Medicine, Faculty of
Cellular and Physiological Sciences, Department of
DSpace
UBCV
Capstick, Valerie
Master of Science - MSc
University of British Columbia
The study of rhabdomyosarcomas induced by nickel sulphide was undertaken in order to investigate their growth and responsiveness characteristics and to evaluate their possible usefulness as experimental tumour systems. A study was also made on the carcinogenic specificity of nickel sulphide. Intramuscular injections of 0.5 to 20 mg. of Ni₃S₂ resulted in tumours after two to six months, which grew fairly rapidly, once palpable. On transplantation they proved to be highly malignant, causing 100 per cent takes within approximately two weeks and death of the host within three to eight weeks. Detailed histological study was performed on many sections of tumour and muscle in order to establish identity of the rhabdomyosarcomas and to observe such cytological phenomena as muscle degeneration, tissue disruption and development of anaplasia. The carcinogenic activity of Ni₃S₂ was emphasized by the rapidity of tumour induction observed after very low doses, while the difference in latent periods suggested some correlation between dose and response. Injections of Ni₃S₂ into body organs illustrated the extreme toxicity of the substance in individual tissues. Although results were somewhat inconclusive within the time limit of the experiment, the general tenor of the data suggested that Ni₃S₂ had a toxic rather than tumourigenic action on most tissues. The appearance of rhabdomyosarcomas when the compound was in contact with abdominal or leg musculature gave evidence that nickel sulphide might have some specificity for striated muscle. Preliminary experiments with NiS indicated that its carcinogenicity, if any, was considerably less than that of Ni₃S₂, with different solubility in muscle being a possible explanation. Several of the induced tumours were used for metabolic and experimental therapy studies with various compounds. Numerous experiments indicated that the tumour was essentially unreliable for such studies since growth rates and response were not reproducible in different tumour generations. However, some general trends were noted. Response to the corticoids ranged from residual to complete inhibition of many tumours during the period of treatment with Cortisol or cortisone. Subcutaneous injections of testosterone showed acceleration of growth in some tumour lines. Tests with the Vinca alkaloids suggested some correlation with clinical anti-tumour effects, indicating an increased effectiveness of both Vinblastine and Vincristine when administered during the first week after transplantation.
A STUDY OF RHABDOMYOSARCOMAS INDUCED BY NICKEL SULPHIDE IN RATS by VALERIE CAPSTICK B . S c , The U n i v e r s i t y o f B r i t i s h Columbia, 1961 A THESIS SUBMITTED IN PARTIAL'FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department, of P h y s i o l o g y We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1963 In presenting this thesis'in p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library sh a l l make i t freely available for reference and study. I further agree that per-mission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his'.representatives..- It i s understood'that copying, or publi-. cation of this thesis for f i n a n c i a l :gain shall not be allowed . without my written permission. Department 0 f Physiology The University of B r i t i s h Columbia, Vancouver 8 , Canada. Date A p r i l , 1963 ABSTRACT The study o f rhabdomyosarcomas induced by n i c k e l s u l p h i d e was undertaken i n order to i n v e s t i g a t e t h e i r growth and r e s p o n s i v e n e s s c h a r a c t e r i s t i c s and to e v a l u a t e t h e i r p o s s i b l e u s e f u l n e s s as experimental tumour systems. A study was a l s o made on the c a r c i n o g e n i c s p e c i f i c i t y o f n i c k e l s u l p h i d e . Intramuscular i n j e c t i o n s of 0.5 to 20 mg. of Ni^S2 r e s u l t e d i n tumours a f t e r two to s i x months, which grew f a i r l y r a p i d l y , once p a l p a b l e . On t r a n s p l a n t a t i o n they proved to be h i g h l y malignant, causing 100 per cent takes w i t h i n a p p r o x i -mately two weeks and death of the h o s t w i t h i n three to e i g h t weeks. D e t a i l e d h i s t o l o g i c a l study was performed on many s e c t i o n s of tumour and muscle i n order to e s t a b l i s h i d e n t i t y of the rhabdomyosarcomas and to observe such c y t o l o g i c a l phenomena as muscle degeneration, t i s s u e d i s r u p t i o n and development of a n a p l a s i a . The c a r c i n o g e n i c a c t i v i t y of Ni^S2 was emphasized by the r a p i d i t y of tumour i n d u c t i o n observed a f t e r v e r y low doses, w h i l e the d i f f e r e n c e i n l a t e n t p e r i o d s suggested some c o r r e l a t i o n between dose and response. I n j e c t i o n s of i n t o body organs i l l u s t r a t e d the extreme t o x i c i t y o f the substance i n i n d i v i d u a l t i s s u e s . A lthough r e s u l t s were some-what i n c o n c l u s i v e w i t h i n the time l i m i t o f the experiment, the g e n e r a l tenor o f the data suggested t h a t N i ^ ^ had a t o x i c r a t h e r than tumourigenic a c t i o n on most t i s s u e s . The appearance of rhabdomyosarcomas when the compound was i n co n t a c t w i t h abdominal or l e g musculature gave evidence t h a t n i c k e l s u l p h i d e might have some s p e c i f i c i t y f o r s t r i a t e d muscle. P r e l i m i n a r y experiments w i t h NiS i n d i c a t e d t h a t i t s c a r c i n o g e n i c i t y , i f any, was c o n s i d e r a b l y l e s s than t h a t o f NigS2, w i t h d i f f e r e n t s o l u b i l i t y i n muscle being a p o s s i b l e e x p l a n a t i o n . S e v e r a l o f the induced tumours were used f o r m e t a b o l i c and experimental therapy s t u d i e s w i t h v a r i o u s compounds. Numerous experiments i n d i c a t e d t h a t the tumour was e s s e n t i a l l y u n r e l i a b l e f o r such s t u d i e s s i n c e growth r a t e s and response were not r e p r o d u c i b l e i n d i f f e r e n t tumour g e n e r a t i o n s . However, some g e n e r a l trends were noted. Response to the c o r t i c o i d s ranged from r e s i d u a l to complete i n h i b i t i o n o f many tumours du r i n g the p e r i o d o f treatment w i t h C o r t i s o l or c o r t i s o n e . Subcutaneous i n j e c t i o n s o f t e s t o s t e r o n e showed a c c e l e r a t i o n of growth i n some tumour l i n e s . T e s t s w i t h the V i n c a a l k a l o i d s suggested some c o r r e l a t i o n w i t h c l i n i c a l anti-tumour e f f e c t s , i n d i c a t i n g an i n c r e a s e d e f f e c t i v e n e s s o f bot h V i n b l a s t i n e and V i n c r i s t i n e when a d m i n i s t e r e d d u r i n g the f i r s t week a f t e r t r a n s p l a n t a t i o n . ACKNOWLEDGEMENTS I would l i k e to express my s i n c e r e a p p r e c i a t i o n and thanks to Dr. R.L. Noble f o r the i n s p i r a t i o n p r o v i d e d by h i s i n v a l u a b l e suggestions and constant encouragement d u r i n g t h i s i n v e s t i g a t i o n . I am a l s o indebted to Dr. A.F. Burton f o r h e l p f u l d e t a i l s r e g a r d i n g s t e r o i d a c t i v i t y and to Dr. W.L. Dunn f o r h i s i n f o r m a t i v e a n a l y s i s o f h i s t o l o g i c a l s e c t i o n s . In a d d i t i o n , I must thank Mr. R.. Finegan, Miss E. I r v i n e and Mrs. R. King f o r t h e i r kindness i n a s s i s t i n g w i t h the t e c h n i c a l aspects o f the experiments. T h i s r e s e a r c h was made p o s s i b l e by a Studentship from the N a t i o n a l Research C o u n c i l and by support from the N a t i o n a l Cancer I n s t i t u t e . The use of l a b o r a t o r y f a c i l i t i e s a t the Cancer Research Centre, U.B.C, i s g r a t e f u l l y acknowledged. TABLE OF CONTENTS PAGE INTRODUCTION 1 Me t a l C a r c i n o g e n e s i s 1 Rhabdomyosarcomas 7 Pres e n t I n v e s t i g a t i o n 11 EXPERIMENTAL MATERIALS AND METHODS 16 M a t e r i a l s 16 Methods 17 RESULTS 19 Tumour I n d u c t i o n S t u d i e s w i t h Ni^S2 19 Tumour I n d u c t i o n S t u d i e s w i t h NiS 24 T r a n s p l a n t a t i o n 28 H i s t o l o g y 30 T h e r a p e u t i c S t u d i e s 44 DISCUSSION 52 CONCLUSIONS 81 APPENDIX 84 BIBLIOGRAPHY 85 LIST OF TABLES TABLE PAGE I . Response of Rats to Intramuscular Doses of N i 3 S 2 i 20 I I . Response of Rhabdomyosarcomas to V i n b l a s t i n e 45 I I I . Response of Rhabdomyosarcomas to V i n c r i s t i n e 47 IV. E f f e c t s o f S t e r o i d s on Rhabdomyosarcomas and body weight. 49 V. E f f e c t o f T e s t o s t e r o n e on Rhabdomyosarcomas 51 LIST OF FIGURES FIGURE PAGE 1. Normal Muscle Showing S t r i a t i o n s 32 2. Ni^S2 Induced Primary Rhabdomyosarcoma 32 3. Ni^S2 Induced Primary Rhabdomyosarcoma w i t h Strap C e l l s 33 4. Induced Rhabdomyosarcoma w i t h Spider-web C e l l s . . 33 5. Pleomorphic Rhabdomyosarcoma w i t h G i a n t C e l l 35 6. l n < ^ u c e c ^ Rhabdomyosarcoma w i t h M i t o t i c F i g u r e s . . . 35 7. Benign F i b r o u s Tumour Induced by Ni^S2 37 8. F i b r o u s Rhabdomyoma Induced by NiS 37 9. D i s r u p t e d Muscle, a f t e r I n j e c t i o n w i t h NiS 39 10. Muscle, E i g h t Weeks a f t e r I n j e c t i o n of NiS 39 11. Attachment of Tumour to P e r i t o n e a l W a l l a f t e r I n t r a -h e p a t i c I n j e c t i o n 41 12. Attachment of Above Tumour to G a s t r o i n t e s t i n a l T r a c t . . 41 13. T e s t i c u l a r T i s s u e S i x Months a f t e r I n j e c t i o n of Ni^S2. 42 14. Macrophages and N i k k e i P a r t i c l e s i n I n t e r s t i t i a l Spaces of Above T i s s u e 42 15. E f f e c t o f V i n c a l e u k o b l a s t i n e on Growth of 2-N-5 44 16. E f f e c t of V i n c r i s t i n e on Growth of 2-N-5 46 17. E f f e c t of C o r t i c o i d s on Growth of 2-N-5 48 INTRODUCTION Metal C a r c i n o g e n e s i s In 1959 Hueper pre s e n t e d a comprehensive review o f the known o c c u p a t i o n a l c a r c i n o g e n s , which number over two hundred, and s t a t e d t h a t s e v e r a l metals, i n c l u d i n g a r s e n i c , chromate, b e r y l l i u m and n i c k e l , e x h i b i t e d p o t e n t i a l c a u s a l r e l a t i o n s h i p to cancer i n humans (50). He emphasized the potency o f the suspected carcinogens by s t a t i n g t h a t i f a person were exposed to a c a r c i n o g e n i c agent the ensuing development of cancer was only a matter o f time. The time l a g between exposure and tumour appearance, or the l a t e n t p e r i o d , has been shown to be c h a r a c t e r -i s t i c a l l y long i n metal c a r c i n o g e n e s i s , w i t h the average l a t e n t p e r i o d f o r a r s e n i c a l cancer b e i n g t w e n t y - f i v e y e a r s ; f o r chromate, f i f t e e n y e a r s ; f o r n i c k e l , twenty-two y e a r s . These f i g u r e s served to re-emphasize the main c h a r a c t e r i s t i c o f environmental and o c c u p a t i o n a l cancers which had been i m p l i e d by P o t t s almost two hundred years ago, when he pr e s e n t e d h i s c l a s s i c a l account of s c r o t a l cancer i n chimney sweeps (41). He was the f i r s t to r e c o g n i z e the long l a t e n t p e r i o d between exposure and develop-ment of the d i s e a s e . As w e l l as time f o r development, the other determining f a c t o r i n metal c a r c i n o g e n e s i s was s a i d to be the 2 amount of exposure. Most of the proven c a r c i n o g e n i c metals ex e r t e d t h e i r d e l e t e r i o u s e f f e c t s a f t e r i n h a l a t i o n , as had been i n d i c a t e d i n the many r e p o r t s of lung and n a s a l c a n c e r s . I t was t h e r e f o r e suggested by Hueper t h a t a man i n a r e f i n e r y who worked harder a t h i s job and breathed- more deeply c o u l d have a g r e a t e r chance of g e t t i n g lung cancer, i f a harmful metal were pr e s e n t i n the dust, than someone on a l e s s p h y s i c a l l y strenuous job i n the same exposed l o c a t i o n (50). Neubauer has s t a t e d t h a t the e a r l i e s t r e p o r t of cancer l i n k e d to a s p e c i f i c metal appeared i n 1822 when P a r i s r e p o r t e d cases o f s k i n cancers i n men working i n copper smelters and t i n r e f i n e r i e s (65). H i s b e l i e f t h a t the neoplasms were due to a r s e n i c i n the p r o c e s s fumes was g e n e r a l l y i g n o r e d f o r many ye a r s . In 1879 some cases o f lung cancer i n Germany were a t t r i b u t e d to the presence of a r s e n i c i n the dust of c e r t a i n f a c t o r i e s . T h i s evidence s t i l l was not accepted, s i n c e i t was f e l t t h a t those a f f l i c t e d had been exposed to' other c a r c i n o -genic agents. S i n c e t h a t time, however, the i m p l i c a t i o n of a r s e n i c as a c a u s a l agent has g r a d u a l l y become b e t t e r e s t a b l i s h e d . Although the number o f cases of lung cancer r e p o r t e d has not been g r e a t , there seemed to be s i g n i f i c a n t l y more deaths from lung cancer among ar s e n i c - e x p o s e d workers than among the average 3 population (43). Arsenic has also been shown to cause skin cancer when ingested (65). Animal experiments with arsenic have not been conclusive, since the latent period required was generally longer than the l i f e span of the animals. A similar struggle ensued to have chromate recognized as a possible metal carcinogen. From 1911 to 1932 there were occasional reports of respiratory system tumours associated with exposure to chromates. The f i r s t lung cancer positively linked with chromate was in 1911 in Germany, when two cases were reported by P f e i l (7). Twenty-four years later he liste d another five cases in the same plant. Machle reviewed the relationship of chromates to cancer in the United States and stated that between 1937 and 1947, forty-two cancer deaths, most of them due to bronchogenic carcinomas, had been reported in men working in the chromium industry (60). With Switzerland and England contributing only occasional cases, Germany and the United States recorded a majority of the 122 cases of respiratory tract cancer reported in chromate workers up to 1950 (7). The exposure time was between sixteen and twenty-two years, and the average age of death was fifty-two years. The death rate for lung cancer in chromate workers in the United States was calculated to be twenty-five times greater than the normal rate (60) and the carcinogenicity of chromate has now been acknowledged. 4 B e r y l l i u m i s one of the metals which as y e t has been c o n s i d e r e d o n l y as a p o s s i b l e c a r c i n o g e n i c hazard to man. C l i n i c a l l y , i n h a l a t i o n of b e r y l l i u m oxide has l e d to a c u t e and c h r o n i c pulmonary inflammations, but not to n e o p l a s t i c changes. E x p e r i m e n t a l l y , however, osteogenic sarcomas have been produced i n r a b b i t s e i t h e r by intravenous i n j e c t i o n or by i n h a l a t i o n of dust c o n t a i n i n g b e r y l l i u m oxide (22). The l a t t e r method i s p a r t i c u l a r l y i n t e r e s t i n g s i n c e i t suggests t h a t the b e r y l l i u m p a r t i c l e s might be phagocytosed i n passage through the lung and c a r r i e d v i a b l o o d and lymph to bone, a p r o c e s s which c o u l d c o n c e i v a b l y occur i n man. . E x p e r i m e n t a l s t u d i e s of the p o s s i b l e c a r c i n o g e n i c e f f e c t s of metals have assumed importance i n view of t h e i r e x t e n s i v e use not o n l y i n i n d u s t r y but a l s o i n common commercial p r o d u c t s . A r s e n i c i s found i n ores of copper, z i n c , s i l v e r , c o b a l t and l e a d , and i s employed as a p e s t i c i d e and i n weed-k i l l e r s . Chromate i s used i n s t a i n l e s s s t e e l , i n k s and p a i n t s , as w e l l as i n the manufacture o f gas t u r b i n e s and j e t engines. B e r y l l i u m has been i n c o r p o r a t e d i n t o e l e c t r i c a l h e a t i n g elements(50) The emergence of n i c k e l as a c a r c i n o g e n i c compound was r e c o r d e d i n 1932 when lung and sinus cancers were noted among men i n the Mond n i c k e l works (22) . Hueper r e p o r t e d t h a t ( i n 1949 the annual r e p o r t of the C h i e f I n s p e c t o r of F a c t o r i e s and 5 Workshops i n B r i t a i n l i s t e d f o r t y - s e v e n cases o f n a s a l cancer and eighty-two of lung cancer i n England d u r i n g the p e r i o d 1923 to 1948 (47). D o l l estimated t h a t t h i s i n c i d e n c e was t h i r t y times above normal f o r n a s a l cancer and f i v e times above normal f o r lung cancer, i m p l y i n g a d e f i n i t e c a r c i n o g e n i c agent somewhere i n the n i c k e l r e f i n e r i e s (19). As w i t h some of the other metals mentioned, the l a t e n t p e r i o d d i d not depend on the i n i t i a l age o f exposure, but on the a c t u a l l e n g t h of the exposure p e r i o d . Passey pr e s e n t e d a theory t h a t s i n c e t h e r e was o f t e n a long p r e c e d i n g h i s t o r y of i r r i t a t i o n or lung damage such as b r o n c h i t i s , there may have been excess mucus b l a n k e t i n g normal exchange of gases and f l u i d s underneath, c a u s i n g n i c k e l to s e t t l e i n t o the a l v e o l i (69). A s i m i l a r h y p o t h e s i s was suggested by Hueper, who f e l t t h a t i n h a l e d n i c k e l c a r b o n y l c o u l d be r a p i d l y decom-posed i n the lungs to carbon monoxide and m e t a l l i c n i c k e l , the l a t t e r then being p r e c i p i t a t e d on r e s p i r a t o r y s u r f a c e s (49). T h i s c i r c u m s t a n t i a l evidence o f the c a r c i n o g e n i c a c t i v i t y of n i c k e l compounds p r e s e n t s an i n t e r e s t i n g problem i n view of the u b i q u i t o u s occurrence of n i c k e l and i t s s a l t s i n everyday circumstances. N i c k e l has been found g e n e r a l l y i n s o i l s and p l a n t s throughout the world. . The l a r g e s t d e p o s i t s f o r i n d u s t r i a l purposes a r e found i n Canada and New C a l e d o n i a , 6 where i t occurs as d e p o s i t s of s u l p h i d e o r e . N i c k e l i s known as a p a r t i c u l a r l y common component o f i n d u s t r i a l p r o c e s s e s and household a r t i c l e s . I n d u s t r i a l l y i t i s used i n p r e p a r i n g a l l o y s w i t h copper, magnewium, z i n c , chromium, i r o n and molybdenum, and i n making c o r r o s i o n - r e s i s t a n t and h e a t - r e s i s t a n t s t e e l and c a s t i r o n (57). Two processes which have developed the most widespread i n d u s t r i a l use of n i c k e l a r e e l e c t r o p l a t i n g , i n which n i c k e l ammonium sulphate Is used as the e l e c t r o l y t i c f l u i d , and hydrogenation of o i l s , i n which pure n i c k e l serves as a c a t a l y s t (23). Some of the more common a r t i c l e s c o n t a i n i n g n i c k e l i n c l u d e s t a i n l e s s s t e e l p r o d u c t s , k i t c h e n u t e n s i l s , l i n o l e u m , p a i n t s , i n k s , o i l , s torage b a t t e r i e s , c o i n s and j e w e l r y (47, 48, 57). Hueper has performed s e v e r a l e xperimental s t u d i e s on the c a r c i n o g e n i c a c t i v i t y o f n i c k e l which was o b t a i n e d from i n d u s t r i a l d i s i n t e g r a t i o n o f n i c k e l c a r b o n y l . I n t e r e s t arose because of c i r c u m s t a n t i a l evidence i n v o l v i n g adverse e f f e c t s of the l a t t e r compound. I n j e c t i o n of f i f t y m i l l i g r a m s of m e t a l l i c n i c k e l i n t o femoral and p l e u r a l areas r e s u l t e d i n 30 per cent tumours i n seven to s i x t e e n months. In some lungs n i c k e l was embedded i n p l e u r a and c a p i l l a r i e s , i n d i c a t i n g t h a t i t c o u l d be p r e c i p i t a t e d t h e r e . Of the femoral a r e a tumours o n l y 25 per cent were of muscular o r i g i n (48). Other tumour 7 types i n c l u d e d o s t e o g e n i c s p i n d l e c e l l sarcomas, squamous c e l l carcinomas and r e t i c u l u m c e l l sarcomas, w h i l e an e a r l i e r exper-iment had shown no muscle tumours a t a l l (46) . He concluded, from the v a r i e t y of h i s t o l o g i c a l neoplasms p r e s e n t , t h a t although n i c k e l d i d have c a r c i n o g e n i c a c t i v i t y , t h e r e was no t i s s u e s p e c i f i c i t y a s s o c i a t e d w i t h n i c k e l c a r c i n o g e n e s i s . T h i s p o i n t of view was i n d i r e c t c o n t r a s t to t h a t of Gilman who f e l t t h a t , e x p e r i m e n t a l l y , n i c k e l showed a d e f i n i t e p r e f e r e n c e f o r s t r i a t e d muscle. Both o b s e r v a t i o n s a r e s u b j e c t to some c r i t i c i s m w i t h r e g a r d to the experimental work. Gilman i n j e c t e d n i c k e l mainly i n t o areas of s t r i a t e d muscle, w i t h o n l y a few animals r e c e i v i n g n i c k e l i n other body areas (30) . The l a t t e r experiments c o n s i s t e d of i n j e c t i o n s i n t o the abdominal c a v i t y which r e s u l t e d i n a few tumours i n v o l v i n g the omentum and diaphragm. Hueper, on the other hand, o b t a i n e d the m a j o r i t y of h i s data from experiments i n v o l v i n g i n j e c t i o n s i n t o the bone marrow of the femur, where i t i s c o n c e i v a b l e t h a t leakage a l o n g the i n j e c t i o n p a t h c o u l d have r e s u l t e d i n d i v e r s e tumours of the surrounding t i s s u e s . Rhab domyo s ar coma s Primary tumours de v e l o p i n g from the mesenchymal element of muscle a r e c o n s i d e r e d to be extremely r a r e . The most 8 malignant type i s known as a rhabdomyosarcoma or tumour of s t r i a t e d muscle. C l i n i c a l l y i t i s s t a t e d t h a t these neoplasms make up ten to f i f t e e n per cent o f sarcomas of the s o f t somatic t i s s u e s , and t h a t approximately tw o - t h i r d s a r i s e i n the lower e x t r e m i t i e s . Other gross c h a r a c t e r i s t i c s have been d e s c r i b e d such as l i m i t e d m o b i l i t y , r a p i d growth and i n v a s i v e n e s s , n e c e s s i t a t i n g e x t e n s i v e removal of t i s s u e or complete limb amputation, i f f e a s i b l e ( 1 ) . The spontaneous appearance o f rhabdomyosarcomas i n animals has o c c u r r e d o n l y r a r e l y . I s o l a t e d r e p o r t s o f such tumours have appeared s i n c e 1896, d e s c r i b i n g neoplasms i n the t a i l o f a s t a l l i o n , the kidney of a hog, the h e a r t o f a cow, and i n t r o u t and c o d f i s h (10). The e a r l i e s t documented r e p o r t of the neoplasm i n rodents was i n 1922 when B u l l o c k and C u r t i s d e s c r i b e d a chondrorhabdomyosarcoma i n the s t e r n a l musculature of a r a t (10). The tumour had the now f a m i l i a r appearance of g i a n t c e l l s and i r r e g u l a r p o l y h e d r a l c e l l s w i t h c r o s s - s t r i a t i o n s . Maddock, Kury and R i l e y r e p o r t e d another spontaneous rhabdomyo-sarcoma i n 1962, t h i s one o c c u r i n g i n the s t e r n a l a r e a as w e l l (61). I t s o r i g i n a l appearance, h i g h l y f i b r o u s w i t h s m a l l round c e l l s , e v e n t u a l l y changed to one of complete pleomorphism, w i t h no r e l a t i o n s h i p to the s t r i a t e d f i b e r s of o r i g i n . 9 Expe r i m e n t a l p r o d u c t i o n o f rhabdomyosarcomas has been e q u a l l y uncommon. In 1954 Heath began t e s t i n g the c a r c i n o g e n i c a c t i o n o f c o b a l t (36). I n j e c t i o n of approximately twenty-eight m i l l i g r a m s of pure c o b a l t i n t o Hooded r a t s r e s u l t e d i n the i n d u c t i o n of 50 per cent rhabdomyosarcomas a f t e r s i x months. F u r t h e r s t u d i e s on the a c t i v i t y o f i n j e c t e d c o b a l t i n muscle l e d to the sugg e s t i o n t h a t t h i s metal seemed to have some s p e c i f i c i t y f o r ca u s i n g s t r i a t e d muscle tumours (37) . . . Another r e p o r t of t h e i r e xperimental i n d u c t i o n by metals f o l l o w e d from Gilman and c o l l e a g u e s a t Guelph, who announced t h a t rhabdomyosarcomas c o u l d be induced i n c e r t a i n s t r a i n s o f experimental animals by n i c k e l s u l p h i d e (29) . Work had begun i n 1960 on t e s t i n g the e f f e c t s o f i n j e c t i o n o f a r e f i n e r y dust which c o n t a i n e d n i c k e l , c o b a l t , copper and i r o n s i l i c o n , i n order to i n v e s t i g a t e the p o s s i b i l i t y t h a t such dust might be c a r c i n o g e n i c (28). I n j e c t i o n of t h i s mixture i n t r a -m u s c u l a r l y i n t o r a t s and mice r e s u l t e d i n approximately 70 per cent tumours a r i s i n g a t the s i t e o f i n j e c t i o n . The l a t e n t p e r i o d i n mice was twice as long as i n r a t s and the tumours were mainly f i b r o s a r c o m a s , w h i l e Xhose i n the l a t t e r s p e c i e s were rhabdomyosarcomas. As an e x t e n s i o n o f t h i s experiment a study on the c a r c i n o g e n i c a c t i v i t y o f v a r i o u s s a l t s o f the heavy metals 10 mentioned p r e v i o u s l y was performed (29) . A standard dose o f twenty m i l l i g r a m s per t h i g h muscle was a d m i n i s t e r e d to r a t s . The response of Hooded and Wistar r a t s to c u p r i c o x i d e , f e r r i c oxide and n i c k e l s u l phate was n e g a t i v e a f t e r 415 days, w h i l e n i c k e l o x i d e , c o b a l t oxide and n i c k e l s u l p h i d e (Ni3S2) showed a p o s i t i v e response. The l a t t e r compound produced the g r e a t e s t number of tumours. Almost a l l of the tumours induced by n i c k e l s u l p h i d e and c o b a l t oxide, and a m a j o r i t y o f those induced by n i c k e l o xide, were rhabdomyosarcomas, h i g h l y c e l l u l a r and pleomorphic. Metastases to both lungs and lymph nodes were noted i n 95 per cent of the r a t s g i v e n n i c k e l s u l p h i d e . In an attempt to c o r r e l a t e tumour i n c i d e n c e w i t h dosage of n i c k e l s u l p h i d e , i t was found t h a t o c c a s i o n a l tumours (3 per cent) appeared i n r a t s g i v e n i n t r a m u s c u l a r doses as low as 0.5 m i l l i g r a m s , w i t h some r e l a t i o n s h i p b e i n g shown between s i z e of i n i t i a l dose and per cent response. The c o n c l u s i o n was made t h a t the observed c a r c i n o g e n i c i t y of c e r t a i n n i c k e l and c o b a l t compounds supported the h y p o t h e s i s t h a t such dusts i n the r e f i n e r y i n d u s t r y might c o n s t i t u t e an i n d u s t r i a l cancer hazard. T h i s would tend to s u b s t a n t i a t e the evidence o b t a i n e d i n r e f i n e r y s t u d i e s , where the m a j o r i t y of tumours have i n v o l v e d lung and n a s a l areas which 11 would l o g i c a l l y be exposed to the dust. F u r t h e r i n t e r e s t has s i n c e been c o n c e n t r a t e d on n i c k e l s u l p h i d e as a means of i n d u c i n g rhabdomyosarcomas s i n c e i t appeared to be the most potent metal compound of those t e s t e d . P r e s e n t I n v e s t i g a t i o n The use of n i c k e l s u l p h i d e as an agent f o r the inducement o f rhabdomyosarcomas i n animals was o r i g i n a l l y adopted f o r the p r e s e n t i n v e s t i g a t i o n i n an attempt to o b t a i n a r e l i a b l e tumour system f o r experimental t e s t i n g of a group of chemo-t h e r a p e u t i c compounds known as V i n c a a l k a l o i d s . R e l a t i v e l y few cases o f treatment o f these neoplasms by chemotherapeutic agents have been c i t e d i n c l i n i c a l l i t e r a -t u r e . However, a r e c e n t a r t i c l e by Whitelaw and Teasdale r e p o r t e d treatment of a rhabdomyosarcoma w i t h an a l k a l o i d used i n treatment of other s o l i d tumours (83) . A six-month o l d boy was examined f o r a lump on h i s b u t t o c k , which was diagnosed as an embryonal rhabdomyosarcoma. R a d i a t i o n had no e f f e c t on the tumour mass and by e i g h t months i t had spread to the r i g h t i n g u i n a l area and the r i g h t lower abdomen. The compound s e l e c t e d f o r chemotherapy, V i n b l a s t i n e , was a d m i n i s t e r e d twice i n doses of 1.5 m i l l i g r a m s , i n j e c t e d i n t r a v e n o u s l y . The tumour r e g r e s s e d , then r e c u r r e d a g a i n a t twelve months. A t t h i s time two doses o f 12 2.5 m i l l i g r a m s were a d m i n i s t e r e d and r e p e a t e d a g a i n i n two weeks. A f t e r t h i s treatment, the tumour disappeared completely and showed no s i g n of r e c u r r e n c e a t s i x t e e n months. V i n b l a s t i n e has been t e s t e d a g a i n s t a l a r g e number of human tumours which have e x h i b i t e d varied responses. Most e f f e c t i v e response has been o b t a i n e d i n Hodgkin's d i s e a s e , cancer of the b r e a s t and lung, and c h o r i o e p i t h e l i o m a . In an e x t e n s i v e review of the V i n c a a l k a l o i d s , Johnson e t a L c i t e d four cases of rhabdomyo-sarcomas which had been t r e a t e d w i t h V i n b l a s t i n e ( 5 7 ) . C l i n i c a l l y the dose has been somewhat l i m i t e d by a l e u k o p e n i c e f f e c t which may cause a g e n e r a l i z e d i n c r e a s e d s u s c e p t i b i l i t y to i n f e c t i o n . The V i n c a a l k a l o i d s a r e o f i n t e r e s t to t h i s Research Centre s i n c e they have been used e x t e n s i v e l y i n experimental tumour s t u d i e s . Although c l i n i c a l i n t r o d u c t i o n of these compounds was not made u n t i l 1958, the o r i g i n a l experimental o b s e r v a t i o n s were r e c o r d e d i n 1949 when Noble was s t u d y i n g the e f f e c t s o f an e x t r a c t of the p e r i w i n k l e p l a n t , V i n c a r o s e a ( L i n n . ) , on carbohydrate metabolism (69). Although p r i m i t i v e f o l k l o r e had suggested i t s v a l u e i n the c o n t r o l o f d i a b e t e s m e l l i t u s , e x t r a c t s o f the p l a n t g i v e n o r a l l y to experimental animals proved to have no e f f e c t on the b l o o d sugar of normal 13 or d i a b e t i c r a t s or r a b b i t s . In an attempt to i n c r e a s e the p o s s i b l e e f f e c t i v e n e s s of the e x t r a c t i t was a d m i n i s t e r e d i n t r a p e r i t o n e a l l y to r a t s . Again t h e r e was no change i n the b l o o d sugar l e v e l but an unexpected s i d e - e f f e c t was observed. In most cases death o c c u r r e d i n f i v e to seven days from a r a p i d , severe i n f e c t i o n shown to be a s s o c i a t e d w i t h a marked r e d u c t i o n i n c i r c u l a t i n g l e u k o c y t e s . Concomitant w i t h the r a p i d l y f a l l i n g white b l o o d count there was a marked d e s t r u c t i o n of g r a n u l o c y t e s and p r o f o u n d l y depressed bone marrow. I t was decided to use the leukopenic e f f e c t i n r a t s as an assay method i n attempting to i s o l a t e an a c t i v e p r i n c i p l e from crude e x t r a c t s of p e r i -w i n k l e . Beer was e v e n t u a l l y a b l e to i s o l a t e a c r y s t a l l i n e a l k a l o i d from a number of a c t i v e substances i n the p l a n t (17). I t was subsequently shown to be of a new c l a s s o f chemical compounds, d i m e r i c i n d o l e a l k a l o i d s , and was c a l l e d V i n c a -l e u k o b l a s t i n e ( V i n b l a s t i n e ) . Concurrent r e s e a r c h c a r r i e d out a t the E l i L i l l y l a b o r a t o r i e s r e s u l t e d i n the i s o l a t i o n o f a number of other a l k a l o i d s , one of which, L e u r o c r i s t i n e ( V i n c r i s t i n e ) , v a r i e d o n l y s l i g h t l y i n chemical composition (51). Both a l k a l o i d s have now been t e s t e d a g a i n s t v a r i o u s animal tumours such as the P1534 leukemia, E h r l i c h a s c i t e s , c h l o r o l e u k e m i a , AKR leukemia and o t h e r s . In many cases the 14 anti-tumour e f f e c t has been l i m i t e d by the leukopenia which r e s u l t s from h i g h e r doses. Although V i n b l a s t i n e and V i n c r i s t i n e have both demonstrated a n t i - m i t o t i c a c t i v i t y i n a l l tumours t e s t e d , these r e s u l t s have not c o r r e l a t e d w i t h anti-tumour a c t i v i t y (51). The a l k a l o i d s appeared to e f f e c t the most s t r i k i n g i n h i b i t i o n of tumour growth or number of takes when ad m i n i s t e r e d soon a f t e r t r a n s p l a n t a t i o n . T e s t i n g of the compounds a g a i n s t new tumour l i n e s has been s t a n d a r d i z e d by g i v i n g ten c o n s e c u t i v e d a i l y i n j e c t i o n s , b e g i n n i n g twenty-four hours a f t e r t r a n s p l a n t a t i o n . I t was b e l i e v e d , t h e r e f o r e , t h a t a r e l i a b l e r a t rhabdomyosarcoma might serve as an experimental model f o r V i n b l a s t i n e and V i n c r i s t i n e , p a r t i c u l a r l y i n view of the observed c l i n i c a l e f f e c t s of the former on rhabdomyosarcomas i n humans. D i f f e r e n t dose regimens and times of a d m i n i s t r a t i o n were planned f o r chemotherapeutic t e s t i n g on s e v e r a l l i n e s of the induced tumours. Because of the s t r i a t e d muscle o r i g i n of rhabdomyo-sarcomas they were c o n s i d e r e d to be u s e f u l f o r s t u d y i n g m e t a b o l i c e f f e c t s of c e r t a i n a n a b o l i c and c a t a b o l i c hormones, a g a i n w i t h the i d e a of attempting to e s t a b l i s h e xperimental models f o r v a r i o u s s t e r o i d hormones. In a d d i t i o n , the p o s s i b l e growth-15 i n h i b i t i n g e f f e cts of corticosteroids on thi s experimental neoplasm were of some therapeutic i n t e r e s t . At the same time, the induction of such tumours for the above studies offered an opportunity to study a phase of metal carcinogenesis. An attempt was made to observe the s p e c i f i c i t y and general carcinogenic a c t i v i t y of n i c k e l sulphide i n r a t s . As well, the use of a r e l i a b l e and potent agent for producing rhabdomyosarcomas was necessary i n order to study th e i r gradual development i n muscle and other organs, as influenced by experimental variables such as dosage, sex, st r a i n and age. More d i r e c t l y , i t was desirable to observe growth c h a r a c t e r i s t i c s and h i s t o l o g i c a l changes during the induction of rhabdomyosarcomas. EXPERIMENTAL MATERIALS AND METHODS 16 M a t e r i a l s N i c k e l Compounds. N i c k e l s u l p h i d e , i n the form of Ni3S2, was prepared by I n t e r n a t i o n a l N i c k e l Company of Canada i n the f o l l o w i n g manner (70): "The f i n e n i c k e l s u l p h i d e was produced by m e l t i n g r e f i n e d n i c k e l metal w i t h b r i g h t y e l l o w s u l p h u r . The molten s u l p h i d e was c a s t , s o l i d i f i e d , crushed i n a l a b o r a t o r y jaw c r u s h e r , ground i n a d i s c p u l v e r i z e r and the ground produced was f u r t h e r comminuted to the d e s i r e d s i z e i n a l a b o r a t o r y pebble m i l l . 1 1 - P a r t i c l e s i z e was approximately f i v e microns. The o r i g i n a l sample was o b t a i n e d from Dr. J.W. Gilman. N i c k e l s u l p h i d e , i n the form of NiS, was prepared by Dr. P.H. J e l l i n c k a t the Cancer Research Centre, U.B.C., i n the f o l l o w i n g manner: To twenty grams of n i c k e l o u s c h l o r i d e (Reagent S p e c i a l ) f i f t e e n ml. of A n a l y t i c a l Reagent grade ammonium s u l p h i d e was added arid the p r e c i p i t a t e o f n i c k e l o u s s u l p h i d e f i l t e r e d . The p r e c i p i t a t e was r e p e a t e d l y washed w i t h warm d i s t i l l e d water u n t i l the f i l t r a t e was c o l o u r l e s s and then washed s u c c e s s i v e l y w i t h warm acetone, e t h y l a l c o h o l , benzene and acetone a g a i n . The powder was d r i e d and p u l v e r i z e d . 17 Compounds were a d m i n i s t e r e d as suspensions i n d i s t i l l e d water or sesame o i l . Intramuscular i n j e c t i o n s were made i n t o the gastrocnemius muscle o f one.leg. I n j e c t i o n s i n t o v a r i o u s organs were performed by s u r g i c a l opening under ether a n e s t h e s i a . V i n c a A l k a l o i d s . V i n c a l e u k o b l a s t i n e and L e u r o c r i s t i n e were s u p p l i e d by E l i L i l l y Company under the trade names of V i n b l a s t i n e Sulphate (VLB) and V i n c r i s t i n e Sulphate (VCR) r e s p e c t i v e l y . Both compounds were d i s s o l v e d i n p h y s i o l o g i c a l s a l i n e and 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 . Hormones. Three c o r t i c o s t e r o i d s , C o r t i s o l , c o r t i s o n e and p r e d n i s o l o n e , were o b t a i n e d from N u t r i e n t B i o c h e m i c a l s C o r p o r a t i o n , and were a d m i n i s t e r e d i n t r a p e r i t o n e a l l y or sub-cutaneously i n water. T e s t o s t e r o n e P r o p i o n a t e was o b t a i n e d from F i s h e r Chemical Company and was a d m i n i s t e r e d subcutaneously i n sesame o i l . Methods Experimenta1 Anima1s. Male and female r a t s o f the f o l l o w i n g s t r a i n s were used: Hooded, F i s c h e r , P.A., Wistar and Sprague-Dawley. The f i r s t t h ree s t r a i n s were from i n b r e d stock maintained i n the l a b o r a t o r y . A l l animals were maintained on Master's Fox Chow d i e t ad l i b i t u m and were examined a t 18 p e r i o d i c i n t e r v a l s f o r tumour f o r m a t i o n . Tumour measurements were made w i t h c a l i p e r s i n two dimensions and tumour weights were c a l c u l a t e d from Shreck's formula, the t h i r d diameter being the mean of the two diameters measured (73). . Techniques o f T r a n s p l a n t a t i o n . Routine t r a n s p l a n t a t i o n was accomplished i n the f o l l o w i n g manner. A s m a l l p i e c e of tumour t i s s u e was e x c i s e d and chopped f i n e l y i n a P e t r i d i s h . I t was then homogenized i n a g l a s s blender w i t h approximately equal volumes of p h y s i o l o g i c a l s a l i n e . Approximately ten m i l l i o n c e l l s , determined by cou n t i n g i n a haemocytometer, were i n j e c t e d i n 0 . 2 c c . of suspension. A #21 needle was used and i n j e c t i o n was made i n t o the gastrocnemius muscle of r e c i p i e n t r a t s . Tumours were a s s i g n e d p r e f i x numbers to denote new tumour l i n e s and a f f i x numbers to i n d i c a t e t r a n s p l a n t g e n e r a t i o n s . Techniques o f H i s t o l o g y . T i s s u e s were f i x e d i n Bouin's f l u i d or 107o f o r m a l - s a l i n e . P a r a f f i n s e c t i o n s were s t a i n e d r o u t i n e l y w i t h haematoxylin and e o s i n . M a l l o r y ' s phospho-t u n g s t i c - a c i d haematoxylin and P e r l ' s s t a i n s were a p p l i e d to muscle, tumour and organ s e c t i o n s f o r s p e c i f i c h i s t o c h e m i c a l s t u d i e s . B a s i c techniques were o b t a i n e d from L i l l i e ' s t e x t -book of h i s t o l o g y ( 5 9 ) . 19 RESULTS Tumour I n d u c t i o n S t u d i e s w i t h ^ 3 8 2 I n j e c t i o n i n t o Muscle. Male and female Hooded r a t s weighing e i g h t y to one hundred grams were i n j e c t e d i n t r a -m u s c u l a r l y w i t h v a r y i n g doses of Ni3S2, r a n g i n g from 0.5 to 20 mg. The animals e x h i b i t e d no t o x i c s i d e - e f f e c t s or l o s s of weight a f t e r r e c e i v i n g the i n j e c t i o n s , and continued growing a t a normal r a t e . Tumours were r e c o r d e d as p a l p a b l e when they were approximately one cm. i n diameter or when an obvious s w e l l i n g of the l e g muscle was apparent. Animals were k i l l e d when tumours reached a s i z e of three to four cm. i n diameter, u s u a l l y one to two months a f t e r the f i r s t appearance. A t the time of death normal l e g f u n c t i o n was g e n e r a l l y i n h i b i t e d . L a t e n t p e r i o d s f o r tumour appearance ranged from ninety-two days f o r the h i g h e s t dose to almost two hundred days f o r the lowest, w i t h the f i r s t tumour appearing a f t e r f i f t y - e i g h t days. A f t e r n i n e months animals which had r e c e i v e d 10 and 20 mg. of Ni<jS2 had 100 per cent tumours; those r e c e i v i n g 2.5 mg. had 66 per cent w h i l e those w i t h 0.5 mg. had 40 per cent tumours. R e s u l t s are summarized i n Tab l e I . Autopsy showed t h a t the tumours g e n e r a l l y arose a t the s i t e o f i n j e c t i o n and were l o c a l i z e d i n the muscle r e g i o n , r e p l a c i n g the gastrocnemius muscle which had degenerated. An TABLE I RESPONSE OF RATS TO INTRAMUSCULAR DOSES OF NioS Dose Number of Tumours Average l a t e n t (mg.) animals Number % p e r i o d * 20 5 5 100 9 2 + 2 4 days 10 3 3 100 1 7 1 + 19 days 2.5 15 10 66 184 i - 21 days 0.5 15 6 40 191 + 23 days * f i r s t p a l p a b l e as hard s w e l l i n g w i t h i n muscle 20} e x c e p t i o n to t h i s statement was seen i n one female r a t i n which the tumour mass had i n f i l t r a t e d through the p e r i t o n e a l w a l l and formed a l a r g e , continuous mass i n the abdominal c a v i t y . P a r t i c l e s of n i c k e l were observable i n most tumour masses, s c a t t e r e d t h i n l y throughout the stroma. Most animals e x h i b i t e d m u l t i p l e nodules i n the lung which were f a i r l y hard and white i n appearance. However, those e x a m i n e d - h i s t o l o g i c a l l y confirmed the gross o b s e r v a t i o n of abcess f o r m a t i o n . No other unusual nodules were found. D e t a i l e d h i s t o l o g i c a l examination of these tumours, to be p r e s e n t e d l a t e r , c o r r o b o r a t e d gross evidence t h a t they were h i g h l y malignant rhabdomyosarcomas. One tumour a r i s i n g eleven months a f t e r i n j e c t i o n demonstrated a d i f f e r e n t growth c h a r a c t e r i s t i c i n t h a t i t was s i t u a t e d as a s m a l l white nodule w i t h i n the normal muscle mass r a t h e r than i n p l a c e of i t , and i t d i d not t r a n s p l a n t . H i s t o l o g y of t h i s tumour w i l l be compared w i t h those d e s c r i b e d above. Female Wistar r a t s , e i g h t y to one hundred grams, were i n j e c t e d w i t h 20 mg. o f Ni3S2 and e x h i b i t e d four tumours among t h i r t y a n imals. Many of the r a t s appeared to have s w e l l i n g s which arose d u r i n g the t h i r d month a t the s i t e o f i n j e c t i o n . However, these a l l r e g r e s s e d w i t h i n four weeks and no s i g n of any tumour formation was seen i n these a r e a s . Autopsy performed a t random on s e v e r a l animals a f t e r ten months r e v e a l e d t h a t no 22 n i c k e l p a r t i c l e s were p r e s e n t i n the i n j e c t e d muscle r e g i o n s . Of the r e s u l t i n g tumours, o n l y one was a t the i n j e c t i o n s i t e , and i t had spread from the muscle r e g i o n i n t o the lower r i g h t abdominal quadrant. T h i s tumour, 7-N, was t r a n s p l a n t e d e a s i l y and grew r a p i d l y , c a using death to the h o s t w i t h i n three weeks. A f t e r e i g h t months one r a t developed a lump on the neck which d i d not take on subcutaneous t r a n s p l a n t a t i o n , w h i l e another r a t developed two tumours a f t e r s i x t e e n months. These a r o s e i n the upper chest r e g i o n and i n the lower l e f t g r o i n ; both were l o o s e l y p a l p a b l e and a t t a c h e d to the s k i n . H i s t o l o g i c a l d e t a i l i s p r e s e n t e d i n another s e c t i o n . I n j e c t i o n i n t o Organs. In order to t e s t the a l l e g e d s p e c i f i c i t y o f n i c k e l f o r i n d u c i n g tumours i n s t r i a t e d muscle, Ni3S2 was i n j e c t e d i n t o v a r i o u s organs i n Hooded r a t s , i n doses of approximately 1 to 2 mg. I n j e c t i o n s were made i n t o l i v e r , s pleen, t e s t i s , p r o s t a t e , u t e r u s and o v a r i a n c a p s u l e . Two out of three animals r e c e i v i n g an i n t r a h e p a t i c dose developed huge tumours a f t e r s i x months which, a t the time of death, f i l l e d the abdominal c a v i t y and were g r o s s l y p a l p a b l e from the e x t e r n a l s u r f a c e . Although p a r t of the tumour was s i t u a t e d deep among the lobes of the l i v e r , the o r i g i n appeared to be the p e r i t o n e a l w a l l immediately o v e r l y i n g the l i v e r . The 23 tumour had spread to the i n t e s t i n a l t r a c t as w e l l , where i t appeared to be w e l l a t t a c h e d . T r a n s p l a n t a t i o n of s m a l l p i e c e s of the tumour was e a s i l y a c h i e v e d f o r s e v e r a l g e n e r a t i o n s . Both the o r i g i n a l tumour and a l l t r a n s p l a n t s were t y p i c a l rhabdomyosarcomas on h i s t o l o g i c a l examination. In another r a t , the n i c k e l was found to be e n c l o s e d i n a s m a l l hard pocket of t i s s u e adhered to the abdominal w a l l , but no tumour forma t i o n was seen. The i n j e c t i o n o f ^382 i n t o t e s t e s d i d not appear to have d e l e t e r i o u s e f f e c t s on the g e n e r a l h e a l t h of the r a t s . Autopsy performed a f t e r s i x and n i n e months showed t h a t the n i c k e l p a r t i c l e s were s t i l l l o c a l i z e d i n the i n j e c t i o n a r e a . G r o s s l y the t e s t i s c o n t a i n e d b l a c k and g r i t t y m a t e r i a l , w i t h the metal completely surrounding the sem i n i f e r o u s t u b u l e s . The u n t r e a t e d t e s t i s showed no a b n o r m a l i t i e s . S e c t i o n s of these and other organs were made f o r h i s t o l o g i c a l study, to be d e s c r i b e d l a t e r . Two r a t s which had r e c e i v e d i n t r a p r o s t a t i c i n j e c t i o n s developed l a r g e lumps a t the s i t e s h o r t l y a f t e r the o p e r a t i o n . Autopsy showed t h a t t h i s was abcess formation around the suture l i n e . A t h i r d r a t had a l a r g e s w e l l i n g o v e r l y i n g the t e s t i c u l a r a rea which was due to g r e a t displacement o f the i n t e s t i n a l t r a c t . 24 The prostate gland was hard and f u l l of n i c k e l , with no evidence of abnormal enlargement, while the testes did not seem to be affected by the i n t e s t i n a l compression. A small white lump, approximately f i v e mm. i n diameter was located i n the spleen of one r a t which had received an i n j e c t i o n i n that area nine months previously. The nodule, which was very hard and showed some n i c k e l p a r t i c l e s i n the center, occupied one end of the spleen and the r e s t of the organ appeared to be normal. Injections into the ovarian capsule and the uterus produced no obvious changes i n the organs when examined grossly a f t e r nine months. In general the animals survived w e l l . No deaths occurred as an immediate r e s u l t of the operation and only two out of eighteen rats succumbed during the nine-month period. Tumour Induction Studies with NiS Injection into Muscle. For comparative purposes a second form of n i c k e l sulphide, NiS, was tested for carcino-genic a c t i v i t y i n r a t s . Intramuscular inj e c t i o n s of NiS were made into a t o t a l of 150 rats i n order to observe the effects of the following factors on tumour induction. S t r a i n differences were tested by i n j e c t i n g NiS into female rats of s i x s t r a i n s : 25 Hooded,. Fischer, P.A., Sprague-Dawley, UBJC. Wistar, Woodlyn Farm Wistar. Approximately h a l f of the Sprague-Dawleys and UBJC. Wis tars died during the f i r s t two months af t e r i n j e c t i o n . The l a t t e r group was composed of younger r a t s , weighing t h i r t y to f o r t y grams, i n contrast to average weights of eighty to one hundred grams i n the other s t r a i n s . After four months, none of the surviving rats showed any evidence of tumours. The e f f e c t of sex on response to NiS was tested by i n j e c t i n g 20 mg. into male and female Hooded and Wistar r a t s , while age differences were studied by the i n j e c t i o n of 5 to 10 mg. into Hooded rats at ages of six hours, two days, nine days, three weeks and four months. In the age experiments, intramuscular inje c t i o n s into newborn and baby rats (up to nine days) proved almost impossible because of the small quantity of muscle a v a i l a b l e . However, an approximate dose, as indicated above, was injected i n 0.05 cc. d i s t i l l e d water into the hind thigh area. The suspension was seen to spread beneath the skin over the entire v e n t r a l leg. Mor t a l i t y was high i n experiments involving young animals, so that many had to be repeated i n order to obtain s i g n i f i c a n t numbers of treated animals. I t was soon apparent that NiS was much more toxic than Ni3S2 and of less carcinogenic potency. After four months 26 o n l y two tumours were v i s i b l e and two others p a l p a b l e as s w e l l i n g s i n the i n j e c t e d muscle r e g i o n . A l l were found i n Hooded r a t s which had r e c e i v e d 20 mg. of NiS a t the age of four months. One tumour was e x c i s e d f o r t r a n s p l a n t a t i o n (which was u n s u c c e s s f u l ) , and showed some clumps of n i c k e l throughout the tumour mass. The f i r m , white nodule was s i t u a t e d a t the edge o f the gastrocnemius muscle and appeared to be c o n t a i n e d i n a f i b r o u s sheath which a c t e d as the p o i n t of attachment to the gastrocnemius. The i n j e c t e d muscle s t i l l had a pocket of n i c k e l which was g r o s s l y v i s i b l e near one s u r f a c e . H i s t o l o g i c a l examination of both tumour and muscle r e v e a l e d s e v e r a l f e a t u r e s of i n t e r e s t which w i l l be p r e s e n t e d i n another s e c t i o n . S e v e r a l subcutaneous s w e l l i n g s o c c u r r e d i n male Wistar r a t s which had r e c e i v e d 20 mg. of NiS. These ar o s e two weeks a f t e r i n j e c t i o n and disappeared a f t e r two months. One which p e r s i s t e d developed i n t o a f i r m mass approximately two cm. i n diameter. Autopsy r e v e a l e d t h a t the gastrocnemius muscle had completely disappeared w h i l e a l a r g e , e n capsulated and f r e e l y movable mass had become s i t u a t e d i n t h a t a r e a . The i n t e r i o r of the c a p s u l e c o n s i s t e d of g r i t t y p a r t i c l e s of n i c k e l mixed w i t h n e c r o t i c , p a l e green m a t e r i a l , the whole b e i n g surrounded by a f i b r o m u s c u l a r c o v e r i n g . 27 Similar toxic reactions to the metal were seen i n several young rats which had received 50 mg. of NiS. This high dose appeared to e l i c i t overly toxic s i d e - e f f e c t s , both systemic and l o c a l . Approximately 30 per cent of the treated rats died within two weeks following i n j e c t i o n , while two young rats developed a s o l i d mass af t e r two weeks, at the s i t e of i n j e c t i o n . These masses persisted for eight weeks, at which . time the animals were k i l l e d and the swellings examined. The size at time of death was about two cm. i n diameter. Autopsy revealed an outer capsule of fibrous tissue surrounding a necrotic mixture of n i c k e l and inflammatory c e l l s and f l u i d . Injection of the same dose into two hundred gram rats caused death to 40 per cent within three weeks af t e r i n j e c t i o n , although none developed similar swellings. S e r i a l H i s t o l o g i c a l Study. In an attempt to study gradual effects of n i c k e l sulphide on muscle, a s e r i a l study was undertaken i n which 20 mg. of NiS was injected into f o r t y -f i v e male and female Hooded r a t s . Then, beginning four weeks after i n j e c t i o n , rats were s a c r i f i c e d at bi-weekly in t e r v a l s and the treated muscles were examined for h i s t o l o g i c a l changes. Such changes began to be sporadically evident aft e r eight w,eeks. Most of the muscles at four, s i x , eight, ten and twelve weeks 28 showed nothing unusual, although on gross observation i t was noted that approximately 80 per cent of the rat s had no observable, n i c k e l p a r t i c l e s i n the muscle area. One muscle at eight weeks had a small pocket of granuloma tissue surrounding the n i c k e l p a r t i c l e s , which bulged up from the external surface. The muscle showed abnormal h i s t o l o g i c a l d e t a i l , to be described l a t e r , which indicated possible malignant changes. Transplantation Several transplant l i n e s were maintained i n three d i f f e r e n t strains of r a t s . Those c a r r i e d i n Hooded and Wistar rats resulted from the present experiments while the Fischer transplants were obtained from Gilman, a l l having been induced by i n j e c t i o n of Ni3S2 • In general the tumours exhibited markedly d i f f e r e n t gross c h a r a c t e r i s t i c s among the three species. The Wistar and both Fischer transplants appeared s o l i d and firm, with l i t t l e evidence of necrosis. : A l l Hooded l i n e s , on the other hand, were firm only around the outside edge, with the inner area being sof t and bloody. As well, l o c a l hemorrhage showed beneath the skin i n that area. A l l transplants k i l l e d the animals i n three to eight weeks and showed extreme necrosis at that time. 29 In a preliminary attempt to determine the minimum range of c e l l s necessary for takes, one Fischer l i n e was injected with d i l u t i o n s of f i v e m i l l i o n to sixteen thousand c e l l s . Only one tumour had appeared i n the f i v e m i l l i o n c e l l range a f t e r f i f t y -f i v e days, while a l l control tumours, which had received ten m i l l i o n c e l l s , appeared a f t e r seven days. A similar experiment was performed on a Hooded l i n e and i t was found that 100 per cent tumours arose a f t e r thirty-nine days when four thousand c e l l s were injected intramuscularly. The f i r s t tumour appeared on day twenty-eight as opposed to day sixteen for controls. Another experiment on thi s same tumour l i n e involved c e n t r i f u -gation and separation of the tumour suspension before i n j e c t i o n . Rats receiving the packed c e l l s developed 100 per cent tumours after sixteen days while those receiving the supernatant, which was supposedly c e l l - f r e e , as determined i n a counting chamber, produced one tumour afte r f i f t y - s e v e n days. The various tumour l i n e s maintained i n the laboratory showed c h a r a c t e r i s t i c growth periods, as indicated by the time necessary to grow to a standard, measurable si z e of approxi-mately one cm. Although some of the l i n e s underwent changes on repeated transplanting, most growth rates were s t a b i l i z e d at d i s t i n c t i v e rates, ranging from s i x to twenty-one days. 30 Histology H i s t o l o g i c a l examination of approximately one hundred sections stained with haematoxylin and eosin were carr i e d out. Most of the sections examined were primary and transplanted tumours, with some attention being paid to various body organs. In addition to regular staining with haematoxylin and eosin (H and E), two sp e c i a l stains were used for i d e n t i f i c a t i o n of s p e c i f i c c h a r a c t e r i s t i c s . One of these, Mallory's phospho-tungstic-acid haematoxylin, was used to s t a i n approximately eighty sections for the presence of muscle elements. Since thi s solution, commonly labeled PTAH, stains s t r i a t e d f i b r i l s blue, the s t a i n was necessary i n order to examine sections for c r o s s - s t r i a t i o n s , not only on the muscle f i b e r s , but also on neoplastic c e l l s . An example of the desired r e s u l t i s shown i n F i g . 1. A second stain which was used for means of i d e n t i -f i c a t i o n was Perl's s t a i n . The Prussian blue reaction r e s u l t i n g from t h i s i s a s p e c i f i c histochemical test for the presence of i r o n . In addition, other metals can be detected, such as n i c k e l , which gives a greenish-white reaction. The l a t t e r s t a i n was used on a few muscle and tumour sections and most injected organs i n order to v e r i f y the continuing presence of n i c k e l . Sections chosen for microphotography are stained with H and E, unless otherwise stated. 31 S e v e r a l e x c e l l e n t r e f e r e n c e s were used to a i d i n d i a g n o s i s o f the p a t h o l o g i c a l s e c t i o n s , i n c l u d i n g pathology-t e x t s by W i l l i s (84), Robbins (72) and Adams, Denny-Brown and Pearson ( 1 ) . In a d d i t i o n , the review on rhabdomyosarcomas by Stout (77) and a r t i c l e s by B u l l o c k and C u r t i s (10) and Gilman (29) were used as sources of comparison f o r h i s t o l o g i c a l p i c t u r e s o f the v a r i o u s tumours. Recent examination of c e r t a i n s e c t i o n s by Dr. W.L. Dunn served to c l a r i f y s e v e r a l c y t o l o g i c a l d e t a i l s . The m a j o r i t y of tumours a r i s i n g i n Hooded r a t s i n j e c t e d w i t h Ni^S2 showed a h i g h l y c e l l u l a r and pleomorphic p i c t u r e . T y p i c a l examples a r e seen i n F i g . 2 and F i g . 3. C e l l s appear l a r g e and i r r e g u l a r l y round or strap-shaped and elongated, and o f t e n e x h i b i t many granules which s t a i n deeply a c i d o p h i l i c w i t h H and E. An a t y p i c a l r a c q u e t c e l l i s seen i n F i g . 2. F a i n t suggestions o f c r o s s - s t r i a t i o n s a r e o c c a s i o n a l l y seen, but a r e p r o b a b l y due to arrangement o f granules i n a l i n e a r f a s h i o n . Among the more p r e v a l e n t c e l l types are l a r g e c e l l s w i t h a foamy, v e s i c u l a t e d appearance. F i g . 4 i l l u s t r a t e s some of these t y p i c a l "spider-web 1 1 c e l l s . O c c a s i o n a l g i a n t c e l l s r esembling muscle f i b e r s are noted and they o f t e n c o n t a i n Figure 1 Showing S t r i a t i o n s . PTAH. x500. Figure 2 M 3 S 2 Induced Primary Rhabdomyosarcoma. x500. Figure 3 M 3 S 2 Induced Primary Rhabdomyosarcoma with Strap C e l l s . x800. Figure 4 N i o S ? Induced Rhabdomyosarcoma with Spider-web C e l l s . x800. 34 m u l t i p l e n u c l e i and f i b r i l l a r s t r a n d i n g , as seen i n F i g . 5 . Another h i s t o l o g i c a l f e a t u r e of the tumours i s seen i n F i g . 6 , which i n d i c a t e s the presence o f many m i t o t i c f i g u r e s . These g e n e r a l l y e x h i b i t e d phases of metaphase, anaphase or t e l o p h a s e and were found more o f t e n i n primary tumours or d u r i n g the f i r s t few g e n e r a t i o n s . One tumour which arose a f t e r eleven months appeared to have bundles of elongated f i b e r s i n t e r s p e r s e d w i t h round c e l l s , as shown i n F i g . 7. In view of i t s gross c h a r a c t e r -i s t i c s which were p r e v i o u s l y d e s c r i b e d and i t s h i s t o l o g i c a l appearance o f l o c a l l y i n v a s i v e f i b r o s i s among muscle c e l l s , a t e n t a t i v e d i a g n o s i s of a benign tumour was made. Only one tumour arose in,Hooded r a t s i n a body area other than muscle. A f i f t e e n month o l d r a t gave r i s e to a l a r g e , s o f t tumour i n the lower r i g h t quadrant which appeared m i c r o s c o p i c a l l y to have g l a n d u l a r f o r m a t i o n . The presence of ducts f i l l e d w i t h s e c r e t i o n suggested the f o r m a t i o n of some k i n d of mammary tumour. Tumours a r i s i n g i n Wistar r a t s c o n s i s t e d of two mammary neoplasms, one carcinoma of squamoid o r i g i n and one u n i d e n t i f i e d tumour. The m u l t i p l e tumours which had developed i n one r a t showed g l a n d u l a r stroma, w i t h ducts and l i t t l e Figure 5 Pleomorphic Rhabdomyosarcoma with Giant C e l l . x500. 36 secretion, i n d i c a t i n g mammary o r i g i n , while the tumour a r i s i n g i n the neck was not i d e n t i f i a b l e . The 7-N tumour a r i s i n g i n the leg region had a h i s t o l o g i c appearance d i s s i m i l a r to rhabdomyosarcomas. C e l l s were larger and rounder with no spindle shapes evident. Nuclei were paler and v e s i c u l a r and c e l l s seemed to be arranged i n broad sheets. Such character-i s t i c s indicated a tumour of squamoid o r i g i n such as a squamous c e l l carcinoma. The sectioned tumour and muscle r e s u l t i n g from i n j e c t i o n of NiS showed several i n t e r e s t i n g features, as seen i n F i g . 8. The tumour appears more f i b r i l l a r than the other c e l l u l a r forms noted previously, and a large amount of the n i c k e l i s s t i l l scattered throughout the stroma. No giant c e l l s and very few mitoses were seen. I t i s possible that the tumour may have been a benign rhabdomyoma, saimilar to F i g . 7. The disruption of the muscle f i b e r s which had been injected, with NiS i s shown in F i g . 9 and indicates the damaging e f f e c t of the metal. Individual f i b e r s show complete disarrangement and are surrounded with many f i b r o b l a s t s and lymphocytes as well as n u c l e i from degenerated muscle f i b e r s . Some multiple n u c l e i i n tandem are seen i n an elongated f i b e r , suggesting myoneural tube formation. Certain h i s t o l o g i c a l r e s u l t s of the s e r i a l study gave tenuous i n d i c a t i o n that a neoplastic process might have occurred Figure 7 Benign Fibrous Tumour Induced by N i ^ S o . x!25. Figure 8 Fibrous Rhabdomyoma Induced by NiS. x500. 38 aft e r eight weeks. Some muscles examined at four weeks had clumps of n i c k e l i n the muscle mass but the tissue i t s e l f did not appear to be affected beyond a s l i g h t increase i n lymphocytes. The lack of n i c k e l noted grossly i n most muscles examined at four to twelve weeks was substantiated by h i s t o l o g i c study of the treated areas. F i g . 10 shows the muscle which had n i c k e l present a f t e r eight weeks and i t i s seen that there i s f i b e r d i s -arrangement somewhat similar to that described i n F i g . 9. The s o l i d clump of n i c k e l was surrounded by a wall of lympho-cytes and did not appear to be interspersed throughout the whole muscle, although histochemical staining did show some p a r t i c l e s i n f i b e r s some distance removed from the s o l i d clump. F i g . 10 shows an area near the n i c k e l focus i n which there seems to be a mingling of c e l l types. Fragments of s t r i a t e d f i b e r s are mixed with i n d i v i d u a l muscle n u c l e i and small c e l l s , i n d i c a t i n g atrophy of f i b e r s . Other i s o l a t e d f i b e r s , not pictured, appeared to be active rather than atrophic, giving the appearance of giant c e l l s i n formation. Nuclei were large and vacuolated with dark n u c l e o l i , and cytoplasm was darkly stained with eosin, i n d i c a t i n g protein a c t i v i t y . Significance of these findings i s discussed l a t e r . 39 Figure 9 Disrupted Muscle, aft e r Injection with NiS. PTAH. x500. Figure 10 Muscle, Eight Weeks af t e r Injection of NiS. PTAH. x500. 40 Injections into various organs resulted i n several unusual h i s t o l o g i c a l findings. Examination of the tumour a r i s i n g i n those rats which had received an intra-hepatic i n j e c t i o n indicated that i t , too, was a rhabdomyosarcoma. The pattern was generally similar to the leg muscle tumours although somewhat less anaplastic. The suggestion of p e r i -toneal o r i g i n of the tumour was confirmed by a section of the tissue adhered to the abdominal wall. As seen i n F i g . 11, the neoplastic tissue i s continuous with the s t r i a t e d layer of the abdominal w a l l . F i g . 12 shows the attachment to the g a s t r o i n t e s t i n a l t r a c t and also demonstrates some of the pleomorphic c e l l types. A comparative examination of testes from rat s which had received d i f f e r e n t treatments indicated the r e l a t i v e potency of n i c k e l sulphide. Although the rats with i n t r a -t e s t i c u l a r i n j e c t i o n s showed no tumour formation, intense damage to t e s t i c u l a r tissue was noted. As seen i n F i g . 13, the seminiferous tubules are almost completely destroyed with only an occasional row of spermatogonia remaining. The c e l l -free tubules have patches of material i n them which exhibit an eosinophilic reaction with periodic acid S c h i f f reagent. The i n t e r s t i t i a l spaces show much phagocytic a c t i v i t y , with many p a r t i c l e s engulfed i n h i s t i o c y t e s , as seen i n F i g . 14. Figure 11 Attachment of Tumour to Peritoneal Wall a f t e r Intra-hepatic Injection. xl25. Figure 12 Attachment of Above Tumour to Ga s t r o i n t e s t i n a l Tract. x500. 42 Figure 13 Testi c u l a r Tissue Six Months a f t e r Injection of N i 3 S 2 . PAS. x50, Figure 14 Macrophages and Nickel P a r t i c l e s i n I n t e r s t i t i a l Spaces of Above Tissue. x500. 43 N i c k e l was demonstrated histochemically to be not only i n the p a r t i c u l a t e form but a c t u a l l y dissolved throughout the macro-phages. A second injected t e s t i s showed similar tubular degeneration but, i n addition, there was an area of s o l i d collagen and f i b r o b l a s t s with no tubules present, i n d i c a t i n g previous complete destruction supplanted by fibrous growth. Around the edge of the fibrous mass was a r i n g of dystrophic or metastatic c a l c i f i c a t i o n . The t e s t i s of the prostate-injected r a t showed s l i g h t tubular atrophy, but of such a degree as to implicate age of the animal rather than e f f e c t of the n i c k e l . The only probable reaction to the metal i n the prostate was the presence of some i n t e r s t i t i a l c e l l edema, such that some Leydig c e l l s were separated by a greater amount of stroma. The t e s t i s of a Hooded r a t injected intramuscularly with NiS showed no abnormalities at a l l . Examination of the splenic nodule revealed several i n t r i g u i n g d e t a i l s . F i r s t of a l l , a large number of m i t o t i c phases were present, including abnormal chromatin bridge figures. Also there were some giant c e l l s with multiple v e s i c l e s which might have been either giant f i b r o b l a s t s or abnormal pre-neoplastic c e l l s . The main bulk of the nodule To Face Page 44 Figure 15 In h i b i t i o n of growth rate of 2-N-5 following administration Vincaleukoblastine during f i r s t week af t e r implantation. 44 consisted of a loose meshwork of f i b r o b l a s t s with some f o c i of c a l c i f i c a t i o n . The injected uterus showed metastatic c a l c i f i c a t i o n surrounding n i c k e l p a r t i c l e s i n various areas. Although the myometrium seemed to have an excessive amount of collagen, the c e l l u l a r appearance was not unusual. Injected ovaries had no observable n i c k e l and appeared normal, with functioning f o l l i c l e s . Therapeutic Studies A l k a l o i d Treatment. Vinblastine was tested at random on several transplant l i n e s . Dose regimens were varied, as were times of administration. Tumour diameters were^measured throughout the i n j e c t i o n period and at four to s i x weeks a f t e r implantation. Beyond this time period, factors such as necrosis tended to confuse r e s u l t s . Table II l i s t s summarized r e s u l t s . Response varied considerably i n the d i f f e r e n t tumour l i n e s , with the best effects being noted i n the 2-N l i n e . The tumours occurring i n treated rats grew during the i n j e c t i o n period but at a slower rate than i n control animals. An example of this i s shown i n F i g . 15. I t i s of i n t e r e s t to note that the most e f f e c t i v e suppression of takes was observed i n those rats bearing the 2-N tumour which received three consecutive 45 TABLE II RESPONSE OF RHABDOMYOSARCOMAS TO VINBLASTINE Tumour l i n e Dose Tumour diameter Number of and generation (mean ± S.D.,cm.) tumours 2-N-5 Controls 2.7 + .2 4/4 0.35 mg/kg days 1-3 2.1 + .1 2/4 days 3-5 1.8 .3 2/4 days 6-9 3.2 .7 4/4 days 14-24 1.9 + .1 3/4 5-N-18 Controls 2.4 ± .1 4/4 0.1 mg/kg days 1-10 2.6 + .5 4/4 10-N-2 Controls 2.2 + .3 4/4 0.3 mg/kg days 1-10 ( a l l rats dead by day 15) 12-N-2 Controls 2.2 .4 5/5 0.1 mg/kg days 1-10 2.2 + .2 5/5 To Face Page 46 I Figure 16 Increase i n latent period for 2-N-5 following administration of V i n c r i s t i n e . 46 i n j e c t i o n s of V i n b l a s t i n e b e g i n n i n g one or t hree days a f t e r t r a n s p l a n t a t i o n . Those animals i n which tumours d i d not occur a t f o u r weeks time had not developed tumours by the t e r m i n a t i o n date of seven weeks. The s i g n i f i c a n c e of these r e s u l t s w i l l be d i s c u s s e d l a t e r . V i n c r i s t i n e . w a s a d m i n i s t e r e d to the same tumour l i n e s i n v a r y i n g dose regiments. R e s u l t s a r e summarized i n T a b l e I I I . An i n c r e a s e d l a t e n t p e r i o d was demonstrated by the i n j e c t i o n of V i n c r i s t i n e to 2-N-5 d u r i n g days one to t h r e e . The e f f e c t was made more emphatic by the f a c t t h a t the f i r s t tumour a r i s i n g from f i v e t r e a t e d r a t s d i d not become p a l p a b l e u n t i l f o r t y -t h ree days a f t e r treatment had been i n i t i a t e d . The r e m a i n i n g ' t r e a t e d r a t s showed s m a l l tumours a f t e r f i f t y - o n e days, thus the t e s t to c o n t r o l r a t i o was 1:6 or 0.17. E f f e c t i v e response to the a l k a l o i d decreased as treatment was delayed up to seven days, i l l u s t r a t e d g r a p h i c a l l y i n F i g . 16. I t i s a l s o seen t h a t growth appeared to be somewhat i n h i b i t e d d u r i n g the f i r s t t h r e e weeks w i t h a p r o l i f e r a t i v e s p u r t b e i n g shown a f t e r t h a t time. Hormon e Admin i s t r a t i on. A l l r a t s bearing t r a n s -plantable rhabdomyosarcomas were t r e a t e d with C o r t i s o l , c o r t i -sone or p r e d n i s o l o n e , i n regimens chosen a t random i n an attempt to f i n d tumour l i n e s s e n s i t i v e to a d r e n a l c o r t i c o i d s . A l l i n j e c t i o n s were begun seven days a f t e r i m p l a n t a t i o n , when most 47 TABLE III RESPONSE OF RHABDOMYOSARCOMAS TO VINCRISTINE Tumour l i n e and generation Dose Tumour diameter (mean + S.D.,cm.) Number of tumour s 2-N-5 Controls 2.7 + .2 4/4 0.1 mg/kg days 1-3 1.7 1/5 0.1 mg/kg days 7-9 1.7 + • 6 4/4 0.1 mg/kg days 14-24 2.2 + .5 4/4 5-N-18 Controls 2.4 + .1 4/4 0.1 mg/kg days 1-10 2.4 + .5 4/4 10-N-2 Controls 3.2 + .3 3/3 0.1 mg/kg days 1-10 2.9 ± .2 4/4 12-N-2 Controls 2.2 + .4 4/4 0.1 mg/kg days 1-10 2,0 ± .1 4/4 To Face Page 48 Suppression of growth of 2-N-5 d u r i n g the i n t r a p e r i t o n e a l i n j e c t i o n of C o r t i s o l or c o r t i s o n e . I n j e c t i o n p e r i o d f o r s t e r o i d s , days seven to seventeen. 48 tumours were j u s t measurable. S e v e r a l dose regimens and r e s u l t s a r e g i v e n i n Tab l e IV. P o s i t i v e response, i n d i c a t e d by v a r y i n g degrees of i n h i b i t i o n o f growth, was ob t a i n e d i n fou r out o f seven tumour l i n e s t e s t e d i n b o t h Hooded and F i s c h e r r a t s . 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 40 mg. of C o r t i s o l and 50 mg. o f cortisone r e s u l t e d i n complete s u p p r e s s i o n of tumour growth i n 2-N-5 du r i n g the i n j e c t i o n p e r i o d , as seen i n F i g . 17, a l t h o u g h s i m i l a r C o r t i s o l treatment of the same tumour i n the twenty-t h i r d g e n e r a t i o n r e s u l t e d i n r e v e r s a l o f e f f e c t . The appear-ance o f tumours i n t r e a t e d r a t s i n the l a t t e r experiment was a c t u a l l y s l i g h t l y l a r g e r than those i n c o n t r o l r a t s ; Sub-cutaneous a d m i n i s t r a t i o n o f the c o r t i c o i d s was adopted i n the m a j o r i t y o f experiments, and some i n h i b i t i o n of growth was ach i e v e d i n a l l experiments u s i n g 2-N, 8-N and 12-N. G e n e r a l l y the i n h i b i t i o n was r e s i d u a l i n t h a t tumour growth was not completely i n h i b i t e d but was slowed d u r i n g the i n j e c t i o n p e r i o d t o t t h e extent t h a t t r e a t e d r a t s had tumours which were o n e - t h i r d the weight of c o n t r o l tumours. Weight g a i n was g e n e r a l l y stopped or r e v e r s e d d u r i n g i n j e c t i o n s , a l t h o u g h r a t s gained weight a f t e r treatment was d i s c o n t i n u e d . T e s t o s t e r o n e was a d m i n i s t e r e d i n a standard dose o f 5 mg. i n j e c t e d subcutaneously f o r ten days to 2-N, 6-N, 7-N, 49 TABLE IV EFFECTS OF STEROIDS ON RHABDOMYOSARCOMAS AND BODY WEIGHT Tumour Number Steroid Dose and % change Tumour size of rats route body weight grams T/C 2-N-5 5 Controls + 39.9 36.30 5 C o r t i s o l 40 mg. I.P. + 13.6 3.61 .09 5 Cortisone 50 mg. I.P. + 18.9 2.83 .08 5 Cortisone 50 mg. S.C. - 13.6 15.85 .44 2-N-8 5 Controls + 3.7 59.05 5 C o r t i s o l 40 mg. S.C. - 9.8 14.16 .24 5 Cortisone 50 mg. S.C. - 17.8 18.59 .31 2-N-16 5 Controls + 12.2 18.57 5 C o r t i s o l 20 mg. S.C. - 7.4 5.44 .29 5 C o r t i s o l 20 mg. S.C. + testosterone 50 mg . +12.2 5.29 .28 2-N-23 5 Controls + 12.9 3.25 5 C o r t i s o l 60 mg. I.P. 5.1 7.33 2.26 4-N-16 5 Controls + 32.0 169.87 5 Prednisolone 20 mg. S.C • -13.2 28.51 .17 8-N-10 5 Controls + 13.0 58.81 5 C o r t i s o l 40 mg. S.C. - 9.0 17.24 .30 12-N-2 5 Controls + 37.6 21.90 5 C o r t i s o l 40 mg. S.C. + 4.0 5.53 .25 50 8-N, 10-N and 12-N. Time of i n i t i a t i o n was an important v a r i a b l e , w i t h most animals r e c e i v i n g the f i r s t i n j e c t i o n on days one or seven a f t e r i m p l a n t a t i o n , as seen i n Tabl e V. Two out o f fou r groups i n j e c t e d from days one to ten showed a c c e l e r a t e d growth of tumours, w h i l e no groups r e c e i v i n g i n j e c t i o n s b e g i n n i n g on days f o u r or seven showed any s i g n i f i -cant d i f f e r e n c e i n s i z e between t e s t and c o n t r o l tumours. One e x c e p t i o n a l r e s u l t was noted i n 2-N-19, when e a r l y treatment w i t h t e s t o s t e r o n e r e s u l t e d i n g r e a t i n h i b i t i o n o f growth, a r e s u l t which was markedly d i f f e r e n t from t h a t o b t a i n e d when the same treatment was g i v e n to the same tumour l i n e t hree gener-a t i o n s e a r l i e r . 2-N-16 received combined treatment o f 2 mg. of C o r t i s o l and 5 mg. of t e s t o s t e r o n e , b o t h g i v e n ten times subcutaneously b e g i n n i n g day seven. T h i s r e s u l t e d i n some i n h i b i t i o n o f tumour s i z e , s i m i l a r to t h a t seen w i t h C o r t i s o l a l o n e , but w i t h an i n c r e a s e i n weight equal to t h a t seen i n c o n t r o l animals (see T a b l e I V ) . 51 TABLE V EFFECT OF TESTOSTERONE ON RHABDOMYOSARCOMAS Tumour l i n e Number of Treatment i n i t i a t e d Response and generation animals (days a f t e r transplant) T/C 2-N-9 5 1 .62 2-N-16 5 4 1.00 2-N=19 5 1 .08 6-N-ll 5 1 1.64 6-N-12 5 7 1.15 7-N-ll 5 1 1.74 8-N-10 5 7 .83 10-N-2 5 1 .76 10-N-3 5 7 .61 12-N-2 5 7 .67 52 DISCUSSION There are cert a i n acknowledged problems attendant i n using animals for tumour studies. Tumour induction p a r t i c u l a r l y can be i n h i b i t e d by environmental factors such as inadequate di e t , general health, infections and so on. Other variables occur i n selection of the species, s t r a i n and sex of the experimental animal chosen, and the age at the s t a r t of the experiment (8). Even i f these factors are c a r e f u l l y regulated, r e s u l t s may s t i l l vary considerably. Hieger states the discouraging f a c t that tumour induction assays are often reproducible only by a factor of two or f i v e (42). That i s , one may f i n d 10 per cent tumours the f i r s t time, and only 5 or 2 per cent the next time, i n spite of i d e n t i c a l technique. When working with a compound of unknown a c t i v i t y , r e l a t i v e l y empirical choice of dose, route and rate of admini-s t r a t i o n i s necessary since i t s general t o x i c i t y to the animal i s not known. I t i s usual to assay a compound for t o x i c i t y before beginning the actual experiments. In the case of Ni3S2 the o r i g i n a l assay tests were performed by Gilman, who found that i n j e c t i o n of 30 mg. into a muscle caused some l o c a l e f f e c ts probably due to trauma (26) . I t was possible to use 20 mg. per muscle without adverse e f f e c t s , although b i l a t e r a l i n j e c t i o n s of this dose sometimes resulted i n general t o x i c i t y . 53 A suggested reason for t h i s was the presence of n i c k e l sulphate, a s a l t which was e a s i l y removed by washing the i n j e c t i o n mixture. The induction of tumours by intramuscular i n j e c t i o n of Ni3S2 proved to be f a i r l y r e l i a b l e . . The short latent period of ninety-two days exhibited i n those rat s which had received 20 mg. suggested that this compound had strong carcinogenic a c t i v i t y i n Hooded r a t s . That the latent periods obtained were shorter than those recorded by Gilman aft e r i n j e c t i o n of the same dose into Wistar rat s might be due to the f a c t that Hooded rats are highly inbred whereas Wistars are not. I t has been found so far that none of the inbred strains i s completely r e s i s t a n t to a carcinogenic stimulus, i f the species i n general i s known to be susceptible to such a stimulus (5). Since genetic factors may control the degree of s u s c e p t i b i l i t y , i t i s f e a s i b l e that a carcinogen may e l i c i t tumours more r e a d i l y i n the highly inbred strains than i n those which are not inbred. There seemed to be some co r r e l a t i o n between amount of n i c k e l sulphide injected and percentage tumour response i n r e l a t i o n to time. The most rapid induction was achieved i n four months when 100 per cent tumours occurred a f t e r i n j e c t i o n of 20 mg. . From Table I, i t was seen that tumour appearance 54 was 100 per cent with 10 mg. as well, but with 2.5 mg. and 0.5 mg. i t decreased. I t may have been that there was not enough n i c k e l a v a i l a b l e a f t e r a certain length of time. Gilman has stated that the compound i s slowly soluble and diffuses out gradually over a long period of time (27). The i n j e c t i o n of higher doses perhaps meant that greater amounts of n i c k e l were present to attack a proportionately greater number of c e l l s , thereby setting up d i f f e r e n t and larger f o c i of a l t e r e d c e l l s throughout the muscle, whereas a small dose 5 might have had only enough n i c k e l to set up one small focus. In the rats given 2.5 mg. and 0.5 mg., the rate of increase i n per cent response appeared to slow down af t e r eight months, possibly because the absolute amount of n i c k e l present at that time was c r i t i c a l . This hypothesis might explain the fac t that the average latent period was not s i g n i f i c a n t l y d i f f e r e n t i n those rats injected with 0.5 to 10 mg. Although the expected delay i n tumour appearance was seen between 10 and 20 mg., among the lower doses only twenty days separated the average latent periods. This again indicated that the absolute amount of n i c k e l was the deciding factor, once the concentration was below a c r i t i c a l l e v e l . Above this t h e o r e t i c a l amount, an 55 increase i n number of n i c k e l p a r t i c l e s might r e s u l t i n yet faster induction of tumours. Whether or not the two-stage mechanism of carcinogenesis (25) could be applied to thi s s i t u a t i o n i s s t r i c t l y a matter of speculation. I t would involve a c r i t i c a l amount of n i c k e l being needed to convert normal c e l l s to subthreshold neoplastic c e l l s ( i n i t i a t i n g stimulus), with a further and larger amount of n i c k e l neede to stimulate such c e l l s to p r o l i f e r a t e into a v i s i b l e tumour (promoting stimulus). As mentioned i n the introduction, tumours of s t r i a t e d muscle o r i g i n have been extremely rare i n experimental animals. The i d e n t i f i c a t i o n of such tumours induced by Ni 3S2 as being rhabdomyosarcomas was made on the basis of several observations. F i r s t , the s i t e of o r i g i n was invaria b l y i n the leg muscle region, with the tumour being situated i n the area of the gastrocnemius muscle. Second, the fac t that they were f a s t -growing, once palpable, and exhibited rapid l e t h a l i t y i i n d i c a t e d their malignant or sarcomatous nature. The t h i r d and most p o s i t i v e means of i d e n t i f i c a t i o n was by h i s t o l o g i c a l techniques. Reference has been made to detailed descriptions of rhabdomyo-sarcomas by Stout (77) and Adams, Denny-Brown and Pearson (1). They have outlined certain s p e c i f i c types of rhabdomyoblast 56 c e l l s which characterize these tumours: (1) large round c e l l s with one to several n u c l e i ; (2) strap c e l l s , with two or more n u c l e i ; (3) racquet c e l l s with a single nucleus at one end and tapering body; (4) giant "spider-web" c e l l s with a single nucleus and peripheral vacuoles separated by d e l i c a t e cytoplasmic threads. Stout noted that c r o s s - s t r i a t i o n s and l o n g i t u d i n a l l y arranged myofibrils or some vague suggestion of their formation were sometimes seen i n strap c e l l s . This l a t t e r c h a r a c t e r i s t i c i s considered to be absolute proof of a rhabdomyosarcoma, according to a u t h o r i t a t i v e pathology texts (72, 84). However, many descriptions of rhabdomyosarcomas have mentioned that s t r i a t i o n s are notoriously hard, i f not impossible, to demonstrate i n some tumours. D i l i g e n t searching of primary and transplanted tumour sections stained s p e c i f i c a l l y for f i b r i l s f a i l e d to reveal such s t r i a t i o n s . Gilman had stated that he found them only i n the o r i g i n a l tumours and not i n any transplant generations (26). The f a c t that certain of the c h a r a c t e r i s t i c c e l l u l a r elements were seen i n a l l induced tumours gave h i s t o l o g i c a l support to the gross observation of rhabdomyosarcomas r e s u l t i n g from i n j e c t i o n of n i c k e l sulphide. 57 The observation that the Wistar r a t s , i n contrast to Hooded^showed almost no response to a similar dose of Ni3S2 was rather curious, e s p e c i a l l y i n view of the fa c t that rhabdomyosarcomas have been induced i n Wistars by Gilman. There i s some question as to the o r i g i n of the Wistar rats supplied at U.B.C. and the lack of tumours i n these Wistars, as opposed to those used by Gilman which had been obtained from Woodlyn Farms at Guelph, might be explained i n t h i s way. The fa c t that no n i c k e l p a r t i c l e s were present i n the injected muscle region suggested that the m e t a l l i c compound may have been handled d i f f e r e n t l y i n this s t r a i n of r a t s . The o r i g i n a l swellings which appeared between eight and twelve weeks might have been a l o c a l reaction of the tissues to the foreign i r r i t a n t , r e s u l t i n g i n i t s complete removal. I t should be noted that the single Wistar r a t which developed a benign tumour i n the leg region had not shown a swelling i n the t h i r d month, nor had the r a t which developed the lump i n the neck area. The n i c k e l i n these animals may have been d i s t r i b u t e d , at least t h e o r e t i c a l l y , throughout the body, thus being able to e l i c i t a systemic action. I t i s possible that i n most Wistar rats the n i c k e l was dissolved and phagocytosed, then absorbed into the blood stream to be disposed of by excretion, although there seems 58 to be no l o g i c a l reason why t h i s should happen i n only one s t r a i n of r a t s . Gilman has made preliminary studies on the excretion of n i c k e l i n urine a f t e r implantation of a disc of N 1 3 S 2 which releases an amount of n i c k e l equivalent to the i n j e c t i o n of 10 mg., and has found that 3.5 per cent of the n i c k e l was excreted a f t e r three months. 5 and 13 mg. of n i c k e l combined With E.D.T.A. i n p e l l e t form resulted i n the excretion of 38.6 and 27.3 per cent of the n i c k e l , r e s p e c t i v e l y . Although the r e s u l t s are equivocal, they suggest that n i c k e l can be f a i r l y e a s i l y excreted through the kidneys, even when in chelated form. The i n j e c t i o n of Ni3S2 into various body organs served to emphasize the destructive p o t e n t i a l i t i e s of t h i s compound, as well as i n d i c a t i n g a possible carcinogenic s p e c i f i c i t y for s t r i a t e d muscle. . The l a t t e r point was suggested i n the r e s u l t s of the intrahepatic i n j e c t i o n s . The extensive tumours which developed were almost c e r t a i n l y not of hepatic o r i g i n since the root of the tumours appeared grossly and h i s t o l o g i c a l l y to be i n the muscle layers of the peritoneal w a l l . I t i s probable that the suspension of n i c k e l p a r t i c l e s was extruded from the l i v e r by leakage and somehow became adsorbed onto the muscle w a l l . The f a c t that i t could adhere to the immediate 59 o v e r l y i n g p o r t i o n of the peritoneum r a t h e r than being d i s -p e r s e d throughout the abdominal c a v i t y i s concomitant w i t h Gilman's h y p o t h e s i s t h a t n i c k e l has a s p e c i f i c i t y f o r s t r i a t e d muscle. The tumours d i d appear to be rhabdomyosarcomas and demonstrated extreme malignancy. Although i n j e c t i o n s i n t o other organs d i d not r e s u l t i n tumours, damage to most t i s s u e s was e x t e n s i v e . T e s t i c u l a r t i s s u e seemed to be p a r t i c u l a r l y s e n s i t i v e to the presence of Ni 3 S 2 l o c a t e d e i t h e r d i r e c t l y i n the area or i n the p r o s t a t i c t i s s u e . The extreme degeneration of the s e m i n i f e r o u s t u b u l e s , accompanied by much macrophage a c t i v i t y , which r e s u l t e d from d i r e c t i n t r a t e s t i c u l a r i n j e c t i o n was understandable as a d r a s t i c response to an extremely i r r i t a t i n g l o c a l f o r e i g n substance. The p a r t i a l e f f e c t on the t e s t e s i n r a t s r e c e i v i n g i n t r a p r o s t a t i c i n j e c t i o n s c o u l d p o s s i b l y have been due to some n i c k e l c i r c u l a t i n g through the organ from the p r o s t a t i c f o c i . However, the m i l d i n t e r s t i t i a l edema observed can be e l i c i t e d by many i r r i t a n t substances and was p r o b a b l y not a s p e c i f i c r e a c t i o n to n i c k e l . The e f f e c t of d i r e c t c o n t a c t of n i c k e l w i t h the t e s t e s i s of i n t e r e s t i n view of r e s u l t s t h a t have been o b t a i n e d w i t h the use of other metals, such as cadmium. Kar and Das 60 have recorded i n d e t a i l the t e s t i c u l a r changes i n rat s following treatment with cadmium chloride (52). They observed that between two and seven days a f t e r subcutaneous i n j e c t i o n the seminiferous tubules shrank and a l l c e l l u l a r elements disappeared, with the i n t e r i o r of most tubules containing only dead c e l l debris. The i n t e r s t i t i a l c e l l s disappeared afte r t h i r t y days and were replaced by f i b r o b l a s t s . No mention was made of phagocytic a c t i v i t y . The above picture of t e s t i c -ular damage i s not unlike that obtained i n the present experi-ments, p a r t i c u l a r l y with reference to the presence of numerous fi b r o b l a s t s and i r r e g u l a r patches of intra-tubular debris. The presence of some abnormal c e l l forms i n n i c k e l -injected testes gives tentative suggestion that such tissue v might have become neoplastic a f t e r a greater length of time, since t e s t i c u l a r tumours have been induced by other metals. As reported by RivieYe, zinc s a l t s are commonly known to produce tumours of the testes i n b i r d s , provided that a gonadotrophic factor i s present (71). Similar tumours have been induced by parenteral i n j e c t i o n of zinc chloride i n rodents. Ri v i e r e found that a 1 to 4 mg. suspension of t h i s s a l t resulted i n eleven tumours, most of them i n t e r s t i t i a l c e l l neoplasms, among one hundred and twenty-five rats a f t e r 61 two y e a r s . I t i s n o t improbable, then, to suggest t h a t tumours might a l s o have been induced by Ni3S2 a f t e r a -longer exposure p e r i o d . The f a c t t h a t none were seen i n the p r e s e n t experiments c o u l d a l s o be due to the t o x i c i t y of the metal, s i n c e the dose of 5 to 10 mg. was o b v i o u s l y too damaging to most of the c e l l s . The tumour i n d u c t i o n s t u d i e s w i t h NiS were intended to be an e x t e n s i o n of the experiments u s i n g Ni3S2> i n order to e l u c i d a t e f u r t h e r v a r i a b l e s a f f e c t i n g metal c a r c i n o g e n e s i s . I t was soon apparent t h a t the d i f f e r e n t composition of t h i s second form o f n i c k e l s u l p h i d e might mean t h a t the compound would have d i f f e r e n t p r o p e r t i e s . NiS was o b v i o u s l y more t o x i c than the f i r s t compound, causing both systemic and l o c a l r e a c t i o n s . The l a t t e r response, m a n i f e s t e d as l o c a l s w e l l i n g , i n d i c a t e d the e f f e c t i v e n e s s of the h o s t ' s defense a g a i n s t a h i g h l y i r r i t a n t substance. Although the o r i g i n a l muscle i n these areas was sometimes completely a t r o p h i e d , the n i c k e l i t s e l f was c o n t a i n e d w i t h i n an i s o l a t i n g c a p s u l e . The presence of muscle f i b e r s i n t e r s p e r s e d among c o n n e c t i v e t i s s u e f i b e r s suggested t h a t the pocket o f i n j e c t e d p a r t i c l e s had p r o b a b l y d i g e s t e d most of the muscle t i s s u e surrounding i t . The appearance o f two d e f i n i t e measurable tumours among twenty Hooded r a t s i n j e c t e d w i t h 20 mg. of NiS gave a 62 tumour response of only 10 per cent aft e r four months, compared with 100 per cent using M 3 S 2 , suggesting that although NiS i s capable of being carcinogenic, i t s potency i s apparently less than that of N i 3 S 2 . An unknown e f f e c t of age may have had something to do with the occurrence of tumours with NiS, since any tumours a r i s i n g to date have been i n rats which were injected as adults. Although only one NiS-induced tumour has been examined h i s t o l o g i c a l l y , i t i s i n t e r e s t i n g to speculate that t h i s com-pound may have a slower and less potent e f f e c t . The suggestion that i t has induced a benign rhabdomyoma, rather than a malignant rhabdomyosarcoma, of f e r s p o t e n t i a l experimental studies. Close surveillance of developing tumours by means of periodic biopsies might indicate whether the tumours could progress from benign to malignant states or whether they would originate i n one state or the other and remain that way. The p o s s i b i l i t y that NiS has d i f f e r e n t carcinogenic a b i l i t i e s w i l l be developed i n future investigations. In attempting to follow the action of NiS injected intramuscularly, d i f f i c u l t i e s were encountered owing to the unpredictable fate of the metal. Unlike M 3 S 2 , NiS did not remain i n the o r i g i n a l muscle area for any length of time, as noted i n the r e s u l t s . In those muscles i n which n i c k e l was 63 s t i l l present six weeks afte r i n j e c t i o n , the accumulation of lymphocytes was undoubtedly e l i c i t e d by the chronic presence of a foreign i r r i t a n t . The r e s u l t s obtained from one muscle which had n i c k e l present at eight weeks indicated that the compound might be exerting carcinogenic c a p a b i l i t i e s at this time. Although there was no gross evidence of tumour formation, the presence of c e l l s d i s t i n c t i v e l y d i f f e r e n t from normal muscle f i b e r s and similar to those seen i n established rhabdo-myosarcomas suggested that the muscle was undergoing pre-malignant changes. Gilman has found a similar picture of an increase i n the number of subsarcolemmal n u c l e i with clus t e r s of n u c l e i i n degenerating giant muscle c e l l s (30). Hueper had observed i n his experiments involving intrafemoral in j e c t i o n s of m e t a l l i c n i c k e l that around the n i c k e l deposits there occurred p r o l i f e r a t i o n of hyperchromatic, i r r e g u l a r c e l l s , which he f e l t represented early sarcomatous f o c i (46). Heath has performed an extensive study on the carcino-genic a c t i v i t y of cobalt i n 'muscle, i n which changes were observed and recorded from one day to twenty weeks af t e r i n j e c t i o n (39). B r i e f l y , these consisted of immediate i n f i l -t r a t i o n of leukocytes and f i b r o b l a s t s between muscle bundles and f i b e r s , followed by rapid necrosis which was manifested by loss of n u c l e i and s t r i a t i o n s i n some bundles within one week. 64 Mononucleated myoblasts were formed and some regeneration was seen but d i f f e r e n t i a t e d f i b e r s did not reappear. By s i x weeks muscle degeneration and f i b r o b l a s t penetration had spread beyond the area of i n j e c t i o n . Between eight and fourteen weeks myo-blasts became large and multinucleated, with many mit o t i c figures apparent, and giant c e l l s were seen. The tumour nodule did not become palpable u n t i l twenty weeks at which time the cobalt was s t i l l present. Heath postulated that cobalt was capable of producing muscle tumours by v i r t u e of a modification of normal regenerative and repair processes due to the presence of the metal. Cobalt ions were probably l i b e r a t e d continuously by slow solution i n tissue f l u i d s or the muscle c e l l s may have been subjected to d i r e c t c a t a l y s i s at the surface of the metal grains. I t would seem that the continued presence of a metal i s a prerequisite for production of tumours. The experimental work on M 3 S 2 and NiS has indicated that, i n the majority of cases, n i c k e l p a r t i c l e s were present i n rats which developed tumours while those rats i n which the n i c k e l had disappeared showed no evidence of muscle abnormality. Gilman has recently reported the subcutaneous implantation of discs of n i c k e l sulphide rather than i n j e c t i o n of the metal as a p a r t i c u l a t e suspension (30). This might be a more r e l i a b l e method of 65 ensuring a small but constant supply of n i c k e l ions. He has noted the presence of myoblast c e l l s a f t e r eight weeks of contact with the metal, but states that transplantation of similar tissue i s not e f f e c t i v e u n t i l a f t e r eleven weeks of exposure. Tumours do not become palpable u n t i l a f t e r sixteen weeks. Such r e s u l t s indicate a c r i t i c a l exposure period of muscle to n i c k e l sulphide. There appears to be no obvious explanation why NiS f a i l e d to remain i n most of the injected muscles. The question of the importance of p a r t i c l e size has not been answered. I t i s conceivable that i f the i n j e c t i o n volume contained p a r t i c l e s small enough to be i n a c o l l o i d a l form, these could possibly d i f f u s e quickly out of the muscle, causing systemic t o x i c i t y and no muscle tumours. This would indicate that i t i s necessary for a c r i t i c a l amount to remain i n the muscle region. However, the p a r t i c l e s of both compounds tested were of a similar size and were not soluble i n the i n j e c t i o n v e h i c l e s , thus both would presumably be located within the muscle as a p a r t i c u l a t e mass. Their further fate i s e n t i r e l y unknown. I t i s possible that only NiS was r a p i d l y excreted by the kidney but again there seems no rati o n a l e for such a statement. Studies on the comparative excretion rates of the two compounds are planned 66 for future experiments i n th i s laboratory. The p o s s i b i l i t y of d i f f e r e n t s o l u b i l i t i e s i n vivo, as indicated by urinary analysis, i s discussed i n greater d e t a i l below. The fa c t that the continuing quantitative mass of the metal may be important suggests that i t s detection i n the urine might indicate differences i n behaviour between the two forms of n i c k e l sulphide. An a l t e r n a t i v e p o s s i b i l i t y to that of p a r t i c u l a t e mass a f f e c t i n g s t a b i l i t y of the compound i s that d i r e c t contact with muscle acids may have some e f f e c t . Once interspersed among the muscle bundles, n i c k e l sulphide would undoubtedly be exposed to a r e l a t i v e l y higher a c i d i t y , due to l a c t i c and pyruvic acids. Whether the two compounds could have d i f f e r e n t s o l u b i l i t y properties i n muscle acids has not been determined, although both compounds were found to have similar s o l u b i l i t i e s i n hydrochloric a c i d . I f NiS were less stable i n a c i d and were situated deep within the b e l l y of the muscle i t might be sub-jected to a greater amount of acid capable of d i s s o l v i n g i t away. . This hypothesis i s t e n t a t i v e l y suggested by the f a c t that the tumour a r i s i n g from NiS arose at the upper external surface of the gastrocnemius and appeared to be connected to i t rather than embedded i n i t . 67 I f l a c t i c acid does have an e f f e c t on n i c k e l sulphide, t h i s same acid environment might also play some r o l e i n the r a p i d i t y of tumour induction i n s t r i a t e d muscle. L a c t i c a c i d i s well known as a common metabolic by-product of r a p i d l y -growing tumours and has been implicated i n t h e i r invasive c h a r a c t e r i s t i c s by the speculation that the release of free acid and decrease i n pH might a i d the destructive action of tumour c e l l s adjoining normal tissues. However, the amount libe r a t e d i s probably quickly converted by the l i v e r to glycogen. An opposing theory has c i t e d that l a c t i c a c i d w i l l diminish the carcinogenic potency of some substances (84). Thus, the true r o l e of this a c i d remains an ambiguity i n the neoplastic processes. To have any carcinogenic e f f e c t on voluntary muscle an agent would have to exert a strong e f f e c t on t h i s d i f f e r -entiated tissue i n order to make i t malignant. The question of whether mature s t r i a t e d muscle f i b e r s are capable of mitotic d i v i s i o n has been open to some discussion. Swann has stated the negative point of view emphatically by remarking that every tissue i n the body of a higher animal shows at least occasional mitoses, with the exceptions of nerve c e l l s and s t r i a t e d muscle (79). Thus even though most d i f f e r e n t i a t e d c e l l s are capable of d i v i d i n g , t h i s a b i l i t y i s l o s t i r r e t r i e v a b l y 68 i n s t r i a t e d muscle. The case has been presented less f i r m l y by W i l l i s i n his description of the lim i t e d capacity of s t r i a t e d muscle for p r o l i f e r a t i o n once i t has attained adult-hood (84). A more thorough analysis of the problem has been given by Adams et a l . i n Diseases of Muscle (1). During the embryological development of the muscle, m i t o t i c d i v i s i o n i s thought to cease aft e r the f i f t h month i n humans and further enlargement i n muscles should be by an increase i n the si z e of the i n d i v i d u a l f i b e r s rather than i n the number of f i b e r s . In r e f e r r i n g to the mature f i b e r , the statement i s made that i n view of the embryonic o r i g i n of s t r i a t e d muscle f i b e r s from myoblasts, which have formed by mitotic d i v i s i o n but which then become multinucleated syncytiums by amitosis, i t i s probable that mitotic d i v i s i o n would occur only i f the muscle f i b e r reverted to a pr i m i t i v e u n i c e l l u l a r or myoblast state. This could be correlated with Heath's study mentioned previously on the ef f e c t s of a m e t a l l i c ion on s t r i a t e d muscle. The o r i g i n a l response of the muscle to the mechanical injury of trauma produced by i n j e c t i o n of the metal p a r t i c l e s would be an attempt at regeneration and r e p a i r . However, i f there were continued action of the m e t a l l i c i r r i t a n t , the muscle might f a i l to complete the regenerative processes and instead 69 continue to ded i f f e r e n t i a t e , being reduced eventually to the embryonic myoblast state. Once at t h i s l e v e l , ensuing mitotic figures might be alt e r e d d i r e c t l y by the n i c k e l ions, such that the f i n a l r e s u l t would be a malignant state. This does not explain, of course, how n i c k e l sulphide i n p a r t i c u l a r would a l t e r the muscle c e l l s or why i t should have any s p e c i f i c i t y for s t r i a t e d muscle. Presumably the tissue reaction to n i c k e l would be more than just the response to a foreign i r r i t a n t since n i c k e l i s not an i n e r t substance. Considered to be the commonest s p e c i f i c skin s e n s i t i z e r , i t s action i n dermatitis i s probably the formation of protein complexes through linkages with sul f h y d r y l groups of amino acids (31, 47). Hueper has advanced a theory that n i c k e l may i n h i b i t the action of free s u l f h y d r y l groups and cause enzymatic disturbances which lead to cancer (47). A similar action has been t e n t a t i v e l y a t t r i b u t e d to metals i n general by Haddow who suggested that introduction of excess metal into a c a r e f u l l y balanced system of metals and metal enzymes could r e s u l t i n interference with c e r t a i n functions such as tissue r e s p i r a t i o n , thus causing c e l l u l a r mutation (35). However, th i s theory i s merely speculative, e s p e c i a l l y i n ap p l i c a t i o n to n i c k e l , since the presence of t h i s metal i n normal body tissues i s not v i t a l . 70 The great v a r i a b i l i t y observed i n growth character-i s t i c s of the d i f f e r e n t rhabdomyosarcomas was not unexpected since the c e l l populations which made up d i f f e r e n t transplant l i n e s would acquire s p e c i f i c behaviour patterns when they f i r s t originated. In most cases, growth rates and amount of d e d i f f e r e n t i a t i o n did not change during transplant generations. The s t a b i l i t y of i n d i v i d u a l c h a r a c t e r i s t i c s which tumours exhibit i s one of their most remarkable properties and shows that neoplasia must r e s u l t from an i r r e v e r s i b l e , hereditable change i n the c e l l s themselves (84). Such changes i n response as did occur af t e r repeated s e r i a l transplants served to exhibit the phenomenon of progression. Foulds has stated that progression depends more on i n t r i n s i c properties of i n d i v i d u a l tumours than on the environment to which they are a l l exposed, and trans-plantation may merely complete a progression whose course was set i n the primary tumour (25). The tumour may continue to become more unresponsive to treatments i n each generation, although maintaining similar growth c h a r a c t e r i s t i c s . Thus any experimental work on transplants must allow for such v a r i a t i o n s . The d e s i r a b i l i t y of having a r e l i a b l e tumour for use as a test system for therapeutic compounds i s considered to be extremely important i n cancer research. In employing s o l i d transplanted tumours for experimental purposes, cert a i n c r i t e r i a 71 for evaluation of e f f e c t had to be set up, as well as regimens for administration of test compounds. The l a t t e r problem depended on empirical choice of route, frequency, dose l e v e l and time at which treatment should be i n i t i a t e d . In evaluating the e f f e c t , the c r i t e r i o n for a p o s i t i v e reaction had to be d e f i n i t e l y stated i n order to avoid confusion as to the comparable effectiveness of p a r t i c u l a r compounds. In the case of the Vinca a l k a l o i d s , i n h i b i t i o n of tumour growth within a certain time period, as determined by measurements and calculated weights, was the desired endpoint. Dose regimens were chosen empirically with regard to amounts administered while the choice of d i f f e r e n t periods of i n j e c t i o n was made i n order to see what e f f e c t the establishment of the tumour had on i t s s u s c e p t i b i l i t y to treatment. With Vinblastine there was l i t t l e difference i n tumour growth when the compound was given aft e r the tumour had become well-established. However, some of the test animals did not develop tumours throughout the entire test period when Vinblastine was administered during the f i r s t week. With V i n c r i s t i n e , also, the response seemed to be somewhat better when treatment was given during the early stages of tumour establishment. The mechanism of action of both alkaloids i s unknown, although anti-tumour effects have been well documented i n 72 s e v e r a l animal experiments (51). In most cases the response has been of two types, as seen i n the p r e s e n t experiments. The most s t r i k i n g e f f e c t i s s u p p r e s s i o n of takes, w i t h i n d e f i -nate s u r v i v a l of the t r e a t e d animals, w h i l e the more common response i s i n h i b i t i o n of growth r e s u l t i n g i n prolonged but l i m i t e d s u r v i v a l . The e f f e c t o f e a r l y treatment w i t h the a l k a l o i d s on percentage of tumour takes has been observed p a r t i c u l a r l y on P1534 leukemia i n mice, where i n i t i a t i o n of treatment s i x days a f t e r i m p l a n t a t i o n r e s u l t s i n absence of takes and i n d e f i n a t e s u r v i v a l . I t i s thought t h a t the s t r i k i n g c o n t r o l of t r a n s p l a n t growth might be due to some i n h i b i t i o n o f the v a s c u l a r supply which i s n e c e s s a r y f o r any tumour development. Both V i n b l a s t i n e and V i n c r i s t i n e a r e capable of causing m i t o t i c a r r e s t i n both i n v i v o and i n v i t r o systems (16, 51). The s p e c i f i c e f f e c t i s thought to be on the s p i n d l e so t h a t the chromosomes a r e h e l d a t metaphase, p r e s e n t i n g a p i c t u r e known as C - m i t o s i s . C u t t s showed t h a t a s i n g l e i n t r a p e r i t o n e a l i n j e c t i o n of V i n b l a s t i n e to animals b e a r i n g tumours caused an i n c r e a s e i n the number of c e l l s i n metaphase, w h i l e m u l t i p l e doses a t d a i l y i n t e r v a l s had an a d d i t i v e e f f e c t on accumulation of metaphases (16). However, Johnson, i n comparing t h i s 73 a c t i v i t y with that of a known spindle i n h i b i t o r such as c o l c h i c i n e , has stated that the s i m i l a r i t y i s probably coincidental and does not advance i t as an explanation of the anti-tumour action (51). Further biochemical studies have suggested a possible anti-metabolic e f f e c t involving amino acids, but as yet no accepted theory has been presented for the mode of action of the Vinca a l k a l o i d s . The testing of experimental tumours with hormones has a double r a t i o n a l e . , The more general reason i s to record any new tumour, regardless of morphology, which responds non-s p e c i f i c a l l y to various hormones. Certain adrenocortical compounds have been used i n cancer therapy with varying degrees of success, depending on the tumour systems tested. They have often been employed as s p e c i f i c a n t i m i t o t i c or a n t i p r o l i f e r a t i v e agents (18). A more s p e c i f i c purpose i s to investigate the p o s s i b i l i t y that the tumour may be hormone-responsive, by v i r t u e of i t s tissue of o r i g i n . In the case of certain steroids, derived from the adrenal cortex and the testes, a study of their effects on tumours of s t r i a t e d muscle was deemed p a r t i c u -l a r l y i n t e r e s t i n g i n view of their physiologic effects on protein metabolism. Voluntary muscle i s known to exhibit completely opposite reactions, with respect to i t s protein metabolism, 74 a f t e r administration of adrenal glucocorticoids on the one hand and androgens on the other. The effects of glucocorticoids have been demonstrated to be severe depletion of tissue protein and a negative nitrogen balance, manifested by muscle atrophy and weight lo s s . Conversely, the administration of androgens r e s u l t s i n a myotrophic or protein anabolic e f f e c t , causing generalized muscular hypertrophy. I t seemed l o g i c a l that a muscle tumour might also exhibit these antagonistic effects i n response to exogenous c o r t i c o s t e r o i d s . At the present time there i s only one p h y s i o l o g i c a l system i n use for large scale testing of testosterone and androgenic derivatives. I t involves the myotrophic action of such compounds on the levator ani muscle i n r a t s . After i n j e c t i o n with appropriate compounds, the muscle i s dissected out and i t s weight i s compared with controls, a twofold increase i n size being i n d i c a t i v e of active anabolic e f f e c t s . I t was thought that a responsive rhabdomyosarcoma might provide another test system for such compounds. The use of a wide v a r i e t y of c o r t i c o s t e r o i d dose regimens i n attempted treatment of rhabdomyosarcomas was necessitated by the lack of other experimental information on such tumours. A l l routes and doses chosen were pharmacological since the r a t has no endogenous secretion of C o r t i s o l or cortisone. 75 The only constant factor was the time of i n i t i a t i o n of treatment, on the seventh day afte r transplantation. This time lapse was allowed i n order for the tumour gr a f t to achieve v a s c u l a r i z a t i o n . I t i s an established fact now that most transplanted tumour c e l l s acquire a blood supply from the host. A l g i r e and Chalkley showed that the vascular system of a tumour owes i t s o r i g i n to the host's response to the tumour transplant (3). Immediately following transplantation, f l u i d and leukocytes accumulate around the foreign c e l l s and adjacent c a p i l l a r i e s begin to d i l a t e during the next few days. By the f i f t h to seventh day d e f i n i t e vessels are present In the tumour, occupying a s t a b i l i z e d 40 to 50 per cent of the t o t a l tumour volume (2). Concomitant with t h i s penetration, the rate of growth of the tumour becomes approximately constant and gross tumour growth i s now evident. The vascular supply of the tumour i s usually 100 per cent greater than that of the surrounding connective tissue, i n d i c a t i n g that the tumour w i l l have the preferred n u t r i t i o n a l status of the two tissues. Since t h i s same phenomenon occurs, although only temporarily, i n wound healing, i t i s thought that the tumour merely appropriates the host vessels which have p r o l i f e r a t e d as they would i n response to any foreign body or wound. 76 The tumour vessels are generally similar to c a p i l l a r i e s i n structure, having only a single endothelial layer, but the diameters are often large. Merwin and H i l l have described the enlarging and convoluting of the host c a p i l l a r i e s (62). The resultant tortuosity and slow blood flow may be s i g n i f i c a n t i n the possible s e n s i t i v i t y of transplanted neoplasms to a n t i -tumour compounds. Since the expected poor oxidation and accumulation of l a c t i c acid might a f f e c t subsequent growth of rhabdomyosarcomas, i n i t i a l v a s c u l a r i z a t i o n was considered to be an important and necessary factor before treatment was i n i t i a t e d . Despite the v a r i a b i l i t y of r e s u l t s obtained with c o r t i c o i d administration, certain i n t e r e s t i n g trends were indicated. The f i r s t of these was the s t a r t l i n g i n h i b i t i o n of growth, without attendant weight loss, r e s u l t i n g from i n t r a -peritoneal i n j e c t i o n s of either C o r t i s o l or cortisone to an early transplant generation of one tumour. Radioactive tracer studies have shown that i n t r a p e r i t o n e a l l y administered c o r t i c o -steroids w i l l reach a peak i n the blood stream within h a l f an hour and be completely removed afte r two hours (13). The lack of protein breakdown, as shown by weight loss, following t h i s route of administration was probably due to the f a c t that there was l i t t l e time for the steroids to act on the body's protein reserves. The repeated attacks of high doses might explain 77 the anti-tumour action, while the rapid disappearance of the injected material might r e s u l t i n the i n h i b i t i o n of growth only during the i n j e c t i o n period. The curious phenomenon noted when a higher dose of i n t r a p e r i t o n e a l l y administered C o r t i s o l proved i n e f f e c t i v e against a tumour l i n e which was o r i g i n a l l y very susceptible served to emphasize the main drawback i n using transplantable tumours as test systems for therapeutic compounds. S n e l l has stated that tumours may change during many transfers due to morphologic or physiologic a l t e r a t i o n s (74). In the former case, responsive normal c e l l s which might o r i g i n a l l y have been carr i e d along with the tumour have now become neoplastic or mutations within tumour c e l l s may have taken place. Physiologic changes i n the tumour may include an increase i n the number of faster-growing c e l l s or an enhanced a b i l i t y to k i l l hosts which were previously r e s i s t a n t . . The fa c t that the h i s t o l o g i c a l composition of the tumour had not changed appreciably indicated true progression, where one c h a r a c t e r i s t i c , hormone s e n s i t i v i t y , had changed independently of growth. . The adoption of subcutaneous inje c t i o n s was decided upon i n order to determine any effects of a more prolonged presence of c o r t i c o i d s . Such compounds injected i n th i s manner should be released slowly from the s i t e of i n j e c t i o n , 78 accumulating i n a depot f o r a few days. T h e r e f o r e one would suppose t h a t the e f f e c t i v e dose should r i s e s l i g h t l y a t the s t a r t o f the i n j e c t i o n p e r i o d and f a l l o f f s l o w l y a t the end. The f a c t t h a t the tumour growth r a t e was i n h i b i t e d o n l y p a r t i a l l y d u r i n g the time of treatment i n d i c a t e d t h a t the s t e r o i d s p r o b a b l y had a slower but more prolonged e f f e c t . The weight l o s s r e s u l t i n g from t h i s treatment was not unexpected, s i n c e the continued presence o f C o r t i s o l or cortisone would l o g i c a l l y r e s u l t i n continued breakdown of the p r o t e i n r e s e r v e s of the body. Another i n t e r e s t i n g p o i n t was i l l u s t r a t e d by the combination treatment of one tumour w i t h both C o r t i s o l and t e s t o s t e r o n e . T h i s was done i n order to determine i f the decreased weight of the t e s t tumours was a r e s u l t o f g e n e r a l systemic weight l o s s . The f a c t t h a t tumour weight was i n h i b i t e d i n animals treated either with C o r t i s o l or with C o r t i s o l p l u s t e s t o s t e r o n e , w h i l e the body weight l o s s was r e v e r s e d i n those r e c e i v i n g both hormones, gave c l e a r - c u t evidence t h a t the a n t i -tumour e f f e c t of C o r t i s o l was d i s t i n c t from i t s catabolic e f f e c t s i n rhabdomyosarcoma-bearing animals. Treatment of some t e s t r a t s w i t h t e s t o s t e r o n e showed r a t h e r e q u i v o c a l e f f e c t s . In an i d e a l s i t u a t i o n , the tumours should 79 have been slower growing so that any increase i n protein anabolism would be more obvious. However, some tentative indications of action could be noted. Although there was no e f f e c t i f the hormone was administered a f t e r the f i r s t week of growth, some examples of accelerated growth were seen when testosterone was injected twenty-four hours aft e r transplantation. This suggested a mode of action d i f f e r e n t from that of other steroids, with t h i s hormone's e f f e c t being mediated by some means other than established v a s c u l a r i z a t i o n . A possible explanation i s that testosterone may have been exerting i t s myotrophic e f f e c t on muscle elements s t i l l present within the rhabdomyosarcoma. On th i s basis, however, one would expect those transplant generations closest to the primary tumour to show the greatest response, since they would r e t a i n a greater amount of o r i g i n a l tissue c h a r a c t e r i s t i c s . This was obviously not the case, with both sens i t i v e l i n e s being i n the eleventh generation. The anti-tumour actions of the adrenal steroids are r e l a t i v e l y unknown, although i t has been suggested that c o r t i c o i d s may i n t e r f e r e with growth of cert a i n mesodermal structures and connective tissue, attacking such elements as f i b r o b l a s t growth (53) . S i m i l a r l y , the action of testosterone 80 has not been demonstrated to s a t i s f a c t i o n , other than by the gross physiologic response of hormone dependency i n certa i n tumours derived from endocrine tissues (68) . Thus the divergent responses obtained i n the present studies do not necessarily mean that the experiments have had e s s e n t i a l l y negative r e s u l t s . In a search for sensitive and r e s i s t a n t animal strains to serve as r e l i a b l e test systems, any i n f o r -mation which r e s u l t s from empirical testing of new tumours i s of some benefit. Because of the v a r i a b i l i t y of response, transplantable r a t rhabdomyosarcomas would probably not be of great value for routine testing of steroi d compounds. 81 CONCLUSIONS A study of the induction of rhabdomyosarcomas i n rats by the i n j e c t i o n of n i c k e l sulphide provided information not only on the carcinogenic a c t i v i t y of n i c k e l sulphide but also on the growth and development of rhabdomyosarcomas i n r a t s . I t was found that intramuscular i n j e c t i o n s of Ni3S2 resulted i n f a i r l y rapid appearance of tumours at the s i t e of i n j e c t i o n , with 20 mg. causing 100 per cent tumours a f t e r four months. These tumours were examined by h i s t o l o g i c a l means and the i r i d e n t i t y established as rhabdomyosarcomas. After their f i r s t appearance as swelling of the muscle region, they grew r a p i d l y , r e s u l t i n g i n complete atrophy of the injected muscle. On transplantation the tumours proved to be highly malignant, with the average host mortality ranging between three and eight weeks. Attempts at experimental tumour therapy proved rather equivocal. Tests with the Vinca a l k a l o i d s seemed to be most e f f e c t i v e i n causing i n h i b i t i o n of growth when both Vinblastine and V i n c r i s t i n e were administered during the f i r s t week afte r implantation. Administration of steroids for a n t i -tumour and metabolic experiments resulted i n a response ranging from r e s i d u a l to complete i n h i b i t i o n of many tumours following 82 c o r t i c o i d treatment, and acceleration of growth of some tumours following testosterone administration. In general the tumours appeared to be e s s e n t i a l l y unreliable for such studies due to unpredictable changes i n response among d i f f e r e n t generations. Preliminary experiments on the carcinogenicity of NiS suggested that i t was more toxic and less carcinogenic than Ni3S2 under similar experimental conditions. A possible reason for this difference i n a c t i v i t y may have been the rapid disappearance of NiS from the injected muscle areas, noted i n most animals. The possible carcinogenicity of d i f f e r e n t metal s a l t s offers many avenues of further experimentation. The problem of s o l u b i l i t y i n muscle acids can be examined from two d i r e c t i o n s . F i r s t , the intramuscular or subcutaneous implantation of weighed metal p e l l e t s would provide a continuous supply of n i c k e l to the muscle, while t h e i r l a t e r removal and weighing would show exactly how much had been released. In addition, the s o l u b i l i t i e s of c l o s e l y r e l a t e d compounds such as NigS2 and NiS could be compared by estimations of the amount of n i c k e l excreted i n the urine. The alleged s p e c i f i c i t y of n i c k e l sulphide for s t r i a t e d muscle might be validat e d by more extensive experiments involving exposure of several body 83 tissues to the compounds for longer periods of time. The rapid induction of rhabdomyosarcomas by n i c k e l sulphide presents many opportunities for the i n v e s t i g a t i o n of early, premalignant changes, since the histogenesis of such tumours i s r e l a t i v e l y unknown. S e r i a l studies of the exposed muscle would provide information on i t s acute and chronic reactions to foreign i r r i t a n t s , followed by possible tissue damage and neoplasia. Periodic biopsies of the muscle and, more p a r t i c u l a r l y , the developing tumour, might give some in d i c a t i o n as to whether the tissue undergoes progressive changes to an ultimate malignant form. The comparative r a r i t y of t h i s tumour of f e r s a challenging experimental model for research purposes. 84 APPENDIX M a l l o r y ' s P h o s p h o t u n g s t i c - a c i d Haematoxylin (PTAH) S t a i n : 1. Remove wax and b r i n g s e c t i o n s to water 2. Mordant s e c t i o n s i n m e r c u r i c c h l o r i d e f o r t h r e e hours a t room temperature 3. Rinse i n d i s t i l l e d water 4. S t a i n w i t h Lugol's i o d i n e , f i f t e e n minutes 5. Rinse i n two changes of 95 per cent a l c o h o l 6. R i n s e i n d i s t i l l e d water 7. S t a i n w i t h 0.25 per cent potassium permanganate, f i f t e e n minutes 8. R i n s e i n d i s t i l l e d water 9. S t a i n w i t h f i v e per cent o x a l i c a c i d u n t i l b l eached white 10. R i n s e i n d i s t i l l e d water 11. S t a i n w i t h M a l l o r y ' s PTAH, eigh t e e n to twenty-fo u r hours 12. R i n s e i n 95 per cent a l c o h o l 13. Dehydrate, c l e a r and mount P e r l ' s P r u s s i a n Blue s t a i n : 1. Remove wax and b r i n g s e c t i o n s to water 2. Mix equal amounts of f r e s h l y made 2 per cent potassium f e r r o c y a n i d e and 2 per cent h y d r o c h l o r i c a c i d ; s t a i n s e c t i o n s one hour 3. Rinse i n d i s t i l l e d water 4. C o u n t e r s t a i n w i t h e o s i n , two minutes 5. Dehydrate, c l e a r and mount 85 BIBLIOGRAPHY 1. ADAMS, R.D., DENNY-BROWN, D., and PEARSON, C.M. Diseases of Muscle. 2nd ed., New York: Harper and Brothers, 1962. 2. ALGIRE, G.H. Vascular Reactions of Normal and Malignant Tissues i n Vivo. J. Natl. Cancer Inst., 15: 483, (1954). 3. ALGIRE, G.H.., and CHALKLEY, H.W. The Vascular Supply of Mammary Gland Carcinomas. In: F.R. Moulton (ed.), Mammary Tumours i n Mice. Lancaster: Science Press, 1945. 4. ALTSCHUL, R. Atrophy, Degeneration and Metaplasia i n Denervated Skeletal Muscle. Arch. Path. 34: 982, 1942. 5. ANDERVONT, H.B. 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Thesis/Dissertation
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Physiology
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A study of rhabdomyosarcomas induced by nickel sulphide in rats.
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