a l i n d u s t r i e s (Dubois and G e i l i n g , 1959) f o r the p r o d u c t i o n of cooking u t e n s i l s , b a t t e r i e s , g l a s s , p a i n t s , p l a s t i c s , e t c . ( F l i c k e t a l . , 1971). Because of the extensive use of cadmium, i n c r e a s i n g awareness has been generated concerning the r o l e of cadmium as an environmental hazard. Many i n v e s t i g a t o r s have s p e c u l a t e d , based on experimental o b s e r v a t i o n s , t h a t cadmium may be i n t i m a t e l y a s s o c i a t e d with v a r i o u s p a t h o l o g i c a l c o n d i t i o n s such as c e r t a i n forms of r e n a l and c a r d i o v a s c u l a r d i s e a s e s o c c u r i n g i n man ( P i s c a t o r , 1962; Perry and E r l a n g e r , 1974). The b i o l o g i c a l e f f e c t s of cadmium, a c c o r d i n g to Dencker (1975)* deserve a t t e n t i o n f o r many reasons. I t s use i n d i f f e r e n t i n d u s t r i a l processes and products has i n c r e a s e d i t s presence i n the environment. I t s e x c e p t i o n a l l y l o n g h a l f - l i f e i n the human body, about 10 t o 30 years ( K j e l l s t r o m e t a l . , 1971), s t r e s s e s the importance of a b e t t e r understanding of i t s a c t i o n . Cadmium a f f e c t s s p e c i f i c t i s s u e s i n the body; f o r example, the s t e r o i d hormone producing organs (Parizek 1957; Kar et a l . , 1959 ; Kaul 6 and Ramaswani, 1970). In l a t e g e s t a t i o n i t causes p l a c e n t a l n e c r o s i s , e s p e c i a l l y i n the pars f e t a l i s , and subsequent f e t a l death. In the mother, i t causes p a t h o l o g i c a l changes with c l i n i c a l and morphological s i m i l a r i t i e s to human toxemia o f pregnancy (Pari z e k , 1965). The metabolism of cadmium has been s t u d i e d by F r i b e r g et a l . (1971) a f t e r exposure v i a r e s p i r a t o r y , g a s t r o i n t e s t i n a l , or i n j e c t i o n r o u t e s . The extent of a b s o r p t i o n i s not known e x a c t l y with regard t o i n h a l e d or i n g e s t e d cadmium, but reasonable estimates are about 10% t o 40% f o r the i n h a l e d and 2% f o r the i n g e s t e d cadmium*. The a b s o r p t i o n of i n g e s t e d cadmium may i n c r e a s e c o n s i d e r a b l y i f the d i e t i s d e f i c i e n t i n cal c i u m , o r p r o t e i n , or both. There i s a l s o c o n s i d e r a b l e i n d i v i d u a l v a r i a t i o n s i n the a b s o r p t i o n of cadmium (Fitzhugh and M e i l l e r , 1941). A f t e r a s i n g l e i n j e c t i o n ( P i s c a t o r and Larson, 1972), cadmium w i l l i n i t i a l l y be found mainly i n the plasma, but d u r i n g the f i r s t 24 hours a f t e r i n j e c t i o n a r a p i d c l e a r a n c e from plasma takes p l a c e , so t h a t e v e n t u a l l y the c o n c e n t r a t i o n i n the e r y t h r o c y t e s w i l l exceed t h a t i n the plasma (Carlson and F r i b e r g , 1957). A f t e r repeated exposure cadmium w i l l mainly be found i n the blood c e l l s , bound t o p r o t e i n s such as m e t a l l o t h i o n e i n and hemoglobin. During c h r o n i c exposure to cadmium there w i l l be a gr a d u a l i n c r e a s e i n blood l e v e l s of cadmium, but a t a c e r t a i n l e v e l a p l a t e a u w i l l be reached and no f u r t h e r i n c r e a s e w i l l be seen* When exposure has ceased, the c o n c e n t r a t i o n i n blood w i l l decrease* F i f t y t o 75% of the t o t a l body burden of cadmium w i l l be found i n the l i v e r and kidneys. Immediately a f t e r a s i n g l e 7 exposure most w i l l be i n the l i v e r * but e v e n t u a l l y r e n a l l e v e l s w i l l exceed l i v e r l e v e l s . . Repeated exposure to small amounts w i l l a l s o r e s u l t i n the r e n a l c o n c e n t r a t i o n s s u r p a s s i n g the l i v e r so l i v e r c o n c e n t r a t i o n s w i l l not exceed r e n a l c o n c e n t r a t i o n s . The accumulation i n the l i v e r and kidneys seems, i n l a r g e p a r t , to be a r e s u l t of the storage of cadmium i n the cadmium-binding p r o t e i n , m e t a l l o t h i o n e i n ( F r i b e r g e t a l . , 1974). Cadmium i s p r i m a r i l y excreted i n the f e c e s and the u r i n e , but other routes of e x c r e t i o n i n c l u d e h a i r * p l a c e n t a , s a l i v a , and s k i n . In a d d i t i o n , the metabolism of cadmium i s i n t i m a t e l y conected with z i n c metabolism* The low molecular weight p r o t e i n , m e t a l l o t h i o n e i n , i s able to bind both cadmium and z i n c , and these two metals w i l l be t r a n s p o r t e d together.,(Bunn et a l . , 1962). Z i n c i s an e s s e n t i a l metal and many exzymes are z i n c dependent.. Cadmium seems to have the a b i l i t y to exchange with z i n c i n d i f f e r e n t organs, causing change i n enzymatic a c t i v i t y ( F r i b e r g e t a l . , 1974). During exposure t o cadmium, organ l e v e l s of z i n c w i l l i n c r e a s e , but the a c t u a l mechanisms r e s p o n s i b l e f o r t h i s are s t i l l not c l e a r ( F r i b e r g et a l . , 1974). TERATOGENIC ACTION: GENEHAL CONSIDERATIONS When experimental mammalian t e r a t o l o g y developed i n the 1930's, the s c i e n c e of experimental t e r a t o l o g y i n lower c l a s s e s of animals was at l e a s t 100 years o l d . Experiments on a v i a n and amphibian eggs were performed s u c c e s s f u l l y d u r i n g the n i n e t e e n t h and e a r l y t w e n t i e t h c e n t u r i e s , and many p r i n c i p l e s and fundamental f a c t s were.already known. However, t h e r e was doubt 8 t h a t the r e s u l t s obtained on eggs of lower forms c o u l d be a p p l i e d t o mammalian or human s i t u a t i o n s . I t was thought t h a t mammalian embryos and f e t u s e s were so w e l l p r o t e c t e d by the maternal organism t h a t they c o u l d not be modified by c o n d i t i o n s shown to be t e r a t o g e n i c f o r embryos of lower animals. I t was thought t h a t adverse environmental c o n d i t i o n s would e i t h e r k i l l the mammalian embryo or l e a v e i t unharmed (Warkany, 1964)., Modern r e p r o d u c t i v e s t u d i e s are concerned with e v a l u a t i n g p o s s i b l e e f f e c t s on f e r t i l i t y , on the zygote, on i t s t r a n s p o r t , on i m p l a n t a t i o n and development, on the t e r a t o g e n i c p o t e n t i a l , on p a r t u r i t i o n , on the newborn, on l a c t a t i o n , on weaning, on care o f the young and on delayed p o s t n a t a l anomalies (Frohberg, 1977). A v a r i e t y of f a c t o r s i n f l u e n c e the outcomes of t e r a t o g e n i c i t y s t u d i e s . In t e r a t o g e n i c s t u d i e s , m i s l e a d i n g n e g a t i v e or p o s i t i v e r e s u l t s can be obtained by u s i n g the wrong animal s p e c i e s * Methotrexate , which i s t e r a t o g e n i c i n man and i n s m a l l r o d e n t s , does not cause malformations i n rhesus monkeys (Wilson and F r a d k i n , 1969; Wilson, 1971).. C a r b a r y l i s t e r a t o g e n i c i n dogs, but not i n r a t s , guinea p i g s , r a b b i t s , hamsters, rhesus monkeys, or man. The r e s u l t obtained i n dogs with c a r b a r y l , however, are not r e l e v a n t t o man as the metabolism of c a r b a r y l i s d i f f e r e n t i n the two s p e c i e s (Smalley e t a l . , 1968; Weil et a l . , 1972, 1973). As d i f f e r e n c e s between s p e c i e s are i n p a r t due to d i f f e r e n c e s i n the metabolism and pharmacokinetics of drugs, animal s p e c i e s used f o r t e r a t o g e n i c s t u d i e s should, i f p o s s i b l e , absorb, d i s t r i b u t e , metabolize, and excrete the t e s t compound i n 9 a manner s i m i l a r t o t h a t i n man. In the case of drugs, t h e i r pharmacodynamic p r o p e r t i e s should be taken i n t o c o n s i d e r a t i o n . Adverse p h a r m a c o l o g i c a l e f f e c t s on the maternal animal, l i k e anorexia o r s e d a t i o n , may have an i n f l u e n c e on f e t a l development (Frohberg, 1977). T h e r e f o r e i n a l l t e r a t o l o g i c s t u d i e s the dams should be subjected to comprehensive c l i n i c a l and post-mortem examination i n order to d e t e c t l e s i o n s which may impair f e t a l development. Although t h e r e are some d i f f e r e n c e s i n recommendations f o r t e r a t o g e n i c i t y t e s t i n g , as f a r as d u r a t i o n of treatment and the time of a d m i n i s t r a t i o n are concerned, the r e l a t i o n s h i p must be c l o s e l y examined, because embryonic d i f f e r e n t i a t i o n proceeds r a p i d l y d u r i n g the p e r i o d of organogenesis (Frohberg, 1977).. In g e n e r a l , drugs and chemicals are a d m i n i s t e r e d d u r i n g the p e r i o d of organogenesis, s i n c e the onset of t e r a t o g e n i c s u s c e p t i b i l i t y occurs at about the time the germ l a y e r s are formed. In mammals t h i s i s s e v e r a l days a f t e r c o n c e p t i o n , about 5 days i n hamster and mouse, 8 days i n r a t , 9 days i n r a b b i t , assumed to be 10 days i n monkey, and could be as e a r l y as 11 or 12 days i n man. Most organs have a p e r i o d of p a r t i c u l a r s u s c e p t i b i l i t y t o t e r a t o g e n s , and t h i s very l i k e l y c o i n c i d e s with e a r l y and c r i t i c a l developmental events i n t h a t organ (Wilson, 1964). Furthermore, a d m i n i s t r a t i o n of a teratogen d u r i n g an e a r l i e r p e r i o d of g e s t a t i o n may cause embryonic death so t h a t malformations l i k e l y to appear as a r e s u l t of l a t e r treatment may be hidden. For t h i s reason Wilson (1973) recommended a d m i n i s t e r i n g suspected teratogens i n s u c c e s s i v e s h o r t segments durin g the p e r i o d of organogenesis* 10 In a d d i t i o n , important i n f o r m a t i o n may be gained from s t u d i e s i n which the drug c o n c e n t r a t i o n i s measured i n the p l a c e n t a and i n other maternal and f e t a l t i s s u e s to determine whether the pharmacokinetics of a chemical d i f f e r i n the pregnant and the non-pregnant animal. P h y s i o l o g i c a l substances normally are accumulated i n f e t a l t i s s u e i n a s i m i l a r way as i n the maternal t i s s u e s . The d i s t r i b u t i o n p a t t e r n w i t h i n the f e t u s sometimes shows a very s t r o n g and s e l e c t i v e accumulation i n one p a r t i c u l a r f e t a l t i s s u e . T e t r a c y c l i n e accumulates s e l e c t i v e l y i n the f e t a l s k e l e t o n , t h i o u r a c i l i n the f e t a l t h y r o i d gland ( O l l b e r g e t a l . , 1970). The d i s t r i b u t i o n p a t t e r n i s i n f l u e n c e d by c h e m i c a l p r o p e r t i e s , such as f a t s o l u b i l i t y and degree o f i o n i z a t i o n . Fat s o l u b l e compounds g e n e r a l l y pass both i n t o the b r a i n and i n t o the f e t u s , while other drugs may be blocked by p l a c e n t a l and perhaps b l o o d - b r a i n b a r r i e r . . However, some substances seem t o cause f e t a l damage without r e a c h i n g the f e t u s (Frohberg, 1974). In a d d i t i o n , one important p o i n t i s the dosage. There i s a lower range which permits normal development, and a higher range which k i l l s a l l the embryos, and the mother a l s o i f extended f a r enough* Between these ranges t h e r e i s a narrow t e r a t o g e n i c zone i n which dosage i s s u f f i c i e n t t o i n t e r f e r e with s p e c i f i c developmental events without d e s t r o y i n g the whole embryo. (Wilson, 1 964) . T h e r e f o r e , the type of malformations induced by a t e r a t o g e n depends on the chemical s t r u c t u r e , the time of a d m i n i s t r a t i o n and, i n p a r t , on the dose* During the e a r l y cleavage s t a g e s , b l a s t o g e n e s i s , the s u s c e p t i b i l i t y of the embryo i s normally l e s s 11 than dur i n g organogenesis* However, th e r e are r e p o r t s showing t e r a t o g e n i c i t y i n e a r l y cleavage stages caused by X-rays (Rugh and Grupp, 1959). A f i n a l c o n s i d e r a t i o n i n t e r a t o g e n i c s t u d i e s may be genotype , the importance o f which has been amply d i s c u s s e d ( F r a s e r , 1961). In s i m p l e s t terms genotype i s important because i t determines the i n h e r e n t s u s c e p t i b i l i t y of an embryo to a given agent a t a given time of development. When organogenesis i s completed, the embryo en t e r s the f e t a l p e r i o d , which i s c h a r a c t e r i z e d p r i n c i p a l l y by growth and f u n c t i o n a l maturation. During t h i s p e r i o d , t e r a t o g e n e s i s , i n the s t r i c t sense does not occur because embryonic processes can no lo n g e r be i n t e r r u p t e d or d i v e r t e d . Any agent s u f f i c i e n t l y potent to a f f e c t the f e t u s would e i t h e r r e t a r d growth or cause pathology of the types t h a t occur i n p o s t n a t a l animals. Most of the l e s i o n s a s s o c i a t e d with c o n g e n i t a l s y p h i l i s should, i n t h i s l i g h t , be regarded as examples of c o n g e n i t a l pathology r a t h e r than malformations* The developing r a t en t e r s t h i s p e r i o d about the seventeenth day postconception.. Human embryogenesis i s completed about the end of the e i g h t h week of i n t r a u t e r i n e l i f e and, developmental d e f e c t s f o r the most part cannot be produced a f t e r t h i s time (Wilson, 1964). CADMIUM AS A TERATOGEN I n c r e a s i n g a t t e n t i o n i s being given t o the r o l e t h a t t r a c e elements may play i n disease processes.. However, r e l a t i v e l y l i t t l e i s known about the r o l e t h a t t r a c e elements may assume i n mammalian r e p r o d u c t i o n . Many of the heavy metals normally 12 present w i t h i n the body i n s m a l l q u a n t i t i e s are: becoming more important c o n s t i t u e n t s of the atmosphere i n areas of heavy i n d u s t r i a l i z a t i o n and, as such, are unknown f a c t o r s which may c o n t r i b u t e t o r e p r o d u c t i v e problems. . I t i s i n t e r e s t i n g t o note that some of the cadmium emitted t o the a i r by i n d u s t r i e s and mines w i l l be i n h a l e d by people and animals, but most of i t w i l l be deposited i n s o i l or water., The cadmium d e p o s i t e d i n water may then i n c r e a s e the c o n c e n t r a t i o n s of cadmium i n a q u a t i c organisms* In the event of f l o o d i n g or i r r i g a t i o n , cadmium i n water might a l s o i n c r e a s e the c o n c e n t r a t i o n s i n s o i l , i n turn causing an i n c r e a s e i n cadmium i n a g r i c u l t u r a l products, such as r i c e and wheat ( F r i b e r g e t a l * , 1974) . In a d d i t i o n , a c c o r d i n g t o Cherian e t a l . (1978) , the l a r g e s t sources of cadmium i n the normal d i e t are c e r e a l and meat, e s p e c i a l l y l i v e r and kidney, where cadmium occurs i n the form of m e t a l l o t h i o n e i n * Since m e t a l l o t h i o n e i n i s heat s t a b l e (Cherian, 1974), t h i s p r o t e i n - m e t a l complex i s probably not destroyed by cooking and thus may be the main d i e t a r y form of cadmium f o r people who consume these organ meats. Previo u s experiments have demonstrated the complex t e r a t o g e n i c i n t e r a c t i o n of cadmium and z i n c (Ferm, 1969). P r e l i m i n a r y s t u d i e s showed t h a t cadmium c o n c e n t r a t i o n s o f 7-18uM/kg of body weight were t e r a t o g e n i c i n r a t s when given on day 9, 10, or 11 of g e s t a t i o n , but excess f e t a l m o r t a l i t y and malformations were not found a f t e r a d m i n i s t r a t i o n of cadmium on day 6, 7, 8, or 12 (Ferm, 1969). Cadmium c o n c e n t r a t i o n s i n excess of 22uM/kg o f t e n k i l l e d pregnant r a t s (Webb, 1972). B e r l i n and D l l b e r g (1963) were unable t o demonstrate t h a t 109-13 cadmium c r o s s e d the mouse.placenta l a t e i n g e s t a t i o n , although Ferm e t a l . . (1969) found s i g n i f i c a n t r a d i o a c t i v i t y i n the f e t u s 24 hours a f t e r cadmium was given i n t r a v e n o u s l y t o pregnant hamsters on the 8th day of g e s t a t i o n . T h e r e f o r e , whether or not cadmium reaches the f e t u s and whether i t i s c o n c e n t r a t e d i n a f f e c t e d s i t e s i s unknown. Furthermore, Ferm e t a l . (1969) showed t h a t 96 hours a f t e r a d m i n i s t r a t i o n * the c o n c e n t r a t i o n of cadmium i n the hamster f e t u s had decreased some 6 0 - f o l d from the c o n c e n t r a t i o n present 24 hours a f t e r i n j e c t i o n . Thus there was a net e l i m i n a t i o n of cadmium from the f e t u s beyond the r e d u c t i o n i n c o n c e n t r a t i o n due to i n c r e a s e i n f e t a l mass. A p p r e c i a b l e amounts of 109-cadmium were found i n the hamster y o l k - s a c and p l a c e n t a , and t h i s may bear on the problem of how the f e t u s e l i m i n a t e s cadmium from i t s body (Ferm, 1969). Ferm (1971) s t a t e d that cadmium has been shown to induce s e r i o u s malformations of the f a c e when adm i n i s t e r e d to pregnant hamsters at s p e c i f i c times i n development. . The time of a d m i n i s t r a t i o n f o r these f a c i a l d e f e c t s c o i n c i d e d with the b e g i n i n g of the p e r i o d of c r i t i c a l embryogenesis* F o r example, the golden hamster undergoes a very r a p i d d i f f e r e n t i a t i o n d u r i n g the 8th day of g e s t a t i o n . Beginning e a r l y on the :8th day i t i s i n an e a r l y p r i m i t i v e s t r e a k stage and a t 8 p.m. on the 8th day 4 t o 6 somites have formed. E a r l y on the 9th day of g e s t a t i o n , the n e u r a l tube has completely c l o s e d and the heart has s t a r t e d t o beat* Thus, the hamster embryo t e l e s c o p e s i t s major stages o f d i f f e r e n t i a t i o n i n t o a 24-hour-period. .The : s i t e s p e c i f i c i t y of a number o f metal t e r a t o g e n s has been demonstrated and the f u l l 14 range of d e l e t e r i o u s e f f e c t s of these metals on developing organ systems should be determined. He a l s o s t a t e d t h a t these molecular teratogens might well exert t h e i r e f f e c t s v i a t h e i r important r o l e s i n the m o i e t i e s of the metalloenzymes and t h i s may y i e l d some v a l u a b l e i n f o r m a t i o n concerning the morphogenesis of s p e c i f i c malformations., In a d d i t i o n , Ferm (1971), using hamsters* has reported t h a t the spectrum of malformations changed as the time of the t e r a t o g e n i c i n s u l t i s s h i f t e d to l a t e r p e r i o d s i n embryonic development. Thus, f a c i a l malformations and exencephaly are found i n embryos t r e a t e d i n the e a r l y stages of development (beginning on the 8th day) while i n j e c t i o n s at l a t e r stages of development ( l a t e r on the 8th day) r e v e a l a decided d e c l i n e i n fac e and head a b n o r m a l i t i e s with a s h i f t to r i b and upper limb d e f e c t s * A b n o r m a l i t i e s o f those embryos t r e a t e d on the 9th day of g e s t a t i o n c o n s i s t e d p r i m a r i l y of r i b and limb d e f e c t s with a change toward d e f e c t s of the lower limbs*.Rib d e f e c t s were p r i n c i p a l l y those of f u s i o n of adjacent ribs.„ A l s o , animals t r e a t e d i n the 9th day of g e s t a t i o n r e v e a l e d severe a b n o r m a l i t i e s of the caud a l r e g i o n c h a r a c t e r i z e d by f u s i o n o f the lower e x t r e m i t i e s , and sympodia. Ferm (1971) a l s o found t h a t embryos t r e a t e d on the 8th day of g e s t a t i o n showed a higher r a t e of death i n utero as well as a higher i n c i d e n c e o f developmental malformation among the l i v i n g embryos* T h e r e f o r e , he o f f e r e d two hypotheses t o e x p l a i n the t e r a t o g e n i c events. The f i r s t was t h a t the teratogen i s n o n s p e c i f i c , the r e s u l t depending on the organogenetic event at the time of the i n s u l t * The second was t h a t c e r t a i n t eratogens 15 might w e l l prove t o be s i t e - s p e c i f i c and induce malformations only i n c e r t a i n organ systems. Barr, (1973) studying r a t s found t h a t when the r a t s were i n j e c t e d with 16 uM/kg body weight of cadmium c h l o r i d e on t h r e e d i f f e r e n t days of g e s t a t i o n (9th r 10th, or 11th), f e t a l weight was depressed. Anophthalmia or microphthalmia on one or both s i d e s was most o f t e n found a f t e r cadmium on day 9, and d y s p l a s t i c or absent ears were found only a f t e r day 9 treatment. Hydrocephalus was observed a f t e r cadmium treatment on day 9 or 11..Halformation of the t a i l , r a n g i n g from complete absence to t i g h t c u r l i n g of the t i p was found i n a high percentage of the s u r v i v o r s f o l l o w i n g i n j e c t i o n on day 11. In t h i s study the author d i d not o f f e r an e x p l a n a t i o n f o r the t e r a t o g e n i c e f f e c t of cadmium, s t a t i n g t h a t the mechanism of cadmium t o x i c i t y i n mammals has not been e s t a b l i s h e d . . T h e t o x i c i t y of cadmium has been shown ( F l i c k e t a l . ,1971) to be i n h i b i t e d by v a r i o u s agents such as selenium and z i n c , l e a d i n g B a r r t o suggest t h a t cadmium was c o m p e t i t i v e with other c a t i o n s , e s p e c i a l l y z i n c , f o r s p e c i f i c enzymes. . In another study, Ferm (1969) made an attempt t o combine the t e r a t o g e n i c e f f e c t of cadmium and lead u sing pregnant hamsters and found t h a t the t e r a t o g e n i c e f f e c t s of both cadmium and l e a d , when i n j e c t e d s e p a r a t e l y i n t o pregnant hamsters, corresponded w e l l with previous data (Ferm and Carpenter, 1968). Cadmium caused a n t e r i o r malformations mainly, while l e a d o n l y caused t a i l malformations. However, the combination of these agents, r e v e a l e d t h a t the frequency and s e v e r i t y of the d e f e c t s i n the l i p and p a l a t e caused by cadmium were reduced i n the 16 presence o f l e a d , while the p o s t e r i o r t a i l malformations caused by l e a d appear to be p o t e n t i a t e d i n the presence of cadmium. Sympodia (union of the feet) was never seen i n the animals t r e a t e d with l e a d only but d i d appear with r e l a t i v e l y high freguency when cadmium was added to the l e a d . T h e r e f o r e , Ferm (1969) s p e c u l a t e d t h a t there, was a t e r a t o g e n i c e f f e c t on embryonic t i s s u e s , a b l o c k i n p l a c e n t a l t r a n s f e r of some e s s e n t i a l m e t a b o l i t e , or an induced d e f e c t i n maternal metabolism which s e c o n d a r i l y a f f e c t s the d i f f e r e n t i a t i n g embryonic t i s s u e . He suggests t h a t , because of t h e i r f u n c t i o n i n s e v e r a l metalloenzymes, heavy metals may play an important r o l e i n embryonic d i f f e r e n t i a t i o n . Thus, i t i s p o s s i b l e that under experimental c o n d i t i o n s employed by Ferm (1969), cadmium and l e a d i n t e r a c t a d d i t i v e l y on c e r t a i n enzyme systems i n the case of the t a i l malformation. However, l e a d b l o c k s the e f f e c t of cadmium on other enzymes, such as those of the d i f f e r e n t i a t i n g v i s c e r a l a r ch systems, preventing the f a c i a l a b n o r m a l i t i e s . , Holmberg e t a l . (1969) were a l s o concerned with determining the i n t e r r e l a t i o n s h i p of t r a c e elements as a cause o f malformations. They s t u d i e d the e f f e c t s of selenium, cadmium, and a r s e n i c on the developing hamster embryos by a n a l y s i n g the f e t a l m o r t a l i t y and c o n g e n i t a l malformations and found t h a t cadmium and a r s e n i c were: independently t e r a t o g e n i c . , A l s o selenium, which was not i t s e l f t e r a t o g e n i c under s i m i l a r c o n d i t i o n s * d i d provide s i g n i f i c a n t p r o t e c t i o n a g a i n s t the malformations induced by cadmium or a r s e n i c when i n j e c t e d s i m u l t a n e o u s l y with e i t h e r of these teratogens. Furthermore, P a r i z e k and Lahor (1956) and Parizek (1957, 17 1960) s t a t e d t h a t one s i n g l e i n j e c t i o n of cadmium i n doses s u f f i c i e n t t o produce complete t e s t i c u l a r n e c r o s i s i n males, would when a d m i n i s t r a t e d t o pregnant r a t s , r e s u l t i n a complete d e s t r u c t i o n of the f e t a l part o f the p l a c e n t a and death of the f e t u s e s * P a r i z e k (1964) s t a t e d that the p l a c e n t a l damage was s p e c i f i c f o r cadmium and was a con s t a n t phenomenon already a t e a r l i e r stages of pregnancy. In a d d i t i o n t o t h i s * during the l a s t four days of pregnancy of r a t s the p a t h o l o g i c changes i n the p l a c e n t a s and f e t u s e s were: accompanied by a d d i t i o n a l p a t h o l o g i c changes a f f e c t i n g the maternal organism. During these days of pregnancy the a d m i n i s t r a t i o n of cadmium, i n a dose w e l l t o l e r a t e d by nongravid or post-partum r a t s , was h i g h l y l e t h a l not only f o r f e t u s e s but f o r a very high p r o p o r t i o n of pregnant r a t s , producing p a t h o l o g i c changes s p e c i f i c f o r t h i s p e r i o d o f pregnancy and g u i t e unusual f o r cadmium i n t o x i c a t i o n i n non-g r a v i d r a t s . P a r i z e k (1968) a l s o s t u d i e d how f a r the p e c u l i a r t o x i c e f f e c t s o f cadmium during pregnancy c o u l d be prevented by the a d m i n i s t r a t i o n of selenium. He i n j e c t e d Wistar r a t s with cadmium and selenium and found, t h a t i n c o n t r a s t t o the p a t h o l o g i c changes observed with cadmium alone* no c h a r a c t e r i s t i c changes were observed i n the p l a c e n t a when cadmium s a l t s were i n j e c t e d s i m u l t a n e o u s l y with selenium. Fetuses s u r v i v e d i n a l l r a t s and no maternal deaths occurred* Hemorrhagic r e n a l n e c r o s i s , t y p i c a l of cadmium i n t o x i c a t i o n d u r i n g the l a s t f o u r days of pregnancy, was not observed i n r a t s t r e a t e d simultaneously with cadmium and selenium. Thus, i n agreement with p r e v i o u s p r e l i m i n a r y r e p o r t s (Parizek, 1967), selenium proved to be h i g h l y e f f e c t i v e i n 18 pr e v e n t i n g the p e c u l i a r t o x i c e f f e c t s of cadmium d u r i n g pregnancy* Another area of study has been the i n t e r f e r e n c e with f a c i a l f o r m a t i o n caused by cadmium.. M u l v i h i l l e t a l . (1970) i n v e s t i g a t e d the e f f e c t of cadmium i n normal and cadmium t r e a t e d hamsters and found t h a t cadmium has a marked d e l e t e r i o u s e f f e c t on the head mesoderm of golden hamsters, causing the pr o d u c t i o n of numerous malformations, i n c l u d i n g u n i l a t e r a l and b i l a t e r a l c l e f t l i p and p a l a t e * Cadmium appears to have marked d e l e t e r i o u s e f f e c t s on c a r t i l a g e and bone f o r m a t i o n . In some animals the v e r t i c a l c a r t i l a g i n o u s n a s a l capsule was e n t i r e l y missing*.In o t h e r s i t was g r e a t l y f o r e s h o r t e n e d and b i f u r c a t e d . Bone formation tended to be r e t a r d e d i n a l l a r e a s . Where c l e f t s were not present, bone formation appeared q u i t e normal*.However, they concluded t h a t the mechanism of a c t i o n of cadmium, whether d i r e c t l y on the d i f f e r e n t i a t i n g embryonic t i s s u e . o r i n d i r e c t l y through a c t i o n on the maternal t i s s u e s , remains to be e l u c i d a t e d . F i n a l l y , Chiguoine (1965) i n v e s t i g a t e d the e f f e c t o f cadmium i n the pregnant a l b i n o mouse and s t a t e d t h a t a s i n g l e i n j e c t i o n of cadmium given t o pregnant mice on any day from the 6th to the 17th day of pregnancy r e s u l t s i n i n t r a u t e r i n e death of the embryos and l o c a l i z e d n e c r o s i s of the p l a c e n t a or adjacent d e c i d u a l t i s s u e * However, he concluded t h a t cadmium d i d not cause permanent damage to the female r e p r o d u c t i v e t r a c t . CADMIUM AND ZINC DISTRIBUTION IN TISSUES The e f f e c t s of i n o r g a n i c cadmium i n humans and animals have 19 a t t r a c t e d c o n s i d e r a b l e a t t e n t i o n i n the l a s t 15 to 20 y e a r s . Much of t h a t has been the r e s u l t of the wide-spread i n t e r e s t generated by the cadmium-induced " I t a i - I t a i " d i s e a s e i n Japan among people exposed to cadmium-contaminated food (Hagino and Yoshioka, 1961).,In the United S t a t e s the d a i l y human i n t a k e o f cadmium has been estimated to be 200-500 ug (Schroeder et a l . , 1967), and the "Standard American Man" has been estimated to have an average body burden of 30 mg of cadmium which may i n c r e a s e with age (Underwood, 1971). A number of t o x i c e f f e c t s i n humans and animals r e s u l t i n g from c h r o n i c cadmium exposure have been r e p o r t e d ( F r i b e r g e t a l . , 1974). T e s t i c u l a r atrophy (Alsberg and Schwartze, 1919; P a r i z e k and Lahor, 1956; Gunn and Gould, 1970), hyp e r t e n s i o n (Schroeder and Vinton, 1962; Perry and E r l a n g e r , 1974), r e n a l d y s f u n c t i o n (Wilson et a l . , 1941; F r i b e r g , 1950, 1952; P i s c a t o r , 1962; Axelsson and P i s c a t o r , 1966), c e n t r a l nervous system i n j u r y (Gabbiani e t a l . , 1967), anemia (Wilson e t a l . , 1941; F r i b e r g , 1950), and h e p a t i c i n j u r y ( F r i b e r g , 1955; Stowe et a l . , 1972; Wagstaff, 1973; Unger and Clausen, 1973: Hadley e t a l . , 1974 S i n g h a l e t a l . , 1974; Johnston e t a l . , 1975) are among the t o x i c e f f e c t s a t t r i b u t e d t o c h r o n i c cadmium exposure* T h e r e f o r e , i t should be i n t e r e s t i n g t o i n v e s t i g a t e how cadmium i s d i s t r i b u t e d i n d i f f e r e n t t i s s u e s . . According to Kotsonis and Klaassen (1977), the c o n c e n t r a t i o n of cadmium i n r a t s 2 days a f t e r a d m i n i s t r a t i o n was h i g h e s t i n the l i v e r , f o l l o w e d by the i n t e s t i n e , kidney, pancreas, h e a r t , lung, t e s t i c l e , muscle, b r a i n , blood, and plasma. A f t e r 14 days most t i s s u e s showed approximately a 50% decrease i n cadmium with notable e x c e p t i o n s , namely, the kidney, 20 which had up t o a three t o fo u r f o l d i n c r e a s e , the l i v e r * which remained t h e same at the h i g h e s t doses, and the i n t e s t i n e , which had up to a 95% decrease. I t was of i n t e r e s t to note t h a t i n t h e i r study only the b r a i n had cadmium c o n c e n t r a t i o n s lower than the blood, presumably due to a blood b r a i n b a r r i e r . . In a d d i t i o n , a f t e r 2 days only 7% of the cadmium i n the blood was l o c a t e d i n the plasma* Kots o n i s and Klaassen (1977), a l s o , s t a t e d t h a t the cadmium i n the blood q u i c k l y c o n c e n t r a t e s i n the e r y t h r o c y t e s , bound probably e i t h e r t o hemoglobin or to a m e t a l l o t h i o n e i n - l i k e p r o t e i n . The high c o n c e n t r a t i o n of cadmium i n the i n t e s t i n e a f t e r 2 days and the dramatic decrease a f t e r 14 days may be ex p l a i n e d by the presence of an i n t e s t i n a l m e t a l l o t h i o n e i n which was subsequently l o s t d u r i n g the normal turnover o f i n t e s t i n a l e p i t h e l i a l c e l l s . They a l s o reported t h a t the c o n c e n t r a t i o n of m e t a l l o t h i o n e i n i n the kidney a t 2 days a f t e r a d m i n i s t r a t i o n o f cadmium was unchanged but a f t e r 14 days had i n c r e a s e d as a f u n c t i o n of dose. In c o n t r a s t , the c o n c e n t r a t i o n of m e t a l l o t h i o n e i n i n the l i v e r a t 2 days had i n c r e a s e d as a f u n c t i o n of dose and was approximately the same a f t e r 14 days. Thus, the r e d i s t r i b u t i o n of cadmium t o the kidney which was observed with time was probably accounted f o r by the longer time p e r i o d r e q u i r e d f o r the i n c r e a s e of m e t a l l o t h i o n e i n l e v e l s i n the kidney* In a d d i t i o n , the un u s u a l l y long h a l f - l i f e of cadmium, estimated to be 200-300 days i n r a t s (Derbin e t a l . , 1957) was probably due t o the presence o f m e t a l l o t h i o n e i n s i n c e most o f the cadmium l o c a t e d i n the l i v e r and kidney was bound to 21 m e t a l l o t h i o n e i n (Kotsonis and Klaassen, 1977). , Sugawara (1977) a l s o s t u d i e d the d i s t r i b u t i o n of cadmium i n mice but was i n t e r e s t e d i n c l a r i f y i n g the i n t e r r e l a t i o n s h i p between i n j e c t e d cadmium and endogenous zi n c i n the l i v e r and kidney* He r e p o r t e d t h a t cadmium was detected i n both organs as e a r l y as 0.5 hours a f t e r the cadmium i n j e c t i o n and was about 14% and 1% o f the i n j e c t e d dose i n the l i v e r and kidney, r e s p e c t i v e l y . . Cadmium c o n c e n t r a t i o n s i n c r e a s e d p r o g r e s s i v e l y with time i n the l i v e r , at 6 days a f t e r i n j e c t i o n , the t o t a l content o f cadmium i n the l i v e r reached a maximum l e v e l of about 54% o f the i n j e c t e d dose. However, t h e r e was no s i g n i f i c a n t d i f f e r e n c e i n the c o n c e n t r a t i o n of the l i v e r cadmium from the 10 hours group to the 20-day group. I n the kidney t h i s c o n c e n t r a t i o n i n c r e a s e d with time and was about 7% of the i n j e c t e d dose at 20 days a f t e r i n j e c t i o n . In the kidney, z i n c c o n c e n t r a t i o n at 20 days i n c r e a s e d s i g n i f i c a n t l y i n comparison with t h a t of the c o n t r o l group. In the l i v e r , z i n c c o n c e n t r a t i o n was not d i f f e r e n t from t h a t i n the c o n t r o l group up to 10 hours, but a f t e r 1 day t h i s c o n c e n t r a t i o n i n c r e a s e d s i g n i f i c a n t l y , and at 6 and 20 days was about two times t h a t observed i n the c o n t r o l group. These r e s u l t s are i n agreement with Webb (1972), who a l s o s t u d i e d the d i s t r i b u t i o n of cadmium i n l i v e r and kidney of r a t s . In a d d i t i o n , Webb examined the q u e s t i o n of whether the s t o r e d cadmium was m o b i l i z e d , and thus became t o x i c t o the:maternal and the f e t a l organism during subsequent pregnancy._ V i r g i n female r a t s were i n j e c t e d subcutaneously with cadmium, which was r e t a i n e d mainly i n l i v e r and kidney. When these: animals became 22 pregnant, Webb found t h a t the s t o r e d cadmium was not m o b i l i z e d and thus, i n c o n t r a s t to " f r e e " cadmium, d i d not induce toxemia o r e x c e s s i v e f e t a l malformations.. Eohrer et a l . (1978) examined the d i s t r i b u t i o n of cadmium i n zinc-normal and z i n c - d e f i c i e n t maternal animals and found t h a t the maternal t i s s u e l e v e l s of cadmium i n c r e a s e d as the maternal dose o f cadmium increased..The i n c r e a s e d l e v e l s were evi d e n t i n a l l t i s s u e s from both the zinc-normal and z i n c -d e f i c i e n t d i e t s groups* In a l l cases f o r the zinc-normal group, the t i s s u e s from pregnant r a t s which r e c e i v e d 1.5 mg/kg body weight dose of cadmium contained s i g n i f i c a n t l y g r e a t e r amounts of cadmium than d i d the same t i s s u e s from pregnant r a t s r e c e i v i n g lower doses of cadmium. A d i f f e r e n t cadmium accumulation p a t t e r n was observed i n the maternal t i s s u e s of the z i n c d e f i c i e n t d i e t group. No c o n s i s t e n t s t a t i s t i c a l d i f f e r e n c e s e x i s t e d i n the cadmium co n t e n t of the maternal t i s s u e s from d i f f e r e n t cadmium dose l e v e l s . These f i n d i n g s suggested t h a t the cadmium b i n d i n g c a p a b i l i t i e s of the maternal t i s s u e s may have been i n f l u e n c e d by a t r a n s i t o r y z i n c d e f i c i e n c y . P i e t r z a k - F l i s e t a l . (1978) i n v e s t i g a t e d the accumulation and d i s t r i b u t i o n of c h r o n i c a l l y administered cadmium i n two g e n e r a t i o n s of r a t s and found t h a t , i n the f i r s t g e n e r a t i o n dams, at the end o f g e s t a t i o n or l a c t a t i o n the cadmium c o n c e n t r a t i o n i n kidneys was twice the l e v e l found i n the l i v e r . A l s o , cadmium c o n c e n t r a t i o n s i n the b r a i n reached a maximum a f t e r 43 days of exposure* In maternal milk, cadmium was d e t e c t a b l e on the second day o f l a c t a t i o n only i n the h i g h e s t group (5 ppm i n the d r i n k i n g water). , Detectable blood cadmium 23 l e v e l s were found only i n a d u l t animals of the 5 ppm exposure group. In the l i v e r and kidney cadmium c o n c e n t r a t i o n s i n c r e a s e d with time and i n c r e a s e d more r a p i d l y d u r i n g the p e r i o d 21 to 50 days than from 50 to 130 days of age. P i e t r z a k - F l i s et a l . (1978) suggested t h a t these r e s u l t s may i n d i c a t e t hat c h r o n i c exposure t o constant l e v e l s of cadmium may r e s u l t i n maximum t i s s u e l e v e l s much e a r l i e r than would be p r e d i c t e d . M i l l e r e t a l . (1975) a l s o s t u d i e d the e f f e c t s of normal and z i n c d e f i c i e n t d i e t s upon the uptake and d i s t r i b u t i o n o f 115m-cadmium i n the golden hamster. Cadmium r e t e n t i o n i n the kidneys of z i n c d e f i c i e n t hamsters was found to be s i g n i f i c a n t l y lower than i n kidneys from normal animals..A t r e n d was noted toward i n c r e a s e d r e t e n t i o n of cadmium by the whole.body and l i v e r o f these animals, which the authors s t a t e d may e x p l a i n the r e l a t i v e decrease i n the kidneys. Cadmium may have re p l a c e d z i n c i n the l i v e r and other s i t e s where z i n c would normally be found, thus r e s u l t i n g i n l e s s cadmium being a v a i l a b l e f o r t r a n s p o r t to the kidneys* . In a d d i t i o n , the r e d u c t i o n i n z i n c i n t a k e observed may have r e s u l t e d i n decreased m e t a l l o t h i o n e i n p r o d u c t i o n , r e d u c i n g t r a n s p o r t of z i n c and cadmium to the kidneys. They a l s o concluded t h a t examination of the replacement of z i n c by cadmium i n the l i v e r and pancreas and i n v e s t i g a t i o n of z i n c d e f i c i e n c y e f f e c t s on m e t a l l o t h i o n e i n l e v e l s may help e x p l a i n the decrease i n kidney cadmium seen i n t h e i r i n v e s t i g a t i o n . C herian et a l . (1978) s t u d i e d the d i s t r i b u t i o n , i n the mouse, of cadmium from o r a l cadmium c h l o r i d e and cadmium-m e t a l l o t h i o n e i n and found t h a t t h e r e was no s i g n i f i c a n t d i f f e r e n c e i n the whole body r e t e n t i o n of cadmium given i n these 24 two forms d u r i n g the experimental p e r i o d of 7 days*.However, 56% of the cadmium c h l o r i d e was found i n the l i v e r compared with only 6.5% of the cadmium-metallothionein* In sharp c o n t r a s t , 63.6% of cadmium accumulated i n the kidneys when given i n the form of cadmium-metallothionein, compared with o n l y 8 .7% when give n as cadmium c h l o r i d e * . More cadmium a l s o accumulated i n bone, h e a r t * lung* s p l e e n , pancreas, and t e s t e s from cadmium c h l o r i d e than from cadmium-metallothionein o r a l a d m i n i s t r a t i o n . These r e s u l t s were s i m i l a r t o the body d i s t r i b u t i o n , e s p e c i a l l y i n l i v e r and kidney, a f t e r p a r e n t e r a l a d m i n i s t r a t i o n of cadmium c h l o r i d e and cadmium-metallothionein r e s p e c t i v e l y as r e p o r t e d by Cherian and Shaikh, (1975). They concluded t h a t s i n c e the kidney was the main t a r g e t organ f o r the c h r o n i c t o x i c e f f e c t s of cadmium* the i n c r e a s e d r e n a l cadmium d e p o s i t i o n a f t e r o r a l a d m i n i s t r a t i o n of cadmium-metallothionein may be of s i g n i f i c a n c e i n d e c i d i n g the maximum a l l o w a b l e amount of cadmium i n food. ZINC AS A TERATOGEN Although the e f f e c t s of many heavy metals on embryonic development i n avian systems have been s t u d i e d * few e s s e n t i a l metals have r e c e i v e d a t t e n t i o n as p o s s i b l e t eratogens a f f e c t i n g mammalian embryos* According to Ferm (1968), i n f a c t , few data are a v a i l a b l e on the e f f e c t s of e s s e n t i a l t r a c e metals on mammalian r e p r o d u c t i o n , and even l e s s i n f o r m a t i o n i s a v a i l a b l e concerning the p e r m e a b i l i t y of the mammalian p l a c e n t a to heavy metals duri n g g e s t a t i o n . T h i s problem i s compounded by the marked v a r i e t y of p l a c e n t a l forms i n mammals, as well as the changing s t r u c t u r e of the p l a c e n t a l membranes durin g g e s t a t i o n 25 i n a l l mammalian s p e c i e s . Z i n c has been repo r t e d to produce o n l y a mild t e r a t o g e n i c response. However, Ferm (1968) s t u d y i n g the i n t e r r e l a t i o n s h i p o f cadmium, z i n c , and copper i n embryogenesis i n hamsters, r e p o r t e d t h a t the i n t r a v e n o u s i n j e c t i o n of z i n c i n amounts g r e a t e r than 30 mg per kg body weight was l e t h a l f o r the maternal animals w i t h i n 24 hours;. Dosage l e v e l s from 10 t o 25 mg per kg body weight were w e l l t o l e r a t e d by the mother and induced a f e t a l r e s o r p t i o n r a t e of only 12 p e r c e n t * . Z i n c even at higher dosage l e v e l , produced few g r o s s malformations, c o n s i s t i n g o f exencephaly and r i b f u s i o n . However, no c o n s i s t e n t p a t t e r n o f malformations was noted i n the embryos t r e a t e d with z i n c . In another study Ferm (1967) i n v e s t i g a t e d the t e r a t o g e n i c e f f e c t s o f cadmium and i t s p r e v e n t i o n by z i n c , and s t a t e d t h a t when pregnant hamsters were t r e a t e d with 2 t o 6 mg/kg of body weight o f z i n c on day 8 of g e s t a t i o n , out of 142 f e t u s e s from 12 mothers o n l y 4 were resorbed and o n l y 2 were malformed. Therefore, he s t a t e d again that z i n c alone provoked a very mild t e r a t o g e n i c response. Chang et a l . (1977) s t u d i e d the t e r a t o g e n i c i t y of z i n c c h l o r i d e i n doses of 20.5 mg and 25 mg/kg body weight* administered i n t r a p e r i t o n e a l l y to mice* They produced s i g n i f i c a n t i n c i d e n c e s of s k e l e t a l d e f e c t s . As the dosage of the z i n c s a l t was reduced maternal and f e t a l t o x i c i t y , r e l a t i v e f e t a l weight r e d u c t i o n , and the i n c i d e n c e of s k e l e t a l anomalies were.correspondingly decreased. The p r i n c i p a l s i t e f o r the t e r a t o g e n i c i t y of z i n c s a l t s a p p a r e n t l y was t h e : s k e l e t a l system and with i n c r e a s i n g doses the m a j o r i t y of the d e f e c t s produced 26 i n v o l v e the r i b cage* Z i n c c h l o r i d e , 20.5 mg/kg body weight, exerted g r e a t e r t o x i c e f f e c t s on the mother and f e t u s e s when admin i s t e r e d on day 10 of g e s t a t i o n than on any other day. Ripple r i b s caused by z i n c f i r s t appeared when t h e ; i n j e c t i o n was given on day 9 of g e s t a t i o n and became more frequent and pronounced a f t e r i n j e c t i o n on day 11 o f g e s t a t i o n . . T h e r e f o r e , they recommended t h a t s i m i l a r s t u d i e s be c a r r i e d out to determine more p r e c i s e l y the re l e v a n c e o f e x c e s s i v e c h a l l e n g e o f z i n c as an e t i o l o g i c f a c t o r i n the development of malformations i n mammals* These s t u d i e s w i l l be very important, because the mechanism of z i n c as a teratogen i s an open q u e s t i o n , as w e l l as i t s r e l a t i o n s t o other t e r a t o g e n s , i . e. i t s s p e c i e s s p e c i f i c i t y , e t c . CADMIUM AND ZINC INTERACTIONS Zinc i s an e s s e n t i a l m e t a l . . I t i s a c o n s t i t u e n t of a number of metalloenzymes, and a c t s as a c o f a c t o r f o r s e v e r a l o t h e r enzymes (Orten, 1966). Zi n c i s e s s e n t i a l f o r normal growth and development (Halsted e t a l . , 1974). There i s a vast l i t e r a t u r e on z i n c d e f i c i e n c y i n animals, documenting, f o r example, impaired growth, t e s t i c u l a r atrophy, and p a r a k e r a t o s i s ( M i l l e r e t a l . , 1958; Robertson and Burns, 1963; Hoekstra, 1969) . Some of these e f f e c t s have a l s o been noted i n z i n c - d e f i c i e n t humans (Halsted e t a l . , 1972). Cadmium on the other hand, i s not e s s e n t i a l , i s t o x i c , and accumulates i n the human body with i n c r e a s i n g age..It has been estimated t h a t r e n a l damage may occur at cadmium c o n c e n t r a t i o n s of over 20 0 ug/g wet weight i n the kidney c o r t e x ( F r i b e r g et 27 a l . , 1974). In Sweden f o r example* the average c o n c e n t r a t i o n of cadmium i n the kidney c o r t e x a t 50 years of age has been found to be around 25 ug/g wet weight ( P i s c a t o r and L i n d 1972, E l i n d e r e t a l * , 1976), whereas much higher v a l u e s have been r e p o r t e d from Japan (Tsuchiya e t a l . , 1972). In animals, i t has been shown t h a t z i n c c o u n t e r a c t s some of the t o x i c e f f e c t s of cadmium (Bunn and Matrone 1966; Banis e t a l . , 1969) and t h a t cadmium enhances the e f f e c t s of z i n c d e f i c i e n c y ( P e t e r i n g e t a l . , 1971). In humans and animals, i n c r e a s e d cadmium l e v e l s i n the kidney have been accompanied by i n c r e a s e d z i n c c o n c e n t r a t i o n s (Schroeder et a l ; , 1967; Anke. and Schneider 1971; P i s c a t o r and Lind 1972; Hammer et a l . , 1973; P i s c a t o r 1974). I t has been p o s t u l a t e d ( P i s c a t o r and L i n d , 1972) t h a t t h i s z i n c i n c r e a s e i s r e l a t e d t o the i n c r e a s e d c o n c e n t r a t i o n o f r e n a l m e t a l l o t h i o n e i n , which c o n t a i n s eguimolar amounts o f cadmium and z i n c . . A c o n c e n t r a t i o n of cadmium i n kidney c o r t e x exceeding 200 ug/g wet weight may g i v e r i s e t o t u b u l a r p r o t e i n u r i a . F r i b e r g e t a l . (1974), Parizek et a l . (1957) and Gunn et a l . (1961) demonstrated t h a t simultaneus subcutaneous a d m i n i s t r a t i o n o f z i n c and cadmium to r a t s p r o t e c t e d a g a i n s t the severe t e s t i c u l a r i n j u r y observed when cadmium was g i v e n alone, Gunn e t al..(1963) r e p o r t e d a l s o t h a t cadmium-induced t e s t i c u l a r tumors were prevented by the a d m i n i s t r a t i o n of z i n c * Schroeder and Buckman (1967) showed t h a t the i n c r e a s e d blood pressure i n r a t s given cadmium could be reduced by i n j e c t i o n of z i n c c h e l a t e . A marginal z i n c i n t a k e by r a t s g i v e s r i s e t o high cadmium a b s o r p t i o n and r e t e n t i o n compared t o animals given excess of z i n c (Campbell et a l . , 28 1978). Furthermore, even a low i n t a k e of cadmium aggravates the symptoms o f z i n c d e f i c i e n c y i n r a t s (Petering e t a l * , 1971)., In a number of i n v i t r o s t u d i e s , cadmium has been shown t o decrease the a c t i v i t y of zinc-dependent enzymes (Immelhoch e t a l . , 1969; V a l l e e , and Olmer, 1972). I t has been proposed t h a t the t o x i c i t y of cadmium, at l e a s t p a r t l y , can be e x p l a i n e d by a c o m p e t i t i o n between cadmium and z i n c at c o f a c t o r s i t e s i n enzymes r e q u i r i n g z i n c , r e s u l t i n g i n decreased a c t i v i t i e s o f these enzymes ( V a l l e e , and Olmer, 1972).. Thus, the t u b u l a r p r o t e i n u r i a which occurs at high r e n a l cadmium c o n c e n t r a t i o n s might be e x p l a i n e d by decreased a c t i v i t i e s of c e r t a i n z i n c r e q u i r i n g enzymes* such as a l k a l i n e phosphatase and l e u c i n e a m i n o p e p t i d a s e , which are thought to be engaged i n the t u b u l a r r e a b s o r p t i o n of p r o t e i n s (Wachsmut and T o r h o r s t , 1974). Reduced a c t i v i t i e s of these enzymes i n kidney has been observed i n p i g s and r a t s p e r o r a l l y exposed to cadmium (Cousins et a l . , 1973; and Washko et a l . , 1975). In h i g h e r mammals, such as man, horse, and pigs z i n c c o n c e n t r a t i o n s i n r e n a l c o r t e x have been shown to i n c r e a s e on a equimolar b a s i s with the i n c r e a s e of cadmium up t o a cadmium l e v e l of about 50 to 70 ug/g above t h i s l e v e l the i n c r e a s e o f z i n c i s l e s s pronounced ( P i s c a t o r et a l . , 1975; E l i n d e r et a l . , 1977; E l i n d e r et a l * , 1978); The b a s a l l e v e l of z i n c i n r e n a l c o r t e x of humans and horses has been estimated t o be about 2 5 ug/g ( P i s c a t o r e t a l . , 1972). The i n c r e a s e : of z i n c at low cadmium l e v e l s i s b e l i e v e d t o be a compensation f o r the i n c r e a s e of cadmium, a mechanism probably i n v o l v i n g the production of a form of m e t a l l o t h i o n e i n which binds both z i n c and cadmium i n a 29 molar r a t i o of 1:1 (Nordberg et a l . , 197 2) . The ; mechanism behind the l e s s marked i n c r e a s e of z i n c i n r e l a t i o n t o cadmium at h i g h c o n c e n t r a t i o n s i s s t i l l not determined. I t may be a t t r i b u t a b l e to the s y n t h e s i s of other forms of m e t a l l o t h i o n e i n with a h i g h e r r a t i o of cadmium t o z i n c or p o s s i b l y t o a r e l a t i v e : d e f i c i e n c y of z i n c i n kidney ( E l i n d e r and P i s c a t o r , 1978). In a d d i t i o n , i n v e s t i g a t o r s (Gunn e t a l . , 1961; Ferm and Carpenter, 1967, 1968), have suggested t h a t .cadmium e x e r t s i t s t o x i c e f f e c t s through the i n a c t i v a t i o n o f z i n c metalloenzymes, as a r e s u l t of i t s exchange with z i n c . The a d m i n i s t r a t i o n o f a d d i t i o n a l z i n c enables i t t o r e t a i n i t s b i n d i n g s i t e s i n the enzymes and thus* prevents the b i n d i n g s i t e s from being f i l l e d by cadmium* I t i s i n t e r e s t i n g t o note the; r e s u l t s of an experiment c a r r i e d out by Dixon and Compher (1977), where z i n c metalloenzyme was shown to be e s s e n t i a l f o r t h e : r e g e n e r a t i o n o f the f o r e l i m b of the a d u l t newt. Cadmium i n h i b i t e d r e g e n e r a t i o n , presumably by i n t e r f e r i n g with z i n c metalloenzymes. T h e r e f o r e , they suggested t h a t z i n c was necessary f o r normal r e g e n e r a t i o n to occur, and i t s replacement i n metalloenzymes c o u l d be d e l e t e r i o u s * Furthermore, i t i s w e l l known t h a t i n the l i v e r and kidney, z i n c i s i n c r e a s e d by the i n j e c t i o n s of cadmium* T h i s i n c r e a s e might be due to the i n d u c t i o n of m e t a l l o t h i o n e i n s y n t h e s i s i n both organs, (Suzuki, 1972). However, the o r i g i n of t h i s i n c r e a s e d z i n c i s s t i l l under d i s c u s s i o n . Winge et a l . , (1975) a t t r i b u t e d the accumulation of z i n c i n part to d e p l e t i o n of blood z i n c . Faeder et a l . (1977), proposed i n c r e a s e d d i e t a r y uptake as an e x p l a n a t i o n of the high z i n c a b s o r p t i o n from the 30 duodenum, due t o low molecular weight c h e l a t e complexes i n the i n t e s t i n e . . Sugawara et a l . (1978) t r i e d to d e f i n e t h i s problem using r a t s and concluded t h a t cadmium b i n d i n g p r o t e i n does not i n f l u e n c e z i n c b i n d i n g to the low molecular weight f a c t o r . The r a p i d accumulation of l i v e r z i n c f o l l o w i n g i n j e c t i o n of cadmium may be due not t o uptake of d i e t a r y z i n c but t o supply of exchangeable z i n c from other organs, and p a r t l y to d e p l e t i o n o f plasma z i n c . Brown and C h a t e l (1978) i n v e s t i g a t e d the i n t e r a c t i o n s of cadmium and z i n c i n ducks from a h e a v i l y p o l l u t e d area o f B r i t i s h Columbia (Canada) and s t a t e d t h a t c o m p e t i t i o n mf z i n c and cadmium f o r s i m i l a r b i n d i n g s i t e s i n t i s s u e was w e l l e s t a b l i s h e d * I t has been shown that t o x i c e f f e c t s of cadmium do not occur u n t i l the b i n d i n g c a p a c i t y of m e t a l l o t h i o n e i n i s exceeded and cadmium occurs i n the high molecular weight p r o t e i n pool (Winge e t a l . , 1974; I r o n s and Smith, 1976). T h e i r s t u d i e s i n d i c a t e t h a t cadmium may appear i n the high molecular weight p o o l , not onl y when the bi n d i n g c a p a c i t y of m e t a l l o t h i o n e i n was exceeded, but a l s o when there was a d e f i c i e n c y o f z i n c i n the high molecular weight p r o t e i n p o o l . Since t o x i c e f f e c t s o f cadmium occur when i t appears i n the high molecular weight p r o t e i n p o o l , i t was l i k e l y t h a t d e f i c i e n c i e s of z i n c i n c r e a s e t o x i c e f f e c t s of cadmium by p e r m i t t i n g an i n c r e a s e of cadmium i n the high molecular weight p r o t e i n p o o l . C o n s i d e r a b l e i n t e r e s t has been generated concerning m e t a l l o t h i o n e i n because of i t s d i s t i n c t i v e p r o p e r t i e s . T h i s low molecular weight c y t o p l a s m i c m e t a l l o p r o t e i n i s c h a r a c t e r i z e d by a high c y s t e i n e s u l f u r content which accounts f o r i t s u n u s u a l l y 31 high b i n d i n g p r o p e r t i e s f o r z i n c , cadmium and mercury (Richards and Cousins, 1 9 7 6 ) . . O r i g i n a l l y m e t a l l o t h i o n e i n was i s o l a t e d and c h a r a c t e r i z e d from l i v e r and kidney derived from animals of unknown n u t r i t i o n a l h i s t o r y (Kagi et a l . , 1960; Nordberg et a l . , 1972). T h i s p r e c l u d e d d e f i n i n g any r o l e t h a t t h i s b i n d i n g p r o t e i n may have i n the metabolism of n u t r i e n t minerals.. To a v o i d t h i s problem l a r g e doses of metals, f o r example z i n c , cadmium and mercury have been i n j e c t e d or fed to animals p r i o r t o m e t a l l o t h i o n e i n i s o l a t i o n (Cousins et a l . , 1974; Chen et a l . , 1974; Weser e t a l . , 1 9 7 3 ) H o w e v e r , such an approach, although v a l u a b l e f o r p r e p a r a t i v e purposes, can only serve a l i m i t e d r o l e i n the e l u c i d a t i o n of the f u n c t i o n or f u n c t i o n s of m e t a l l o t h i o n e i n i n v i v o because the normal p o p u l a t i o n may never be exposed to such l a r g e amounts of metals (Richards and Cousins 1976) . S e v e r a l f u n c t i o n s have been p o s t u l a t e d f o r m e t a l l o t h i o n e i n , l a r g e l y based on s t u d i e s t h a t employed i n j e c t e d doses of z i n c or cadmium..These i n c l u d e r o l e s i n a b s o r p t i o n (Richards and Cousins 1975a), h e p a t i c storage (Richards and Cousins 1975a, b), and d e t o x i c a t i o n of heavy metals (Squibb, 1974; Shaikk and L u c i s , 1972). Repeated a d m i n i s t r a t i o n of low doses of cadmium produce t o l e r a n c e to the subsequent i n j e c t i o n of higher, u s u a l l y l e t h a l , doses of the metal (Kimura et a l . , 1974). T h i s t o l e r a n c e has been a t t r i b u t e d t o the p r o t e c t i v e e f f e c t of m e t a l l o t h i o n e i n , which was s y n t h e s i z e d i n response t o exposure to cadmium. M e t a l l o t h i o n e i n has a high a f f i n i t y f o r the b i n d i n g of cadmium and has been suggested as a p o s s i b l e means of d e t o x i c a t i o n o f the metal (Kimura et a l . , 1974). 32 Probst et a l . (1977) have presented data t o support the p r o t e c t i v e r o l e of m e t a l l o t h i o n e i n i n mice..These workers have demonstrated a dose-response r e l a t i o n s h i p between administered cadmium and h e p a t i c c o n c e n t r a t i o n s of the p r o t e i n . They a l s o found that the LD 50 value f o r subsequently administered cadmium was d i r e c t l y r e l a t e d t o h e p a t i c m e t a l l o t h i o n e i n c o n c e n t r a t i o n . In another study, Ghafghazy and Mennear (1973) i n v e s t i g a t e d the e f f e c t s of acute and subacute a d m i n i s t r a t i o n of cadmium on carbohydrate metabolism i n mice* These workers r e p o r t e d t h a t a s i n g l e dose of the metal produced a s i g n i f i c a n t r e d u c t i o n i n p a n c r e a t i c i n s u l i n s e c r e t i o n * whereas repeated a d m i n i s t r a t i o n di d not* They suggested t h a t t h i s apparent t o l e r a n c e was a r e f l e c t i o n of the presence of m e t a l l o t h i o n e i n i n the p a n c r e a t i c b e t a - c e l l * The r e g u l a t i o n of i n t e s t i n a l z i n c a b s o r p t i o n i s an important aspect o f z i n c homeostasis. The nature of t h i s mechanism i s s t i l l not c l e a r l y d e f i n e d , but Starcher (1969) re p o r t e d the presence of a m e t a l l o t h i o n e i n i n the c h i c k i n t e s t i n e which was capable of b i n d i n g r a d i o n u c l i d e s of copper and z i n c * I t was proposed t h a t t h i s p r o t e i n f u n c t i o n e d d i r e c t l y i n the t r a n s p o r t of these metals from the i n t e s t i n a l lumen to the plasma. Chen et a l * (1977) showed that z i n c accumulated e n t i r e l y i n m e t a l l o t h i o n e i n i n l i v e r s of r a t s fed d i e t s c o n t a i n i n g 1000 and 2000 ppm o f d i e t a r y z i n c f o r 2 weeks, p r o v i d i n g f u r t h e r evidence f o r a r e l a t i o n s h i p between m e t a l l o t h i o n e i n and z i n c metabolism. Thus, m e t a l l o t h i o n e i n was proposed t o play a r o l e as a s t o r a g e p r o t e i n f o r z i n c * 33 Although the exact b i o l o g i c a l r o l e of t h i s p r o t e i n i s not w e l l understood, i t s p h y s i c a l and chemical c h a r a c t e r i s t i c s are c o n s i s t e n t with a r o l e i n a wide range of biochemical and p h y s i o l o g i c a l mechanisms such as c a t a l y s i s , t r a n s p o r t , s t o r a g e , immune phenomena and heavy metals d e t o x i c a t i o n * . CADMIUM IN HUMAN HEALTH As the l a s t p o i n t i n t h i s review, i t w i l l be i n t e r e s t i n g to mention some of the s t u d i e s which r e l a t e cadmium t o x i c e f f e c t s and human h e a l t h . In 1950 F r i b e r g showed that one of the main symptoms i n c h r o n i c cadmium p o i s o n i n g was p r o t e i n u r i a . . R e s u l t s of i n v e s t i g a t i o n s of cadmium-poisoned men ( F r i b e r g * 1957) have shown t h a t i n c h r o n i c poisoning kidney damage a r i s e s , e s p e c i a l l y i n the t u b u l e s . . As has been pointed out by B u t l e r and Flym (1958), however, p r o t e i n u r i a does not necessary occur i n a l l t u b u l a r damage but only damage t o s p e c i a l p a r t s of the t u b u l e s . T h e r e f o r e t h e r e may be s e v e r a l reasons f o r the t u b u l a r damage caused by cadmium, a c c o r d i n g to P i s c a t o r (1962). Probably what happens i s t h a t cadmium, which i s e s p e c i a l l y d e p o s i t e d i n the kidneys, i n h i b i t s enzyme systems which are necessary f o r r e a b s o r p t i o n * In agreement with t h i s hypothesis C larkson and Kench (1956) found an i n c r e a s e d e x c r e t i o n of amino a c i d s i n cadmium workers, which c o u l d be caused by decreased r e a b s o r p t i o n * Another p o s s i b i l i t y ( F r i b e r g , 1950) i s t h a t p r o t e i n d e p o s i t i o n i n the t u b u l e s from i n c r e a s e d p r o t e i n c o n c e n t r a t i o n of the glomerular f i l t r a t e was a r e s u l t o f i n c r e a s e d p r o d u c t i o n of p r o t e i n i n the body due t o the t o x i c a c t i o n of cadmium. 34 Another harmful e f f e c t of cadmium f o r humans has been suggested by Schroeder (1966) and C a r r o l l (1966), who s a i d t h a t abnormal l e v e l s of cadmium may be a c a u s a t i v e f a c t o r i n heart f a i l u r e and death.. Hawley and Kopp (1975) induced c a r d i a c problems i n r a t s by dosages b r a c k e t i n g the ranges f o r cadmium found i n human blood* Voors and Shuman (1976) i n v e s t i g a t e d , by means of autopsy s t u d i e s , the h y p o t h e s i s t h a t cadmium exposure i s a s s o c i a t e d with c a r d i a c problems, and found a p o s i t i v e c o r r e l a t i o n between l i v e r c o n c e n t r a t i o n s of cadmium and death from heart d i s e a s e . However, t h e i r r e s u l t s are not i n accordance with those of T i p t o n (1960) who s t u d i e d a d i f f e r e n t p o p u l a t i o n and found no s i m i l a r a s s o c i a t i o n . They did not g i v e any e x p l a n a t i o n f o r the d i f f e r e n c e i n r e s u l t s * However* t h e i r sample po p u l a t i o n was very s m a l l (28 p a t i e n t s ) and even though they s t u d i e d smoking h a b i t s , which seems to be a major source of the body cadmium burden (Lewis et a l * , 197 2; Shuman e t a l . , 1974), no a s s o c i a t i o n was noted between r e p o r t e d c i g a r r e t t e smoking and death from heart d i s e a s e or between smoking and cadmium l i v e r c o n c e n t r a t i o n . I t i s important t o keep i n mind the f a c t t h a t h y p e r t e n s i o n has been a s s o c i a t e d with high kidney l e v e l s of cadmium i n c e r t a i n s t u d i e s (Morgan, 1969) cannot be used t o form c o n c l u s i o n s concerning c a u s a l i t y . The data of most of these s t u d i e s , a c c o r d i n g to Morgan (1969) were obtained by a n a l y s i s o f kidneys from people who died from v a s c u l a r d i s e a s e , and the i n t e r p r e t a t i o n of the data i s s u b j e c t to the usual d i f f i c u l t i e s met i n t h i s type of e p i d e m i o l o g i c a l a n a l y s i s , i . e . c o n c l u s i o n s are ambiguous, and are based on the a s s o c i a t i o n o f 35 c a r d i o v a s c u l a r d i s e a s e with d u s t f a l l data ( F r i b e r g , 1950) or with cadmium c o n c e n t r a t i o n i n a i r ( F r i b e r g , 1974). In c e r t a i n areas of Japan where there has been a c o n s i d e r a b l e exposure t o cadmium f o r decades, h y p e r t e n s i o n has not been a s s o c i a t e d with cadmium exposure (Nogawa and Kawano, 1969). In a d d i t i o n , t h e r e are no r e p o r t s showing t h a t workers exposed to cadmium have a higher prevalence of hypertension than other groups (Tsuchiya, 1971). T h e r e f o r e , a c c o r d i n g to F r i b e r g (1974) the a v a i l a b l e data do not support the hypothesis t h a t cadmium i s c a u s a l l y a s s o c i a t e d with c a r d i o v a s c u l a r d i s e a s e i n man. However, t h e r e are reasons t o study the q u e s t i o n f u r t h e r * , The f i n d i n g s , p a r t i c u l a r l y i n the animal s t u d i e s , but even i n the e p i d e m i o l o g i c a l s t u d i e s i n human beings, merit f u r t h e r a t t e n t i o n with p a r t i c u l a r emphasis upon the mechanisms of cadmium induced h y p e r t e n s i o n , because the reasons f o r d i f f e r i n g r e s u l t s r e p o r t e d by v a r i o u s i n v e s t i g a t o r s are not c l e a r . Lauwerys et a l . . (1978) undertook a survey among 500 pregnant women l i v i n g i n d i f f e r e n t areas of Belgium, i n order t o e v a l u a t e the extent of exposure of heavy metals ( l e a d , mercury and cadmium), during f e t a l l i f e , t h e i r p o s s i b l e b i o l o g i c a l e f f e c t s , and the e p i d e m i o l o g i c a l f a c t o r s which may i n f l u e n c e the i n t e n s i t y of exposure. They found t h a t the cadmium accumulation i n the newborn blood was 50 percent lower than i n the mother's blood. T h i s suggests t h a t the p l a c e n t a p l a y s a b a r r i e r r o l e : f o r the t r a n s f e r of cadmium. However* Baglan e t a l . (1974) who analyzed 100 maternal and f e t a l blood samples from persons l i v i n g i n Tennessee, found* v a l u e s 10 times h i g h e r than those 36 found by Lauwerys et a l . (1978) . They e x p l a i n e d t h a t the di s c r e p a n c y was probably due to methodological d i f f e r e n c e s r a t h e r than t o v a r y i n g degrees of cadmium exposure of the p o p u l a t i o n s t u d i e d , s i n c e i t i s now g e n e r a l l y r e c o g n i z e d t h a t the normal c o n c e n t r a t i o n of cadmium i n the blood of the g e n e r a l p o p u l a t i o n i s below 1.0ug/100 ml (Lauwerys et a l . * 1976; Ulander and Axelson, 1974). . F u r t h e r , Buchet et a l . (1973) continued the p r e v i o u s s t u d i e s of Lauwerys e t a l . (1978) of the p o s s i b l e i n f l u e n c e of v a r i o u s e p i d e m i o l o g i c a l f a c t o r s ( r e s i d e n c e , age, smoking h a b i t s , d r i n k i n g h a b i t s , occupation) on the exposure of the pregnant women and t h e i r newborns t o some heavy metals, i n c l u d i n g cadmium;. They s t a t e d t h a t of the parameters s t u d i e d , o n l y smoking h a b i t s had an i n f l u e n c e on the amount of cadmium found i n the blood of the mother* The d i s t r i b u t i o n of blood-cadmium l e v e l s i n mothers was s i g n i f i c a n t l y d i f f e r e n t between smokers and non-smokers* The high values of blood-cadmium i n smokers were c o n s i s t e n t with the presence of t h i s p o l l u t a n t i n c i g a r e t t e smoke* However, t h a t i n c r e a s e d blood-cadmium due to c i g a r e t t e smoking was not a s s o c i a t e d with a s i m i l a r i n c r e a s e i n blood-cadmium i n the newborns. This was e x p l a i n e d by the o b s e r v a t i o n that cadmium c o n c e n t r a t i o n i n the p l a c e n t a was on the average 25 percent h i g h e r i n smokers than i n non-smokers (Soels et a l . , 1978). S i n c e cadmium i s a well-known inducer of m e t a l l o t h i o n e i n * i t i s p o s s i b l e t h a t i n d u c t i o n of m e t a l l o t h i o n e i n s y n t h e s i s i n the p l a c e n t a was r e s p o n s i b l e f o r the higher cadmium accumulation i n the: smoker's p l a c e n t a and hence f o r a decreased t r a n s f e r of the metal to the newborn. , 37 Eoels et a l . (1978) r e p o r t e d another study with the same group of women i n which p l a c e n t a l l e v e l s of heavy metals (among them cadmium) were analyzed, with the aim of f i n d i n g out whether they were r e l a t e d to the l e v e l s i n maternal and cord blood and whether the d i f f e r e n t e p i d e m i o l o g i c a l f a c t o r s c i t e d above c o u l d a l s o i n f l u e n c e the metal accumulation i n p l a c e n t a . . The most s i g n i f i c a n t o b s e r v a t i o n made du r i n g t h i s study was the marked accumulation of cadmium i n placenta ( i n comparison with i t s l e v e l s i n maternal b l o o d ) . T h i s accumulation was r e l a t e d t o the r o l e of the pl a c e n t a as a b a r r i e r f o r the t r a n s f e r of cadmium t o the f e t u s * T h i s o b s e r v a t i o n however, d i d not allow them to conclude t h a t cadmium was not hazardous t o the f e t u s , accumulation of cadmium i n the p l a c e n t a c o u l d a l t e r i t s f u n c t i o n s and thus be embryotoxic* Indeed, as p r e v i o u s l y r e p o r t e d , P a r i z e k , (1965) has shown that a f t e r maternal cadmium i n j e c t i o n s of 2 to 1 mg/kg the p l a c e n t a c o u l d be r a p i d l y damaged, l e a d i n g t o death of the embryos i n u t e r o . However, th e r e was no evidence t h a t the s l i g h t accumulation of cadmium i n p l a c e n t a found i n the human p o p u l a t i o n had any d e l e t e r i o u s a c t i o n on the pregnancy.. F i n a l l y , among the v a r i o u s e p i d e m i o l o g i c a l f a c t o r s s t u d i e d by Eoels et a l * (1974), only smoking was found to i n c r e a s e cadmium accumulation i n p l a c e n t a (approximately 3 p e r c e n t ) . I t must* however, be recognized t h a t i n the g e n e r a l p o p u l a t i o n the d i e t c o n s t i t u t e s the main source o f cadmium i n t a k e : and d i e t a r y h a b i t s of the pregnant mothers were not i n v e s t i g a t e d during t h i s survey. In a d d i t i o n , i n t h e i r study the d i e t of the mother or the i n g e s t i o n of a p a r t i c u l a r food by the mother ( l i k e l i v e r or 38 kidney) i n l a r g e q u a n t i t i e s may be a f a c t o r which i n f l u e n c e d the accumulation of cadmium i n the p l a c e n t a or i n other maternal t i s s u e s , even though t h i s p o s s i b i l i t y may be remote* T h e r e f o r e , the present study, based on the above i n f o r m a t i o n , was concerned with two o b j e c t i v e s * The f i r s t was to i n v e s t i g a t e the e f f e c t s of z i n c on the t e r a t o g e n i c a c t i o n o f cadmium. The second was t o examine the i n t e r a c t i o n i n the d i s t r i b u t i o n of the metals, by a d m i n i s t e r i n g them together or i n d i v i d u a l l y t o pregnant r a t s . Then the e f f e c t s of z i n c on cadmium d i s t r i b u t i o n i n the maternal t i s s u e s and f e t u s , o r cadmium on z i n c d i s t r i b u t i o n i n the maternal t i s s u e s and f e t u s were e v a l u a t e d , using 115m-cadmium or 6 5 - z i n c . 39 CHAPTER I I I MATERIALS AND METHODS Young adult female Wistar rats (purchased from Canadian Breeding Laboratories, Quebec), weighing about 250 to 350 g, were kept overnight with males (1 male with 3 females). The following morning the males were separated from the females and vaginal washings were examined for sperm. The presence of sperm indicated day zero of pregnancy* The pregnant rats were caged separately, with Purina Rat Chow and water supplied ad libiturn. The animal room had fluorescent l i g h t s with a l i g h t i n g cycle . of 12 hours l i g h t , from 600 to 1800, and 12 hours darkness, from 1800 to 600 and the temperature:of the room was 72±2 degrees Fahrenhait. Wistar rats were used for these experiments because i n preliminary studies i t was found that they were more sensitive than some other s t r a i n s of rats to the teratogenic action of cadmium* In addition, the greater variety of malformations produced in Wistar rats makes t h i s s t r a i n more useful for studying the teratogenic effects of cadmium. (Parzyck et a l . , 1978). EXPERIMENT J Treatment of Experimental Animals On day 9 of pregnancy each rat was injected i n t r a p e r i t o n e a l l y , under l i g h t ether anaesthesia* with one of 40 the f o l l o w i n g : 1 1) Cadmium c h l o r i d e , 16uH/kg body weight i n s a l i n e c o n t a i n i n g 10-15uCi c a r r i e r - f r e e : 115m-cadmium c h l o r i d e . 2) Cadmium c h l o r i d e plus z i n c c h l o r i d e , 16uM/kg body weight of each, i n s a l i n e c o n t a i n i n g 10-15uCi c a r r i e r -f r e e 115m-cadmium c h l o r i d e . 3) Z i n c c h l o r i d e , 16uM/kg body weight i n s a l i n e c o n t a i n i n g 10-15uCi c a r r i e r - f r e e 65-zinc c h l o r i d e * 4) Z i n c c h l o r i d e plus cadmium c h l o r i d e , 16uM/kg body weight of each, i n s a l i n e c o n t a i n i n g 10-15uCi c a r r i e r -f r e e 65-zinc c h l o r i d e . A l l s o l u t i o n s were prepared t o a c o n c e n t r a t i o n such t h a t each animal r e c e i v e d approximately 1 ml of s o l u t i o n . , A f t e r the i n j e c t i o n of each r a t , a standard was prepared, c o n s i s t i n g o f the same amount of s o l u t i o n as was given t o the r a t . Day 9 was chosen f o r the day of i n j e c t i o n on the b a s i s o f the s t u d i e s of Barr (1973) and Parzyck (1978), i n which i t was demonstrated t h a t a g r e a t e r number and v a r i e t y of malformations occ u r r e d f o l l o w i n g i n j e c t i o n o f cadmium on day 9 than o c c u r r e d f o l l o w i n g i n j e c t i o n of cadmium on day 8, 10, or 11.. T h i s was confirmed i n p r e l i m i n a r y s t u d i e s i n t h i s l a b o r a t o r y , but the R a d i o i s o t o p e s were obtained from New England Nuclear Corp., as c a r r i e r - f r e e products. 41 data are not i n c l u d e d here. The e f f e c t i v e n e s s of cadmium g i v e n on day 9 may be expected as t h i s i s d u r i n g the p e r i o d of most r a p i d organogenesis i n the r a t (Barr, 1973; Holmberg e t a l . , 1969; Frohberg, 1974; Wilson, 1964). The amount of cadmium to be i n j e c t e d was s e l e c t e d on the b a s i s of p r e l i m i n a r y s t u d i e s which showed t h a t a cadmium dose of 16uM/kg of the body weight was t e r a t o g e n i c when giv e n on day 9 of pregnancy. Amounts of cadmium i n excess of 22uM/kg of body weight k i l l e d many pregnant r a t s (Barr, 1973), and 32uH/kg of body weight k i l l e d a l l pregnant dams. In a d d i t i o n , i t has been r e p o r t e d (Ferm and Carpenter, 1967, 1968) t h a t the p r o t e c t i v e e f f e c t of z i n c a g a i n s t the t e r a t o g e n i c e f f e c t s of cadmium was noted when administered i n e q u a l amounts simultaneously with cadmium or 15 minutes to 6 hours a f t e r the i n j e c t i o n of cadmium.,Therefore, i n groups 2 and 4 (cadmium-zinc and zinc-cadmium groups) z i n c c h l o r i d e was i n j e c t e d s i m u l t a n e o u s l y with cadmium c h l o r i d e . . Examination and C o l l e c t i o n pf T i s s u e s The f o u r groups of animals (9 t o 12 i n each group) were s a c r i f i c e d on day 20 of pregnancy. The r a t s were a n e s t h e t i z e d with ether and blood was drawn d i r e c t l y from the heart with a h e p a r i n i z e d s y r i n g e . Approximately 5 cc of blood was obtained from each animal and placed i n 3 tubes* One tube was used f o r the measurement of whole blood r a d i o a c t i v i t y , the second was c e n t r i f u g e d and the plasma was removed f o r r a d i o a c t i v e c o u n t i n g , and the t h i r d sample was used f o r d e t e r m i n a t i o n of the hematocrit (Brown, 1976). 42 The thorax of the animal was opened and the h e a r t was removed f o r counting* The abdominal wall was then opened and the whole uterus was d i s s e c t e d , from ovary to ovary. , The u t e r i n e w a l l was opened and each embryonic sac separated and examined. The empty u t e r i n e w a l l was examined c a r e f u l l y f o r r e s o r p t i o n s i t e s , which were recorded f o r each animal. Each embryonic sac was opened and the f e t u s separated from the p l a c e n t a . The f e t u s e s were then c l e a n e d , weighed, and f i x e d i n Bouin's s o l u t i o n 1 * each f e t u s was placed i n an i n d i v i d u a l counting tube* One of the p l a c e n t a s , chosen a t random, from each animal was then cleaned i n d i s t i l l e d water, weighed and p l a c e d i n a counting tube* The r e s t of the placentas from each animal were pooled and, a f t e r c l e a n i n g i n d i s t i l l e d water and weighing, were placed i n c o u n t i n g tubes. The same t h i n g was done f o r the embryonic sacs and f o r the u m b i l i c a l cords*_In a d d i t i o n , the number o f f e t u s e s , both dead and a l i v e , was recorded. The l i v i n g f e t u s e s were c a r e f u l l y examined f o r g r o s s e x t e r n a l malformations, and these were recorded. The whole l i v e r , the h e a r t , the lungs, the l e f t k idney, both a d r e n a l s , the spleen* the upper p a r t of the s m a l l i n t e s t i n e , (approximately 3 cm), about 1 g of f a t from the abdominal c a v i t y , the l e f t femur, approximately 1 g of muscle from the l e f t lower limb, the whole b r a i n , and approximately 3 cm of uterus were then d i s e c t e d from the mother, washed i n d i s t i l l e d water, and weighed. . Each t i s s u e was placed i n a i B o u i n ' s s o l u t i o n contained 25 cc s a t u r a t e d p i c r i c a c i d , 25 cc o f f o r m a l i n (40% formic a c i d ) , and 5 cc of g l a c i a l a c e t i c a c i d . . 43 separate tube f o r r a d i o a c t i v e counting* EXPEEIMENT 2 A f t e r a n a l y z i n g the r e s u l t s of the f i r s t experiment i t was found t h a t even though t h e r e was a l a r g e range of malformations i n group 1, some s m a l l amount i n group 3, and none i n groups 2 and 4, almost no r a d i o a c t i v e cadmium was found i n the f e t u s e s o f the 115m-cadmium t r e a t e d groups and there were very s m a l l d i f f e r e n c e s i n the d i s t r i b u t i o n of cadmium i n t h e : 115m-cadmium t r e a t e d animals. T h e r e f o r e , i t was decided t o c a r r y out a second experiment based on the hy p o t h e s i s t h a t cadmium goes to the f e t u s i n the e a r l y hours a f t e r i n j e c t i o n s and i s then e x c r e t e d . This h y p o t h e s i s i s supported by r e p o r t s t h a t cadmium c r o s s e s the p l a c e n t a and causes damages i n the pars f e t a l i s of the p l a c e n t a s of t r e a t e d animals (Parizek e t a l . , 1968; Ferm et a l . , 1969; Bruce et a l . , 1977; L u c i s et a l . , 1971). T h e r e f o r e , young a d u l t female Wistar r a t s weighing about 250 to 350 g were mated as d e s c r i b e d above., Treatment of Experimental Animals At 1100 on day 9 of pregnancy each pregnant r a t 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 with a s o l u t i o n of e i t h e r cadmium c h l o r i d e p l u s 115m-cadmium c h l o r i d e , or cadmium c h l o r i d e p l u s z i n c c h l o r i d e plus 115m-cadmium c h l o r i d e , prepared as d e s c r i b e d above. Six groups of animals* 5 animals per group, were e s t a b l i s h e d . Three groups (groups 5, 7, and 9) were i n j e c t e d with cadmium c h l o r i d e plus 115m-cadmium, and s a c r i f i c e d between 44 1000 t o 1200 on days 10, 11, and 12 r e s p e c t i v e l y of pregnancy; and the other 3 groups (6, 8, and 10) were i n j e c t e d with cadmium c h l o r i d e p l u s z i n c c h l o r i d e plus 115m-cadmium c h l o r i d e , and s a c r i f i c e d between 1000 to 1200 on days 10, 11, and 12 o f pregnancy. Examination and C o l l e c t i o n of T i s s u e s A l l animals were anest h e s i z e d with ether and s a c r i f i c e d as d e s c r i b e d above. The manner of t r e a t i n g the t i s s u e s was as d e s c r i b e d i n the f i r s t experiment, except f o r the treatment given t o the uterus and f e t u s . The uterus was d i s s e c t e d from ovary t o ovary and the u t e r i n e w a l l was opened* the embryonic sacs were separated and counted as a u n i t with f e t u s and placentas* Because o f the s m a l l s i z e of these t i s s u e s a t these e a r l y s t a g e s of pregnancy i t was very d i f f i c u l t t o i d e n t i f y the d i f f e r e n t parts* However, i n t h i s case most o f the uterus was cleaned, weighed and placed i n t o a c o u n t i n g tube. In a d d i t i o n , the t e r m i n a l end of the r i g h t horn of the uterus, with one embryo, was d i s s e c t e d together as a u n i t and placed i n a tube f o r c o u n t i n g and the new t i s s u e was c a l l e d "Uterus p l u s Fetus". In t h i s experiment i t was not p o s s i b l e t o i d e n t i f y and keep a r e c o r d of the l i v e f e t u s e s , dead f e t u s e s , r e s o r p t i o n s i t e s or number of malformations due to the s m a l l s i z e and s i m i l a r appearence of a l l embryos at these e a r l y stages of g e s t a t i o n . , COUNTING OF EADIOISOTOPES Tubes c o n t a i n i n g the r a d i o a c t i v e t i s s u e s were placed i n a P i c k e r Autowell Gama Spectrometer f o r the measurement of 115m-45 cadmium and 6 5 - z i n c . In the case of 115m-cadmium the spectrometer was set t o record d i s i n t e g r a t i o n s with e n e r g i e s between 0.820 and 1.1 MeV and f o r 65-zinc d i s i n t e g r a t i o n s with e n e r g i e s between 0.06 and 0.07 MeV were recorded. Each t i s s u e was counted twice f o r 5 minutes, t o g e t h e r with the standards o f the i n j e c t e d dose* The standards were prepared f o r each animal at the time of i n j e c t i o n . Counts (115m-cadmium and 65-zinc) per minute per gram were c a l c u l a t e d f o r each t i s s u e * using the wet weight of each sample f o r the c a l c u l a t i o n s ; . Then the counts as percent of the t o t a l amount i n j e c t e d i n t o each animal were a l s o c a l c u l a t e d . T h i s was c a l c u l a t e d from the standards. STATISTICAL ANALYSIS S t a t i s t i c a l a n a l y s es were performed using a standa r d program ( S t a t i s t i c a l Package For The S o c i a l S c i e n c e s , SPSS) at the Computer Center of the O n i v e r s i t y of B r i t i s h Columbia. Means, standard e r r o s , and standard d e v i a t i o n s were c a l c u l a t e d . Student's t t e s t was employed i n order t o compare means o f p a i r s of e xperimental groups. One-way A n a l y s i s of Variance and M u l t i p l e Range t e s t s were used t o compare the v a r i a b l e s "Hematocrit", " F e t a l Height", and "Malformations" among the f o u r treatment groups i n Experiment 1.,Two-way A n a l y s i s of V a r i a n c e was used t o compare the two treatment and t h r e e treatment p e r i o d s i n Experiment 2. For s t a t i s t i c a l a n a l y s i s a l l the f e t u s e s from each dam were pooled, so t h a t the means of f e t a l weight, counts per minute per gram, and percent of i n j e c t e d dose were c a l c u l a t e d f o r each dam 46 and r e p o r t e d as "Fetus". Furthermore, because the only d i f f e r e n c e i n the procedures employed i n these two experiments was the day of pregnancy on which the animals were s a c r i f i c e d , i t was p o s s i b l e t o compare the 10 groups s t a t i s t i c a l l y * A n a l y s i s of v a r i a n c e and m u l t i p l e range t e s t s were used i n comparing the f o l l o w i n g v a r i a b l e s : the hematocrit of the 10 groups; the maternal t i s s u e s and the f e t u s e s of the groups t r e a t e d with cadmium c h l o r i d e plus 115m-cadmium o r the groups t r e a t e d with cadmium c h l o r i d e p l u s z i n c c h l o r i d e plus 115m-cadmium. 47 CHAPTER IV RESULTS Gross e x t e r n a l malformations were seen at e x t e r n a l examination of the f e t u s e s * In the cadmium t r e a t e d group, out o f 12 l i t t e r s , 10 contained some malformed f e t u s e s . In the z i n c group out of 10 l i t t e r s only 1 l i t t e r contained 2 malformed f e t u s e s , whereas no malformations were found i n the cadmium-zinc groups. In the cadmium group 28.8 percent o f the f e t u s e s presented some kind of a b n o r m a l i t i e s (Table I ) . D y s p l a s t i c t a i l and absent t a i l were the predominant d e f e c t s , f o l l o w e d by t h i n abdominal w a l l , d y s p l a s t i c neck, d y s p l a s t i c eye, exencephaly, d y s p l a s t i c e a r s , d y s p l a s t i c limb, and stunted body. In the z i n c group both malformed f e t u s e s were exencephaly. , In a d d i t i o n , s e v e r a l other parameters of f e t a l h e a l t h were examined (Table I I ) . They were: r e s o r p t i o n s i t e s * dead f e t u s e s , l i t t e r s i z e and f e t a l weight. I t was found t h a t the cadmium t r e a t e d f e t u s e s were s i g n i f i c a n t l y s m a l l e r than the cadmium-zinc t r e a t e d and z i n c t r e a t e d f e t u s e s . When the other t h r e e parameters were compared i n the 4 d i f f e r e n t t r e a t e d groups no s i g n i f i c a n t d i f f e r e n c e was found* Another parameter s t u d i e d was the maternal hematocrit. A s i g n i f i c a n t d i f f e r e n c e was found between the z i n c and z i n c -cadmium groups, where cadmium seems t o have caused the volume percent of c e l l s t o be s m a l l e r . However, t h i s r e s u l t i s probably not meaningful, s i n c e no d i f f e r e n c e s were found between the cadmium and cadmium-zinc groups*.Also the v a r i a n c e s between the means o f these 4 groups were smal l (Table I I I ) . 48 TABLE I E f f e c t o f Zinc on Cadmium-Induced Malformations, and Cadmium on Zinc-Induced Malformations of Fetuses of Wistar Rats I n j e c t e d on Day 9 and Examined on Day 20 of G e s t a t i o n Percent and Number of Malformed Fetuses Cadmium Cadmium-Zinc Zinc Zinc-Cadmium No. of Fetuses 156 109 129 110 T o t a l Malformed 28.84 (45) 0 1.55(2) 0 Absent T a i l 7.69 (12) 0 0 0 D y s p l a s t i c T a i l 10.25(16) 0 0 0 Exencephaly 1.?2( 3) 0 1.55(2) 0 D y s p l a s t i c Neck 5.76 ( 9) 0 0 0 D y s p l a s t i c Ears 1.28( 2) 0 0 0 Thin Abdominal Wall 6.41 (10) 0 0 0 D y s p l a s t i c Limbs 0.64 ( 1) 0 0 0 D y s p l a s t i c Eyes 3.20( 5) 0 0 0 Stunted Body 0.64( 1) 0 0 0 ( )=Number of Malformed Fetuses 49 TABLE I I E f f e c t of Zinc on Cadmium-Induced C h a r a c t e r i s t i c s * and Cadmium on Zinc-Induced C h a r a c t e r i s t i c s of 20 Day Old Fetuses o f Wistar Rats Cadmium Cadmium-Zinc Zinc Zinc-Cadmium No* Of 156 109 129 110 Fetuses No. of 12 9 10 9 L i t t e r s Resorption 1.17±0.61* 0.78±0.32 1. 30±0.42 1. 22+0.36 S i t e s 3 Dead 0.17±0.11 0.1H0.11 0.10±0.10 0.44±0.24 F e t u s e s 3 L i t t e r 13.00±0.70 12.1U0.63 12.90±0.64 12. 22±0.79 S i z e 3 F e t a l 2. 89±0. 062 3.26±0.11 3.47±0. 08 3;21±0. 11 Weight (g) * 1 =Mean±Standard E r r o r 2 = S i g n i f i c a n t d i f f e r e n c e (p<0.05) from other groups 3=Mean number per l i t t e r *=Average of l i t t e r means 50 TABLE I I I E f f e c t o f Zinc and Cadmium on the Hematocrit of Wistar Rats I n j e c t e d on Day 9 and Sampled on Day 20 of G e s t a t i o n . . A l l Chemicals were I n j e c t e d a t 16uM/kg of Body Weight. Treatments No. of Animals Hematocrit (%) Cadmium 12 32. 2 ± 0. 8* Cadmium-Zinc 9 32.0 ± 0.9 Zinc 10 33. 4 ± 0.4 Zinc-Cadmium 9 31.0 ± 0.52 l=Mean±Standard E r r o r 2 = S i g n i f i c a n t d i f f e r e n c e (p<0.05) from the z i n c group 51 Table IV shows the recovery of 115m-cadmium, as percent o f the i n j e c t e d dose, i n s e v e r a l t i s s u e s of animals given cadmium c h l o r i d e alone or cadmium c h l o r i d e p l u s z i n c c h l o r i d e . In these t i s s u e s , t o t a l organ weight was recorded, thus, the f i g u r e s i n d i c a t e t o t a l organ recovery. There were no s i g n i f i c a n t d i f f e r e n c e s between the cadmium and cadmium-zinc groups f o r any t i s s u e except the f e t u s e s . However, t h i s was probably not a meaningful d i f f e r e n c e , as the r e c o v e r y i n the cadmium-zinc group was only 2 or 3 counts per minute, compared with zero i n the cadmium group. The l a r g e s t accumulations of 115m-cadmium were found i n the l i v e r (49.52% i n the cadmium group and 49.47% i n the cadmium-z i n c group), f o l l o w e d by the.kidney (3.19% i n the cadmium group and 3.51% i n the cadmium-zinc group), where t h e . a d d i t i o n of z i n c s l i g h t l y , but not s t a t i s t i c a l l y s i g n i f i c a n t l y , i n c r e a s e d the amount of r a d i o a c t i v i t y d e p o s i t e d i n t h i s organ by day 20 of g e s t a t i o n . Only s m a l l amounts of r a d i o a c t i v e cadmium were found i n a l l the other organs examined as shown i n Table IV. Table V l i s t s the percent of i n j e c t e d dose: recovered per gram of t i s s u e , s i n c e the t o t a l t i s s u e weight was not known, the r e s u l t s a re expressed per gram o f t i s s u e , as percent of dose administered. In the f a t and uterus the accumulation of 115m-cadmium was s i g n i f i c a n t l y g r e a t e r (p<0.05) i n the cadmium group than i n the cadmium-zinc group* For the other t i s s u e s the d i f f e r e n c e s between the two groups were not s t a t i s t i c a l l y s i g n i f i c a n t . Table VI shows the recovery of 6 5 - z i n c , as percent of the i n j e c t e d dose, i n s e v e r a l t i s s u e s of animals given z i n c c h l o r i d e 52 TABLE IV E f f e c t o f Zinc on Cadmium D i s t r i b u t i o n i n D i f f e r e n t Organs of Wistar Hats I n j e c t e d on Day 9 and Sampled on Day 20 Of G e s t a t i o n . A l l Chemicals were I n j e c t e d at 16uM/kg of Body Weight. Percent o f I n j e c t e d Dose:Recovered Cadmium Cadmium-Zinc No. of Animals 12 9 L i v e r 49.5253 ± 1. 0861 49.4702 ± 1.086 Kidney 3: 1938 ± 0. 145 3.5173 + 0.05 2 B r a i n 0.0096 ± 0. 002 0.0170 ± 0.004 Adrenals 0.0238 ± 0. 003 0.0214 ± 0.003 Heart 0. 1296 ± 0. 006 0.1569 ± 0.015 Lung 0. 1625 ± 0. 012 0. 1614 ± 0.010 Spleen 0.2850 ± 0. 011 0.2809 ± 0.010 Femur 0.0476 ± 0. 005 0.0612 ± 0.007 Pl a c e n t a 0.0055 ± 0. 001 0.0064 ± 0.002 Placentae 0.1140 ± 0. 011 0.1012 ± 0.006 U m b i l i c a l cord 0.0021 ± 0. 001 0.0012 ± 0.001 Embryonic sac 0.0223 ± 0. 004 0.0212 ± 0.004 Fetus 0.0000 ± 0. 000 0.0015 ± 0.0012 1 =Mean±Standard E r r o r 2 = S i g n i f i c a n t d i f f e r e n c e (p<0.05) from corresponding group without z i n c 53 TABLE V E f f e c t of Zinc on Cadmium D i s t r i b u t i o n i n D i f f e r e n t Organs of Wistar Rats I n j e c t e d on Day 9 and Sampled on Day 20 o f G e s t a t i o n . . A l l Chemicals were I n j e c t e d at 16uM/kg of Body Weight.. Percent o f I n j e c t e d Dose Recovered per Gram of T i s s u e No. of Animals Whole Blood Small I n t e s t i n e Fat Uterus Muscle Plasma Cadmium 12 0.0133 ± 0.002* 0.2852 ± 0.021 0.2966 ± 0.106 0. 1888 ± 0.012 0.0114 ± 0.002 0.0017 ± 0.001 Cadmium-Zinc 9 0.0170 ± 0.004 0.2372 ± 0.017 0.0487 ± 0.0102 0.1397 ± 0.0092 0.0071 ± 0.002 0.0008 ± 0.001 1 =Mean±Standard E r r o r 2 = S i g n i f i c a n t d i f f e r e n c e (p<0.05) from corresponding group without z i n c 54 TABLE VI E f f e c t of Cadmium on Zinc D i s t r i b u t i o n i n D i f f e r e n t Organs o f Wistar Rats I n j e c t e d on Day 9 and Sampled on Day 20 of Ge s t a t i o n . A l l Chemicals were I n j e c t e d at 16uM/kg of Body Weight. Percent of I n j e c t e d Dose Recovered Zinc Zinc-Cadmium No. of Animals 10 9 L i v e r 3.3568 ± 0. 2291 6.4016 ± 0.3342 Kidney 0.3324 ± 0. 022 0.5614 ± 0.0312 Br a i n 0.2612 ± 0. 015 0. 2940 ± 0.012 Adrenals 0.0063 ± 0. 001 0.0147 ± 0.0022 Heart 0.1537 ± 0. 009 0.1914 ± 0.0102 Lung 0.2115 + 0. 010 0.2983 ± 0.0122 Spleen 0. 1220 ± 0. 008 0.1748 ± 0.0102 Femur 0.3001 ± 0. 016 0.3079 ± 0.025 Pla c e n t a 0.0590 ± 0. 004 0.0778 ± 0.0032 Placentae 0.6067 ± 0. 03 9 0.7826 ± 0.0662 O m b i l i c a l cord 0.0295 + 0. 003 0.0338 ± 0.003 Embryonic sac 0.2557 ± 0. 018 0.3114 + 0.028 Fetus 0.5064 + 0. 034 0.6470 ± 0.0302 »=Mean±Standard E r r o r 2 = s i g n i f i c a n t d i f f e r e n c e from corresponding group without cadmium (p<0. 05) 55 alone or z i n c c h l o r i d e p l u s cadmium c h l o r i d e * In t h i s t i s s u e s , as i n the case of Table IV, t o t a l organ weight was known, so the f i g u r e s i n d i c a t e t o t a l organ recovery. There were s i g n i f i c a n t d i f f e r e n c e s (p<0.05) between the z i n c and zinc-cadmium groups i n the l i v e r , kidney, a d r e n a l , h e a r t , lung, spleen, p l a c e n t a , and f e t u s , where the a d d i t i o n of cadmium i n c r e a s e d the amount of 65-z i n c d e p o s i t e d i n those organs. . As i n the cadmium groups* the l i v e r was the organ i n which the l a r g e s t amount of 65-zinc was accumulateed (3.35% i n the z i n c group and 6.40% i n the zinc-cadmium group).. T h i s was f o l l o w e d by the kidney (0.33% i n the z i n c group and 0.56% i n the zinc-cadmium group). R a d i o a c t i v i t y was found i n a l l t i s s u e s examined. Table VII l i s t s the percent of i n j e c t e d dose recovered per gram of t i s s u e , s i n c e the t o t a l t i s s u e weight was not known the r e s u l t s were expressed per gram of t i s s u e , as percent o f dose admin i s t e r e d . In f a t , uterus, muscle, and plasma, the accumulation of 65-Zinc was s i g n i f i c a n t l y g r e a t e r (p<0 . 0 5 ) i n the z i n c group than i n the zinc-cadmium group..The d i f f e r e n c e s between the two groups f o r whole blood and s m a l l i n t e s t i n e were not s t a t i s t i c a l l y s i g n i f i c a n t . Due to the f a c t t h a t gross e x t e r n a l malformations were found i n the cadmium group when the animals were i n j e c t e d on day 9 and sampled on day 20 of g e s t a t i o n , but no r a d i o a c t i v i t y was dete c t e d i n the f e t u s e s , a second experiment was c a r r i e d out i n order t o determine whether cadmium reaches the f e t u s i n the e a r l y hours a f t e r i n j e c t i o n . The animals w e r e . i n j e c t e d with cadmium c h l o r i d e plus 115m-cadmium, or with cadmium c h l o r i d e and 56 TABLE VII E f f e c t o f Cadmium on Zinc D i s t r i b u t i o n i n D i f f e r e n t Organs o f wis t a r Rats I n j e c t e d on Day 9 and Sampled on Day 20 of G e s t a t i o n . A l l Chemicals were I n j e c t e d a t 16uM/kg of Body Weight. Percent of I n j e c t e d Dose Recovered per Gram of T i s s u e Zinc Zinc-Cadmium No. of Animals 10 9 Whole Blood 0.0507 ± 0.0031 0.0574 ± 0.004 Small I n t e s t i n e 0.1865 ± 0.015 0.2144 ± 0.010 Fat 0.0162 ± 0.002 0.0285 ± 0.002 2 Oterus 0.1825 ± 0.010 0.2209 ± 0.011 2 Muscle 0. 1080 ± 0.007 0. 1267 ± 0.005 2 Plasma 0.0033 ± 0.000 0.0081 ± 0.001 2 i=Mean±Standard E r r o r 2 = S i g n i f i c a n t d i f f e r e n c e (p<0.05) from corresponding group without cadmium 57 z i n c c h l o r i d e p l u s 115m-cadmium on day 9 (as d e s c r i b e d above) and sampled on day 10, 11 and 12 of g e s t a t i o n . _ A s i g n i f i c a n t d i f f e r e n c e i n 115m-cadmium (p<0.05) was found between the cadmium and cadmium-zinc groups on day 10 i n the uterus, uterus p l u s f e t u s , and f e t u s . In a l l these t i s s u e s the amount of 115m-Cadmium was l e s s i n the cadmium-zinc group than i n the cadmium group. No d i f f e r e n c e s were found i n the ot h e r t i s s u e s (Table V I I I ) . . With r e s p e c t to those t i s s u e s where recovery was c a l c u l a t e d as percent of i n j e c t e d dose per gram of t i s s u e , a s i g n i f i c a n t d i f f e r e n c e was found i n the f a t and plasma (Table I X), where recovery was l e s s (p<0.05) i n the cadmium-zinc groups than i n the cadmium group* When the animals were sampled on day 11 the amount of r a d i o a c t i v i t y recovered i n the a d r e n a l s and i n the s m a l l i n t e s t i n e was s i g n i f i c a n t l y l e s s i n the cadmium-zinc group than i n the cadmium group (Tables X and X I ) . In the case of the animals sampled on day 12 the o n l y s i g n i f i c a n t d i f f e r e n c e found was i n the femur, where the amount of cadmium t h a t was recovered was l e s s i n the cadmium-zinc group than i n the cadmium group (Tables XII and XIII) . The cadmium t r e a t e d group was a l s o s t udied using one-way a n a l y s i s o f v a r i a n c e s f o r days of g e s t a t i o n (10, 11, and 1 2 ) . , I t was found t h a t the amount of 115m-cadmium d e p o s i t e d i n the kidney expressed as percent of i n j e c t e d dose recovered i n c r e a s e d with time* and i n the uterus decreased with time as shown on Tables XIV and XV. However, when one-way a n a l y s i s o f v a r i a n c e was c a r r i e d out 58 TABLE VIII E f f e c t of Zinc on Cadmium D i s t r i b u t i o n i n D i f f e r e n t Organs o f Wistar Rats I n j e c t e d on Day 9 and Sampled on Day 10 o f G e s t a t i o n . . A l l Chemicals were I n j e c t e d a t 16uM/kg of Body Weight* Percent o f I n j e c t e d Dose Recovered Cadmium Cadmium-Zinc No. of Animals 5 5 L i v e r 48.5201 ± 4.2521 46.6721 ± 1. 327 Kidney 2. 1737 ± 0. 148 2.2039 ± 0. 168 B r a i n 0.0185 ± 0.003 0.0167 ± 0.003 Adrenal 0.0405 ± 0.010 0.0186 ± 0.003 Heart 0. 1760 ± 0.004 0. 1763 ± 0.009 Lung 0. 1938 ± 0.022 0.1975 ± 0.011 Spleen 0.2700 ± 0.018 0.2685 ± 0.025 Oterus 0.7795 ± 0. 150 0.3226 ± 0.0762 Femur 0.0862 ± 0.006 0.0748 ± 0.008 Oterus+Fetus 0.0870 ± 0.007 0.0451 + 0.0072 Fetus 0.0152 ± 0.001 0;0094 ± 0.0012 i=Mean±Standard E r r o r 2 = S i g n i f i c a n t d i f f e r e n c e (p<0.05) from corresponding group without cadmium 59 TABLE IX E f f e c t o f Zinc on Cadmium D i s t r i b u t i o n i n D i f f e r e n t Organs o f Wistar Rats I n j e c t e d on Day 9 and Sampled on Day 10 o f G e s t a t i o n . A l l Chemicals were Administered at 16uM/kg of Body Weight Percent of I n j e c t e d Dose.Recovered per Gram o f T i s s u e Cadmium Cadmium-Zinc No. of Animals 5 5 Whole Blood 0.0242 ± 0.0041 0.0193 ± 0.004 Small I n t e s t i n e 0.5731 ± 0.104 0.4960 ± 0.024 Fat 0.0867 ± 0.018 0.0290 ± 0.0042 Muscle 0.0233 ± 0.003 0.0148 ± 0.003 Plasma 0.0068 ± 0.001 0.0031 ± 0.0012 i=Mean±Standard E r r o r z = s i g n i f i c a n t d i f f e r e n c e (p<0.05) from corresponding group without z i n c 60 TABLE X E f f e c t o f Zinc on Cadmium D i s t r i b u t i o n i n Organs of Wistar Rats I n j e c t e d on Day 9 and Sampled on Day 11 of G e s t a t i o n . . A l l Chemicals were I n j e c t e d At 16uM/kg of Body Weight.. Percent of I n j e c t e d Dose Recovered Cadmium Cadmium-Zinc No. of Animals 5 i 5 L i v e r 50.9920 ± 2. 149* 48.6335 ± 0.559 Kidney 2. 1210 ± 0.009 2.2162 ± 0. 194 Br a i n 0.0313 + 0.013 0.0527 ± 0.014 Adrenals 0.2 999 ± 0.004 0.0198 ± 0.0012 Heart 0. 1714 ± 0.007 0.1579 ± 0.012. Lung 0. 2519 ± 0.040 0.1941 ± 0.025 Spleen 0.3185 ± 0.033 0.2823 ± 0.022 Uterus 0.6178 ± 0.062 0.3706 ± 0.061 Femur 0. 0697 ± 0.007 0.0549 ± 0.011 Oterus+Fetus 0,0736 ± 0.011 0.0448 ± 0.007 Fetus 0.0139 ± 0.003 0.0086 ± 0.002 *=Mean+Standard E r r o r z = S i g n i f i c a n t d i f f e r e n c e (p<0.05) from corresponding group without cadmium 61 TABLE XI E f f e c t o f Zinc on Cadmium D i s t r i b u t i o n i n D i f f e r e n t Organs o f Wistar Rats I n j e c t e d on Day 9 and Sampled on Day 11 of Ge s t a t i o n . . A l l Chemicals were I n j e c t e d at 16uM/kg of Body Weigit. Percent o f I n j e c t e d Dose Recovered per Gram of T i s s u e Cadmium Cadmium-Zinc No. of Animals 5 5 Whole Blood 0.0332 ± 0.009* 0.0245 ± 0.005 Small I n t e s t i n e 0.5958 ± 0.052 0.3826 ± 0.0272 Fat 0.0825 ± 0.034 0.0288 ± 0.004 Muscle 0,0271 ± 0.006 0.0137 ± 0.003 Plasma 0. 0039 ± 0.002 0.Q023 ± 0.001 1 =Mean±Standard E r r o r 2 = S i g n i f i c a n t d i f f e r e n c e (p<0.05) from corresponding group without z i n c 62 TABLE XII E f f e c t of Zinc on Cadmium D i s t r i b u t i o n i n D i f f e r e n t Organs of Wistar Rats I n j e c t e d on Day 9 and Sampled on Day 12 of G e s t a t i o n . . A l l Chemicals were I n j e c t e d a t 16uH/kg of Body Weight. Percent of I n j e c t e d Dose Recovered Cadmium Cadmium-Zinc No. of Animals i 5 5 L i v e r 53.8880 ± 2.553* 47.4111 ± 2. 171 Kidney 2.7313 ± 0. 197 2. 5588 + 0.039 B r a i n 0.0485 ± 0.014 0.0433 ± 0.014 Adrenals 0.0174 ± 0.003 0.0206 ± 0.004 Heart 0. 1693 ± 0.014 0.1818 + 0.006 Lung 0.2221 ± 0.020 0.1815 ± 0.011 Spleen 0.2925 ± 0.021 0.2403 ± 0.030 Oterus 0.3324 ± 0.082 0*4404 + 0.076 Femur 0.0741 ± 0.001 0.0593 ± 0.00 32 Oterus+Fetus 0.077 0 ± 0.031 0.0583 + 0.011 Fetus 0.0086 + 0.003 0.0101 + 0.003 *=Mean±Standard E r r o r 2 = s i g n i f i c a n t d i f f e r e n c e (p<0.05) from corresponding group without cadmium 63 TABLE XIII E f f e c t o f Zinc on Cadmium D i s t r i b u t i o n i n D i f f e r e n t Organs o f Wistar Rats I n j e c t e d on Day 9 and Sampled on Day 12 o f G e s t a t i o n . A l l Chemicals were I n j e c t e d at 16uH/kg of Body Weight. Percent o f I n j e c t e d Dose Recovered per Gram of T i s s u e Cadmium Cadmium-Zinc No* of Animals 5 5 Whole Blood 0.0328 ± 0.004* 0.0292 ± 0.003 Small I n t e s t i n e 0.3480 ± 0.050 0.2844 ± 0.014 Fat 0.0433 ± 0.013 0.0391 ± 0.010 Muscle 0.0141 ± 0.005 0.0176 ± 0.001 Plasma 0.0030 ± 0.002 0.0036 ± 0.Q01 1Mean±Standard E r r o r TABLE XIV Cadmium D i s t r i b u t i o n i n D i f f e r e n t Organs o f Wistar Rats I n j e c t e d on Day 9 and Sampled on Day 10, 11, and 12 o f G e s t a t i o n . Cadmium C h l o r i d e was I n j e c t e d at 16uM/kg of Body Weight* Percent of I n j e c t e d Dose Recovered Sampling Day 10 11 12 No..of Animals 5 5 5 L i v e r 48.5201±4.2522* 50.9920±2.}489 53.8880±2. 5529 Kidney 2. 1737±0.1476 2.1210±0.0089 2.7313±0. 19702 B r a i n 0.0185±0.0026 0.0313±0.0126 0.0485±0.0140 Adrenals 0.0405±0.0098 0.0299±0.0037 0.0174±0.0027 Heart 0. 1760±0.0040 0.1714±0.0073 0.1693±0.0140 Lung 0. 1930±0.0224 0.2519±0.0399 0.2221+0.0205 Spleen 0.2700±0.0180 0.3185±0.0328 0.2925±0.0209 Oterus 0.7795±0.1504 0.6178±0.0620 0.3324±0. 08183 Femur 0.0862±0.0056 0.0697±0.0072 0.0741±0.0013 Uterus* Fetus 0.0870±0.0072 0.0736±0.0106 0.0770±0.0308 Fetus 0.0152±0.0009 0.0139±0.0035 0.0086±0.0029 J=Mean±Standard E r r o r 2 = s i g n i f i c a n t d i f f e r e n c e (p<0.05) from day 11 group 3 = S i g n i f i c a n t d i f f e r e n c e (p<0.05) from day 10 group 65 TABLE XV Cadmium D i s t r i b u t i o n i n D i f f e r e n t Organs of Wistar Rats I n j e c t e d on Day 9 and Sampled on Day 10, 11, and 12 of G e s t a t i o n . Cadmium C h l o r i d e was I n j e c t e d a t 16uM/kg of Body Weight. Percent of I n j e c t e d Dose Recovered per Gram of Ti s s u e Sampling 10 11 12 Day Noi. of 5 5 5 Animals Whole 0.0242±0.00431 0.0332±0.0085 0.0328±0.0039 Blood Small 0.5731±0.1041 0.5958±0.0524 0.3480±0.0496 I n t e s t i n e Fat 0.0867±0.0181 0.0825±0.0336 0.0433±0.0126 Muscle 0.0233±0.0033 0.0271±0.0058 0.0141±0i0050 Plasma 0.0068±0.0009 0.0039+0.0018 0.0030±0.0018 i=Mean±Standard E r r o r 66 on the cadmium-zinc group f o r days o f g e s t a t i o n (10, 11 and 12), no s i g n i f i c a n t d i f f e r e n c e s were found i n those t i s s u e s when recovery was expressed as percent of i n j e c t e d dose recovered i n the whole organ (Table XVI). When the same a n a l y s i s was done on those organs i n which recovery i s expressed as dose recovered per gram of t i s s u e , a s i g n i f i c a n t d i f f e r e n c e was found i n the s m a l l i n t e s t i n e , where i t appeared t h a t with the a d d i t i o n of z i n c the amount of cadmium recovered i n t h i s organ decreased with time (Table XVII). Maternal hematocrit, l i t t e r s i z e , and f e t a l weight were a l s o s t u d i e d , as parameters t h a t r e f l e c t o v e r a l l f e t a l h e a l t h . . No s i g n i f i c a n t d i f f e r e n c e s were found between days or between groups (Table XVIII). F i n a l l y , one-way a n a l y s i s of v a r i a n c e by days was used to compare days 10, 11, 12, and 20 i n the cadmium and the cadmium-z i n c groups f o r v a r i o u s t i s s u e s . In t h e case of the l i v e r no s i g n i f i c a n t d i f f e r e n c e s were found between days or between groups. Approximately 5055 of the i n j e c t e d dose reached the l i v e r 24 hours p o s t - i n j e c t i o n and t h i s remained unchanged u n t i l day 20 of g e s t a t i o n * Furthermore, the treatment (cadmium versus cadmium-zinc) d i d not change t h i s p a t t e r n , as can be seen i n F i g u r e I. The kidney presented a d i f f e r e n t p a t t e r n . .In both treatment groups the amount of cadmium deposited i n t h i s organ i n c r e a s e d s i g n i f i c a n t l y with time (Figure II) . In the b r a i n the amount of cadmium was l e s s on day 20 than at day 12 i n the cadmium t r e a t e d animals, but no d i f f e r e n c e was found i n the cadmium-zinc group (Figure I I I ) . 67 TABLE XVI E f f e c t o f Zinc on Cadmium D i s t r i b u t i o n i n D i f f e r e n t Organs o f Wistar Rats I n j e c t e d on Day 9 and Sampled on Day 10, 11, and 12 o f G e s t a t i o n . A l l Chemicals were I n j e c t e d at 16uM/kg of Body Weight. . Percent of I n j e c t e d Dose Recovered Sampling Day 10 11 12 No. of Animals 5 5 5 L i v e r 46.6721±1.32741 48.6335±0.5588 47.4111±2.1708 Kidney 2.2039±0.1678 2.2162±0.1937 2.5588±0.0395 Br a i n 0.0167±0.0025 0.0527±0. 0136 0.0433±0.0144 Adrenals 0.0186±0.0027 0.0198±0.0013 0.0206±0.0040 Heart 0. 1763±0.0093 0.1579±0.0123 0. 1816±0.0057 Lung 0. 1975±0.0113 0.1941±0.0251 0.1815±0.0112 Spleen 0.2685±0.0250 0.2823±0.0221 0.2403±0.0299 Oterus 0.3226±0.0757 0.3706±0.0606 0.4404±0.0757 Femur 0.0748±0.0085 0.0549±0.0107 0.0593±0.0028 Oterus+ Fetus 0.045U0.0073 0.0448±0.0069 0.0583±0.0109 Fetus 0.0094±0.0012 0.0086±0.0081 0.0101±0.0034 i=Hean±Standard E r r o r 68 TABLE XVII E f f e c t of Zinc on Cadmium D i s t r i b u t i o n i n D i f f e r e n t Organs o f Wistar Rats I n j e c t e d on Day 9 and Sampled on Day 10, 11, and 12 of G e s t a t i o n . A l l Chemicals were I n j e c t e d at 16uM/kg of Body Weight. Percent of I n j e c t e d Dose Recovered per Gram of T i s s u e Sampling Day No» of Animals Whole Blood Small I n t e s t i n e Fat Muscle Plasma 10 11 12 0*0193±0.00411 0.0245±0.0047 0;0292±0.0029 0.4960±0i0238 0.3826±0.0266 0.2844±0.01422 0.0290±0.0036 0.0228±0.0041 0.0391±0.0104 0.0148±0.0033 0.0137±0.0031 0.0176±0.0010 0.003U0.0010 0.0023±0.0009 0. 0036±0. 0011 i=Mean±Standard E r r o r 2 = S i g n i f i c a n t d i f f e r e n c e (p<0.01) from corresponding 10, and 11 groups. 69 T A B L E X V I I I E f f e c t of Zin c and Cadmium on the Maternal Hematocrit, L i t t e r S i z e , and F e t a l Weight of Wistar Hats Sampled on Day 10, 11, and 12 of G e s t a t i o n . Each Group Contained 5 Animals* Hematocrit L i t t e r S i z e F e t a l Weight Day 10 Cadmium 39.500+0.3871 14.000H.304 0.04310.003 Cadmium-Zinc 39.80010.583 10.000i3.130 0.045i0.006 Day J l Cadmium 36.400l1.077 13.200i1.281 0.055±0.008 Cadmium-Zinc 36.400±0.927 11.400l1.913 0.075i0.006 Ml 12. Cadmium 38.400l1.030 5.800H.393 0.11010.025 Cadmium-Zinc 38.400i1.122 11.400i2.293 0.13810.020 *=MeaniStandard E r r o r 60 10 11 12 13 14 15 16 17 18 19 20 21 DAY OF GESTATION F i g . i A c c u m u l a t i o n o f 115m-Cadmium i n l i v e r o f a n i m a l s g i v e n Cadmium o r Cadmium+Zinc on day 9 of g e s t a t i o n . 4 0 C d = C d + Zn = W/- 1 J L i I I I I I I I 1 0 1 1 1 2 1 3 1 8 1 9 2 0 2 1 1 4 1 5 1 6 1 7 D A Y O F G E S T A T I O N F i g . I I A c c u m u l a t i o n o f 1 1 5 m - C a d m i u m i n k i d n e y s o f a n i m a l s g i v e n C a d m i u m o r C a d m i u m + Z i n c o n d a y 9 o f g e s t a t i o n , * = s i g n i f i c a n t l y d i f f e r e n t ( p < 0 . 0 5 ) f r o m d a y 1 0 . 0. 06 H CQ Z Q W W > o u w K cn o Q < z H c H o. c 0. 05 0. 04 0. 03 0. 02 0. 01 I ' • 1 1 L L 1 1 J I 10 11 12 13 14 15 16 17 18 19 20 21 DAY O F G E S T A T I O N F i g I I I Accumulation of 115m-Cadmium i n b r a i n s of animals g i v e n Cadmium or Cadmium+Zinc on day 9 of g e s t a t i o n , * = s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from day 12. C/3 < Z W PS Q D W w > o U W W w o Q t-3 * ! Z H o H o fa o OP 0.045 0.030 0.015 Cd Cd + Zn L//-J—L 10 11 1 1 J L L 1 1 1 I I 12 13 17 18 19 20 21 14 15 16 DAY OF GESTATION F i g . I V Accumulation of 115m-Cadmium i n a d r e n a l s of animals g i v e n Cadmium or Cadmium+Zinc on day 9 of g e s t a t i o n , * = s i g n i f i c a n t l y d i f f e r e n t (p<0.05) f r o m day 10. u> 74 In t h e a d r e n a l s the amount of cadmium decreased s i g n i f i c a n t l y with time: i n t h e cadmium t r e a t e d group. ( F i g u r e I V ) , however, i t appeared unchanged i n the cadmium-zinc group.. In the h e a r t , a l s o , the amount of cadmium decreased with time i n the cadmium t r e a t e d animals, where a s i g n i f i c a n t d i f f e r e n c e was found between day 10 and 20 of g e s t a t i o n , but no d i f f e r e n c e s were found i n the cadmium-zinc group (Figure V).. The amount of cadmium i n the lung i n c r e a s e d s l i g h t l y but not s t a t i s t i c a l l y s i g n i f i c a n t l y between day 10 and 11, and decreased s i g n i f i c a n t l y t h e r e a f t e r i n the cadmium t r e a t e d animals, however the changes i n the cadmium-zinc group were not s i g n i f i c a n t (Figure V I ) . In the sp l e e n no s i g n i f i c a n t change was found among days i n e i t h e r group, or between the two treatments (cadmium and cadmium-zinc), as i s shown i n F i g u r e VII. In the femur there was a s i g n i f i c a n t d i f f e r e n c e between day 10 and 20. The amount of cadmium de p o s i t e d i n the femur was l e s s on day 20 i n the cadmium t r e a t e d animals, whereas no d i f f e r e n c e s were found i n the cadmium-zinc group (Figure V I I I ) . . In the case of the u t e r u s , the a n a l y s i s compared onl y days, 10, 11, and 12 because the day 20 valu e s f o r t h i s organ were c a l c u l a t e d as percent of i n j e c t e d dose per gram of t i s s u e . N e v e r t h e l e s s , a s i g n i f i c a n t d i f f e r e n c e was found i n the cadmium group where the amount of cadmium d e p o s i t e d decreased with time. In the cadmium-zinc group, although the amount of cadmium i n c r e a s e d with time, the change was not s i g n i f i c a n t . . In a d d i t i o n , the amount of cadmium accumulated i n the uterus 24 hours p o s t - i n j e c t i o n was s i g n i f i c a n t l y g r e a t e r i n the cadmium ,y I I I I | | | | | I I | 10 11 12 13 14 15 16 17 18 19 20 21 DAY OF GESTATION Fi g . V Accumulation of 115m-Cadmium i n hearts of animals g i v e n Cadmium or Cadmium+Zinc on day 9 of g e s t a t i o n , * = s i g n i f i c a n t l y d i f f e r e n t (p<0 from day 10 . Cd + Zn = \,>> i i i I I i i I I I I I 10 11 12 13 14 15 16 17 18 19 20 21 DAY OF GESTATION F i g . V I A c c u m u l a t i o n of 115m-Cadmium i n lungs of a n i m a l s g i v e n Cadmium or Cadmium+Zinc on day 9 o f g e s t a t i o n , * = s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from day 10. 0 .40 z w w CA 0.35 0.30 0.25 0.20 0.15 0.10 > o CJ w <* w cn o Q Hi < Z H e> M « o fa o * 0.05 Cd Cd + Zn I i I i I I I I I I I I 10 11 13 18 19 20 21 14 15 16 17 DAY OF GESTATION F i g . V I I Accumulation of 115m-Cadmium i n spleens of animals given Cadmium or Cadmium+Zinc on day 9 of g e s t a t i o n . 0.100 H 0.075 0.050 0 .025 Cd = Cd + Zn = 1 1 1 1 1 J I L 1 J I 10 11 12 13 17 18 19 20 21 14 15 16 DAY OF GESTATION F i g . V I I I A c c u m u l a t i o n o f 115m-Cadmium i n femurs o f a n i m a l s g i v e n Cadmium o r Cadmium+Zinc on day 9 o f g e s t a t i o n , * = s i g n i f i c a n t l y d i f f e r e n t ijXO.05) from day 10. oo to w En D Q W > O u w CO o Q 25 o o OP L . G O 0 .90 0 .80 0 .70 0.60 0 .50 0 .40 3 0.30 0 .20 0 .10 r 1 10 11 12 DAY OF GESTATION Fig.IX A c c u m u l a t i o n of 115m-Cadmium i n u t e r u s of a n i m a l s g i v e n Cadmium or Cadmium+Zinc on day 9 of g e s t a t i o n , * = s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from day 10, ** = s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from c o r r e s p o n d i n g group w i t h Z i n c . 80 group than i n the cadmium-zinc group (Figure I X ) . . The t i s s u e c a l l e d "Uterus p l u s F e t u s " was analysed i n the same manner as the uterus and no d i f f e r e n c e s were found i n e i t h e r treatment group, comparing d i f f e r e n t days..However, as i n the case o f the uterus the amount of 115m-cadmium recovered was s i g n i f i c a n t l y g r e a t e r 24 hours p o s t - i n j e c t i o n i n the cadmium group than i n the cadmium-zinc group (Figure X) .. When the f e t u s e s were s t u d i e d , i t was found t h a t the amount of 115m-cadmium recovered was s i g n i f i c a n t l y decreased i n both groups with time. In a d d i t i o n , the amount of 115m-cadmium t h a t reached the f e t u s 24 hours p o s t - i n j e c t i o n was s i g n i f i c a n t l y g r e a t e r i n the cadmium group than i n the cadmium-zinc group (Figure X I ). The amount of 115m-cadmium found i n the blood was s i g n i f i c a n t l y g r e a t e r on day 11 or 12 than on day 20 i n the cadmium group but no d i f f e r e n c e s were found i n the cadmium-zinc group (Figure X I I ) . In the s m a l l i n t e s t i n e the amount of 115m-cadmium recovered decreased with time i n both groups..There was a s i g n i f i c a n t decrease i n the cadmium group between day 10 or 11 and 20; and between days 11 and 12* In the cadmium-zinc group the main d i f f e r e n c e was found between day 10 and 11 (Figure X I I I ) . . In f a t the amount of 115m-cadmium i n c r e a s e d i n the cadmium group between day 12 and 20, but the d i f f e r e n c e was not s i g n i f i c a n t . In the cadmium-zinc group no change was d e t e c t e d (Figure XIV). In the muscle a s i g n i f i c a n t difference.was found i n the cadmium group between day 11 and day 20, the amount of 115m-CO D E-i W fa W E3 Q W w > o CJ w cn O Q H ! M O M PH o fa o CAP 0.10 0.09 0.08 0.07 0 .06 0 .05 0 .04 0.03 0.02 0.01 0 ** I 1 I Cd = Cd + Zn = 10 11 12 DAY OF GESTATION F i g . X A c c u m u l a t i o n of 115m-Cadmium i n u t e r u s p l u s f e t u s of a n i m a l s g i v e n Cadmium or Cadmium+Zinc on day 9 o f g e s t a t i o n , ** = s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from c o r r e s p o n d i n g group w i t h Z i n c . oo 0.020 CO E-i W 10 11 12 13 14 15 16 17 18 19 20 21 DAY OF GESTATION Fi g . X I Accumulation of 115m-Cadmium i n f e t u s e s of animals given Cadmium or Cadmium+Zinc on day 9 of g e s t a t i o n , * = s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from day 10, ** = s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from corresponding group with Z i n c . 0 .050 Q O O t-H1 PQ W O H D W Ctf W > o u w cn o Q < H o H Pi o fa o OP 0.045 0.040 0.035 0.030 0.025 0.020 0.015 0.010 0.005 r~ Cd = Cd + Zn = J L 1 J I L 1 J L 1 J I 10 11 12 13 14 15 16 . 17 18 19 20 21 DAY OF GESTATION F i g . X I I A c c u m u l a t i o n o f 115m-Cadmium i n b l o o d of a n i m a l s g i v e n Cadmium o r Cadmium+Zinc on day 9 of g e s t a t i o n , * = s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from day 11 and 12. oo U) H o M o o dp 0.15 0.10 0.05 J I I L 1 J I L Cd Cd + Zn J L 10 11 12 13 17 18 19 20 21 14 15 16 DAY OF GESTATION F i g . X I I I Accumulation of 115m-Cadmium i n small i n t e s t i n e of animals g i v e n Cadmium or Cadmium+Zinc on day 9 of g e s t a t i o n , * = s i g n i f i c a n t l y d i f f e r e n t (p<0»0^) *^ from day 10, ** = s i g n i f f i c a n t l y d i f f e r e n t (p<0.05) from day 11. GO DAY OF GESTATION Fig.XIV A c c u m u l a t i o n of 115m-Cadmium i n f a t o f ani m a l s g i v e n Cadmium o r Cadmium+Zinc on day 9 of g e s t a t i o n , * = s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from c o r r e s p o n d i n g group w i t h Z i n c . 0.030 w u to D g Q W w > o u K W to O D S3 H o H o O dp 0.025 0 .020 0.015 0.010 0 .005 Cd = Cd + Zn = ** 1 1 1 1 J I I I I I 11 12 13 17 18 19 20 21 14 15 16 DAY OF GESTATION Fig.XV A c c u m u l a t i o n o f 115m-Cadmium i n muscles of a n i m a l s g i v e n Cadmium o r Cadmium+Zinc on day 9 of g e s t a t i o n , * = s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from day 11, ** = s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from day 12. oo CTl 87 cadmium being s m a l l e r on day 20. In the cadmium-zinc group a s i g n i f i c a n t d i f f e r e n c e was found between day 12 and day 20, again the amount o f 115m-cadmium being s m a l l e r on day 20 ( F i g u r e XV). In the plasma, as w e l l , the amount of 115m-cadmium decreased with time i n the cadmium group, being s i g n i f i c a n t l y l e s s on day 20 than on day 10 o f g e s t a t i o n . However, the changes i n the cadmium-zinc group were not s i g n i f i c a n t ( F i g u r e XVI) . The maternal hematocrit was a l s o analysed i n the same manner and i t was found t h a t there was a s i g n i f i c a n t decrease i n the maternal h e m a t o c r i t with time i n both groups ( F i g u r e XVII). CO < PH Q w P4 W > o CJ w P4 W cn O Q < 53 H a M P< o Cn O 0.007 0 .006 0.005 0.004 0.003 0 .002 0.001 Cd = Cd + Zn = J L 1 I I L J I I I 10 11 12 13 18 19 20 21 14 15 16 17 DAY OF GESTATION F i g . X V I A c c u m u l a t i o n o f 115m-Cadmium i n plasma o f a n i m a l s g i v e n Cadmium or Cadmium+Zinc on day 9 o f g e s t a t i o n , * = s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from day 10. oo 00 w EH H O EH 30 25 20 15 10 Cd = Cd + Zn = I I I I I I I I I I 1 1 10 11 12 13 17 18 19 20 21 14 15 16 DAY OF GESTATION F i g . X V II E f f e c t o f Cadmium o r Cadmium+Zinc i n t h e m a t e r n a l h e m a t o c r i t o f a n i m a l s i n j e c t e d on day 9 of g e s t a t i o n , * = s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from day 10. oa 90 CHAPTER V DISCUSSION The present r e s u l t s show t h a t cadmium causes gross e x t e r n a l f e t a l malformations when i t i s administered to pregnant Wistar r a t s on day 9 of gestation,. Even though a l l of these animals were 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 at approximately the same time (in the e a r l y hours of the 9th day of gestation) the f e t u s e s presented a wide range of malformations t h a t i n c l u d e d d e f e c t s o f the head, the neck, the abdominal w a l l * the limbs, the t a i l , and the whole body. Within a l i t t e r some f e t u s e s were s i g n i f i c a n t l y s m a l l e r than the r e s t of the l i t t e r m a t e s , which i n d i c a t e s d i f f e r e n t i a l e f f e c t s . I t i s not known whether these t o x i c m a n i f e s t a t i o n s are the r e s u l t of the a c t i o n of cadmium on the maternal t i s s u e s or on the p l a c e n t a , or whether they r e p r e s e n t a d i r e c t a c t i o n of cadmium on the f e t u s , or a combination of these f a c t o r s . Although the p l a c e n t a i s an e f f e c t i v e b a r r i e r a g a i n s t the t r a n s f e r o f s m a l l amounts ( l e s s than 4uM/kg body weight) of maternally a d m i n i s t e r e d cadmium ( B e r l i n and U l l b e r g , 1963) , l a r g e r (more than 10 uM/kg body weight) amounts w i l l enter the f e t u s (Ferm e t a l . , 1969). I t should be noted t h a t the l a t t e r (16uM/kg body weight) was the case i n these s t u d i e s . . The wide range of malformations seen i n these experiments may be e x p l a i n e d by a g e n e r a l hypotheses proposed by Ferm (1971), to account f o r the a c t i o n of s o - c a l l e d "broad-spectrum t e r a t o g e n s " . He suggested t h a t these t e r a t o g e n s are non-s p e c i f i c , t h a t i s to say, the r e s u l t depends p r i m a r i l y on the organogehetic event at the time of the i n s u l t . Thus, a wide 91 spectrum o f malformations should r e s u l t from any t e r a t o g e n i c st i m u l u s given d u r i n g the time of c r i t i c a l embryogenesis. T h e r e f o r e , these would be " n o n - s p e c i f i c " i n t h e i r e f f e c t s . I t was shown i n F i g u r e XI t h a t 115m-cadmium was present i n the f e t u s e s i n an a p p r e c i a b l e amount even 72 hours p o s t - i n j e c t i o n . T h i s c o u l d e x p l a i n t h a t i t was found a broad spectrum of d e f e c t s , even though only one i n j e c t i o n was administered t o these animals, cadmium was present i n the embryonic s t r u c t u r e s , at l e a s t , d u r i n g three important days of organogenesis, when the development of the r a t embryo goes from the h e a r t primodium to the p o s t e r i o r limb bud stage (Schneider,and Norton,1979)., Nonetheless, an a l t e r n a t i v e hypothesis (Ferm, 1971) s t a t e s t h a t c e r t a i n t e r a t o g e n s might w e l l prove to be s i t e s p e c i f i c and induce malformations only i n c e r t a i n developing organ systems. This mechanism ( s i t e - s p e c i f i c teratogens) would suggest t h a t a s p e c i f i c organ-teratogen r e l a t i o n s h i p e x i s t s which could best be e x p l a i n e d by i n t e r f e r e n c e with a p a r t i c u l a r enzymatic event of development. The metals should be good examples of s i t e - s p e c i f i c t e r a t o g e n s , f o r they e n t e r i n t o a v a r i e t y of r a t h e r s p e c i f i c enzymatic r e a c t i o n s (Ferm, 1971)., The f a c t t h a t , when the animals were examined w i t h i n a s i n g l e l i t t e r the same kind of malformation was found i n s e v e r a l f e t u s e s , may be evidence f o r the s p e c i f i c i t y of the t e r a t o g e n i c e f f e c t of cadmium. That i s t o say, t h a t when t h i n abdominal w a l l was the d e f e c t found i n the f i r s t f e t u s examined i n one p a r t i c u l a r l i t t e r ; t h a t was the predominant d e f e c t i n the whole l i t t e r . However t h i s may not be the case, s i n c e s p e c i f i c i t y can be e x p l a i n e d i n another way. 92 T h i s f a c t c o u l d b e t t e r be e x p l a i n e d by v a r i a t i o n i n the a c t u a l time of mating of the animals. For some animals the time of i n j e c t i o n , c o n s i d e r e d to be the e a r l y hours o f the 9th day of g e s t a t i o n , might a c t u a l l y have been the l a t e hours of the 8th day or the l a t e r hours of the 9th day of g e s t a t i o n . . T h e r e f o r e , the a c t u a l d e f e c t found c o u l d be the r e s u l t of the organogenetic event t h a t was t a k i n g p l a c e i n the:moment of i n j e c t i o n , and i t d i d not depend on the s p e c i f i c i t y of the t e r a t o g e n . , In a d d i t i o n * the maternal-embryonic r e l a t i o n s h i p may be d i s t u r b e d s i n c e i t was found t h a t 115m-cadmium accumulates i n the p l a c e n t a , embryonic sac, and the u m b i l i c a l cord. T h i s d e c i d u a l and p l a c e n t a l uptake may i n d i c a t e some i n t e r f e r e n c e with embryonic n u t r i t i o n and be the cause of s i g n i f i c a n t l y s m a l l e r f e t u s e s found i n the cadmium-treated group. T h i s i s supported by the f a c t t h a t some massively damaged p l a c e n t a s were found i n the cadmium-treated group. These p l a c e n t a s were s i m i l a r t o the ones t h a t P a r i z e k (1964) d e s c r i b e d as "...a c l o t o f blood". Nonetheless, the p l a c e n t a serves as an e f f i c i e n t b a r r i e r to the t r a n s f e r of cadmium from mother t o f e t u s , as evidenced by the r e l a t i v e l y high p l a c e n t a l and low f e t a l c o n c e n t r a t i o n s of cadmium found i n these experiments..An e x p l a n a t i o n f o r t h i s has been o f f e r e d by L u c i s e t a l . (197 2), who found t h a t cadmium i n the p l a c e n t a was bound to macromolecules, so t h a t the t r a n s f e r a c r o s s the p l a c e n t a i s r e s t r i c t e d or i n h i b i t e d . . The r e s u l t s of t h i s study, do not support the:hypotheses o f P a r i z e k (1964) and Dencker (1974) , t h a t cadmium i n t e r f e r e s with the s t e r o i d producing organs and t h a t t h i s may cause an 93 i n t e r r u p t i o n of pregnancy, s i n c e , t h e r e was no d i f f e r e n c e i n the mean number of r e s o r p t i o n s i t e s i n the 4 treatment groups. That i s to say, although cadmium does not cause an i n c r e a s e i n r e s o r p t i o n s i t may s t i l l a f f e c t the s t e r o i d producing organs, s i n c e i t has been mentioned above t h a t p l a c e n t a l damage was seen* However, as Dencker (1974) s t a t e d , a p o s s i b l e enzyme a f f i n i t y f o r cadmium may i n f l u e n c e the normal maternal-embryonic r e l a t i o n s h i p , d i s t u r b i n g metabolic pathways and r e s u l t i n g i n i n h i b i t e d i m p l a n t a t i o n , and an i n c r e a s e i n malformations. Thus, t e r a t o g e n i c e f f e c t s o f s p e c i f i c chemical agents i n mammalian development may be a t t r i b u t e d t o one:of three g e n e r a l a c t i o n s , o r perhaps t o a combination of them. F i r s t , t here may be an a l t e r a t i o n of some f a c t o r s i n the maternal system which s e c o n d a r i l y a f f e c t s embryonic d i f f e r e n t i a t i o n (Ferm e t a l . , 1969). As an example of t h i s p o s s i b i l i t y , the t e r a t o g e n i c i t y of cadmium might be due t o a l t e r e d p r o t e i n s t r u c t u r e , as r e p o r t e d by Kench and Sutherland (1966). In cases of human cadmium i n t o x i c a t i o n , i f the p r o t e i n s t r u c t u r e i s a l t e r e d i t i s p o s s i b l e t h a t the embryo w i l l show e x t e r n a l malformations* . Second, cadmium t e r a t o g e n i c i t y may be r e l a t e d to the b l o c k i n g of p l a c e n t a l t r a n s f e r of some e s s e n t i a l m a t e r i a l necessary f o r normal embryonic d i f f e r e n t i a t i o n . T h i r d , the t e r a t o g e n i c e f f e c t of cadmium may be due to a d i r e c t e f f e c t of t h i s metal upon s p e c i f i c embryonic t i s s u e s (Ferm et a l . , 1969).,. A review of the l i t e r a t u r e on the a c t i o n of cadmium on the r e p r o d u c t i v e process can only l e a d t o the c o n c l u s i o n t h a t while the e f f e c t i s c l e a r l y documented, the a c t u a l mechanism of a c t i o n 94 of cadmium i o n s i s very much an open g u e s t i o n . These i n v e s t i g a t i o n s have a l s o shown the p r o t e c t i v e e f f e c t of z i n c on cadmium-induced malformations* s i n c e : no e x t e r n a l d e f e c t s were found i n the cadmium-zinc t r e a t e d animals. However, the r e s u l t s do not agree with Ferm et a l . (1969) who r e p o r t e d t h a t the p l a c e n t a l t r a n s f e r of cadmium was not a f f e c t e d when z i n c was a d m i n i s t e r e d to pregnant animals together with cadmium. I t was shown here t h a t at 24 hours p o s t - i n j e c t i o n the amount o f cadmium i n the f e t u s e s of the cadmium-zinc t r e a t e d group was s i g n i f i c a n t l y l e s s than t h a t i n the cadmium t r e a t e d animals, even though t h i s d i f f e r e n c e was not s t a t i s t i c a l l y s i g n i f i c a n t 48 or 72 hours a f t e r i n j e c t i o n * However, the means by which z i n c p r o t e c t s a g a i n s t t e r a t o g e n e s i s cannot be r e l a t e d to a complete block i n the t r a n s f e r o f cadmium a c r o s s the p l a c e n t a s i n c e the present data shows t h a t the simultaneous i n j e c t i o n of an amount of z i n c equimolar to t h a t of cadmium does not completetely prevent the t r a n s f e r of cadmium from maternal t o embryonic t i s s u e s although the t e r a t o g e n i c e f f e c t of cadmium was a b o l i s h e d . I t i s p o s s i b l e t h a t z i n c r e p l a c e s cadmium at c e r t a i n embryonic s i t e s * . T h u s , although cadmium t r a n s f e r i s not impeded, those s i t e s s p e c i f i c a l l y r e l a t e d to the p r o d u c t i o n of a t e r a t o g e n i c l e s i o n by cadmium c o u l d be occupied by z i n c , cadmium being bound elsewhere i n the embryo a t n o n - s p e c i f i c s i t e s (Ferm et a l . , 1969) . In a d d i t i o n , as Ferm (1967) s t a t e d , the p r o t e c t i v e e f f e c t of z i n c c o u l d be r e l a t e d t o a c r i t i c a l p h y s i o l o g i c a l cadmium:zinc r a t i o . The importance of t h i s has been 9 5 demonstrated, f o r example, by Schroeder (1967), who has shown that high r e n a l r a t i o s of cadmium:zinc are a s s o c i a t e d with a r t e r i a l h y p e r t e n s i o n i n r a t s and t h a t t h i s hypertension can be reversed by z i n c * . F u r t h e r i n v e s t i g a t i o n s on the p e r m e a b i l i t y of the mammalian pla c e n t a to heavy metals and t h e i r l o c a l i z a t i o n i n s p e c i f i c d i f f e r e n t i a t i n g embryonic t i s s u e s are necessary i n order to i d e n t i f y the exact mechanisms of these s i t e s p e c i f i c malformations as w e l l as the complex i n t e r a c t i o n of these t e r a t o g e n i c agents with p r o t e c t i v e agents. When the d i s t r i b u t i o n of 115m-cadmium was examined i n the maternal organs, with and without the a d d i t i o n of z i n c , i t was found t h a t z i n c d i d not change the p a t t e r n s of cadmium d i s t r i b u t i o n except i n the case of the f a t , u t e r u s , s m a l l i n t e s t i n e , and femur. That i s t o say i n general the simultaneous a d m i n i s t r a t i o n of z i n c does not change the p a t t e r n of cadmium d i s t r i b u t i o n and accumulation i n r a t t i s s u e s . In the case o f f a t , s m a l l i n t e s t i n e , and femur the r e s u l t s may not be very meaningful s i n c e d i f f e r e n c e s were found i n only one group of animals f o r each of the t i s s u e s (Tables V, V I I , IX, XI, and XII) . I t i s i n t e r e s t i n g to n o t i c e t h a t f o r most of the organs examined the accumulation of 115m-cadmium decreased with time, but the accumulation of 115m-cadmium i n f a t i n c r e a s e d with time. T h e r e f o r e , i t may be t h a t cadmium i s to some extent not e x c r e t e d from the system, but i s deposited i n f a t t y t i s s u e . The a d d i t i o n of z i n c seems to decrease s i g n i f i c a n t l y the amount of cadmium accumulated i n f a t t y t i s s u e , however i t appears t h a t z i n c does 96 not change the accumulation o f cadmium i n most of the organs examined, so t h a t cadmium may be excreted out of the system. . The a d d i t i o n of z i n c a l s o decreases the amount of 115m-cadmium d e p o s i t e d i n the uterus, and t h i s may be the reason why the amount of cadmium t h a t reaches the f e t u s e s i s s m a l l e r i n the cadmium-zinc group than i n the cadmium group. On the other hand, when the other two groups, ( z i n c and zinc-cadmium) were examined the o p p o s i t e s i t u a t i o n was found, the a d d i t i o n of cadmium seems to i n c r e a s e the: amount o f z i n c deposited i n most organs. T h i s c o u l d be e x p l a i n e d by c o m p e t i t i o n of z i n c and cadmium f o r s i m i l a r b i n d i n g s i t e s i n t i s s u e , which i s w e l l e s t a b l i s h e d . I t i s a l s o known t h a t cadmium e x e r t s t o x i c e f f e c t s v i a i t s e f f e c t on z i n c metalloenzymes (Horvath, 1976), and t h a t high l e v e l s of z i n c prevent or reduce the t o x i c e f f e c t s of cadmium ( F l i c k e t a l . , 1971) . In a d d i t i o n , i t has been shown t h a t * i n the l i v e r and kidney, endogenous z i n c i s i n c r e a s e d by the i n j e c t i o n of cadmium (Sugawara et a l . , 1978). T h i s i n c r e a s e might be due to the i n d u c t i o n of m e t a l l o t h i o n e i n s y n t h e s i s i n both organs (Suzuki, and Yoshikawa, 1972). The o r i g i n of t h i s i n c r e a s e d z i n c i s s t i l l under d i s c u s s i o n , but Winge et a l . , (1975) a t t r i b u t e d the accumulation of z i n c , i n p a r t , to d e p l e t i o n of blood z i n c . However, the present study does not agree with t h a t e x p l a n a t i o n s i n c e the amount of 65-zinc found i n blood was s l i g h t l y but not s i g n i f i c a n t l y i n c r e a s e d by the a d d i t i o n of cadmium* Sugawara et a l . (1978) suggested t h a t the r a p i d accumulation of organ z i n c f o l l o w i n g the a d m i n i s t r a t i o n of 97 cadmium may be not due t o uptake of d i e t a r y z i n c but to a r e d i s t r i b u t i o n of exchangeable z i n c from other organs. T h i s e x p l a n a t i o n a l s o seems d i f f i c u l t t o understand s i n c e , i n the present study the accumulation of a d m i n i s t e r e d 65-zinc s i g n i f i c a n t l y . increased i n most of the organs s t u d i e d . . Therefore, at the present moment the mechanism by which the accumulation of z i n c i n c r e a s e s i n the presence.of cadmium seems to be not very w e l l understood. The data presented i n t h i s study r e v e a l t h a t a f t e r i n t r a p e r i t o n e a l i n j e c t i o n , 115m-cadmium i s predominantly c o n c e n t r a t e d i n the l i v e r , presumably bound to m e t a l l o t h i o n e i n , ( L u c i s et a l . , 1972; Webb, 1975; K o t s o n i s and Klaassen, 1975).. T h i s p a t t e r n does not change by 24, 48, 72 hours, or 11 days a f t e r i n j e c t i o n . A f t e r 11 days most t i s s u e s showed a decrease even though not s i g n i f i c a n t i n t h e i r content of 115m-cadmium. The e x c e p t i o n to t h i s were kidney and f a t , which had an i n c r e a s e ; the s p l e e n which remained the same, and the s m a l l i n t e s t i n e which had a s i g n i f i c a n t decrease. , I t i s of i n t e r e s t t o note t h a t the 115m-cadmium content o f the plasma was very low, which i n d i c a t e s t h a t the c o n c e n t r a t i o n of 115m-cadmium found i n whole, blood was i n the c e l l s ( e r y t h r o c y t e s ) presumably bound t o a p r o t e i n l i k e hemoglobin (Carson and F r i b e r g , 1975; Nordberg e t a l . , 1971). This a l s o agrees with the r e s u l t s of L u c i s et a l . (1972) who s t a t e d t h a t i n experimental animals cadmium i s r a p i d l y c l e a r e d from the c i r c u l a t i o n and deposited i n t r a c e l l u l a r l y so t h a t o n l y t r a c e s o f cadmium remain i n blood plasma or serum. T h i s may be one of the f a c t o r s which p l a y s a r o l e i n the low t r a n s p l a c e n t a l passage of 98 t h i s element, together with the f a c t t h a t the r a t p l a c e n t a s a l s o c o n t a i n i n t r a c e l l u l a r cadmium-binding macromolecules ( L u c i s e t a l . , 1972). The b r a i n had a lower 115m-cadmium c o n c e n t r a t i o n than d i d the plasma, p o s s i b l y due t o the b l o o d - b r a i n b a r r i e r . The high c o n c e n t r a t i o n of cadmium i n the i n t e s t i n e 48 hours p o s t -i n j e c t i o n , and the dramatic decrease a f t e r 11 days may be e x p l a i n e d by the presence of an i n t e s t i n a l m e t a l l o t h i o n e i n which i s subsequently l o s t d u r i n g the normal turnover of i n t e s t i n a l e p i t h e l i a l c e l l s (Richards, and Cousins, 1975; Kotsonis and Klaassen, 1977). F i n a l l y , s e v e r a l i s s u e s i l l u s t r a t e d by these experimental data need t o be c o n s i d e r e d . F i r s t , as P e t e r i n g (1978) s t a t e d , we need t o be more concerned about the n u t r i t i o n a l f a c t o r s which may play a r o l e i n the host response to i n g e s t i o n of heavy metals, as they may be extremely important, both i n experimental and c l i n i c a l e v a l u a t i o n of the t o x i c i t y of cadmium and o t h e r t o x i c metals. I t has been suggested t h a t the acute i n g e s t i o n of cadmium may l e a d t o a l t e r a t i o n s of z i n c metabolism. T h i s i s an important i s s u e , s i n c e i t b r i n g s i n t o f o c u s the importance of a good n u t r i t i o n a l s t a t u s as a host defense mechanism; I t a l s o i n d i c a t e s a p o s s i b l e important p r e v e n t i v e measure: which needs f u r t h e r study, s i n c e cadmium exposure w i l l be with us f o r a long time ( P e t e r i n g , 1978). In a d d i t i o n , even though the l e v e l s of the two metals used i n these experiments exceed the u s u a l l e v e l s of environmental contamination, two important p o i n t s should be kept i n mind. The 99 f i r s t i s t h a t t h e r e i s always the p o s s i b i l i t y of a c c i d e n t a l exposure to high l e v e l s of one of these metals d u r i n g e a r l y human g e s t a t i o n . Secondly, t h e r e a l s o may be marked s p e c i e s v a r i a t i o n i n the t e r a t o g e n i c response t o a given t e r a t o g e n , the example of t h a l i d o m i d e being a case i n p o i n t * T h i s compound, i n r e l a t i v e l y low t h e r a p e u t i c doses, caused s e v e r a l malformations i n the human embryos, but i t took extremely high l e v e l s of the same compound to produce malformations i n experimental animals. Therefore, i t i s p o s s i b l e t h a t a s i m i l a r s i t u a t i o n may e x i s t , and u n t i l i n d i v i d u a l s p e c i e s s e n s i t i v i t y t o v a r i o u s teratogens i s determined a l l p o t e n t i a l t eratogens should be c a r e f u l l y e v a l u a t e d (Holmberg, and Ferm, 1969). 100 BIB LIOGEAPHY A l s b e r , C. L., and Schwartze, E..W. Pharmacological a c t i o n of cadmium. J . Pharmacol. _Exp. Ther*.13:504, 1919.. Anke, Mi, and Schneider, H. J . . 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