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Cytotoxic responses to aflatoxins on fertilized oyster eggs, Crassostrea gigas Park, Hyun Chul 1978

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CYTOTOXIC RESPONSES TO AFLATOXINS ON FERTILIZED OYSTER EGGS, CRASSOSTREA GIGAS by HYUN CHULLP/ARK B. Sc., Seoul N a t i o n a l U n i v e r s i t y , 1963 M. Sc., U n i v e r s i t y o f Oklahoma, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF. MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (THE DEPARTMENT OF FOOD SCIENCE) We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA February, 1978 (cT) HYUN CHUL PARK, 1978 In presenting t h i s t h e s i s in p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the 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 or reference and study. I f u r t h e r agree that permission for extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s re p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of Food S c i e n c e The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date Feb. 20. 1978 - i i -ABSTRACTS Aflatoxins B.^ B^, and G^, and a crude preparation of f r e s h l y prepared a f l a t o x i n produced by Aspergillus parasiticus NRRL 2999 were added to the f e r t i l i z e d oyster, Crassostrea gigas, eggs. The addition of 2, 3, 4 and 5 ug/ml of a f l a t o x i n i n h i -bited the f i r s t c e l l d i v i s i o n of f e r t i l i z e d oyster eggs by 10, 30, 70 and 90 percent, respectively. F i r s t c e l l d i v i s i o n did not appear to be i n h i b i t e d by 5 ug/ml of either aflatoxins B 2, G 1 or G 2. A change i n the f i n e structure of the f e r t i l i z e d oyster eggs was observed following the addition of 5 ug/ml of a f l a t o x i n B^. V i s i b l e changes or a l t e r a t i o n s i n the a f l a t o x i n treated zygotes were not r e a d i l y d e t e c t i b l e i n the electron micrographs except fo r the segregation of the f i b r i l l a r and granular components of the nucleolus, an apparent decrease of the v e s i c l e s surrounding the c i s t e r n a l rough endoplasmic r e t i -culum, and the f i n a l i n h i b i t i o n of the m i t o t i c d i v i s i o n process before f i r s t c e l l d i v i s i o n . The data i s consistant with the observed cytotoxic e f f e c t s suggesting an i n h i b i t i o n at the c e l l DNA r e p l i c a t i o n , RNA t r a n s c r i p t i o n and protein t r a n s l a t i o n a l l e v e l s i n the a f l a t o x i n B, treated oyster zygotes. - i i i -TABLE OF CONTENTS Page TITLE . i ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES i v LIST OF ILLUSTRATIONS V ACKNOWLEDGEMENTS v i INTRODUCTION 1 MATERIALS AND METHODS 13 CULTURES 13 CULTURAL METHODS AND MEDIA FOR AFLATOXIN PRODUCTION. . 13 EXTRACTION OF AFLATOXINS AND STANDARD AFLATOXINS . . . 14 SEPARATION 0F AFLATOXINS BY THIN LAYER CHROMATOGRAPHY (TLC) AND THEIR QUANTITATIVE DETERMINATION 15 SPECTROPHOTOMETRIC METHOD FOR DETERMINATION OF AFLATOXIN 16 BIOASSAY OF AFLATOXINS 17 SPAWNING OF OYSTER 17 APPLICATION OF AFLATOXIN TO FERTILIZED EGGS 18 PREPARATION OF THE ELECTRON MICROGRAPHS 18 RESULTS 22 AFLATOXIN BIOASSAY WITH B. MEGATERIUM 22 BIOLOGICAL EFFECTS OF AFLATOXINS ON FERTILIZED EGGS. . 24 ELECTRON MICROGRAPHIC OBSERVATIONS 26 DISCUSSION 48 BIBLIOGRAPHY . . . 57 - i v -LIST OF TABLES Tables page 1. The components of the embedding p l a s t i c mixture . 20 2. Inh i b i t i o n of c e l l d i v i s i o n i n the a f l a t o x i n B., treated oyster.zygote 25 -v-LIST OF ILLUSTRATIONS Figures Page 1. Structures of aflatoxins 3 2. Suggested mechanism for metabolic a c t i v a t i o n of a f l a t o x i n with nucleic acid i n rat l i v e r . . . 9 3. The s e n s i t i v i t y of B. megaterium to a f l a t o x i n B^, B 2, G 1 and G 2 23 4. The s e n s i t i v i t y of B. megaterium to a f l a t o x i n B^. 23 5. The comparison of the s e n s i t i v i t y between pure af l a t o x i n B^ and a crude af l a t o x i n 23 6. U n f e r t i l i z e d oocyte of oyster, Crassostrea gigas. 27 7. U n f e r t i l i z e d oocyte of oyster 28 8. Two spermatozoa confined to the perpheral aspects of the oyster zygote 30 9. A spermatozoon just p r i o r to incorporation. . . . 30 10. Dispersion of the t i g h t l y packed chromatin of a incorporated spermatozoon 32 11. Vesiculation of germinal v e s i c l e 32 12. Seven nuclei of f a i r l y uniform size and structure i n a polyspermic zygote 33 13. Cisternaes of rough endoplasmic reticulum . . . . 35 14. Incorporation of small v e s i c l e or vacuole to plasma membrane 35 15. Furrow formation and the two c e l l stage, and the four c e l l stage 36 16. Dispersion of chromatin materials throughout nucleus 36 17. Irregular shape of nucleus at the early stage of nuclear formation 37 18. Rough endoplasmic reticulum 37 19. Furrow membrane formation and c o n t r a c t i l e . . . . 38 - v i -F i g u r e s page 20. M u l t i c e l l u l a r stage 40 21. I r r e g u l a r shape o f n u c l e o l i (chromatin d i s p e r s i o n throughout nucleus) . . . . . 40 22. C i s t e r n a e form o f RER and v e s i c l e form o f RER surrounding c i s t e r n a e s . 41 23. C i s t e r n a e and v e s i c l e forms of rough endoplasmic r e t i c u l u m 42 24. M u l t i n u c l e i i n the a f l a t o x i n B. t r e a t e d o y s t e r zygote . . 43 25. N u c l e o l a r s e g r e g a t i o n i n the a f l a t o x i n B. t r e a t e d zygote 43 26 N u c l e o l a r s e g r e g a t i o n t o f i b r i l l a r and g r a n u l a r components. . 44 27. C i s t e r n a e form o f rough endoplasmic r e t i c u l u m . i n the a f l a t o x i n B^ t r e a t e d zygote 45 28. Ribosomes on the rough endoplasmic r e t i c u l u m . . . 46 29. General scheme o f c l a s s I f e r t i l i z a t i o n . . . . . 49 - v i i -ACKNOWLEDGEMENTS I wish to express ray sincere appreciation to Dr. P. M. townsley, whose d i r e c t i o n , advice and many hours of discussion as an educator, coupled with his l i m i t l e s s enthusiasm and imagination, provided the impetus to complete t h i s work, and whose companionship and friendship over the years proved to be invaluable. Sincere thanks are also extended to Drs. W. D. Powrie, S. Nakai and B. J . Skura, not only f o r c r i t i c i s m i n the preparation of t h i s manuscrip, but also for t h e i r help which they gave on more than one occasion. Spe c i a l l y , to my wife, Soon Hee, and my daughter, Anne Misun, loving thanks for t h e i r patience, encourage, and devotion. -1-CYTOTOXIC RESPONSES TO AFLATOXINS ON FERTILIZED OYSTER EGGS, CRASSOSTREA GIGAS INTRODUCTION In 1960, p o u l t r y farms i n England s u f f e r e d from severe l o s s e s of young t u r k e y s (more than 100,000) i n the course o f a few months. T h i s unknown d i s e a s e was t e n t a t i v e l y c a l l e d Turkey X d i s e a s e (Blount, 1961) .„ A s i m i l a r type o f d i s e a s e was a l s o r e p o r t e d i n d u c k l i n g s (Aisplin and Carnaghan, 1961), p i g s and c a t t l e (Loosmore and Harding, 1961; Loosmore and Markson, 1961). Common f a c t o r s i n t h i s d i s e a s e were found t o be the p r e -sence i n the feed of m a t e r i a l from B r a z i l i a n groundnut meal and were i d e n t i f i e d as m e t a b o l i t e s o f the common fungus contaminant, A s p e r g i l l u s f l a v u s . Since then the t o x i c f a c t o r s have been termed c o l l e c t i v e l y " a f l a t o x i n " t o i n d i c a t e t h e i r o r i g i n . The organisms which produce the a f l a t o x i n are mainly A s p e r g i l l u s oryzae, A. f l a v u s , A. p a r a s i t i c u s • A l s o s t r a i n s o f A. n i g e r , A. w e n t i i , A. ruber, A. ochraceous, P e n i c i l l i u m  puberulum, P. citrnum, P. v a r i b l e , P. frequentans have been r e p o r t e d to produce a f l a t o x i n , but t h e r e i s some c o n t r o v e r s y on the i d e n t i f i c a t i o n of a f l a t o x i n i n c u l t u r e e x t r a c t s . These -2-organisms are commonly found i n a i r and s o i l , and are ab l e t o grow on a wide v a r i e t y o f a g r i c u l t u r a l commodities such as pea-nuts, peanut-butter, u n r e f i n e d o i l , c o r n , wheat, cottonseed, sweet p o t a t o , cassava, r i c e , soybean, e t c . . A l l o f the organisms s t u d i e d g e n e r a l l y produce a mixture o f forms which belong t o the a f l a t o x i n f a m i l y . N e s b i t t e t a l . (1962) i s o l a t e d two f l u o r e s c e n t spots on alumina chromatoplates when developed w i t h c h l o r o f o r m : methanol (98.5 : 1.5) under u l t r a v i o l e t l i g h t ; one, a blue v i o l e t f l u o -r e s c e n t s p o t , w i t h an o f 0.6 and the o t h e r , a green f l u o r e s -cent spot, o f lower R^. They d e s i g n a t e d " a f l a t o x i n B" f o r the blue v i o l e t spot and " a f l a t o x i n G" f o r the green spot. A year l a t e r H a r t l e y e t al_. (1963) separated f o u r c l o s e l y r e l a t e d com-pounds from a mixture o f a f l a t o x i n s on a s i l i c a g e l t h i n l a y e r chromatoplate u s i n g c h l o r o f o r m : methanol (98 : 2) as the deve-l o p i n g s o l v e n t and de s i g n a t e d the f l u o r e s c e n t spots as B^, B 2, G^, G 2 i n or d e r o f d e c r e a s i n g R^ v a l u e s . The mol e c u l a r formulae of f o u r a f l a t o x i n s have been determined as c i 7 H i 2 ° 6 ' C 1 7 H 1 4 ° 6 ' C 1 7 H 1 2 ° 7 a n d C 1 7 H 1 4 ° 7 f o r B l ' B 2 ' G l a n d G 2 r e s P e c t i v e l v • B 2 and G 2 were e s t a b l i s h e d t o be the r e s p e c t i v e 2 , 3 - d i h y d r o - d e r i v a -t i v e s o f B^ and G^. Asao e t a l . (1965) determined the s t r u c t u r e s o f the a f l a t o x i n s (Figure 1 ) . These f o u r t o x i n s c o n s i t i t u t e - the important t o x i n s o f the a f l a t o x i n f a m i l y . A f l a t o x i n B^ i n p a r t i c u l a r i s u s u a l l y p r e s e n t i n l a r g e amounts and i s h i g h l y t o x i c . In a d d i t i o n t o these compounds, a f l a t o x i n B 2 a and G 2 a have been i s o l a t e d and i d e n t i f i e d as the 2-hydroxy d e r i v a t i v e s o f a f l a t o x i n B 2 and G 2, r e s p e c t i v e l y (Dutton and Heathcote 1966). - 4 -The m i l k t o x i n , which was d e s i g n a t e d as " a f l a t o x i n M", was found i n m i l k o f cows f e d w i t h groundnut meals c o n t a i n i n g a f l a t o x i n s ( A l l c r o f t and Carnaghan 1 9 6 2 ; 1 9 6 3 ) . T h i s t o x i n was a l s o i s o l a t e d from A s p e r g i l l u s f l a v u s growth medium and i d e n t i -f i e d as a m e t a b o l i t e of a f l a t o x i n i n s e v e r a l animal s p e c i e s , such as sheep, r a t and human. H o l z a p f e l e t a l . ( 1 9 6 6 ) separated two components, a f l a t o x i n s and M 2, from the m i l k t o x i n . A f l a t o x i n was found t o be h y d r o x y l a t e d a f l a t o x i n B^. The chemical s t r u c t u r e s of these a f l a t o x i n s are shown i n F i g u r e 1 . The b i o l o g i c a l response to the a f l a t o x i n s has been observed to be one of t o x i c i t y , c a r c i n o g e n i c i t y , t e r a t o g e n i c i t y , or m u t a g e n i c i t y (Ong, 1 9 7 5 ) . The t o x i c i t y of a f l a t o x i n s has been s t u d i e d i n d u c k l i n g s , r a t s , rainbow t r o u t , hamsters, guinea p i g s , r a b b i t s , dogs, e t c . . The r e l a t i v e t o x i c i t y of v a r i o u s a f l a t o x i n s has been s t u d i e d w i t h one day o l d d u c k l i n g s (Brown, 1 9 6 9 ; Carnghan e t a l . , 1 9 6 3 ; Townsend, 1 9 6 7 ) . The o r a l 7 day L D ^ Q v a l u e s on a 5 0 g body weight male d u c k l i n g were 1 8 . 2 ug f o r B^; 8 4 . 4 ug f o r B 2; 3 9 . 2 ug f o r G^- 1 8 2 . 5 ug f o r G 2; 1 6 . 6 ug f o r M 1; 6 2 . 0 ug f o r M 2; l e s s than 1 2 0 0 . 0 ug f o r B 2 a ; and l e s s than 1 6 0 0 . 0 ug f o r G 2 a » G e n e r a l l y , a f l a t o x i n s were found t o be more t o x i c to young animals than to the o l d . A f l a t o x i n B^ i s c o n s i d e r e d to be the most abundant and the most t o x i c . The t o x i c i t y decreases i n the o r d e r o f G^, B 2, and G 2. The t o x i c i t y of a f l a t o x i n B^ was observed i n many animal s p e c i e s . The s i n g l e o r a l LE>^Q o f a f l a t o x i n B^ f o r one day o l d d u c k l i n g s i s 0 . 3 7 - 0 . 5 7 mg/kg, f o r male r a t s 1 . 0 mg/kg, f o r rainbow t r o u t 1 . 5 mg/kg, f o r 5 kg apes 3 . 7 mg/kg, f o r 2 1 day -5-o l d male r a t s 5.5 mg/kg, f o r 21 day o l d female r a t s 7.2 mg/kg, f o r 30 day o l d male hamsters 10.2 mg/kg (Waart e t a l . , 1974). The t o x i c i t y o f a f l a t o x i n has a l s o been found i n t i s s u e c u l -t u r e systems, such as, human embryonic c e l l s (Legator e t a l . , 1965), Chang l i v e r c e l l s , duck embryo primary c u l t u r e d c e l l s , HeLa c e l l s ( G a b l i k s e t a l . , 1965), and human embryo l i v e r c e l l s (Sullman e t a l . , 1970). The c a r c i n o g e n i c a c t i v i t i e s o f a f l a t o x i n have been s t u d i e d i n many, animals such as i n r a t s , d u c k l i n g s , rainbow t r o u t , mice and rhesus monkeys. When the r a t s were i n j e c t e d t w i ce weekly w i t h doses o f 50 ug and 500 ug o f a mixture o f B^ and a t each i n j e c t i o n f o r 60 and 8 weeks, r e s p e c t i v e l y , the animals developed sarcomas o r fibrosarcomas a t the i n j e c t i o n s i t e (Dickens and Jones, 1964). A l s o h e p a t o c a r c i n o g e n i c i t y o f a f l a t o x i n s i n r a t s was demonstrated a f t e r b e i n g f e d r e p e a t l y w i t h contaminated meals and d i e t s . A d i e t a r y l e v e l as low as 15 ppb was r e p o r t e d t o induce hepatocarcinomas i n a l l 12 male r a t s a f t e r 68 weeks and i n a l l 13 female r a t s a f t e r 80 weeks (Wogan and Newberne, 1967). In the study of the c a r c i n o g e n i c a c t i v i t y o f a f l a t o x i n i n ducks, a d i e t a r y l e v e l o f 30 ppb o f a f l a t o x i n was r e p o r t e d t o develop h e p a t i c tumors i n 14 months (Carnaghan, 1965). In the rainbow t r o u t , 96 per c e n t o f t h e t r o u t developed hepatoma when the animals were f e d f o r 20 months on a d i e t c o n t a i n i n g 20 ppb a f l a t o x i n (Halver, 1969). A l e v e l o f 0.005 ppb was i n e f f e c t i v e i n i n d u c i n g hepatoma; 0.1 ppb induced 10 percent tumor i n c i d e n c e . As s t a t e d , a f l a t o x i n has shown h i g h l e v e l s o f c a r c i n o g e n i c a c t i v i t y when ad m i n i s t e r e d i n low concen--6-t r a t i o n s and has been suggested t o be one o f the most potent hepatocarcinogens found i n the environment. F i n e s t r u c t u r a l changes a t the c e l l u l a r l e v e l have been r e p o r t e d when acute c o n c e n t r a t i o n s o f a f l a t o x i n are i n t r o -duced t o v a r i o u s c e l l s and animals ( B u t l e r , 1966; F l o y d e t a l . , 1968; Pong and Wogan, 1970; Rae e t a l . , 1974; Reynier e t a l . , 1975; Svoboda e t a l . , 1966; Unuma e t a l . , 1967). For example, n u c l e o l a r m a t e r i a l was segregated i n t o f i b r i l l a r and g r a n u l a r components; n u c l e a r i n t e r c h r o m a t i n granule numbers were i n c r e a s e d , and the d i s s o c i a t i o n o f ribosomes from rough endoplasmic r e t i c u l u m (RER) was observed. S i m i l a r l y , an i n c r e a s e i n the amount o f smooth endoplasmic r e t i c u l u m (SER) was e v i d e n t , and t h i s was accompanied by c e n t r o l o b u l a r m i t o c h o n d r i a l s w e l l i n g , an i n c r e a s e i n the number of lysosomes, a l o s s o f glycogen, and an i n c r e a s e i n f a t d r o p l e t s . As a consequence o f the a d m i n i s t r a t i o n o f a f l a t o x i n t o an animal, the t o x i n expressed i t s e l f as a ve r y potent c a r c i -nogen. However, i t i s not c l e a r whether the parent t o x i n or a compound d e r i v e d from the a d m i n i s t e r e d t o x i n i s the a c t i v e form f o r the c a r c i n o g e n e s i s response. Goodal and B u t l e r (1969) suggested a m e t a b o l i t e o f the ad m i n i s t e r e d a f l a t o x i n was respon-s i b l e f o r the c a r c i n o g e n i c i t y . Garner e t a_l. (1971) demonstrated t h a t s t r a i n s TA 1530 and TA 1531, but not s t r a i n s C 207 and G 46, of Salmonella typhimurium were k i l l e d when incubated w i t h r a t l i v e r homogenate and a f l a t o x i n B^ but not w i t h e i t h e r component a l o n e . They i n s i s t e d t h a t the l e t h a l e f f e c t r e q u i r e d the micro-somal f r a c t i o n o f the l i v e r and i t was dependent on oxygen and -7-NADPH. S t r a i n s TA 1530 and TA 1531 of the b a c t e r i a are h i s t i d i n e a u totrophs, w i t h a major d e l e t i o n . G 46 i s a h i s t i d i n e r e q u i r i n g base p a i r s u b s t i t u t i o n mutant whereas C 207 i s a h i s t i d i n e r e q u i -r i n g f r a m e s h i f t mutant. In f u r t h e r s t u d i e s c a r r i e d out by Garner et a l . (1972), l i v e r homogenates from r a t , guinea p i g , mouse, hamster and human c o u l d a l l c o n v e r t a f l a t o x i n t o a t o x i c m e t a b o l i t e f o r TA 1530 o f S. typhimurium. T o x i c m e t a b o l i t e s f o r TA 1530 were a l s o formed i f a f l a t o x i n B^ was r e p l a c e d by e i t h e r a f l a t o x i n G^ o r s t e r i g m a t o c y s t i n i n the microsomal mediated t o x i c i t y assay. From these r e s u l t s they suggested t h a t the 2-3 double bond of the a f l a t o x i n s was c r u c i a l f o r m e t a b o l i c a c t i v a t i o n and 2-3 epoxide of a f l a t o x i n B^ was the a c t i v e m e t a b o l i t e of the a f l a t o x i n . A m o l e c u l a r o r b i t a l c a l c u l a t i o n of a f l a t o x i n B^ i n d i c a t e d t h a t the 2-3 pi-bond has the h i g h e s t bond order o f the a f l a t o x i n molecule. Thus the molecule should be s u s c e p t i b l e t o e l e c t r o p h i l i c a t t a c k and i s the most probable l o c a t i o n of the K r e g i o n * The 2-3 pi-bond, t h e r e f o r e , i s c o n s i d e r e d t o be the most r e a c t i v e bond w i t h i n the a f l a t o x i n molecule (Heathcote and H i b b e r t , 1974). Recently, Garner and Wright (1974) and Swenson et a l . (1973; 1974; 1975; 1977) have pr o v i d e d chemical evidence t h a t a f l a t o x i n 2-3 oxide was most probably m e t a b o l i c a l l y d e r i v e d from a f l a t o x i n i n r a t l i v e r i n v i v o , and from human and hamster l i v e r microsomes i n v i t r o . M i l d a c i d h y d r o l y s i s o f the c o v a l e n t l y bound B^ m e t a b o l i t e w i t h DNA and ribosome RNA y i e l d e d a compound i n d i s t i n g u i s h a b l e from 2-3 dihydro-2,3-dihydroxy a f l a t o x i n B,. - 8 -These r e s u l t s s t r o n g l y suggested t h a t the c o v a l e n t l y bound forms o f a f l a t o x i n r e s u l t e d from r e a c t i o n s o f the m e t a b o l i c a l l y generated e l e c t r o p h i l e a f l a t o x i n B^-2,3-oxide w i t h the nucleo-p h i l i c n i t r o g e n and oxygen atoms i n the DNA and RNA, c o n t r i b u t e d probably l a r g e l y by the guanine r e s i d u e s . The mechanism by which a f l a t o x i n s r e a c t w i t h DNA, RNA and p r o t e i n has been proposed by Garner and Wright (1974) and Swenson e t a l . (1974; 1975; 1977) (Figure 2). Garner (1975) r e p o r t e d t h a t the amount o f a f l a t o x i n B^ b i n d i n g to n u c l e i c a c i d s was f a r g r e a t e r than t h a t to p r o t e i n . Rat l i v e r DNA bound ten times and r-RNA twenty times more t o x i n than p r o t e i n . There were a l s o d i f f e r e n c e s i n the amount of the t o x i n bound t o r a t l i v e r n u c l e i c a c i d s as compared t o those of the hamster, the l a t t e r s p e c i e s b i n d i n g lower amount of a f l a t o x i n B^. The r e a c -t i o n of a f l a t o x i n w i t h n u c l e i c a c i d s c o u l d i n h i b i t DNA s y n t h e s i s ( r e p l i c a t i o n ) , v R N A s y n t h e s i s ( t r a n s c r i p t i o n ) and p r o t e i n synthe-s i s ( t r a n s l a t i o n ) . De Recondo e t a l . (1966) r e p o r t e d t h a t a f l a -t o x i n B^ a t 100 u g / r a t i n h i b i t e d thymidine i n c o r p o r a t i o n i n t o l i v e r DNA by 95 p e r c e n t w i t h i n 12 hours. I t was demonstrated t h a t the enzymes r e s p o n s i b l e f o r DNA s y n t h e s i s were not a f f e c t e d by the t o x i n . From these data i t was p o s t u l a t e d t h a t a f l a t o x i n B^ a f f e c t e d DNA s y n t h e s i s by impairment o f the a b i l i t y of DNA to a c t as a template. T h i s i n h i b i t i o n o f DNA s y n t h e s i s suppressed m i t o s i s i n E s c h e r i c h e a c o l i (Wragg e t a l . , 1966). A f l a t o x i n s a l s o i n t e r f e r e w i t h RNA metabolism. Sporn e t a_l. (1966) r e p o r t e d t h a t a f l a t o x i n B^ binds t o DNA i n v i t r o , and t h a t , i n v i v o , a f l a t o x i n B 1 r a p i d l y i n h i b i t s i n c o r p o -/ 2-3 OXIDE 2 \ * 0 < Liver a. in vivo b. microsome +• NADPH + 02 AFLATOXIN By / DNA r-RNA \ HO. DNA" or \ r-RNA DI HYDRODIOL mild acid HO hydrolysis HO-NUCLEIC ACID ADDUCTS vPr-NH2 HO. 0 ^ HQ- 0 OH HCK H® Pr HN^ i Pr H 0HV Pr-NH2 0 o-F i g u r e 2. PROTEIN ADDUCTS (SCHIFF BASES) a c i d i n r a t l i v e r . Pr : p r o t e i n . i Suggested mechanism f o r metabolic a c t i v a t i o n of a f l a t o x i n w i t h n u c l e i c -10-3 r a t i o n of cy t i d i n e H into r a t l i v e r nuclear RNA and lowers the r a t i o of nuclear RNA to DNA. Friedman and Wogan (1966; 1967) studied the time course and dose response c h a r a c t e r i s t i c s of af l a t o x i n e f f e c t s on nuclear RNA metabolism. They found that a single L D ^ Q dose of a f l a t o x i n to rats strongly i n h i b i t e d precursor incorporation into l i v e r nuclear RNA i n les s than 15 minutes a f t e r a p p l i c a t i o n . In vivo studies with l i v e r - s l i c e preparations have demonstrated s i m i l a r i n h i b i t o r y actions of 14 a f l a t o x i n B^ on RNA metabolism. Orotic acid-C incorporation into t o t a l c e l l u l a r RNA was strongly suppressed when r a t l i v e r s l i c e s were incubated i n v i t r o i n the presence of a f l a t o x i n B^. That impaired RNA synthesis was att r i b u t a b l e to i n h i b i t i o n of RNA polymerase a c t i v i t y has been demonstrated ( C l i f f o r d and Ree, 1967; Gelboin et a l . , 1966; Harley e t a l . , 1969). I t was found that the suppression of RNA polymerase a c t i v i t y was related to nucleolar f i n e s t r u c t u r a l a l t e r a t i o n s . F i f t e e n minutes a f t e r treatment, hepatocyte n u c l e o l i showed decreased prominance of nucleonema, and nucleolar microsegregation was observed. Macrosegregation of granular and f i b r i l l a nucleolar components (nucleolar capping) was demonstrated within one hour a f t e r t r e a t -ment and persisted up to 12 hours (Pong and Wogan,"1970). Afl a t o x i n B^ may cause ribosome d i s s o c i a t i o n from RER i n r a t hepatocytes (Friedman and Wogan, 1966). In the time course of a l t e r a t i o n of l i v e r polysome p r o f i l e s of rats treated with LDgQ dose of the toxin (Pong and Wogan, 1969), the t r e a t -ment caused marked but rev e r s i b l e disaggregation of l i v e r poly-somes. Large increases i n monomer and dimer fracti o n s and dec--11-reases i n polysome areas were e v i d e n t a t t h r e e hours a f t e r dosing but not a t 0.5 hour. T h i s a l t e r a t i o n i n n u c l e a r RNA and RER suggested t h a t the observed e f f e c t s were r e l a t e d t o a l t e r a t i o n s i n both RNA and p r o t e i n metabolism. Hayes e t a_l. (1975) found t h a t a s i n g l e i n j e c t i o n o f 1.5 mg a f l a t o x i n per kg body weight produced a l a r g e amount o f monosomeres from polysomes w i t h i n 18 hours. No p e p t i d y l s t r u c t u r e s were found t o be w i t h i n the monosomes. Recently S a r a s i n and Moule (1975; 1976) r e p o r t e d an i n h i b i t i o n of the t r a n s l a t i o n a l step i n the p r o t e i n s y n t h e s i s by a f l a t o x i n B^ i n r a t l i v e r polysomes. They demonstrated t h a t the i n h i b i t i o n c o u l d be d i v i d e d i n t o two s t e p s . Up t o 5 hours i n c u b a t i o n w i t h a f l a t o x i n B^, the t o x i n b l o c k e d p r o t e i n s y n t h e s i s d i r e c t l y and s p e c i f i c a l l y a t the polysome l e v e l . I n h i b i t i o n would appear t o m a n i f e s t i t s e l f i n the e l o n g a t i o n and/or t e r m i -n a t i o n steps d u r i n g p r o t e i n s y n t h e s i s . Beyond 7 hours i n c u b a t i o n the p r o t e i n s y n t h e s i s i n h i b i t i o n appeared c h i e f l y as a consequence of t r a n s c r i p t i o n impairment. The pr e s e n t study was designed t o determine the e f f e c t of a f l a t o x i n on f e r t i l i z e d o y s t e r eggs membrane s y n t h e s i s . Townsley and Lee (1967) r e p o r t e d a f l a t o x i n B^ i n h i b i t e d c e l l c l eavage i n f e r t i l i z e d eggs o f the m o l l u s c , Bankia s e t a c e a , without p r e v e n t i n g f e r t i l i z a t i o n or n u c l e a r d i v i s i o n . A c c o r d i n g to t h e i r i n v e s t i g a t i o n , the r e a c t i o n was very dramatic and p o s i -t i v e s i n c e f e r t i l i z e d eggs i n the presence of a f l a t o x i n were m u l t i n u c l e a r , whereas the c o n t r o l s had m u l t i p l i e d t o m u l t i c e l l u l a r l a r v a e . They a l s o r e p o r t e d t h a t the a d d i t i o n of a f l a t o x i n i n the -12-range of 0.05 - 40 ug/ml prevented the appearance o f c e l l membranes without p r e v e n t i n g sperm m o b i l i t y , f e r t i l i z a t i o n , o r the d i v i s i o n o f the nu c l e u s . T h i s h i g h s e n s i t i v i t y o f d i v i d i n g m o l l u s c eggs t o a f l a t o x i n was unique and i n v i t e d f u r t h e r study. Abedi and McKinley (1968) used zebra f i s h eggs as a f l a t o x i n b i o a s s a y t e s t organisms. Although t h i s organism was s e n s i t i v e t o sub-^microgram q u a n t i t i e s o f the t o x i n , the a n t i m i -t o t i c r e a c t i o n of t h i s t o x i n which o c c u r r e d i n Bankia setacea as r e p o r t e d by Townsley and Lee (1967) was not observed i n the case o f zebra f i s h eggs. In o r d e r t o more c l e a r l y observe i n an attempt t o e x p l a i n the i n t e r f e r e n c e o f a f l a t o x i n w i t h f e r t i l i z e d m o llusc egg development, the b i o l o g i c a l i n t e r a c t i o n o f a f l a t o x i n w i t h the eggs was s t u d i e d m o r p h o l o g i c a l l y w i t h the a i d of h i g h r e s o l u t i o n microscopy. -13 -METHODS AND MATERIALS CULTURES Aspergillus p a r a s i t i c u s (formally c a l l e d Aspergillus  flavus) NRRL 2999 was obtained from Dr. D. I. Fennell of Northern Regional Research Laboratory i n Peoria, I l l i n o i s , and served as a source of crude a f l a t o x i n . This s t r a i n i s i d e n t i c a l to A. flavus IMI 91019b deposited i n the Commonwealth Mycological I n s t i t u t e . Live fresh oysters (Crassostrea gigas) were purch-ased from l o c a l market. B a c i l l u s megaterium NRRL B-1368, v/as used as the bioassay t e s t organism for a f l a t o x i n , was purchased from Ameri-can Type Culture C o l l e c t i o n (ATCC 10716). CULTURAL METHODS AND MEDIA FOR AFLATOXIN PRODUCTION Difco potato dextrose agar was used for the prepa-r a t i o n of A. p a r a s i t i c u s spores. The fungus culture was inocu-lated on to a slant of potato dextrose agar and incubated for one week at 28° C. A uniform suspension of spores was obtained from a heavy crop of green conidia by adding 5 ml of 0.005 percent T r i t o n X-100 to the slant and scraping the surface of the culture with a loop. This spore suspension was used as a sourse of inoculum for the production of a f l a t o x i n s . -14-A modification of Shotwell's method (1966) was used for the production of the a f l a t o x i n s . About 25-30 ml of tap water was added to 300-ml Erlenmeyer f l a s k s containing 50 g of long grain r i c e obtained from l o c a l grocery stores. The wet r i c e was permitted to absorb the water completely. The f l a s k s were then steam-sterilized at 121°' C for 15 minutes and subse-quently cooled. It was very important that each r i c e kernel was kept separated from i t s neighbor, thereby preventing the formation of compact mass with mycelium. Whenever the r i c e showed a sign of clump formation, i t was loosened by vigorous shaking of the f l a s k . The fl a s k s were then inoculated with 0.5 - 1.0 ml of the spore suspension and incubated at 28° c for 5 days u n t i l the mold was heavily sporulated. The f l a s k s were steamed b r i e f l y i n order to k i l l the fungus, thus preventing laboratory contamination during a f l a t o x i n extraction. In the preliminary studies used f o r obtaining the stock a f l a t o x i n , Townsley and Lee's method (1967) which was a modification of the Adye's method (1964) was applied. Since t h i s method employed a mineral medium and was considered to be less e f f i c i e n t than the Shotwell's method i n producing the toxin, i t was subsequently abandoned. EXTRACTION OF AFLATOXINS AND STANDARD AFLATOXINS The aflatoxins produced by fungus on r i c e were extracted by soaking the molded grain i n 250 ml chloroform (ACS grade) overnight at room temperature (Shotwell et a l . , 1966). The chloroform extracts were f i l t e r e d through cheesecloth, and subsequently:passed through anhydrous sodium sulphate -15-column (2.4 x 10 cm) to remove moi s t u r e . The dehydrated c h l o -roform e x t r a c t s were co n c e n t r a t e d a t 40° C i n a r o t a r y vacuum evaporator t o a volume of 100 ml. Hexane (ACS grade) was added dropwise u n t i l ' t h e s o l u t i o n became cloudy. A f t e r s e t t l e m e n t , the p r e c i p i t a t e was recovered by d e c a n t a t i o n and vacuum d r i e d . The d r i e d p r e c i p i t a t e was d i s s o l v e d i n 5 ml o f f r e s h c h l o r o f o r m . T h i s s o l u t i o n served as the source o f "crude a f l a t o x i n " . S h o t w e l l et al_. + (1966) r e p o r t e d t h a t the crude a f l a t o x i n produced by her procedure c o n t a i n e d B^, B^, and a t r a t i o s of 1.00 : 0.15 : 0.22 : 0.02. More than 1 mg o f a f l a t o x i n per gram of r i c e was re c o v e r e d . Standard pure a f l a t o x i n s B^, B^, G^ and G 2 were purchased from A p p l i e d Science L a b o r a t o r i e s Inc., S t a t e C o l l e g e , Penn t o serve as r e f e r e n c e compounds. A f l a t o x i n B^ was a l s o o b t a i n e d from Calbiochem, Los Angeles, C a l i f o r n i a . SEPARATION OF AFLATOXINS BY THIN LAYER CHROMATOGRAPHY (TLC) AND THEIR QUANTITATIVE DETERMINATION The O f f i c i a l Methods o f A n a l y s i s o f the A s s o c i a t i o n of O f f i c i a l A n a l y t i c a l Chemists (1975) d e s c r i b e s the s e p a r a t i o n and q u a n t i t a t i v e d e t e r m i n a t i o n o f a f l a t o x i n s on TLC. F o r the p r e p a r a t i o n o f the p l a t e s , 30 g of s i l i c a g e l G-HR (Machery, Nagel and Co., 516 Duren, Germany) was put i n t o a 300-ml g l a s s stoppered Erlenmeyer f l a s k , along w i t h 60 ml d i s t i l l e d water. The f l a s k was shaken v i g o r o u s l y f o r not more than one minute i n order t o o b t a i n an even s l u r r y . The s l u r r y was poured i n t o an a p p l i c a t o r w i t h a l a y e r t h i c k n e s s s e t at 0.25 mm and spread on f i v e 20 x 20 cm g l a s s p l a t e s . These p l a t e s were c o o l e d and s t o r e d -16-i n a cabinet or desiccator at room temperature. For the separation of the toxins, the samples were spotted on a l i n e 4 cm from the bottom of plate with a micro-pipette (Corning Cat. No 7099-S). The spotted plates were developed i n an unlined, unequilibrated glass tank containing 150 ml of acetone : Chloroform mixture (1 : 9 v/v). The plates were withdrawn a f t e r solvent front had advanced about 15 cm above the spotting l i n e and permitted to dry at room temperature. The toxins were located by illuminating the plates under a long wave u l t r a v i o l e t lamp i n a dark room. Eppley (1966) reported that an unline, unequilibrated tank used for separation of aflat o x i n s remarkably improved the resolution of the toxins. In his data, Rf values of afla t o x i n s i n acetone : chloroform (1 : 9), i n an unequilibrated vessel were : 0.70, B 2 : 0.61, : 0.52 and G 2 • 0.44 compared to R^ values i n an.equilibrated vessel of B^ : 0.36, B 2 : 0.30, G 1 : 0.24 and G 2 : 0.20. Quantitative determination of a f l a t o x i n on TLC was done by a v i s u a l comparision of the fluorescent i n t e n s i t i e s between an unknown quantity of sample and a known quantity of the reference compound on the sample plate. SPECTROPHOTOMETRY METHOD FOR DETERMINATION OF AFLATOXINS The spectrophotometric method for the determination of the afla t o x i n s was based on the i n t e n s i t y of u l t r a v i o l e t absorption at 363 mu (the long wavelength absorption maximum) of the purified.toxins (Nabney and Nesbitt, 1965). Standard known aflatoxins were used for c a l i b r a t i o n . -17-BIOASSAY OF AFLATOXINS Many b i o a s s a y s f o r a f l a t o x i n s have been d e s c r i b e d (Armbrecht and F i t z h u g h , 1964; V e r r e t t e t a l . , 1964; Abedi and McKinley, 1969). In t h i s experiment, the b a c t e r i o l o g i c a l assay method f o r a f l a t o x i n B^, based on growth i n h i b i t i o n of B a c i l l u s  megaterium NRRL B-1368 (Burmeister and H e s e l t i n e , 1966; L i l l e h o j and C i e g l e r , 1968; Clements, 1969), was used. T h i s technique was adapted from the c l a s s i c a l a n t i b i o t i c zone i n h i b i t i o n methods. B. megaterium was maintained on n u t r i e n t agar and t r a n s f e r r e d t o n u t r i e n t b r o t h which was used as the inoculum t h e r e a f t e r . The inoculum was used when the c u l t u r e was i n the l o g phase o r e a r l y s t a t i o n a r y phase. One ml of the inoculum c u l t u r e was t r a n s f e r r e d t o 9 ml p o r t i o n o f h a l f s t r e n g t h GTY (glucose : t r y p t o n e : y e a s t e x t r a c t : agar = 1 : 5 : 2.5 : 15 g/1) and kept a t 45° C. A f t e r thorough mixing, t h i s 10 ml seed agar medium was poured i n t o a GTY p l a t e ( f u l l s t rengh medium), d i s t r i b u t e d evenly by t i l t i n g and subsequently a l l o w i n g the b a c t e r i a l suspesion t o g e l by p l a c i n g the p e t r i p l a t e s on a l e v e l s u r f a c e . A known q u a n t i t y o f a f l a t o x i n d i s s o l v e d i n c h l o r o -form was a p p l i e d to a 1/4 i n c h diameter Whatman No. 1 paper d i s k (punched w i t h a notebook puncher). A f t e r the c h l o r o f o r m had evaporated completely, the d i s k was p l a c e d on B. megaterium seeded GTY agar p l a t e s , and incubated a t 37° C f o r 24 hours. SPAWNING.OF OYSTER The procedure used f o r the spawning of the o y s t e r s was d e s c r i b e d by G a l t s o f f (1964), Loosanoff (1954), Loosanoff -18-and Davis (1963). S e x u a l l y matured o y s t e r s were p l a c e d i n g l a s s spawning d i s h e s (600 ml beaker). Each d i s h was f i l l e d w i t h sea water of same temperature as t h a t i n which m o l l u s c s were c o n d i -t i o n e d . These d i s h e s were s e t i n a l a r g e water bath which was f i l l e d w i t h hot water thus q u i c k l y r a i s i n g the temperature i n the d i s h e s t o about 30° C. The o y s t e r s were maintained a t t h i s temperature u n t i l spawning (about 1 - 2 h o u r s ) . APPLICATION OF AFLATOXIN TO FERTILIZED EGGS A known q u a n t i t y of a f l a t o x i n i n c h l o r o f o r m was added t o 0.5 ml propylene g l y c o l and the c h l o r o f o r m removed by evapo-r a t i o n under n i t r o g e n gas. Oyster eggs and sperm suspensions were mixed w i t h each o t h e r i n the r a t i o o f 9 : 1. One hundred ml of f e r t i l i z e d egg suspension was added t o the a f l a t o x i n i n propylene g l y c o l f o r t o x i c i t y t e s t s and f o r e l e c t r o n m i c r o g r a p h i c examination. E q u i v a l e n t amounts of propylene g l y c o l were added t o c o n t r o l samples. For the t o x i c i t y t e s t , the samples were observed under an i n v e r t e d phase c o n t r a s t microscope a t c e l l stages e q u i v a l e n t t o the f i r s t , second and t h i r d c e l l d i v i s i o n . PREPARATION OF THE ELECTRON MICROGRAPHS Egg samples were f i x e d i n the 6.3 percent p u r i f i e d g l u t a r a l d e h y d e i n 0.1 M sodium phophate b u f f e r pH 7.2 f o r 6 hours. The eggs sank to the bottom o f the v i a l s f a c i l i t a t i n g t h e i r s e p a r a t i o n from the l i q u i d phase. The f i x e d eggs were washed t h r e e times w i t h 0.1 M sodium phosphate b u f f e r and then s t o r e d i n the b u f f e r a t 5° C f o r 10 t o 12 hours. At t h i s stage o f f i x a t i o n the eggs may be s t o r e d f o r weeks p r o v i d i n g the b u f f e r -19-i s changed every few days. The prefixed eggs were postfixed for one hour i n one percent osmium tetroxide i n 0.07 M sodium phophate buffer (pH 7.2). The samples were washed simply by pouring o f f the f i x a t i v e and repacing i t with one or two changes of d i s t i l l e d water. Five percent uranyl acetate i n 0.07 M sodium phophate buffer (pH 7.2) was added. The eggs were allowed to soak f o r one hour i n the uranyl acetate solution i n order to enhance the membrane structures of the c e l l and DNA f i b r i l s i n the nucleus. The dehydration of the fixed c e l l s was accomplished by passing the sample through a series of ethanol solutions of increasing strength as follows; 50 percent ethanol for 5 minutes 70 percent ethanol for 10 minutes 80 percent ethanol for 10 minutes 90 percent ethanol for 10 minutes with two changes 100 percent ethanol for 20 minutes with three changes of acetone (dried with Na2SO^) for 10 minutes The dehydated sample was embedded i n Spurr's epon (1969). The components of the. epoxy r e s i n mixture are shown i n Table 1. Each ingredient was added i n the order as l i s t e d . i n Table 1 to an Erlenmeyer f l a s k placed on a balance. Mixing was accomplished by s w i r l i n g the f l a s k a f t e r each addition. The f i n a l embedding solution could he stored i n a r e f r i g e r a t o r f o r up to 7 days. A 1 : 1 solution of Spurr's epon and acetone was prepared and t h i s was added to the egg samples. This mixture was decanted from the c e l l s a f t e r two hours at room temperature, and fresh Spurr's epon embedding mixture added for overnight impregnation at room temperature. The specimen was then trans-ferred to fresh embedding solution;and held u n t i l i n f i l t r a t i o n -20-Table 1. The components, of the.plastic,embedding mixture UNOX Epoxide 206 or ERL 4206 (Vinyl cyclohexene dioxide) 10.2 g Hardener NSA (Nonenyl succinic anhydride) 26.0 g P l a s t i c i z e r DER 736 (Diglycidyl ether of propylene glycol) 6.0 g Accelerator S - l (DMAE : Dimethylamino ethanol) 0.4 g -21-of the c e l l s completed ( 2 - 3 ho u r s ) . F i n a l embedding was accomplished by t r a n s f e r r i n g the c e l l s i n t o a mold f i l l e d w i t h a f r e s h p l a s t i c m i x t u r e. The p l a s t i c c o n t a i n i n g the embedded c e l l s was hardened by i n c u b a t i o n i n a 70° C oven f o r 24 hours. The embedded c e l l s w i t h i n the s o l i d i f i e d p l a s t i c were s e c t i o n e d w i t h the ultramicrotome (OM U3, C, R e i c h e r t , A u s t r i a ) . Ultramicrotome s e c t i o n s were p l a c e d on 200 mesh copper g r i d s and s t a i n e d by f l o a t i n g them s e c t i o n s i d e down on s i n g l e drops o f the u r a n y l a c e t a t e , d i s s o l v e d t o s a t u r a t i o n i n 50 per c e n t methanol, f o r 45 minutes on a covered wax s u r f a c e . These p r e p a r a t i o n s were washed i n d i s t i l l e d water and s t a i n e d f o r 2 minutes i n l e a d c i t r a t e (lead n i t r a t e : 1.33 g, sodium c i t r a t e : 1.76 g, d i s t i l l e d water : 30 ml), washed again, and s t u d i e d under the e l e c t r o n microscope (AEI C o r i n t h 275). RESULTS AFLATOXIN BIOASSAY WITH B. MEGATERIUM The a f l a t o x i n bioassay using B. megaterium i s a r e l a t i v e l y rapid and simple method. The s e n s i t i v i t y of the bacteria:to a f l a t o x i n B^, B^# G^ and are shown i n Figure 3. When four ug of each toxin were applied to each disk, B^ was more toxic than B^ to the bacteria, and G^ a n d . w e r e not i n h i b i t o r y . Four ug of B^ was also the minimum amount of a f l a -toxin required to exhib i t i n h i b i t i o n of the b a c t e r i a l growth i n bioassay. I t i s also of in t e r e s t that.Bj was more toxic than G^. Generally G^ has been considered to be more toxic than B 2 i n the one day old duckling and human embryo l i v e r c e l l s . The s e n s i t i v i t y of the bioassay to a f l a t o x i n B^ i s shown i n Figure 4. One ug of the toxin was enough to i n h i b i t growth and thus, l e f t a cle a r zone around the disk. Increasing the toxin up to 4 ug increased i n a proportional manner the diameter of the cle a r zone around the disk. The comparison of the s e n s i t i v i t y of B. megaterium to pure a f l a t o x i n B^ and to a crude a f l a t o x i n extracted from A. par a s i t i c u s NRRL 2999 i s shown i n Figure 5. The amounts of a crude a f l a t o x i n equivalent to pure a f l a t o x i n B^ which were placed on the disk were calculated q u a n t i t a t i v e l y . According F i g u r e 3. The s e n s i t i v i t y o f B, megaterium t o a f l a t o x i n B^, , G^and • Each d i s k c o n t a i n s 4 ug o f the toxin as indicated. F i g u r e 4. The s e n s i t i v i t y o f B, megaterium to a f l a t o x i n B^. Each number i n the d i s k i n d i c a t e s ug of the t o x i n . F i g u r e 5. The comparison of the s e n s i t i v i t y between pure a f l a t o x i n B^ and a crude a f l a t o x i n . C : crude a f l a t o x i n , B^ : a f l a t o x i n B n, Disk 1, 2, 3 and 4 c o n t a i n 1, 2, 3 and 4 ug crude t o x i n , r e s p e c t i v e l y . Disk 5 and 6 c o n t a i n 2.5 and 4 ug of B^r r e s p e c t i v e l y . t o Shotwell e t a l . (1966), the crude a f l a t o x i n (50 percent) from r i c e c o n s i s t e d of : B 2 : G^ : G 2, i n the r a t i o o f 1.00 : 0.15 0.22 : 0.02. T h e r e f o r e , the a p p l i c a t i o n o f a crude a f l a t o x i n e q u i v a l e n t t o 2 ug of a f l a t o x i n B^ based on the above assumption to the d i s k might be assumed t o c o n t a i n 0.30 ug of B 2, 0.44 ug o f G^ and 0.04 ug of G^. The crude a f l a t o x i n c o n t a i n e d some minor f l u o r e s c e n t compounds which had a s m a l l e r v a l u e than G 2« However, the r e s u l t i n F i g u r e 5 shows no d i f f e r e n c e of s e n s i t i v i t y of the b a c t e r i a t o the crude a f l a t o x i n and pure a f l a t o x i n B^. BIOLOGICAL EFFECTS OF AFLATOXINS ON FERTILIZED EGGS The f e r t i l i z e d eggs i n sea water c o n t a i n i n g 0.5 percent propylene g l y c o l were observed t o undergo the f i r s t d i v i s i o n a t approximately 90 minutes a f t e r i n s e m i n a t i o n , and the second d i v i s i o n , a f t e r approximately 120 minutes. A f t e r 7 hours p o s t - i n s e m i n a t i o n the zygotes were i n a moving stage. F i v e ug of the a f l a t o x i n s B^, B 2, G^ ^ and G 2 were i n t r o d u c e d t o the inseminated eggs s e p a r a t e l y . The eggs t r e a t e d w i t h a f l a t o x i n B^ d i d not d i v i d e d whereas the eggs t r e a t e d w i t h a f l a t o x i n s B 2, G^ and G 2 c o n t i n u e d what appeared to be normal c y t o k i n e s i s . The e f f e c t s of v a r i o u s amounts of a f l a t o x i n B^ on c e l l d i v i s i o n w i t h i n inseminated eggs i s shown i n Table 2. At low dosage l e v e l s , the f i r s t d i v i s i o n of the f e r t i l i z e d eggs was a f f e c t e d t o a l e s s e r extent than l a t e r d i v i s i o n s . The zygotes t r e a t e d w i t h 1 ug/ml of a f l a t o x i n d i v i d e d without i n h i -b i t i o n , however o n l y approximately one h a l f o f the f e r t i l i z e d -25-Table 2. I n h i b i t i o n o f c e l l d i v i s i o n i n the a f l a t o x i n B t r e a t e d o y s t e r zygotes A f l a t o x i n B. I n h i b i t e d stage o f c e l l d i v i s i o n (ug/ml) s i n g l e c e l l two c e l l trochophore 0 . 1 _ + 0.5 - + + 1 .0 ± + (50) + 2 . 0 + K 1 0 ) + (<90) + 3 . 0 + (30) + (90) + 4 . 0 + (70) + (100) + 5 . 0 + (90) + (100) + 6 . 0 + (99) + (100) + + I n h i b i t i o n obvious + I n h i b i t i o n i n d e f i n i t e No i n h i b i t i o n ( ) Estimated percent of the t o t a l number o f zygotes i n h i b i t e d -26-eggs were capable of succeeding t o the f o u r c e l l stage. In the zygotes t r e a t e d w i t h 2 ug/ml of the t o x i n , more than 90 p e r c e n t of the eggs d i v i d e d t o the two c e l l stage, a t which time, 80 to 90 percent of a l l eggs were observed to have ceased d i v i s i o n . Three ug/ml of a f l a t o x i n were v e r y h i g h l y t o x i c t o the zygotes. In t h i s case about 70 percent of the eggs d i v i d e d to the two c e l l stage, and 90 percent of the t o t a l eggs were obsereved to have ceased d i v i s i o n a t the two c e l l stage. None of f e r t i l i z e d eggs proceeded beyond the l a t t e r f o u r c e l l stage. Higher l e v e l s o f the t o x i n r e s u l t e d i n a g r e a t e r i n h i b i t i o n of c e l l d i v i s i o n . In the zygotes t r e a t e d w i t h 4 ug/ml a f l a t o x i n B^, 70 percent of the t o t a l eggs were seen to remain i n a s i n g l e c e l l stage. With 5 ug/ml B^ added, 90 p e r c e n t o f the t o t a l eggs were i n h i b i t e d , whereas w i t h 6 ug/ml o f the t o x i n added, almost a l l o f the eggs were prevented from d i v i d i n g . The zygotes t r e a t e d w i t h v e r y low c o n c e n t r a t i o n ( 0.1 ug/ml) of a f l a t o x i n B^ developed to the f r e e swimming stage. However, the s u r v i v a l r a t e up to t h i s stage and beyond depended to a l a r g e e x t e n t on the degree of a s e p s i s of the a r t i f i c i a l c o n d i t i o n s . When crude a f l a t o x i n was added to the inseminated eggs, i t was observed t h a t t h e r e was no s i g n i f i c a n t q u a n t i t a t i v e d i f f e r e n c e i n the i n h i b i t i o n of c e l l d i v i s i o n between the pure and crude t o x i n s . ELECTRON MICROGRAPHIC OBSERVATIONS The inseminated eggs f o r the c o n t r o l were sampled a t 5, 90, 120 and 180 minutes f o l l o w i n g f e r t i l i z a t i o n . The a f l a t o x i n 27 -F i g u r e 6. U n f e r t i l i z e d oocyte of o y s t e r , C r a s s o s t r e a g i g a s . V : v i t e l l i n e c o a t , M : mitochondria, NU : n u c l e o l u s , RER : rough endoplasmic r e t i c u l u m . (11,000 X) F i g u r e 7. U n f e r t i l i z e d oocyte of o y s t e r . N : n u c l e u s , RER : rough endoplasmic r e t i c u l u m . (32,000 X) -29-t r e a t e d zygotes were f i x e d a t 180 and 220 minutes. A l s o u n f e r t i l i z e d eggs were f i x e d . F i n e s t r u c t u r a l changes o r a l t e r -a t i o n s were observed and compared i n these samples. Micrographs o f u n f e r t i l i z e d eggs The f i n e s t r u c t u r e o f the u n f e r t i l i z e d o ocyte i s shown i n F i g u r e s 6 and 7, and was d e s c r i b e d by D a n i e l s e t a l . (1973). The oocytes c o n t a i n e d a l a r g e s p h e r o i d nucleus commonly r e f e r r e d to as the germinal v e s i c l e . A l a r g e s p h e r o i d , dark and homogeneous n u c l e o l u s was a l s o p r e s e n t . The cytoplasm of the oocyte c o n t a i n e d l a r g e numbers of conspicuous y o l k g r a n u l e s and l i p i d g l o b u l e s . D a n i e l s e t a l . (1973) r e p o r t e d t h a t l i p i d g l o b u l e s were o f t e n l a r g e r than y o l k g r a n u l e s , but t h e i r s i z e s v a r i e d c o n s i d e r a b l y . I t was d i f f i c u l t t o d i s t i n g u i s h the two types of g l o b u l e s i n the d e v e l o p i n g egg of the o y s t e r shown i n F i g u r e s 6 and 7. M i t o c h o n d r i a and r i b o -somes were a l s o abundant throughout the cytoplasm. RER was comprised predominantly o f t u b u l a r c i s t e r n a e s arranged i n bundles (Figure 7). A s e c t i o n o r p a r t o f some of the bundles were compacted w i t h t u b u l a r c i s t e r n a e and ribosomes. The RER memb-rane showed risbosomes att a c h e d to t h e i r membrane s u r f a c e s . A l l c i s t e r n a e s were not n e c e s s a r i l y e q u i d i s t a n t and o f t e n had d i s s i m i l a r l e n g t h . However, the ER v e s i c l e s were seldom observed i n the micrographs. Micrographs o f 5 minute postinseminated u n t r e a t e d eggs Eggs were seen t o be i n c o r p o r a t e d by multisperms ( F i g u r e s 8 and 9). F u r t h e r development o f sperm n u c l e i would appear t o depend on some unknown f a c t o r s i n the eggs. Some eggs -30-F i g u r e 8. Two spermatozoa c o n f i n e d t o the p e r i p h e r a l a s p e c t s of the o y s t e r zygote. V : v i t e l l i n e coat, SN : sperm n u c l e i , N : nucleus. (11,000 X) Fi g u r e 9. A spermatozoon j u s t p r i o r t o i n c o r p o r a t i o n . Sp : sperm, SM : sperm mitochondria, SN : sperm n u c l e i , A : acro-some. (15,000 X) -31-were found w i t h sperm e n t e r i n g through the membrane and ot h e r s were seen i n a stage of breakdown of t h e i r n u c l e a r envelope and r e o r g a n i z a t i o n o f the sperm chromatin ( d i s p e r s i o n of the t i g h t l y packed chromatin o f the sperm nucleus) ( F i g u r e 10). About t h i s time, the n u c l e a r envelope (germinal v e s i c l e s ) becomes v e s i c u l a r r e s u l t i n g i n i t s d i s s i p a t i o n . E v e n t u a l l y the v e s i c l e s and c i s t e r n a e s became s c a t t e r e d throughout the zygote (Figure 11). Chromatin and n u c l e o l i were not seen. Changes of the c y t o p l a s m i c o r g a n e l l e s and i n c l u s i o n s , immediately f o l l o w i n g i n s e m i n a t i o n , were not observed. Micrographs o f 90 minute postinseminated u n t r e a t e d eggs V a r i o u s stages Of the development o f the zygotes were seen a t t h i s time, some showing m u l t i n u c l e i (Figure 12), some showing m i g r a t i o n o f chromosomes and some i n a completed two c e l l s t a g e . The p r o n u c l e i which were seen i n the m u l t i n u c l e i stage were of uniform s i z e and shape, and were prese n t a t the ce n t e r o f egg. Longo (1973 a) r e p o r t e d t h a t the p r o n u c l e i o f polyspermic zygotes were approximately the same s i z e as the male and female p r o n u c l e i o f monospermic zygotes i n the s u r f clam, S p i s u l a s o l i d i s s i m a . The RER appeared as e i t h e r s i n g l e c i s t e r n a e o r c i s -t e r naes i n bundles and v e s i c l e s (Figure 13). The m a j o r i t y o f the RER presented c i s t e r n a e s a compact i n bundle form. F r e -q u e n t l y v e s i c l e s were seen around c i s t e r n a e s . Most of these v e s i c l e s were seen w i t h ribosomes a t t a c h e d t o t h e i r membrane. Other v e s i c l e s and/or v a c u o l e s were noted which c o n t a i n e d few, i f any, ribosomes. -32-F i g u r e 10. D i s p e r s i o n of the t i g h t l y packed chromatin of a i n c o r p o r a t e d spermatozoon. SC : sperm chromatin. (26,000 X) F i g u r e 11. V e s i c u l a t i o n of germinal v e s i c l e . V : v e s i c l e s . (6,000 X) -33--34-The presence of furrows and membranes was observed i n t h i s stage. C o n t r a c t i l e r i n g s were found below the furrow membrane and vacuoles were seen i n the membrane form a t i o n area which might i n d i c a t e a p o s s i b l e a s s o c i a t i o n w i t h membrane forma t i o n (Figure 14) Micrographs of 120 minute postinseminated u n t r e a t e d eggs Furrow fo r m a t i o n and the two c e l l stage of the zygote, as w e l l as, the f o u r c e l l stage were observed i n t h i s time ( F i g u r e 15). Some n u c l e i were round and had s e v e r a l n u c l e o l i which were round, dark and v e r y dense. Chromatin m a t e r i a l s were d i s p e r s e d throughout nucleus (Figure 16). The other n u c l e i were i r r e g u l a r and a l s o c o n t a i n e d round dense n u c l e o l i . The presence of i r r e g u l a r n u c l e i might be due t o a stage i n n u c l e a r f o r m a t i o n i n which many chromatin are found t o occur along w i t h v e s i c l e s ( F i g u r e 17). The development o f the RER was more progressed than a t e a r l i e r s t a g e s . C i s t e r n a e s were found t o occur together i n bundles or as s i n g l e c i s t e r n a e s . Many v e s i c l e forms were a l s o observed around c i s t e r n a e s and throughout the cytoplasm (Figure 18). F i g u r e 19 shows the furrows as w e l l as the c o n t r a c t i l e r i n g s which appear as a t h i c k band below the furrow membrane. Micrographs of 18 0 minute postinseminated u n t r e a t e d eggs At t h i s time i n the development o f the zygotes, the organism was m u l t i c e l l u l a r , c o n s i s t i n g o f more than 8 c e l l s . The s i z e o f the zygote was not changed, thus each c e l l was smal-l e r as the zygote developed (Figure 20). V a r i o u s n u c l e a r shapes were due to d i f f e r e n t stages i n the development o f n u c l e i . -35-F i g u r e 13. C i s t e r n a e s o f rough endoplasmic r e t i c u l u m (RER). Y : y o l k , M : mi t o c h o n d r i a , L : l i p i d . (24,000 X) F i g u r e 14. I n c o r p o r a t i o n of small v e s i c l e or v a c u o l e t o plasma membrane. V : vacuole, Vs : v e s i c l e , Mb : plasma membrane. (36,900 X) F i g u r e 15. Furrow formation and the two c e l l stage, and the four c e l l stage. F : furrow. (21,000 X) F i g u r e 16. D i s p e r s i o n of chromatin m a t e r i a l s throughout nucleus. Nu . n u c l e o l u s , Ch : chromatin, N : n u c l e u s . (15,300 X) F i g u r e 20* Rough endoplasmic r e t i c u l u m . R : rough endo-plasmic r e t i c u l u m . vR : v e s i c l e form o f RER, cR : c i s t e r n a e form o f RER. (24,700 X) -39-Some o f n u c l e i were i r r e g u l a r and c o n t a i n e d a n u c l e a r b l e b ( F i g u r e 21) and a lammella membrane a s s o c i a t e d t o the n u c l e a r envelope. The n u c l e o l i appeared both i n the form o f an i r r e g u l a r shape w i t h chromatin d i s p e r s e d through nucleus and as a round dense s t r u c t u r e (Figure 21). In the c'ytoplasm, many v e s i c l e s were observed. Many of these v e s i c l e s were seen w i t h fewer ribosomes than c i s -t e r n a e s . The c i s t e r n a e RER was present as a s i n g l e RER or i n bundles of RER (Fig u r e s 22 and 23). Micrographs o f the a f l a t o x i n t r e a t e d eggs a t 180 and 220 minutes  f o l l o w i n g f e r t i l i z a t i o n No s t r u c t u r a l d i f f e r e n c e s were observed between the 180 minutes and 220 minutes postinseminated a f l a t o x i n t r e a t e d eggs. M u l t i n u c l e i were seen i n the both samples (Figure 24). Some of the n u c l e i were i r r e g u l a r , w h i l e o t h e r s were round. Small n u c l e i which are very s i m i l a r i n appearance t o promale n u c l e i were observed. These l a t t e r o b s e r v a t i o n s i n d i c a t e t h a t c e l l d i v i s i o n was stopped a t the m i t o t i c step b e f o r e the f i r s t c e l l d i v i s i o n . Some n u c l e o l i were seen i n which the f i b r i l l a r and the g r a n u l a r components were segregated (Figures'25 and 26). In the cytoplasm, RER were very s i m i l a r t o t h a t o f the u n f e r t i l i z e d eggs or t o the very e a r l y stages i n the develop-ment of the zygote (Figure 27). Most of RER were present as c i s t e r n a e s i n bundles. Few, i f any, v e s i c l e s were observed around the c i s t e r n a e or through the cytoplasm. The ribosomes were c l o s e l y a s s o c i a t e d w i t h the membrane of the RER (F i g u r e 28) and no s e p a r a t i o n o f ribosomes from the membrane was observed. Figure 21. Irregular shape of n u c l e o l i (chromatin dispersion throughout nucleus). Nu : nucleolus. (15,200) NB : nuclear bleb, LM : lammella membrane. F i g u r e 22. C i s t e r n a e form of RER and v e s i c l e form o f RER around c i s t e r n a e s . N : nucleus, vR : v e s i c l e form o f RER, R : RER, M : m i t o c h o n d r i a . (30.900 X) F i g u r e 23. r e t i c u l u m . C i s t e r n a e and v e s i c l e forms o f rough endoplasmic vR : v e s i c l e form of RER. (120,000 X) - 4 3 -Figure24. M u l t i n u c l e i i n the a f l a t o x i n B.. t r e a t e d o y s t e r zygote. PN : p r o n u c l e u s . (3,600 X) F i g u r e 25. N u c l e o l a r s e g r e g a t i o n i n the a f l a t o x i n B. t r e a t e d zygote. C : cytoplasm, f : f i b r i l l a r component o f n u c l e o l u s , g : g r a n u l a r component of n u c l e o l u s . (40,000 X) -44-F i g u r e 26. N u c l e o l a r s e g r e g a t i o n t o f i b r i l l a r and g r a n u l a r components, f : f i b r i l l a r component, g : g r a n u l a r component (120,000 X) - 4 6 -- 4 7 -Other c y t o p l a s m i c o r g a n e l l e s such as the m i t o c h o n d r i a or g o l g i bodies were very s i m i l a r i n morphology to the c o n t r o l samples. -48-DISCUSSION Longo (1973 b) r e p o r t e d i n a review a r t i c l e t h a t t h e r e e x i s t s e s s e n t i a l l y f o u r c l a s s e s o r stages of m e i o s i s a t which the eggs o f most animals are inseminated. In the f i r s t c l a s s , the spermatozoa e n t e r s the egg b e f o r e the germinal v e s i c l e breaks. The eggs t h a t make up t h i s c l a s s are found i n v a r i o u s nematodes, m o l l u s c s , a n n e l i d s and c r u s t a c e a n s . The eggs of some m o l l u s c s , a n n e l i d s and i n s e c t s are grouped i n t o c l a s s I I , which are inseminated a t the f i r s t m e i o t i c metaphase. C l a s s I I I i n s e m i n a t i o n takes p l a c e a t the second m e i o t i c metaphase. The eggs of most v e r t e b r a t e s are found i n t h i s type. Eggs b e l o n g i n g to c l a s s IV are r e p r e s e n t e d by v a r i o u s echinods and are inseminated a t the p r o n u c l e a r stage. In F i g u r e 8, two spermatozoon n u c l e i can be seen w i t h i n i n the cytoplasm o f the o y s t e r egg b e f o r e the germinal v e s i c l e breaks. T h i s i n d i c a t e s the o y s t e r egg i s f e r t i l i z e d a c c o r d i n g t o c l a s s I. The g e n e r a l scheme of c l a s s I f e r t i l i -z a t i o n i s shown on F i g u r e 29. Longo and Anderson (1970 a; b) s t u d i e d the f i n e m o r p h o l o g i c a l s t r u c t u r e of the s u r f clam egg, Sposula s o l i d i s s i m a , which f a l l s i n c l a s s I . The s u r f clam eggs are inseminated p r i o r t o the onset of t h e i r m e i o t i c d i v i s i o n s , i . e . p r i o r t o p o l a r body form a t i o n , when the egg i s i n the vesicle Nucleolus Nuclear envelope Second polar body Female pronucleus First meiotic apparatus Paternally derived chromatin Maternally derived chromatin First polar body Second meiotic apparatus B Vesiculation of pronuclear , k envelope (3D ' Pronuclear envelope Male pronucleus Figure 29. Mitotic apparatus D Blastomere nuclei Cytokinesis L_| General scheme of class I f e r t i l i z a t i o n germinal v e s i c l e stage (Figure 29-A). Insemination induces the break down o f the l a r g e egg nucleus (germinal v e s i c l e ) , conden-s a t i o n of chromatin and disappearance o f the n u c l e o l u s . The f i r s t m e i o s i s takes p l a c e i n the c e n t e r o f the zygote (Figure 29-B). In t h i s f i r s t m e i o s i s , one a s t e r becomes l o c a l i z e d w i t h i n the c o r t e x t o form the f i r s t p o l a r body (Figure 29-C) while the second a s t e r i s more c e n t r a l l y p l a c e d i n the zygote. During t h i s time, the n u c l e a r envelope surrounding the spermatozoon nucleus breaks down. The p r o n u c l e a r envelope does not develop around the p a t e r n a l l y d e r i v e d chromatin u n t i l the second m e i o s i s has completed. A f t e r the formation of the second p o l a r body, f o l l o w i n g the second m e i o s i s (Figure 29-D), a n u c l e a r envelope i s developed i n d i v i d u a l l y around male and female chromatin. Both n u c l e i then migrate to the c e n t e r of the egg, and the f i r s t m i t o t i c d i v i s i o n begins (Figure 29-G). The cleavage of the zygote a t t h i s stage y i e l d s two blastomeres of unequal s i z e (Figure 29-H) ( A l l e n , 1953). The t r a n s m i s s i o n e l e c t r o n micrograph, F i g u r e 12, shows the presence of m u l t i n u c l e i a t 90 minutes f o l l o w i n g the e n t r y of spermatozoon i n t o the egg. These are most l i k e l y male p r o n u c l e i developed from the spermazoon nucleus. C e l l membrane was a l s o observed i n t h i s stage and i n d i c a t i n g the f o r m a t i o n two c e l l s tage. Egg d i v i s i o n was not synchronized throughout the t o t a l f e r t i l i z e d egg sample. Amemiya (1926) r e p o r t e d t h a t o y s t e r egg (zygote), C r a s s o s t r e a v i r g i n i c a , developed a t room temperature as f o l l o w s : The f i r s t p o l a r body o c c u r r e d a t 40 -51-to 50 minutes f o l l o w i n g i n s e m i n a t i o n ; the second p o l a r body a t 45 to 60 minutes; the f i r s t cleavage a t 75 t o 100 minutes; the second cleavage a t 80 to 130 minutes; the r o t a t i n g b l a s t u l a a t 6 1/2 hours; the f r e e swimming g a s t r u l a a t 8 hours; and f i n a l l y the trochophore a t 24 hours. Figures.8-and 9, taken 5 minutes a f t e r egg insemi-n a t i o n , show the presence of multisperms w i t h i n the cytoplasm of the egg. F i g u r e 12, 90 minutes a f t e r i n s e m i n a t i o n o f the eggs, shows a m u l t i n u c l e a r egg. These r e s u l t s i n d i c a t e the o y s t e r , C r a s s o s t r e a g i g a s i s p o l y spermic. An u l t r a s t r u c t u r a l a n a l y s i s o f polyspermy i n the s u r f clam, S p i s u l a s o l i d i s s i m a , was done by Longo (1973 a) . He r e p o r t e d t h a t f e r t i l i z a t i o n o f an egg by two or more sperm, normally r e f e r r e d t o as "patholo-g i c a l polyspermy", occurs as normal polyspermic f e r t i l i z a t i o n ( p h y s i o l o g i c a l polyspermy) i n a number of animals. The process of polyspermy appears t o i n v o l v e the same s e r i e s o f events as seen d u r i n g monospermy. Multisperm i n c o r p o r a t i o n b r i n g s about the f o r m a t i o n of p r o n u c l e i subsequent t o t h e i r m i g r a t i o n t o the c e n t r a l r e g i o n o f the zygote. T h i s i s f o l l o w e d by the appear-ance of chromosomes which become arranged on a m u l t i p o l a r s p i n d l e i n p r e p a r a t i o n f o r the f i r s t cleavage d i v i s i o n . C l ose to the f i n a l stage of cleavage, v e s i c l e s and c i s t e r n a e s are seen to aggregate along the margin of the chromosomes l o c a t e d a t the r e g r e s s i n g a s t r a l r e g i o n s . The f u s i o n of these membraneous elements around the i n d i v i d u a l chromosomes produces a number of karyomeres. At each a s t r a l r e g i o n , the karyomeres fused w i t h oneanother t o produce a n u c l e u s . Some a s t r a l r e g i o n s may have -52-more than one f o c i of karyomere coalescence so that several nuclei are frequently formed i n proximity to one another. It was reported that a f l a t o x i n i n h i b i t e d c e l l membrane synthesis but not nuclear d i v i s i o n i n the f e r t i l i z e d eggs of Bankia setacea, a mollusc, and the animals were found to be s e n s i t i v e to 0.05 ug/ml to 40 ug/ml of a f l a t o x i n B^ (Townsley and Lee, 1967). In the:application of t h e i r observa-t i o n to the oyster, a close r e l a t i v e to Bankia setacea, the toxin treated oyster zygotes could be expected to respond i n some morphological manner si m i l a r to that observed i n Bankia  setacea: i . e . interference i n c e l l membrane formation or c e l l d i v i s i o n (cytokinesis). Therefore multinuclear c e l l s could be expected to be seen i n the treated zygotes and the number of nuclei within a multinuclear c e l l should be observed to increase with the increasing time subsequent to f e r t i l i z a t i o n . From the observation made with the l i g h t microscope at 150 X, f i r s t c e l l d i v i s i o n of the inseminated eggs of oyster, Crassostrea gigas, were prevented or i n h i b i t e d by the addition of more than 1 ug/ml of a f l a t o x i n B^ to the medium. The trochophore stage, on the other h a n d c o u l d be prevented by the addition of 0.5 ug/ml of a f l a t o x i n B^. The oyster eggs were approximately 20 foifcd more r e s i s t a n t to v i s u a l damage by a f l a t o x i n B^ than the corresponding eggs from Bankia setacea (Townsley and Lee, 1967). The explanation for t h i s difference i n s e n s i t i v i t y between the two mollusc species remains unexplained. Limited studies using the eggs from Bankia setacea suggested that there was l i t t l e difference between the s e n s i t i v i t y of the two species of m o l l u s c s . However, the d i f f i c u l t y i n o b t a i n i n g h e a l t h y , mature Bankia setacea specimens prevented t h i s i n v e s t i g a t i o n from r e s o l v i n g t h i s problem. Although c e l l membrane s y n t h e s i s and subsequently c e l l d i v i s i o n of the e a r l y stage zygote exposed to h i g h l e v e l s of a f l a t o x i n B 1 ( 1 - 5 ug/ml) was the r e s u l t of a d d i t i o n of the t o x i n , " t r u e " n u c l e a r m u l t i p l i c a t i o n , as such, was not observed i n t r e a t e d u n d i v i d e d c e l l s . Using the magni-f i c a t i o n power a v a i l a b l e w i t h the phase c o n t r a s t microscope ( i . e . 1000 X) the o y s t e r eggs inseminated and subsequently t r e a t e d w i t h some doses o f a f l a t o x i n B^ appeared t o show an i n c r e a s e i n n u c l e a r bodies i n agreement w i t h the p r e v i o u s i n v e s -t i g a t i o n (Townsley and Lee, 1967). However on c l o s e examination w i t h e l e c t r o n microscopy, i t was observed t h a t the "apparent n u c l e a r d i v i s i o n " was not n u c l e a r d i v i s i o n but the formation of p o s s i b l e p r o n u c l e i . The o r g a n e l l e , p r o n u c l e i which can be seen w i t h i n a f e r t i l i z e d egg are the r e s u l t o f polyspermic i n s e m i -n a t i o n as shown i n F i g u r e s 8, 9 and 12, and e x p l a i n e d i n F i g u r e 29. T h e r e f o r e , the r e s u l t s o f the p r e s e n t t h e s i s are more c o n s i s t e n t w i t h a p r o p o s a l of a g e n e r a l c y t o t o x i c e f f e c t of a f l a t o x i n B^ on the t r e a t e d o y s t e r zygotes r a t h e r than the s e l e c t i v e b l o c k i n g of biomembrane s y n t h e s i s per se. Numerous i n v e s t i g a t o r s have shown t h a t , i n e u k a r y o t i c c e l l s , t r a n s c r i p t i o n of ribosomal genes lea d s to a ribosomal p r e c u r s o r 45 S (Svedbergs) RNA i n the n u c l e o l u s t h a t must be c l e a v e d a t s p e c i f i c s i t e s t o g i v e to 18 S and 28 S r-RNA 1s on maturation; newly s y n t h e s i z e d r i b o s o m a l RNA 1s emerge i n t o i n the cytoplasm as f r e e 60 S and 40 S s u b u n i t s . Reviews o f these -54-o b s e r v a t i o n s have been p u b l i s h e d by Busch and Smetana (1973), and Smetana and Busch (1974). N u c l e o l a r s e g r e g a t i o n o r an a l t r a t i o n i n the morphology of the n u c l e o l u s , i s c h a r a c t e r i z e d by a rearrangement o f the g r a n u l a r and f i b r i l l a r components i n t o two or more d i s -t i n c t zones. These o b s e r v a t i o n s were r e p o r t e d i n r a t l i v e r by Svoboda e t a l . (1966) and Reynier et al_. (1975), and i n monkey l i v e r by Svoboda et al. (1966). N u c l e o l a r s e g r e g a t i o n was observed a l s o i n some of the a f l a t o x i n B^ t r e a t e d o y s t e r zygotes i n t h i s t h e s i s ( F i g u r e s 25 and 26). The a l t e r a t i o n o f the morphology may r e f l e c t an i n h i b i t i o n - o f n u c l e a r RNA s y n t h e s i s . Reddy and Svoboda (1968) r e p o r t e d t h a t a f l a t o x i n B^ markedly 3 i n h i b i t e d the i n c o r p o r a t i o n of u r i d i n e H i n t o n u c l e a r RNA which was more e v i d e n t a t the l e v e l of n u c l e o l a r RNA when the n u c l e o l a r s e g r e g a t i o n was pronounced. In a d d i t i o n to an i n h i b i t i o n of RNA s y n t h e s i s , the a c t i v i t y o f the enzyme, RNA polymerase, i n segregated n u c l e o l i was c o n s i d e r a b l y d i m i n i s h e d . A f l a t o x i n B^, t h e r e f o r e , has been shown to a c t a t the t r a n s c r i p t i o n a l l e v e l by d i r e c t a c t i o n on RNA polymerase (Gelbion e t a l . , 1966; C l i f f o r d and Ree, 1967; Friedman and Wogan, 1967; Moule and F r a y s s i n e t , 1968; King and N i c h o l s o n , 1969; Edward and Wogan, 1970). A f l a t o x i n can a l s o i n t e r v e n e a t the p o s t - t r a n s c r i p t i o n a l l e v e l a f f e c t i n g RNA matu-r a t i o n and the development of i t s form of t r a n s p o r t . Moule (1973) showed t h a t a f l a t o x i n B^ a l t e r s the p r e c e s s i n g of r i b o -somal RNA. While 18 S RNA c o n t i n u e s t o appear i n the cytoplasm as 40 S, p a r t i c l e s , the emergence i n the cytoplasm of 60 S -55-p a r t i c l e s and o f 213 S RNA decreases g r e a t l y . A f l a t o x i n may a c t , a c c o r d i n g to the l a t t e r r e f e r e n c e , a t the l e v e l of the 60 S p a r t i c l e s which become l e s s s t a b l e and- thus p a r t l y destroyed i n the nucleus. Based on the e a r l y s t u d i e s of S i e k e v i t z and Palade (1960) and extended by Redman e t a l . (1966) and Redman (1969), i t i s now c l e a r t h a t e x p o r t a b l e p r o t e i n s are s y n t h e s i z e d on a t t a c h e d polysomers of rough endoplasmic r e t i c u l u m . The p r o t e i n s are segregated i n t o the c i s t e r n a l space o f the RER ( B l o b e l , 1977). A f t e r s e g r e g a t i o n i n the c i s t e r n a l spaces of the RER, the p r o t e i n s move to the t r a n s i t i o n a l elements of the RER. These elements are l o c a t e d a t the boundary o f the RER w i t h a s p e c i a l morphology, i . e . they have a t t a c h e d polysomes on most o f t h e i r s u r f a c e , or are smooth and p r o v i d e d with v e s i c l e s or v a c u o l e s (Jamieson and Palade, 1977). The RER i n the a f l a t o x i n t r e a t e d zygotes resembles the u n f e r t i l i z e d eggs or e a r l y postinseminated eggs i n t h a t the v e s i c l e s o r vacuoles around the RER are absent o r s m a l l i n s i z e . T h i s o b s e r v a t i o n may i n d i c a t e t h a t the RER i n t r e a t e d zygotes were not developed t o produce p r o t e i n s as would be seen at the e a r l y stage o f f e r t i l i z a t i o n . Recently, S a r a s i n and Moule (1975; 1976) r e p o r t e d t h a t p r o t e i n s y n t h e s i s was i n h i b i t e d a t the trans'lati-on step by a f l a t o x i n B^. A f l a t o x i n B^ b l o c k s p r o t e i n s y n t h e s i s d i r e c t l y and s p e c i f i c a l l y a t the polysome l e v e l ( t r a n s l a t i o n step) from zero to 5 hours a f t e r the t o x i n was a d m i n i s t e r e d J . Beyond 7 hours a f t e r a d m i n i s t r a t i o n , p r o t e i n s y n t h e s i s i n h i b i t i o n appears c h i e f l y as a consequence o f t r a n s c r i p t i o n impairment. Also, they observed that a f l a t o x i n (1 mg/kg) i n h i b i t e d elongation and/or termination of poly-peptide i n rat l i v e r . However, when the toxin concentration was increased, i n addition to i n h i b i t i o n of elongation and/or termination of the polypeptides, there was a decrease i n the i n i t i a t i o n rate of polypeptides. Therefore, i t would appear that zygotes treated with 5 ug/ml of a f l a t o x i n B^ cease production of protein at an early stage of development (translation l e v e l ) . Also the i n h i b i t i o n of protein synthesis may be r e s u l t of impaired nuclear RNA synthesis (transcription l e v e l ) . The observed i n h i b i t i o n of mit o t i c d i v i s i o n of the a f l a t o x i n B^ treated oyster eggs, Crassostrea gigas, before f i r s t c e l l d i v i s i o n might be related to r e p l i c a t i o n i n h i b i t i o n . It was concluded i n t h i s investiga-t i o n that added a f l a t o x i n B^ to developing f e r t i l i z e d oyster eggs causes a cytotoxic e f f e c t i n oyster zygotes through impair-ment of nucleic acid and protein synthesis. However, c o n f i r -mation of the hypothesis would require further biochemical, cytochemical and u l t r a s t r u c t u r a l : s t u d i e s . -57-BIBLIOGRAPHY 1. Abedi, Z. H., and W. P. MeKinley. 1968. Zebra f i s h eggs and larvae as a f l a t o x i n bioassay test organism. J . Assoc. O f f i c . A g r i . Chem. 51:902-905. 2. Adye, J . , and R. I. Mateles. 1964. 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