The effect of ethyl carbamate (urethane) on the egg of the brown trout (Salmo trutta fario L.)

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E ffi EFFECT OF ETHYL CABBAMATE (UEETHANE) car THE EGG- OF THE BEOWN TBCHJT (Salmo t r u t t a f a r i o L . )

by MAEGABET AMTE LAHJG

S ubm itted i n p a r t i a l f u lf ilm e n t o f th e re q u ire m e n ts f o r th e d eg ree o f MASTEE OF SCIENCE

OF WESTEEN ONTABIO LOUDON, CANADA

u h it o e s it t

1950

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UMI Number: EC54086

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Approved, f o r th e D epartm ent o f Z o o lc ^ r7

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Uhie i n v e s t ig a ti o n was made p o s s ib le th ro u g h ; i g ra n t from th e H a tio n a l Cancer I n s t i t u t e o f Canada,

Ih e

w r i t e r w ish es to e x p re ss h e r a p p r e c ia tio n f o r t h i s a s s is ta n c e .

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ACET0WLEDGME1TTS The w r i t e r w ish es to e x p re s s h e r s in c e r e th an k s t o P r o f e s s o r H elen I . B a t tle , D epartm ent o f Zoology, f o r s u g g e s tin g th e problem , f o r h e r i n t e r e s t and a d v ice d a rin g th e c o a rs e o f th e work, and f o r h e r c o n s tr u c tiv e c r i t i c i s m and guidance d a rin g th e w r iti n g o f th e th e s is * Her thanks a r e a ls o ex ten d ed to P r o fe s s o r A. W. A. Brown, Head o f th e Department o f Zoology, f o r m a tin g a v a ila b le th e f a c i l i t i e s o f th e d ep artm en t; to Mr. J . W. A nderson, D epartm ent o f Lands and f o r e s t s , fo r su p p ly in g th e t r o u t eg g s; to Mr. C h a rle s J a r v i s , D epartm ent o f Anatomy, f o r p r e p a r a tio n o f th e p h o to g ra p h s; and to Mr. Ken H isaoka f o r c a r in g f o r th e eggs d u rin g th e C hristm as h o lid a y s .

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V —

CONTENTS

ACKNOWiEDGMENTS................................................................................................

iv

l i s t of m

n

s ..................................................................................................

ILLUSTRATIONS.....................................

v iii

ABSTRACT................................................................................................................

ix

INTRCDUCTI OS'....................................................................................................... H i s t o r i c a l Background............................

1 3

MATERIAL AND METHODS............................................................................

6

EXPERIMENTAL DATA............................................................................................ Experim ent I , 19*4S.......................................................... Experim ent IIA and IIB , 19*4S................................................ Experim ent I and U I, 1914-9..................................................... Experim ent I I and U II, 19% ................................................. Experim ent U I I I , I 9I+9.............................................................. Experim ent UIV, 19*J9................................................................

9 9 12 15 29 35 *45

P ro lo n g ed Exposure E xperim ents 1 and 2 .................................... Experim ent 1 ................................................................................. Experim ent 2 a and Sb................................................................

50 50 55

ATYPICAL CELLULAR CONFIGURATIONS............................................................ H is to r ic a l Background........................................................................ P r e s e n t I n v e s tig a t io n ........................................................................

57 57 60

DISCUSSION...........................................................................................................

66

SUMMARY..................................................................................................................

78

LITERATURE CITED..............................................................................................

SO

VITA

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- vi LIST OF TABLES T able 1. 2.

Page M o r ta lity of u re th a n e t r e a t e d t r o u t eg g s. E xperim ent I , 19^S.......................................................................................

10

M ito s is in u re th a n e tr e a te d t r o u t - m u l t i c e l l u l a r "blastoderm . E xperim ent I , 19^S.......................................................

13

M o r ta lity o f u re th a n e t r e a t e d t r o u t eggs. E xperim ent IIA , 1 9 ^ ...............................................................................

16

M o r ta lity o f u re th a n e t r e a t e d t r o u t eg g s. E xperim ent IIB , 19^S...............................................................................

17

M ito s is i n u re th a n e t r e a t e d t r o u t - m u l t i c e l l u l a r b la sto d e rm . Experim ent IIA .................................................................

IS

M o r ta lity o f u re th a n e t r e a t e d t r o u t e g g s. E xperim ent I , 1 9 % .................................................... ...............................

21

M o r ta lity o f u re th a n e t r e a t e d t r o u t eggs. Experim ent U I, 1 9 % .................................................................................

23

M ito sis i n u re th a n e tr e a te d t r o u t - m u l t i c e l l u l a r b la sto d e rm . Experim ent U I, S e r ie s U2, 1 9 % ..............................

25

M ito s is i n u re th a n e tr e a t e d t r o u t - m u l t i c e l l u l a r b la sto d e rm . Experim ent U I, S e r ie s Uty, 19^9..............................

2J

M ito sis i n u re th a n e tr e a t e d t r o u t - m u l t i c e l l u l a r b la sto d e rm . Experim ent U I, S e r ie s U5, 1 9 % ..............................

J i­

M o r ta lity o f u re th a n e t r e a t e d t r o u t e g g s. E xperim ent I I , 1 9 % .................................................. ..............................

32

12.

M o r ta lity o f a g i t a t e d e g g s, 1 9 % ....................................................

33

13 .

M o r ta lity o f u re th a n e t r e a t e d t r o u t eggs. Experim ent U II, 1 9 % ...............................................................................

3^

Average diam eter o f c e l l s o f urethane tr e a ted tr o u t. E xperim ent U II, S e r ie s U12, 19^9.....................................................

35

M ito sis i n u re th a n e tr e a t e d t r o u t - m u l t i c e l l u l a r b la sto d e rm . E xperim ent U II, S e rie s 1 3 , 1 9 % ............................

37

3.

5. 6. 7. S. 9. 10. ll.

lh .

15 .

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- v ii LIST OF TABLES (continued) Table

Page

16 .

M ortality o f urethane treated trout eggs* Experiment TJIII, 19^9....................................................................

17.

M ito s is in urethane trea ted trout - m u ltic e llu la r blastoderm. Experiment TJIII, S eries U20,..19^9...............

^-1

M itosis in urethane treated trou t - m u ltic e llu la r blastoderm. Experiment TJIII, S eries U21, 19U9 ..............

*+3

M itosis in urethane treated trout - m u lticellu la r blastoderm. Experiment TJIII, S eries TJ22, I9H9 ...............

^

M ortality o f urethane treated trout eggs. Experiment TJIY, 19^9.....................................

V?

18. 19. 20. 21.

M itosis in urethane treated trout - m u ltic e llu la r blastoderm. Experiment TJIY, Series TJ^22, 19^9 . . . . . . • •

22.

M itosis in urethane treated trout - m u ltic e llu la r blastoderm. Experiment TJIY, S eries ^ 23,....19^9...............

52

R elationship between concentration o f urethane and r e la tiv e growth of embryo, I 9U9 ...............................................

53

23.

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- v iii ILLUSTEATI01TS F ig u re 1.

2* 3.

4. 5. 6.

Pag® M o r ta lity o f u re th a n e t r e a t e d t r o u t eg g s. E xperim ents I , IIA and IIB , 1 9 4 8 ,........................................................ M ito s is in u re th a n e t r e a t e d t r o u t , m u l t i c e l l u l a r ■blastoderm. E xperim ent I , 1948, 6 and 24 h o u rs e x p o s u re ..

11

14

M ito s is i n u re th a n e t r e a t e d t r o u t , m u l t i c e l l u l a r b la sto d e rm . E xperim ent IIA , 1948, 48 and 72 h o u rs e x p o su re ..............................................................................

19

M o r ta lity o f u re th a n e t r e a t e d t r o u t eg g s. E xperim ents I , UI and U I I I , 1949............................................................

22

Average d ia m e ter o f c e l l s o f eggs t r e a t e d w ith u re th a n e . Experim ent U I, 1949, S e rie s U2, U4 and U5.......................................

26

M ito sis in u re th a n e t r e a t e d t r o u t , m u l t i c e l l u l a r b la sto d e rm . E xperim ent U I, 1949, S e rie s U4 and U5 ................

28

M o r ta lity o f u re th a n e t r e a t e d t r o u t eg g s. E xperim ents I I , U II, and UIY, 19U9 ...................................................

30

Average d ia m e te r o f c e l l s o f eggs t r e a t e d w ith u re th a n e . Experim ent U II, 1949, S e rie s U12 and U13......................................

36

M ito s is in u re th a n e t r e a t e d t r o u t , m u l t i c e l l u l a r b la sto d e rm . E xperim ent U II, 194-9, S e rie s U I 3 ...........................

38

Average d ia m e ter o f c e l l s o f eggs t r e a t e d w ith u re th a n e . Experim ent U I I I , 1949, S e rie s U20, U21 and U22.........................

42

M ito sis in u re th a n e t r e a t e d t r o u t , m u l t i c e l l u l a r b la sto d e rm . E xperim ent U I I I , 1949, S e rie s U21 a n d U 2 2 . . . .

4-4

M ito s is in u re th a n e t r e a t e d t r o u t , m u l t i c e l l u l a r b la sto d e rm . E xperim ent UIY, 1949, S e rie s U^22 and U-jj23.. .

49

Average d ia m e ter o f c e l l s of eggs t r e a t e d w ith u re th a n e . E xperim ent UIY, 1949, S e rie s U -^2 and U^23.................................

51

P ro lo n g ed exposure experim ent 1 , 1949. B e la tiv e growth o f em bryonic a x i s .....................................................

54

15.

Camera lu c id a draw ings o f a ty p ic a l n u c le a r c o n f ig u r a tio n s .

62

1 6.

Camera lu c id a draw ings o f c e l l s showing norm al chromosome number........................................................................................

64

7. 8. 9. 10. 11. 12. 13. 14.

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ABSTHA.CT

Hie e y to lo g ic a l e f f e c t s o f the water solu ble carcinogen, eth yl carbamate, in concentrations o f 0.1 to 1 .0 percent are described for the egg of the brown trout ( Salmo tr u tta fa r io L .) subjected during e a r ly cleavage and m u ltic e llu la r blastodermic sta g es.

A c r itic a l

period e x is t s at the in it ia t io n of g a stru la tio n , when the egg i s more s e n s itiv e , as in d icated by the sudden r i s e in m o rta lity .

A

temporary stim u lation o f m ito sis i s follow ed by a p a r tia l in h ib itio n and accompanied by an increase in the numbers o f a ty p ica l nuclear co n fig u ra tio n s.

These abnorm alities, including "amitotic" con­

s tr ic t io n , b in u clea te to m ultinucleate giant c e l l s , m ultipolar m itoses, p o lyp loid chromosome numbers, pycnotic n u clei and k a ry o ly sis, resemble those ty p ic a l o f neoplasms and those a lso produced a fte r treatment with other p h y sical and chemical agen ts.

Prolonged ex­

posure to low concentrations o f urethane a c c elera tes growth o f the embryonic a x is .

High concentrations produce a retardation of growth

and cause other abnorm alities.

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IITEOHJCTIOT Growth, i s a phenomenon c h a r a c te r istic o f a l l liv in g m atter, and i s la r g e ly a resu lta n t o f increased c e l l numbers brought about by m ito sis or in d ir e c t c e l l d iv is io n .

The f e r t i l i z e d egg i n i t i a l l y undergoes

m ito tic d iv isio n , a process which continues through cou n tless generations o f c e l l s , u n til the adult con d ition i s reached.

At th is tim e, growth in

the sense o f increase in body s iz e together with c e llu la r d iffe r e n tia tio n cea ses, but c e l l d iv isio n i s by no means arrested . Early d iv isio n s of the f e r t i liz e d egg r e s u lt prim arily in the production o f c e l l s without apparent d iffe r e n tia tio n .

On the in it ia t io n

o f g a stru la tio n th ese c e l l s come under the in flu en ce o f organizers, and as a r e s u lt , p rim itiv e c e llu la r groups se t asid e w ith varying stru ctu ral c h a r a c te r istic s and fu nctional p o t e n t ia lit ie s become in creasin gly more d is c r e te .

As m ito tic d iv isio n s continue, d iffe r e n tia tio n occurs and

in volves development in to m orphologically d istin gu ish ab le e n t it ie s , termed t is s u e s . Although, with the attainment o f the adult s ta t e , growth as regards increase in s iz e and d iffe r e n tia tio n has ceased, the cap acity o f c e l l s to divide i s not l o s t .

During the whole l i f e o f an organism there i s

always some breakdown o f the co n stitu en t t is s u e elem ents, w ith subsequent regeneration by m ito s is , which i s co n tro lled by and in balance with the organism as a u n it.

S im ilarly, i f a tis s u e i s wounded, regeneration i s

brought about by stim u lation o f m ito sis in the adjacent c e l l s .

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This

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a b i l i t y o f t i s s u e s to r e g e n e ra te d im in is h e s w ith th e o n s e t o f o ld age* O c c a sio n a lly th e re may ap p ear a n o th e r ty p e o f grow th w hich r a t h e r th a n b e in g o r d e r ly i s to some e x te n t, f r e e o f th e c o n tr o l o f th e organism . T his type o f grow th i s f r e q u e n tly b o th u n lim ite d and c o n tin u o u s and p ro ­ c ee d s a t a r a t e f a r in e x cess o f th e n o rm al.

I t p r o g r e s s i v e ly in v a d e s,

r e p la c e s and d e s tro y s n e ig h b o u rin g t i s s u e s u n t i l , i f unchecked, p ro v e s l e t h a l to th e organism a s a w hole.

The m icro sco p ic a p p earan ce o f th e

t i s s u e may v a ry c o n s id e ra b ly , many o f th e o r ig i n a l c h a r a c t e r i s t i c s may be r e ta in e d , or th e r e may be a r e v e r s i o n to th e em bryonic, u n d i f f e r e n t i a t e d s ta te .

The p a t t e r n o f th e c e l l s o f th e s e grow ths i s o f te n d is o r d e r ly , th e

c e l l s d e g e n e ra te and u n d i f f e r e n t i a t e d , and th u s s e rv e s a s a n i d e n ti f y in g c h a r a c t e r i s t i c o f th e phenomenon known a s c a n c e r. S ince th e grow th c a p a c ity o f c an c e ro u s ti s s u e i s autonomous o r n e a r ly autonom ous, b e in g a p p a r e n tly in d ep en d en t o f th e organism , i t i s an e s s e n t i a l l y unique p ro p e rty o f th e tumour i t s e l f .

T h is i s th e m ost s t r i k i n g c h a r a c t e r i s ­

t i c o f tum ours a s a c l a s s , and w ith in th e f a c t o r s r e s p o n s ib le f o r t h i s p ro ­ p e r t y , l i e s th e s e c r e t o f i t s c o n t r o l.

T h e re fo re , s u b s ta n c e s which a l t e r

th e ty p ic a l m ito tic p i c t u r e a r e o f i n t e r e s t i n e lu c i d a tin g problem s o f norm al c e l l u l a r and neoplasm ic grow th. E th y l carb am ate, a member o f th e u re th a n e s e r i e s , i s a w id e ly known h y p n o tic and n a r c o tic , which i s u sed e x te n s iv e ly a s a l a b o r a to r y a n a e s th e ti c . I t h a s been shown to p o s s e s s c a rc in o g e n ic p r o p e r t i e s ( K e ttle s h ip , Henshaw and Meyer, 19^+3) and i s s p e c if ic f o r in d u c in g tum ours o f th e pulm onary ty p e . V arious a u th o rs have r e p o r te d a s tim u la to r y and i n h i b i t o r y e f f e c t on c e l l d iv is io n in normal and m a lig n a n t c e l l s .

I n a d d itio n u re th a n e i s one o f th e

few p ro v en c a rc in o g e n s which a re r e a d i l y s o lu b le i n w a te r ( 1 : 0.5 g m .).

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- 3 Because o f i t s extrem e s o l u b i l i t y , i t s c a rc in o g e n ic p r o p e r t i e s and i t s r e p o r te d e f f e c t s on c e l l d iv i s i o n , e t h y l carbam ate was chosen a s th e a g e n t fo r til i s in v e s t i g a t i o n .

HISTORICAL BACKG-RQUILD K e ttle s h ip , Henshaw and Meyer (19^3) f i r s t n o te d t h a t e th y l carbam ate when a d m in is te re d o r a l l y o r i n t r a p e r i t o n e a l l y i n s u b a n a e s th e tic do ses h a s te n e d and r a i s e d th e in c id e n c e o f pulm onary tum ours in A and CjH s t r a i n s o f m ice.

Henshaw and Meyer ( 19^+) showed t h a t a s in g le a n a e s th e tic dose

i s s u f f i c i e n t to induce pulm onary tum ours in s t r a i n A m ice.

In I 9I+5 th e y

found t h a t subcutaneous im p la n ta tio n s o f p e l l e t s o f 1 mg. o r o r a l adm inis­ t r a t i o n s o f a 1 p e r c e n t s o lu tio n s p ro d u ced tum ours a t an e a r l y age i n t h i s s t r a i n , w ith th e number o f in d u ced tum ours b e a r in g a d i r e c t r e l a t i o n s h i p t o th e d osage.

L arsen , Rhoads and Weed (19^6) c o rro b o ra te d th e s e f in d in g s f o r

s t r a i n A m ice, and in d ic a te d t h a t th e e th y l d e r iv a tiv e alo n e was e f f e c t i v e . J a f fe f in 19^6 found t h a t u re th a n e when fe d t o , o r 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 in to r a t s , w hich have a n o rm ally h ig h r e s i s t a n c e to th e developm ent o f p u l­ monary o r h e p a tic tum ours, dev elo p ed a h ig h p e rc e n ta g e o f tum ours o f th e s e ty p e s .

Cowen (igl+7) showed t h a t u re th a n e a ls o p ro d u ced pulm onary tum ours i n

B U I , and CIA s t r a i n and i n C57 s t r a i n w hich a re n o rm a lly r e s i s t a n t to spontaneous pulm onary tum ours, th u s e x te n d in g th e c a rc in o g e n ic e f f e c t s o f u re th a n e to o th e r s t r a i n s o f m ice.

O rr (19^7) a ls o found t h a t u re th a n e

c a u s e s lu n g tumours in many s t r a i n s o f m ice.

Noble and M illa r (19^8) i n

p r e lim in a r y ex p erim en ts n o te d t h a t r a p i d l y grow ing h ig h ly m a lig n a n t tum ours were p ro d u ced i n mice fo llo w in g i n j e c t i o n s o f s tr o n g u re th a n e and z in c a c e ta te .

They a ls o found t h a t s u sp e n sio n s o f th e lu n g s o f m ice which h ad

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- 4 r e c e iv e d sm all d o ses o f u re th a n e , when i n j e c t e d s u b c u ta n e o u sly in to o th e r m ic e , produced adenom ata o f th e lu n g s i n a h ig h p e rc e n ta g e o f th e t r e a t e d a n im a ls . In c o n tr a s t t o th e s e f in d i n g s , Haddow and S exton (1946) showed t h a t e th y l carbam ate cau sed tem porary r e t a r d a t i o n o f sp o n tan eo u s mammary c a n c e r i n th e mouse and i n th e W alker carcinom a 256 o f r a t s .

I n 1946 P a te rs o n ,

Haddow, ApThomas and W atkinson su g g e ste d i t s u se a s a m it o t i c p o is o n i n th e chem otherapy o f m a lig n a n t g ro w th .

H uggins, Yd and Jo n es (1946) found

t h a t u re th a n e h a s an in h i b i t o r y e f f e c t on p r o s t a t i c c a n c e r. The e f f e c t s o f u re th a n e on m ito s is have b een r e p o r te d by a number o f in v e s tig a to rs .

Warburg (1910) showed t h a t p h e n y lu re th a n e i n i f 2000 norm al

c o n c e n tr a tio n r e ta r d e d c le a v a g e , and i n 1/ 5OO c o m p le te ly i n h i b i t e d i t i n th e eggs o f th e s e a u rc h in S tro n g y lo c e n tro tu s l i v i d u s .

L i l l i e (1914)

re p o r te d a s im ila r r e ta r d in g e f f e c t a f t e r t r e a t i n g A rb a cia p u n c tu la ta eggs w ith eth y l u re th a n e .

C lark (1933) n o te d an a l l o r none e f f e c t o f

e th y l carbam ate on c e l l d iv is io n o f s e a u rc h in e g g s.

I t s s u p p re s s iv e

a c t i o n on c le a v a g e h a s a ls o b een o b serv ed f o r th e egg o f A rb a cia punc t u l a t a ( F is h e r and Henry 1944) f o r th e c i l i a t e , Tetrahym ena g e l e i i (Ormsbee and F is h e r , 1944) and f o r th e c i l i a t e Colpoda s t e i n l i ( B u rt, 1945) .

Levan

and O ste rg re n (1943) and O ste rg re n (1944) c o n sid e re d t h a t e th y l carb am ate c au se d c - m ito s is i n th e r o o t c e l l s o f A llium c e p a .

S im ila r ly , Hohl (1947)

n o te d t h a t th e r o o t c e l l s o f TB.cla fa b a and A llium c e p a s u b je c te d t o a

2 p e r c e n t s o lu tio n o f u re th a n e , e x h ib ite d a low ered r a t e o f m i to s is , w ith a stopp age i n th e m etaphase and a c o in c id e n t s h o rte n in g and th ic k e n in g o f th e chromosomes. Ludford (193&) found t h a t u re th a n e , when a p p lie d t o c u l t u r e s o f mouse carcinom a 63 , a r r e s t e d m ito s is i n some c u l t u r e s , b u t h ad l i t t l e

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- 5 e f f e c t on o th e r s .

Gayer and C laus (19*4-7) u s in g i n t r a p e r i t o n e a l i n j e c t i o n s

o f u re th a n e , 1 c c . o f a 10 p e r c e n t s o lu tio n p e r 100 gm. hody w e ig h t, alm o st w h o lly a b o lis h e d m ito s is i n th e c o rn e a l e p ith e liu m o f r a t s and m ice.

In

l a t e r e x p erim en ts lu n g tum ours o f th e adenom atous ty p e a p p e a re d i n 75 o f

86 a n im als t r e a t e d . D u stin (l9 ^ 7 a ) n o te d t h a t i n t r a p e r i t o n e a l in j e c t i o n s o f u re th a n e m o d ified c e l l u l a r d iv is io n i n th e i n t e s t i n e o f th e mouse, c a u s in g low ered m ito ti c a c t i v i t y , w ith marked p y c n o tlc d e s tr u c ti o n .

He a ls o n o te d (19*47**)

t h a t u re th a n e h a s some i n h i b i t i n g e f f e c t on th e grow th o f a n a c u te leu k aem ia in C57 m ice, and t h a t i t te m p o ra rily i n h i b i t s th e d iv is i o n o f e r y t h r o b la s ts in ra b b its .

A s in g le i n j e c t i o n o f 0 .2 and 1 gm. p e r k g . d e c r e a s e s th e

number o f r e t i c u l o c y t e s i n th e b lo o d o f th e r a b b i t betw een 2 and 3 days a f t e r th e i n j e c t i o n . Von M o ellen d o rf ( 193S) t r e a t e d c u ltu r e s ° ^ a b b it su b cu tan eo u s t i s s u e w ith 1.03 p e r c e n t u re th a n e and o b serv ed an i n i t i a l in c r e a s e i n m it o s is , fo llo w e d by a d e c re a s e w ith a su b se q u e n t in c r e a s e .

Bucher (19*49) r e p o r te d

a s tim u la to r y a c ti o n o f u re th a n e on m ito s is i n c u l tu r e s o f r a b b i t f i b r o c y te , a f t e r s h o rt e x p o su res, fo llo w e d by an i n h i b i t i o n o f m ito s is a f t e r lo n g e x p o su re s.

L a s n itz k i (19*4-9) r e p o r te d a s t r i k i n g s tim u la to r y e f f e c t

o f O .b p e rc e n t s o lu tio n o f u re th a n e on th e outgrow th and m ito s is i n a c u ltu r e o f carcinom a 63 and t o a l e s s e r degree i n C57 sarcom a o f th e mouse, i n marked c o n tr a s t to th e i n h i b i t i o n o f c e l l d iv i s i o n and grow th i n com parable c u ltu r e s o f norm al t i s s u e , a f t e r th e same dosage o f u re th a n e .

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- 6 -

MATEHIAL MD METHODS fa rio The egg o f the "brown t r o u t , Salmo t r u t t a AL ., was used, f o r a l l e x p e ri­ m e n ta l w ork.

I t i s o f a t e l o l e c i t h a l ty p e , hence th e y o lk , "being com­

p l e t e l y s e p a ra te from th e p ro to p la s m ic p o r ti o n , does n o t i n t e r f e r e w ith c e l l u l a r c le a v a g e .

T his f a c to r f a c i l i t a t e s th e h a n d lin g o f th e f ix e d egg,

s in c e th e b la s to d is c can be r e a d i l y removed from th e y o lk p r i o r to s e c tio n ­ in g .

The egg i s a p p ro x im a te ly 5*5 mm* i n d ia m e te r ., and i s su rro u n d ed by

a heavy tr a n s lu c e n t membrane, th e c h o rio n .

The y ello w to orange c o lo u re d

y o lk a p p e a rs a s a l a r g e s p h e r ic a l g lo b u le m easu rin g 5 *1+5mm* i n d ia m e te r a t r i g h t a n g le s to the b l a s t o d i s c .

The b lasto d ex m ic e e l l s p a r t i c u l a r i l y d u rin g

e a r l y clea v a g e s ta g e s a re r e l a t i v e l y la r g e and c o n ta in c l e a r w e ll d e fin e d n u c le i.

B la s tu la e o f th e Salm onidae fa m ily do n o t p o s s e s s a d i s c r e t e

se g m e n ta tio n c a v it y .

S ince th e l a t t e r i s r e p r e s e n te d by sp ac e s betw een

■the in d iv id u a l c e l l s o f th e b la sto d e rm , a t t h i s p e r io d each c e l l i s e s s e n t i a l l y a s p h e r ic a l u n i t , and r a r e l y com pressed b y a d ja c e n t c e l l s . Because o f th e s e c h a r a c t e r i s t i c s and th e p e r m e a b ility o f th e c h o rio n , t h i s type o f egg i s id e a l f o r d e te rm in in g e f f e c t s o f a w a te r s o lu b le c a rc in o g e n such a s e th y l carbam ate on th e c e l l . D aring 19hS th e eggs were o b ta in e d from th e O n ta rio P r o v in c ia l Government F is h H a tc h e rie s a t Mount P le a s a n t and H orm andale.

In m ost

in s ta n c e s th e y were re c e iv e d s h o r t l y a f t e r f e r t i l i z a t i o n , s in c e t h i s was known to b e one o f th e s a f e s t p e r io d s f o r s h ip p in g su ch f r a g i l e eg g s. The s to c k su p p ly was k e p t i n p l a s t i c c o a te d w ire s c r e e n tr a y s i n ru n n in g

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- 7 ta p w a te r.

The te m p e ratu re was m a in ta in e d betw een 1 3 -l6 ° C . where d ev elo p ­

ment p ro v ed normal s in c e a number o f th e eggs were s u c c e s s f u lly b ro u g h t through to h a tc h in g . I n I 9H9 , th e eggs w ere o b ta in e d e x c lu s iv e ly from th e h a tc h e ry a t Normandale, and were tr a n s p o r te d d i r e c t l y a f t e r f e r t i l i z a t i o n .

They

were s u p p o rte d on c h e e s e c lo th tr a y s suspended about one in c h below th e s u rfa c e o f th e w a te r.

The te m p e ratu re was m a in ta in e d w ith in a ra n g e o f

12- 15®C. i n an i c e r e f r i g e r a t o r . U rethane (U .S .P .) was p re p a re d i n s to c k s o lu tio n o f 10 and 30 p e rc e n t in tap w a te r, from which th e e x p e rim e n ta l ran g e o f c o n c e n tra tio n s was made up by d i l u t i o n .

At d i f f e r e n t i n t e r v a l s a f t e r f e r t i l i z a t i o n , th e eggs were

s u b je c te d to graded c o n c e n tr a tio n s o f e th y l carbam ate f o r v a ry in g p e r io d s of tim e .

Samples o f eggs were removed and f ix e d i n D a v id so n 's fo r m a lin -

g ly c e r in e - a c e tic - a l e o h o l f i x a t i v e a t th e d e s ir e d tim es and l a t e r tr a n s ­ f e r r e d to D a v id so n 's fo rm a lin -g L y c e rin e -a lc o h o l p r e s e r v a t i v e . The b la sto d e rm s were d is s e c te d o f f w ith a minimum o f y o lk , d e h y d ra te d i n a lc o h o l, c le a r e d i n t o l u o l , and embedded i n t i s s u e mat (M.P. 6 0 -6 2 ° C .). S e r ia l s e c tio n s were p re p a re d a t 5 m icro n s and th e m a jo r ity were s ta in e d w ith H e id e h h a in 's ir o n h em ato x y lin w ith o u t c o u n te r s ta in in g .

L a te r i t

p ro v e d d e s i r a b l e i n some in s ta n c e s to s t a i n s p e c i f i c a l l y f o r thym onucleic a c id (d e s o x y rib o n u c le ic a c i d ) .

S e p a ra te b la sto d e rm s from th e same con­

c e n tr a ti o n s o f u re th a n e w ere s ta i n e d w ith F e u lg e n 's and c o u n te r s ta in e d w ith P a s t G reen, o r th e one h a l f o f a b la sto d e rm was s ta i n e d w ith th e h e m ato x y lin , th e o th e r h a l f w ith T e u lg e n 's to o b ta in a more d i r e c t check.

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g

-

-

A minimum o f th r e e "blastoderm s from each c o n c e n tr a tio n was examined m ic ro s c o p ic a lly u s in g a n o i l im m ersion le n s and a x lh o c u la r .

The

m ito tic f ig u r e s and th e a b n o rm a litie s were c o u n ted i n ev ery f i f t h s e c tio n , th e re b y e x c lu d in g th e p o s s i b i l i t y o f c o u n tin g th e same c e l l tw ice s in c e th e n u c le i never m easured more th a n 125 m ic ro n s.

Counts

i n to to up to 500 c e l l s p e r b la sto d e rm were made where p o s s ib le and S ince i n th e I 9 H9 e x p erim en ts many

av erag ed on a p e rc e n ta g e b a s is .

o f th e eggs were in e a r ly s ta g e s and m ost o f th e c e l l s w ere d iv id in g , i t p ro v ed d e s ir a b le to p ro v id e a n o th e r more d e f i n i t e c r i t e r i o n o f th e c e l l u l a r m u ltip l ic a tio n r a t e .

M easurem ents were made o f th e d ia m e te rs

o f 20-30 c e l l s p e r b la sto d erm a t each c o n c e n tra tio n o f u re th a n e and f o r each exposure and the r e s u l t s a v era g e d .

In a la te r experiment in 19^9 a fte r 11U and lhO hours exposure r e s p e c tiv e ly the eggs were transferred to fresh aerated tap water and allowed to continue development.

A fter ep ib oly had commenced, samples

were fix e d a t varying in te r v a ls.

The amount of overgrowth and the

length of the embryonic axis was measured in f iv e embryos from each concentration and the r e s u lts averaged.

The embryos then were d isse c te d

o ff the yolk , stain ed in to to in alum cochin eal, cleared in creo so te, mounted whole and examined for comparative growth and for gross d eviation s from normal development.

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_ 9 -

IXPERMEHTAi DATA Experiment _I, lglj-g Source o f eg g s:

Mount P le a s a n t,

Age a t 'beginning o f e x p e rim e n t:

Ho t .

S, 19US.

A pproxim ately 30 h o u rs

( m u l t i c e l l u l a r b la s to d e rm ). P e rc e n ta g e c o n c e n tra tio n s o f u re th a n e :

0 .0 , 0 .1 , 0 .2 5 , 0»5» 0»75»

1 . 0 , 2 .0 . Duration o f experiment:

^ days.

A pproxim ately 65 eggs were p la c e d i n each o f sev en p i n t s e a l e r s , c o n ta in in g IJOO c c . o f th e e x p erim en tal s o lu tio n s .

The s e a l e r s w ere c lo s e d

and p la c e d i n a b a th o f ru n n in g w a te r a t ab o u t 15°C.

At 6 and 2k h o u rs ,

sam ples o f eggs from each c o n c e n tr a tio n w ere f ix e d f a r c y to lo g ic a l s tu d y . The dead were removed d a i l y th ro u g h o u t th e c o u rse o f th e e x p erim en t. The m o r t a lit y o f th e eggs over a 6 day p e r io d was e x p re sse d a s a p e rc e n ta g e o f th o se l i v i n g a t th e te r m in a tio n o f th e e x p e rim e n t.

The

g rap h s w ere p l o t t e d on a cu m u lativ e p e rc e n ta g e b a s i s o f th e s e c a l c u l a t i o n s . The eggs i n th e 2 .0 p e r c e n t s o lu tio n showed th e h ig h e s t m o r t a l i t y (T able 1 , f ig u r e l ) .

Over 90 p e r c e n t w ere dead by fe e end o f th e second d a y .

The

m o r t a l i t y o f th e eggs i n th e 0.25 p e r c e n t s o lu tio n was h ig h , a lth o u g h t h i s may have been c au sed by rough h a n d lin g , s in c e a f t e r 6 h o u rs exposure t h i s j a r was b roken, and o v er h a l f th e eggs l o s t .

The number o f eggs dead i n

th e c o n tr o l j a r was h ig h e r th a n in th e 0 . 5 , 0 . 75 i

1*0 p e r c e n t s o lu tio n s ,

f o r which no e x p la n a tio n can be o f f e r e d .

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- 10 -

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission

- 12 Reference to Table 2 and fig u re 2 w ill in d icate th at eggs exposed to concentrations of 0 .1 to 1 .0 percent urethane fo r 6 hours show a decided increase in the numbers o f metaphases, anaphases and telop h ases in the higher concentrations.

A fter 2k hours exposure the number o f m ito tic

fig u res d eclin es considerably in the higher concentrations in comparison to the lower concentrations and the c o n tr o ls. In t h is and the ensuing experiments, the eggs removed fo r c y to lo g ic a l stu d ies were taken before the m o rta lity had sta rted to r i s e . Experiment IIA and IIB, 19^8 Source o f eggs!

Noraiandale, Nov. 13, I9U8.

Age a t beginning o f experiment % A - approximately 6 hours ( l- 2 c e lle d stages) B - 15 days (m u ltic e llu la r blastoderm) Percentage concentrations of urethane! Duration o f experiment!

0 .0 , 0 .1 , 0 .2 5 , 0«5i 0 .7 5 .

1 ,2 5 .

6 days.

The 15 days old eggs were s t i l l in a com paratively ea rly m u ltic e llu la r blastodermic sta g e, sin ce they had been kept at a low temperature (approxi­ m ately 10°C.) in the Normandaie hatchery.

-Approximately 100 eggs were

placed in hOO c c . o f so lu tio n in p in t s e a le r s .

The eggs o f the la t e r stage

were from natural trou t, and were larger and the yolk o f an orange colour rather than yellow as those from hatchery reared tro u t.

These se a le r s were

kept a t a f a i r l y constant temperature in a sim ilar way to Experiment I . The 2 .0 percent so lu tio n was not used in th is s e r ie s because in the previous experiment the m o rtality had been e x c e s siv e ly high in t h is con cen tration . Eggs were preserved at in te r v a ls o f h8, and 72, hours, and the dead were removed and recorded d a ily .

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1




113

164 -

r---------------- c

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TABLE MITOSIS

21

IN URETHANE TREATED TROUT EXPOSURE TIME -

20

HOURS

-

MULTICELLULAR BLASTODERM (SERIES

U 22) b

9------------- 3------------------C---------------- c---------------- c---------------- ?--------------- ---------------- f3---------------- 3------------- 3-------------C------------- o of TOTAL AVERAGE % % % % % % % % p URETHANE INTERPHASES METAPHASES ANAPHASES METAPHASES 2 NUCLEI 3+ NUCLEI MULTIPOLAR PICNOTIC ATYPICAL DIAMETTR FROPHASES TELOPHASES ANAPHASES SPINDLE NUCLEI FIGURES OF CELLSj: TELOPHASES MM. .1

9 2 .8

3 .1

3 .5

6 .6

0 .2







0 .2

0 .0 2 4

.25

95.7

2 .0

2 .2

4 .2

0 .1







0 .1

0 .023

K •s

8 7 .9

2 .2

2 .3

4 .5

3 .9

3 .0

0 .7



7 .6

0 .0 3 0

.75

9 6 .4

1 .7

1 .3

3 .0

0 .4

0 .1

0 .1



0 .6

0 .0 2 8

1 .0

95.3

2.3

1 .4

3 .7

0 .4



0 .4

0 .2

1 .0

0 .0 2 8

Control

95.1

2.5

2 .1

4 .6

0 .2

0 .1





0 .3

0.021

3-------------