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A study of the growth of transplantable tumors in alloxanized Wistar rats Tilser, George 1955

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A STUDY OF THE GROWTH OF  TRANSPLANTABLE TUMORS IN  ALLOXANIZED WISTAR RATS GEORGE TILSER A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n the Department o f BIOLOGY AND BOTANY We accept t h i s t h e s i s as conforming to the standard r e q u i r e d from candidates f o r the degree of MASTER OF ARTS. Members o f the Department o f THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1955. ABSTRACT Blood sugar l e v e l s , and tumor s i z e and i n c i d e n c e were determined f o r a l l o x a n i z e d W i s tar r a t s b e a r i n g subcutaneous and i n t r a p e r i t o n e a l t r a n s p l a n t s o f N o v i k o f f hepatoma and Walker 256 carcinoma, and h i s t o l o g i c a l examination o f the pancreas was undertaken to determine the extent o f the damage to the b e t a - c e l l s of the i s l e t s o f Langerhans. Experiments were conducted i n which animals were a l l o x a n i z e d 14 days, 7 days, 4 days, and 24 hours p r i o r t o , and 24 hours and 4 days a f t e r t r a n s p l a n t i n g . The incidence and r a t e o f growth o f i n t r a p e r i t o n e a l tumors was a p p r e c i a b l y decreased, and the r a t e of growth o f subcutaneous tumors was s l i g h t l y decreased i n a l l o x a n i z e d animals, as compared w i t h tumor-bearing c o n t r o l s . In tumor-bearing a l l o x a n i z e d animals, the h i g h blood sugar l e v e l s c h a r a c t e r i s t i c o f a l l o x a n - d i a b e t e s were reduced to normal i n some animals, and c o n s i d e r a b l y ameliorated i n o t h e r s , as compared w i t h a l l o x a n - d i a b e t i o c o n t r o l s * I n t r a p e r i -t o n e a l tumors were more e f f e c t i v e than subcutaneous tumors i n reducing blood sugar l e v e l s , and r e l i e f was more pronounced i n animals a l l o x a n i z e d j u s t before or a f t e r t r a n s p l a n t i n g than i n those a l l o x a n i z e d two weeks before t r a n s p l a n t i n g . H i s t o l o g i c a l examination i n d i c a t e d t h a t i n t r a p e r i t o n e a l tumor t i s s u e invading the pancreas o f a l l o x a n ! z e d r a t s exerted a p r o t e c t i v e or regenerative e f f e c t on the b e t a - c e l l s o f the i s l e t s of Langerhans, which are s e l e c t i v e l y destroyed by a l l o x a n . The p o s s i b l e r o l e o f s u l p h y d r y l groups i n the ame* l i o r a t i o n o f a l l o x a n - d i a b e t e s i n tumor-bearing animals, and i n the r e d u c t i o n o f tumor s i z e and i n c i d e n c e i n a l l o x a n - d i a b e t i e animals i s discussed* This work was reported i n p a r t i n Proceedings o f the American A s s o c i a t i o n f o r Cancer Research, J2;20, 1955* ACKNOWLEDGEMENTS I am indebted to Dr. E.S. Goranson, tinder whose d i r e c t i o n t h i s i n v e s t i g a t i o n was undertaken, f o r h i s t h o u g h t f u l guidance, sound advice, and constant i n t e r e s t throughout the course o f the study. To Dr. A.H. Hutchinson and Dr. T.M.C. Tay l o r I am g r a t e f u l f o r s t i m u l a t i n g encouragement during the p r e p a r a t i o n o f t h i s t h e s i s . I s i n c e r e l y a p p r e c i a t e the s e r v i c e s rendered by Dr. P a r i s Constantinides and the Department o f Anatomy, F a c u l t y o f Medicine, f o r the a s s i s t a n c e , advice, and use o f equipment without which the h i s t o l o g i c a l s t u d i e s would have been impos-s i b l e . Dr. W.S. H a r t r o f t o f the M e d i c a l F a c u l t y of the Un i v e r * s i t y of Toronto k i n d l y s u p p l i e d d e t a i l s on the s t a i n i n g pro-cedure f o r the b e t a - c e l l s . To my f e l l o w - s t u d e n t s , J.R. McBride and R. Ghopal* Singh, I am Indebted f o r a s s i s t a n c e i n making blood sugar determinations. This work was supported by a G r a n t * i n ~ A i d from the N a t i o n a l Cancer I n s t i t u t e o f Canada. TABLE OF CONTENTS I . INTRODUCTION A. R e l a t i o n between a l l o x a n - d i a b e t e s and tumor Incidence and growth 1 B. H i s t o l o g y of the i s l e t s o f Langerhans i n the normal pancreas o f the r a t •• 3 C. Changes induced by a l l o x a n i n the i s l e t s o f Langerhans and other t i s s u e s 5 I I . METHODS A. Experimental procedures H B. H i s t o l o g i c a l procedures 12 I I I . RESULTS A. E f f e c t of a l l o x a n on blood sugar l e v e l s i n tumor-bearing r a t s 1, A l l o x a n administered to animals b e a r i n g i n t r a p e r i t o n e a l t r a n s p l a n t s of tumors • • 16 'Z,' A l l o x a n administered p r i o r to i n t r a -p e r i t o n e a l i m p l a n t a t i o n o f tumors .., 16 3. A l l o x a n administered to animals b e a r i n g subcutaneous tumor t r a n s p l a n t s 21 4, A l l o x a n administered p r i o r to the sub-cutaneous i m p l a n t a t i o n of tumors 2o B. E f f e c t of a l l o x a n ! z a t i o n on tumor growth i n r a t s t r a n s p l a n t e d w i t h N o v i k o f f hepatoma and Walker 2j?6 carcinoma • 2o C. E f f e c t o f a l l o x a n ! z a t i o n on tumor incidence i n r a t s t r a n s p l a n t e d w i t h N o v i k o f f hepatoma • >1 D. H i s t o l o g y of the pancreas 1. The i s l e t s of Langerhans i n the pancreas of the normal r a t D# H i s t o l o g y o f the pancreas (cont'd.) 2. The i s l e t s o f Langerhans i n a l l o x a n i z e d rats • • 33 3» The i s l e t s o f Langerhans i n tumor-b e a r i n g r a t s .. 37 4. The i s l e t s o f Langerhans i n r a t s a l l o x a -n i z e d 2 weeks p r i o r to t r a n s p l a n t i n g w i t h Walker 2j?o carcinoma "subcutaneously * 40 5. The i s l e t s o f Langerhans i n r a t s t r a n s - . p l a n t e d subcutaneously w i t h Walker 256 carcinoma 4 days p r i o r to a l l o x a n i z i n g • 40 6* The i s l e t s o f Langerhans i n r a t s a l l o x a -n i z e d one day p r i o r to t r a n s p l a n t i n g t i n t r a p e r i t o n e a l l y w i t h N o v i k o f f hepatoma 43 7. The i s l e t s o f Langerhans i n r a t s t r a n s -p l a n t e d i n t r a p e r i t o n e a l l y w i t h Walker 2j?6 carcinoma 4 days p r i o r to a l l o x a n i z i n g . 45 E. Summary o f r e s u l t s • •••••• 50 IV. DISCUSSION A. The i n h i b i t i o n o f a l l o x a n - d i a b e t e s by tumor . 52 B. P r e v e n t i o n and a l l e v i a t i o n o f a l l o x a n - d i a b e t e s by s u l p h y d r y l compounds 55 C. The i n f l u e n c e o f a l l o x a n - d i a b e t e s on tumor growth and i n c i d e n c e 59 V. SUMMARY 62 LITERATURE CITED 65 I . INTRODUCTION. A. R e l a t i o n between alloxan.diabetes,and tumor Incidence  and growth. During the course o f experiments designed to determine the e f f e c t o f the metabolic disturbances a s s o c i a t e d w i t h diabetes on tumor development and growth, i t was discovered that diabetes induced by a l l o x a n and growth o f t r a n s p l a n t a b l e N o v i k o f f hepatoma were mutually antagonistic ( 3 5 ) , . The h i g h blood sugars o f a l l o x a -n i z e d r a t s were reduced towards normal l e v e l s i n tumor-bearing animals, and the incidence and s i z e of.tumors were diminished i n a l l o x a n i z e d animals. Dunning, C u r t i s , and Friedgood (25),reported:that the appearance of benzpyrene-induced sarcomas was n e i t h e r delayed nor prevented i n r a t s o f three s t r a i n s p r e v i o u s l y made d i a b e t i c w i t h a l l o x a n . However t h e i r data show th a t tumor inc i d e n c e was reduced from between 90 and 100 p e r - e e n t - i n the case-of.non-d i a b e t i c a l l o x a n - r e f r a o t i v e o r normal c o n t r o l s to &7 per cent i n d i a b e t i c animals. They d i d not i n v e s t i g a t e the h i s t o l o g y o f the pancreas, nor d i d they present data'on*the blood-sugar l e v e l s . C a r r i e and Ham (13) reported t h a t sarcoma 37 t r a n s -planted subcutaneously i n t o r a t s p r e v i o u s l y made d i a b e t i c w i t h a l l o x a n d i d not reduce blood sugar l e v e l s below those observed i n a l l o x a n i z e d c o n t r o l s , and that tumors grew Just as r a p i d l y i n d i a b e t i c r a t s . a s i n non-diabetic c o n t r o l s . Goranson et a l (35) suggest that the f a i l u r e of a l l o x a n to e x h i b i t any e f f e c t on s u r v i v a l time or tumor development i n these cases may be due to the f a c t that the tumors used were sarcomas, and a l s o suggest th a t the s i t e o f tumor development may be a c o n t r i b u t o r y f a c t o r . The instances c i t e d below, i n which a l l o x a n d i d reduce tumor growth and inc i d e n c e , and a l s o prolonged s u r v i v a l time were a l l oases i n which the tumors i n v o l v e d were carcinomas. Salzberg and G r i f f i n (67) s t u d i e d the e f f e c t o f feeding the carcinogenic azo dye >-methyl-4~dimethylaminoazobenzene to r a t s p r e v i o u s l y made d i a b e t i c w i t h a l l o x a n . One week a f t e r the i n j e c t i o n o f a l l o x a n , r a t s were placed on a d i e t c o n t a i n i n g 0.06 per cent o f the carcinogenic dye, and were maintained on i t f o r a p e r i o d o f three months. On s a c r i f i c i n g or laparotomy 4 to 8 weeks a f t e r the carcinogenic d i e t was d i s c o n t i n u e d , 90 to 100 per cent o f the normal c o n t r o l s had developed hepatomas, whereas o n l y one of 14 a l l o x a n i z e d r a t s showed evidence of hepatoma, and two others had moderate l i v e r n e c r o s i s . These r e s u l t s were a l l the more s t r i k i n g because d i a b e t i c animals consumed about twice as much o f the carcinogenic agent as normal,controls, due to t h e i r increased food consumption. Salzberg has a l s o obtained confirmatory evidence of the i n h i -b i t i o n of growth o f tr a n s p l a n t e d N o v i k o f f hepatoma i n a l l o x a -n i z e d Sprague-Dawley r a t s (pers. comm.). j e h l and McKee (49) found that the s u r v i v a l time of - 5 -a l l o x a n - d i a b e t i c mice i n o c u l a t i o n s o f Ehrlich»s mouse a s c i t e s carcinoma was longer than that o f mice r e c e i v i n g the a s c i t e s carcinoma alone. These s t u d i e s i n d i c a t e a; l i n k between the diabetes induced by a l l o x a n and the genesis and growth of tumors. I n no i n s t a n c e , however, has h i s t o l o g i c a l i n v e s t i g a t i o n of the pancreas been undertaken to determine the e f f e c t o f tumor on the i s l e t s o f Langerhans, and o n l y i n the experiments conducted by Goranson et a l was the i n f l u e n c e of tumor on d i a b e t i c hyperglycemia observed. The present s e r i e s o f experiments was undertaken to f u r t h e r e l u c i d a t e t h i s phenomenon, w i t h p a r t i c u l a r reference to the h i s t o l o g i c a l p i c t u r e i n the pancreas. B. H i s t o l o g y of the i s l e t s o f Langerhans In the normal  pancreas o f the r a t . Gomori (31) demonstrated that the c e l l s of the pan-c r e a t i o i s l e t s o f Langerhans o f the r a t c o n s i s t o f two main types, which d i f f e r i n g r a n u l a t i o n and i n t h e i r r e a c t i o n to va r i o u s s t a i n s , but not i n nuclear s t r u c t u r e . The type c o n s t i -t u t i n g the m a j o r i t y of c e l l s occupies almost a l l of the I s l e t except the p e r i p h e r a l r e g i o n . Although v a r i a t i o n s e x i s t i n the s t a i n i n g p r o p e r t i e s of these o e l l s , they are not s u f f i c i e n t l y great to warrant d i v i d i n g the o e l l s i n t o two types. These are the b e t a - c e l l s , which are r e s p o n s i b l e f o r the s e c r e t i o n o f i n s u l i n . I t i s u s u a l l y assumed that the granules observed i n them represent i n s u l i n or i t s p r e c u r s o r , s i n c e metabolic s i t u a t i o n s inducing i n s u l i n s e c r e t i o n tend to cause temporary degranulation o f the c e l l s . The second c e l l type c o n s t i t u t e s the m i n o r i t y of c e l l s , and occupies the p e r i p h e r a l r e g i o n o f the i s l e t . They d i f f e r from the f i r s t type o n l y i n the d i f f e r e n t s t a i n i n g r e a c t i o n s o f t h e i r cytoplasmic granules and are known as a l p h a - c e l l s . I t i s sug-gested that theynseorete the hormone "glucagon*, which i s hyper-glycemic and thus a n t a g o n i s t i c to i n s u l i n i n i t s a c t i o n (26). . A r n o l d (2) however s t a t e s that there i s a s i z e d i f -ference between the n u c l e i o f alpha- and b e t a - c e l l s , the n u c l e i o f the former averaging 62 cu. i n volume, as compared w i t h 88 cu. f o r the n u c l e i o f the l a t t e r . However, the range o f v a r i a t i o n i n both types i s such that l a r g e a l p h a - c e l l s and s m a l l b e t a - c e l l s overlap i n s i z e . Hard (40) s t a t e s that the b e t a - c e l l s develop during the l a t t e r h a l f of embryonic l i f e and the f i r s t week of post-n a t a l l i f e . The f i r s t three or four i s l e t s d i f f e r e n t i a t e from -the s o l i d p a n c r e a t i c cord. Other i s l e t s o r i g i n a t e from the pancreatic tubules and from the s e c r e t o r y duct system at the base of the a c i n i , and are o c c a s i o n a l l y observed w i t h i n the a c i n i proper. I s l e t s o r i g i n a t i n g i n the l a t t e r way .develop o n l y a f t e r b i r t h . The only c e l l type to d i f f e r e n t i a t e during embryonic l i f e i s -the b e t a - c e l l , beta-granules appearing f i r s t on the 19th day, and s e c r e t o r y a c t i v i t y o c c u r r i n g on the 2.1st day o f embryonic l i f e . The f i r s t a l p h a - c e l l s mere detected on the second day of p o s t n a t a l l i f e and mere a c t i v e l y s e c r e t i n g by the 5th day. C. Changes induced by a l l o x a n i n the i s l e t s of Langerhans  and other t i s s u e s . Dunn, Sheehan, and McLetchie (24) attempted to produce r e n a l damage by the i n j e c t i o n of a l l o x a n i n r a b b i t s . They observed an i n i t i a l r i s e i n blood sugar l e v e l , f o l l o w e d by intense hypoglycemia which proved f a t a l to a l l animals. Upon h i s t o l o g i c a l examination they noted s e l e c t i v e d e s t r u c t i o n o f the i s l e t s of Langerhans i n the pancreas. Brunschwlg et a l (12) reported hyperglycemia sustained i n dogs f o r s e v e r a l weeks f o l l o w i n g a l l o x a n i n j e c t i o n . B a i l e y and B a i l e y (4) produced permanent diabetes i n r a b b i t s w i t h the intravenous i n j e c t i o n o f 150 to 200 mg. o f a l l o x a n per kg. o f body weight. Dunn and McLetchie (23) produced permanent dia b e t e s " accompanied by n e c r o s i s of the i s l e t s of Langerhans i n r a t s by subcutaneous i n j e c t i o n of 150 to 200 mg. o f a l l o x a n per kg. o f body weight. Goldner and Gomori (2.8) produced diabetes and i s l e t -c e l l n e c r o s i s i n dogs by a s i n g l e intravenous I n j e c t i o n of a l l o x a n . They a l s o produced permanent diabetes i n c a t s , pigeons, r a t s and r a b b i t s . E a r l y workers (9, 10) r e p o r t e d that l a r g e doses o f - 6 -a l l o x a n f a i l e d to produce diabetes or i s l e t c e l l damage i n humans. However, i t was shown l a t e r that i s l e t c e l l d e s t r u c t i o n ocourred i n a human case f o l l o w i n g the i n j e c t i o n o f a l a r g e dose of a l l o x a n (16). The h i s t o l o g i c a l changes i n the i s l e t s of Langerhans a f t e r the i n j e c t i o n of a diabetogenic dose of a l l o x a n have been described by s e v e r a l authors (5, 6, 9, 20, 21, 22, 23, 24, 29, 33, 48, 30). T h e i r observations have been summarized by Duff (19) as f o l l o w s : »The h i s t o l o g i c a l changes i n the i s l e t s o f Langerhans f o l l o w i n g a s i n g l e i n j e c t i o n of a l l o x a n .... appear to be p r a c t i c a l l y i d e n t i c a l i n r a b b i t s and i n r a t s . In both s p e c i e s , even as e a r l y as 5 minutes a f t e r the i n j e c t i o n of a diabetogenic dose of a l l o x a n , s l i g h t but d e f i n i t e changes are d i s c e r n i b l e i n the n u c l e i and cytoplasm of the b e t a - c e l l s w i t h a suggestion o f some diminution of t h e i r s p e c i f i c granules. At from 10 to 15 minutes a f t e r i n j e c t i o n there i s a d e f i n i t e r e d u c t i o n of granules. These changes a f f e c t f i r s t the b e t a - c e l l s at the centers of the l a r g e r i s l e t s . Before the end o f one'hour there i s some shrinkage of the a f f e c t e d c e l l s which appear more c l o s e l y paoked and there i s a corresponding widening o f the p e r i c a p i l l a r y spaoes. By the end of one to two hours d e f i n i t e pyknosis of n u c l e i i s evident, and t h i s becomes p r o g r e s s i v e l y more con-spicuous and extensive i n the next few, hours. At the end of three hours some of the a f f e c t e d c e l l s may be detached from one another and rounded In shape w i t h homogeneous, e o s i n o p h i l i c cytoplasm. In the ensuing hours the cytoplasm may become f u r t h e r shrunken or may show conspicuous s w e l l i n g . Signs of d i s i n t e g r a t i o n of the cytoplasm w i t h coalescence of the most a f f e c t e d c e l l s begin to appear. While shrunken, pycnotic n u c l e i may remain v i s i b l e f o r 16 to 24 hours or more, i n c r e a s i n g numbers of n u c l e i begin to show k a r y o l y s i s and there i s evidence of complete d i s i n t e g r a t i o n and disappearance of i n d i v i d u a l c e l l s from 5 hours onwards. These changes continue, u n t i l at the end of 20 to 24 hours the centers of the i s l e t s are occupied o n l y by pale s t a i n i n g g r a n u l a r d e b r i s i n which the shadows of n u c l e i and o c c a s i o n a l n e c r o t i c c e l l s can b a r e l y be recognized. The time o f f i n a l disappearance o f the s p e c i f i c granules of the b e t a - c e l l s i s v a r i o u s l y reported at from 15 minutes to 2 or 3 days. "While the changes described are proceeding i n the b e t a - c e l l s , the p e r i p h e r a l l y placed a l p h a - c e l l s remain f o r the most part undamaged though they may show some s w e l l i n g and evidence of s l i g h t degenerative change. A c c o r d i n g l y , many of the i s l e t s show a n e c r o t i c center surrounded by a l a y e r of s u r v i v i n g a l p h a - c e l l s . Some of the a l p h a - c e l l s , however, may s u f f e r g r e a t e r i n j u r y l e a d i n g to n e c r o s i s of s i n g l e c e l l s here and there. According to some d e s c r i p t i o n s , a l l of the c e l l s of both alpha- and beta-types are apparently destroyed i n prac-t i c a l l y a l l of the i s l e t s . "The extent of the damage i n the i n i t i a l stages seems to determine what may be observed l a t e r . In any case the necrotic debris i s r a p i d l y removed so that no trace of i t i s l e f t a f t e r three to f i v e days and there i s a corresponding collapse of the i s l e t structure. When destruction of i s l e t tissue has been extensive there i s frequently recorded at the end of 5 days or more afte r the i n j e c t i o n the impression of great s c a r c i t y of i s l e t s i n the pancreatic tissue even though small i s l e t s may be i d e n t i f i e d here and there on closer examination. In both rabbits and rats a f t e r more prolonged periods there are recorded i n s ^ tances i n which i t was impossible to f i n d any i s l e t tissue at a l l . On the other hand, a peripheral layer of alpha-cells i s frequently spared and even a few of the b e t a - c e l l s may escape destruction. In this event, when the debris of necrotic c e l l s has disappeared, the i s l e t s may be found i n normal numbers and even of normal appearance when examined with ordinary stains, though they are frequently reduced i n s i z e . However, stains for s p e c i f i c granules show that the i s l e t s consist either of alpha-c e l l s exclusively or of alpha c e l l s with a few non-granular c e l l s of i n d i f f e r e n t type. I t has been suggested that the alpha-c e l l s a c t u a l l y increase i n number, but mitotic figures have been only r a r e l y observed amongst the surviving c e l l s . 1 * That the diabetes r e s u l t i n g from the administration of alloxan i s due to the d i r e c t selective destructive action of alloxan on the b e t a - c e l l s was demonstrated i n the experiments of Goldner and Gomori (34) who observed that l i g a t i o n of the a r t e r i e s supplying a portion of a dogs pancreas for a period o f s i x minutes a f t e r the i n j e c t i o n o f a l l o x a n completely pro-t e c t e d the b e t a - c e l l s i n that r e g i o n and prevented the develop-ment o f d i a b e t e s . A l l o x a n i n j e c t e d s i m u l t a n e o u s l y w i t h i n s u l i n does not i n a c t i v a t e the i n s u l i n ; nor does i n s u l i n prevent the d i a b e t o -genic a c t i o n o f a l l o x a n (29, 30). On the oth e r hand, A l l e g r e t t i and F i s t e r (1) r e p o r t that i n s u l i n p o t e n t i a t e s the d i a b e t o g e n i c a o t i o n o f a l l o x a n and markedly i n c r e a s e s the i n c i d e n c e o f di a b e t e s f o l l o w i n g the a d m i n i s t r a t i o n o f subdiabetogenic doses o f a l l o x a n . Thus the s e l e c t i v e a c t i o n o f a l l o x a n on the be t a -c e l l s can not be due to a r e a c t i o n with i n s u l i n . Houssay (43) suggests three p o s s i b i l i t i e s f o r the mechanism o f the a c t i o n o f a l l o x a n i n s e l e c t i v e l y d e s t r o y i n g the be t a -c e l l s : (1) . A l l o x a n may compete wit h a c h e m i c a l l y s i m i l a r substance necessary f o r the f u n c t i o n i n g o f the b e t a - c e l l s . (2) . A l l o x a n may be accumulated s e l e c t i v e l y i n the b e t a -c e l l s and cause g r e a t e r damage here than i n o t h e r c e l l s . (3) . A l l o x a n may i n a c t i v a t e s p e c i f i c SH groups which are necessary f o r the enzymatic f u n c t i o n o f the b e t a - c e l l s . T h i s h y p o t h e s i s was a l s o suggested by P a l a y and Lazarow (65) and by Sen and Bhattacharya (68). The p r o t e c t i o n o f f e r e d by s u l p h y d r y l - c o n t a i n i n g com-pounds a g a i n s t the d i a b e t o g e n i c a c t i o n o f a l l o x a n i s r e p o r t e d -10-by the three groups mentioned i n the preceeding paragraph and a l s o by B a i l e y (3), Lazarow (51, 5a, 53, 54, 55), Lazarow, P a l a y and Levy (5&), P a l a y and Lazarow-(65), Mart i n e z (58, 59), Houssay, L o t t and M artinez (44), and Ueno (70). Diabetogenic amounts of a l l o x a n a l s o cause damage to organs other than the pancreas. Damage to the e p i t h e l i u m of the convoluted tubules of the kidney has been reported by s e v e r a l authors (5, 6, 22, 23, 24, 33). A l l o x a n a l s o produces n e c r o s i s and yellow atrophy of the l i v e r , according to Wilder (78), P a l a y and Lazarow (65), and Cruikshank (17)» I t i s suggested (52) that the v u l n e r a b i l i t y of c e l l s to the d e s t r u c t i v e a c t i o n of a l l o x a n i s i n v e r s e l y r e l a t e d to the l e v e l of compounds such as g l u t a t h i o n e which counteract the s u l p h y d r y l b i n d i n g a c t i o n of a l l o x a n . The high s p e o i f i c i t y of a c t i o n of a l l o x a n i n the i n s u l a r apparatus of the pancreas i s explained on t h i s b a s i s . e -11-II. METHODS. A. Experimental Procedures. l l s t a r r a t s weighing from 120 to 220. grams were used as experimental animals. Most o f the experiments were performed u s i n g v i r g i n females, but males were used f o r one s e r i e s o f experiments. The animals were fed ad l i b i t u m throughout the experiments on a standard P u r i n a chow d i e t and had f r e e access to water. The tumors used i n these experiments were the t r a n s -p l a n t a b l e N o v i k o f f hepatoma and the Walker 256 carcinoma. For t r a n s p l a n t i n g , non-necrotic p o r t i o n s o f i n t r a p e r i t o n e a l hepatoma and subcutaneous Walker carcinoma o f stock t u m o r - c a r r i e r animals were used. F o r t y to f i f t y per cent tumor homogenates i n 0.9 per cent s a l i n e were prepared by g r i n d i n g the tumors i n P o t t e r -Elvehjem homogenizing tubes by hand under s t e r i l e c o n d i t i o n s . One-half or one ml. aliquo.ts o f tumor homogenates were i n j e c t e d i n t r a p e r i t o n e a l l y , or subcutaneously ( i n the l a t e r a l subscapular region) using a Luer syringe and an 18 gauge needle. A l l o x a n was i n j e c t e d i n t r a p e r i t o n e a l l y (and In some instances subcutaneously) as a 3 per cent s o l u t i o n of a l l o x a n monohydrate (Eastman) i n a dose of 175 mg. per kg. o f body weight, without previous f a s t i n g . P r i o r to s a c r i f i c e the animals were f a s t e d f o r 24 -12-hours. Body weights were recorded i n i t i a l l y and at the time o f s a c r i f i c e . Under ether anaesthesia the abdominal c a v i t y was opened-and 1 ml. o f blood was withdrawn from the p o s t e r i o r vena cava f o r the blood sugar determinations. The f a s t i n g blood sugar l e v e l was determined by-the method of Nelson ( 6 4 ) . Tumors were then d i s s e c t e d out and weighed. The mean, standard d e v i a t i o n , and standard e r r o r o f blood sugar l e v e l s and tumor weights (expressed as per cent o f body weight) were c a l c u l a t e d f o r each group o f animals, and the s i g n i f i c a n c e of d i f f e r e n c e between mean blood sugar l e v e l s o f a l l o x a n c o n t r o l and tumor-bearing a l l o x a n i z e d animals, and between mean tumor weights o f tumor c o n t r o l and tumor-bearing a l l o x a n i z e d animals were c a l c u l a t e d (27)• B. H i s t o l o g i c a l Procedures. Samples of pa n c r e a t i c t i s s u e were removed and f i x e d f o r 1 2 to 24 hours i n Zenker's f l u i d or i n a m o d i f i c a t i o n o f Bouin's f i x a t i v e c o n t a i n i n g 5 per cent t r i c h l o r o a c e t i c a c i d (TCA). Tissues-preserved i n Zenker's f i x a t i v e were dehydrated and embedded i n F i s h e r Tissuemat through chloroform. Tissues f i x e d i n Bouin's TCA were washed w i t h s e v e r a l changes of 95 per cent e t h y l a l c o h o l to remove excess p i c r i c a c i d . They were then dehydrated and embedded i n tissuemat through n - b u t y l a l c o h o l according to the method o f McClung ( 6 2 ) . Sections were cut a t 5 microns u s i n g a Spencer Rotary microtome, and were mounted using Haupt's adhesive. -13^ S t a i n i n g o f the b e t a - c e l l s f o l l o w e d Wilson's m o d i f i -c a t i o n (W.S. H a r t r o f t , pers.oomm.) o f Gomori»s aldehyde-fuchsin method (32). The s t a i n was prepared as f o l l o w s : B a s i c fuchs i n 0.5 gms. 6of. e t h y l a l c o h o l 100 ml. Gone. HC1 • 1 ml. Paraldehyde • 3 ml* The i n g r e d i e n t s were mixed, and the s t a i n was incubated over-n i g h t i n an oven at 40 to 45 degrees Centigrade. The counter-s t a i n used was Halmits s t a i n (39)» which was made up as f o l l o w s : L i g h t green • 0.2 gms. Orange G 1.0 gms. Chromotrope 2R 0.5 gms. Phosphotungstic a c i d 0.5 gms. G l a c i a l a c e t i c a c i d 1.0 ml. D i s t i l l e d water 100 ml. The aldehyde-fuchsin s t a i n used i n i t s o r i g i n a l concen*-t r a t i o n was found to produce too intense c o l o r a t i o n i n the s t a i n i n g time o f four minutes which was recommended. One p a r t of the stock s t a i n prepared by the above method was t h e r e f o r e d i l u t e d w i t h four p a r t s of 60 per cent e t h y l a l c o h o l , and the s t a i n i n g time was reduced to f o u r d i p s . The procedure found to be most s a t i s f a c t o r y was as f o l l o w s : (1) . Decerate and b r i n g through a l c o h o l s to water. (2) . Oxidize i n a c i d permanganate f o r 2 minutes (0.3 per cent potassium permanganate i n 0.3 per cent s u l p h u r i c a c i d . . ...... (3) » Decolorize i n 4 per cent-sodium.bisulphite. (4) . Wash f o r 1 minute.in running.tap water.. (5) « Rinse i n 93 per ce n t , a l c o h o l . - . (6) . S t a i n i n aldehyde-fuchsin by dipping the s e c t i o n 5 to 10 times i n the s t a i n . (7) . Rinse i n 95 per cent a l c o h o l u n t i l s e c t i o n s are almost f r e e of excess s t a i n . (8) . C l e a r completely ln„clean absolute e t h y l , a l c o h o l . (9) . Rinse vigorously.in.running.tap.water u n t i l s e c t i o n s are f a i n t p u r p l i s h to c o l o u r l e s s . . » (10). Dehydrate, c l e a r i n x y l o l , - a n d examine m i c r o s c o p i c a l l y . At t h i s stage the s e c t i o n s should be c o l o u r l e s s on v i s u a l i n s p e c t i o n . M i c r o s c o p i c a l l y , the i s l e t s should stand out sha r p l y due to the purple c o l o u r of the b e t a - c e l l granules. I f -the s t a i n was not s u f f i c i e n t l y i n t e n s e , the s e c t i o n s were taken back to tap water and s t a i n e d by r e d i p p i n g i n aldehyde-fuchsin s e v e r a l times. Once the number o f dips was e s t a b l i s h e d f o r a p a r t i c u l a r s e r i e s of s e c t i o n s , microscopic examination was made a f t e r r i n s i n g w i t h tap water o n l y . The f o l l o w i n g steps were then executed: (1) . S t a i n i n Halmi's s t a i n f o r 4 minutes. (2) . Remove excess s t a i n w i t h acetone and dehydrate i n two -15-a d d i t i o n a l changes o f acetone. ( I f necessary, remove excess s t a i n i n 2 per cent a c e t i c acid.) (3). C l e a r i n x y l o l and mount i n "balsam. . In the t i s s u e s f i x e d i n Bouin's. TCA, the b e t a - c e l l s were s t a i n e d s p e c i f i c a l l y a deep p u r p l e . The cytoplasm o f the a c i n a r c e l l s , the a l p h a - c e l l s of the i s l e t s , and the degranu-l a t e d b e t a - c e l l s s t a i n e d green. The n u c l e i , i f s t a i n e d , were l i g h t r u s t on a green background. In most ins t a n c e s i t was found impossible to prevent f a i n t purple s t a i n i n g o f a c i n a r c e l l s w i t h aldehyde-fuchsin. The a c i n a r c e l l s then have a greenish-brown c o l o u r a f t e r o o u n t e r s t a i n i n g . In t i s s u e s f i x e d i n Z e n k e r ' s , F l u i d , a c i n a r t i s s u e s s t a i n e d purple w i t h aldehyde-f u c h s i n as w e l l as the b e t a - c e l l granules..However the b e t a - c e l l granules were more i n t e n s e l y s t a i n e d , and t h e i r c o l o u r was more b l u i s h . Oounterstaining w i t h Halmi's s t a i n o r other c o u n t e r s t a i n s was unsuccessful because the a c i n a r t i s s u e was a l r e a d y s t a i n e d w i t h the aldehyde-fuchsin. The general s t r u c t u r e of the p a n c r e a t i c t i s s u e and i s l e t s was observed w i t h the combination of a 40X L e i t z apo-chromatic o b j e c t i v e and a 10X p e r i p l a n a t i c eyepiece. For c e l l u l a r d e t a i l a L e i t z 90X o i l immersion o b j e c t i v e w i t h a 10X p e r i p l a n a -t i c eyepiece was employed. For g r e a t e r c e l l u l a r d e t a i l a l l i s l e t s were observed under phase c o n t r a s t w i t h both the above l e n s combinations. Photomicrographs were obtained w i t h the same combinations. -16-I H . RESULTS. A. E f f e c t o f a l l o x a n on blood sugar l e v e l s In tumors-bearing r a t s . 1. A l l o x a n administered to animals bearing i n t r a -p e r i t o n e a l t r a n s p l a n t s of tumors. When tumor homogenates ( e i t h e r Walker 25& carcinoma or N o v i k o f f hepatoma) were i n j e c t e d i n t r a p e r i t o n e a l l y one to four days p r i o r to the a d m i n i s t r a t i o n o f a diabetogenic dose of a l l o x a n (175 mg. per kg. of body weight), The marked hypeiv glycemia c h a r a c t e r i s t i c of a l l o x a n diabetes f a i l e d to develop, and the f a s t i n g blood sugar l e v e l s of the animals t e s t e d remained w i t h i n the normal range (see TABLE I , andFIG-URE I ) . Two groups of animals i n j e c t e d w i t h a l l o x a n alone had blood sugar l e v e l s averaging 189.4 mg. per cent (S.E. 23 .89) and 223.2 mg. per cent (S.E. 28 . 92 ) r e s p e c t i v e l y . Animals t r a n s p l a n t e d w i t h tumor (Walker carcinoma and N o v i k o f f hepatoma) and subsequently a l l o x a n i z e d , however, had an average f a s t i n g sugar l e v e l o f 72.8 mg. per cent (S.E. 10.12) and 105 mg. per cent (one animal). Extremely h i g h m o r t a l i t y r a t e s i n these groups reduced t h e i r numbers con s i d e r a b l y , but i n s p i t e of t h i s , d i f f e r e n c e s between animals r e c e i v i n g both tumor and a l l o x a n and those r e c e i v i n g o n l y a l l o x a n are h i g h l y s i g n i f i c a n t . 2. A l l o x a n administered p r i o r to i n t r a p e r i t o n e a l i m p l a n t a t i o n of tumors. When tumor was i n j e c t e d i n t r a p e r i t o n e a l l y 24 hours a f t e r a diabetogenic dose of a l l o x a n , m o d i f i c a t i o n of the TABLE I. Difference between blood sugar levels of alloxanized and non-alloxanized rats with and without transplants of Novikoff hepatoma and Walker 256 carcinoma. Site Substance Injected F i r s t Mean Blood Glucose Level (mg. Time Alloxanized Tumor-bearing Interval Controls Alloxanized | Probability of Significance Tumor-bearing of Difference .Controls AC-TA TA-TC2 Novikoff intraperit alloxan 1 day 210.2i 8.43^ 148.0 ± 17.47 109.8 ± 1.35 <0.01 < 0.01 intraperit alloxan 7 days 289.7 1 34.95 139.1-14.48 102.lt 14.28 <0.01 > 0.05 subcut alloxan 1 day 255.0± 11.40 128.8 * 13.50 108.4+ 4.20 <0.01 > 0.05 subcut tumor 1 day 239.1i 14.64 173.11 10.64 105.2± 1.83 <0.01 < 0.01 intraperit tumor 1 day 189.41 23.98 105 4 104.3± 4.67 - -Walker intraperit tumor 4 days 225.2i 28.92 72.8 ±10.12 66.5 + 1.00 <0.01 > 0.05 subcut tumor 4 days 225.2i 28.92 263.7 i 0.84 119.81 26.63 >o.05 < 0.01 subcut alloxan 14 days 225.21 28.92 273.0+ 7.02 119.81 26.65 > 0.05 < 0.01 ^Between alloxanized controls and tumor-bearing alloxanized rats. iBetween tumor-bearing alloxanized animals and tumor-bearing controls. ^Standard error of the mean. One animal. FIGURE I. Comparison of blood sugar lev e l s and tumor weights of rats transplanted i n t r a p e r i t o n e a l l y with Walker 25& carcinoma 4 days p r i o r to al l o x a n i z i n g with those of alloxanized and tumor-bearing controls. FIGURE I I . Comparison of blood sugar lev e l s and tumor weights of rats transplanted i n t r a p e r i t o n e a l l y with Novikoff hepatoma 24 hours aft e r alloxanizing with those of alloxanized and tumor-bearing controls. FIGURE I -18-A l l o x a n C o n t r o l T u m o r C o n t r o l A l l o x a n T u m o r s e ; 2 8 9 2 s e t 0 5 4 s e i 0 - 6 5 s e t I O O s e i IO-I2 % b o d y w t . IO 5 mean t u m o r -1 l_ I O O 2 0 0 3 0 0 mg% m e a n f a s t i n g b l o o d g l u c o s e FIGURE I I . A l l o x a n C o n t r o l T u m o r C o n t r o l A l l o x a n T u m o r s e i I 0 6 I L % b o d y w t . IO 5 mean tumor s e t 8 4 3 s e i 1747 J L I O O 2 0 0 3 0 0 mg% mean fasting blood g l u c o s e -19-d i a b e t o g e n i c a c t i o n was l e s s than i n the case where tumor was i n j e c t e d p r i o r to a l l o x a n . However some animals e i t h e r f a i l e d to develop d i a b e t e s , o r e l s e r e c o v e r e d from i t co m p l e t e l y i n the week b e f o r e the animals were s a c r i f i c e d . In oth e r i n s t a n c e s , d i a b e t e s developed, but was much l e s s severe than i n the c o n t r o l animals which were g i v e n a l l o x a n a l o n e . Rats which r e c e i v e d 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 N o v i k o f f hepatoma homogenate 24 hours a f t e r a d i a b e t o g e n i c dose o f a l l o x a n f a i l e d to become hyperglycemic i n 53 per cent o f the cases, and an a d d i t i o n a l 25 per cent developed o n l y m i l d d i a b e t e s . Seventy-four per cent o f c o n t r o l r a t s r e c e i v i n g o n l y a l l o x a n showed moderate to severe d i a b e t e s (see TABLE I I ) . B l o o d sugar l e v e l s o f animals r e c e i v i n g both a l l o x a n and tumor (148.0 mg. pe r cent, S.E. 17.47) were i n t e r m e d i a t e between those o f animals r e c e i v i n g a l l o x a n alone (210.2 mg. per cent, S.E. 8.43) and those o f animals r e c e i v i n g tumor alone (109.8 mg. per cent, S.E. 1.35)» and were s i g n i f i c a n t l y d i f f e r e n t from both o f these groups at the 1 per cent l e v e l (see TABLE I, and FIGURE I I ) . Thus tumor m a t e r i a l i n j e c t e d 24 hours a f t e r the a d m i n i s t r a t i o n o f a l l o x a n was not as e f f e c t i v e as tumor p r e -v i o u s l y implanted i n p r e v e n t i n g the development o f d i a b e t e s . N o v i k o f f hepatoma 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 7 days a f t e r the i n j e c t i o n o f a di a b e t o g e n i c dose o f a l l o x a n a l l e v i a t e d TABLE I I . S e v e r i t y o f diabetes i n r a t s a l l o x a n i z e d 24 hours before t r a n s p l a n t i n g w i t h i n t r a p e r i t o n e a l N o v i k o f f hepatoma. Tumor A l l o x a n - A l l o x a n Blood Sugar L e v e l C o n t r o l s Tumor C o n t r o l s Normal lOOf. 53?o 4f. (under 130 mg. per cent) M i l d Hyperglycemia Z% 12$ (131-190 mg. per cent) Moderate Hyperglycemia l6f. 10$ (191-250 mg. per cent) Severe Hyperglycemia 4f. (over 250 mg. per cent) TABLE I I I . S e v e r i t y o f diabetes i n r a t s a l l o x a n i z e d 7 days before t r a n s p l a n t i n g w i t h i n t r a p e r i t o n e a l N o v i k o f f hepatoma. Blood Sugar L e v e l Tumor Co n t r o l s Alloxan-'' Tumor A l l o x a n C o n t r o l s Normal (under 130 mg. per cent) M i l d Hyperglycemia (131-190 mg. per cent) Moderate Hyperglycemia (191-250 mg. per cent) Severe Hyperglycemia (over 250 mg. per cent) 57f. 22f. 17% 21f. 32$ 42f. -21-the hyperglycemia and reduced blood sugar l e v e l s to normal o r near normal values i n most animals, but a few remained s e v e r e l y d i a b e t i c . F a s t i n g blood glucose l e v e l s o f a l l o x a n i z e d tumor-bearing animals averaged 139.1 mg. per cent (S.E. 14.48) as compared w i t h 102.1 mg. per cent (S.E. 14.28) f o r n o n - a l l o x a -n i z e d tumor-bearing animals and 289.7 mg. per cent (S.E. 34.95) f o r a l l o x a n i z e d animals not i n j e c t e d w i t h tumor (see FIGURE I I I ) . Blood sugar l e v e l s o f a l l o x a n i z e d tumor-bearing animals were s i g n i f i c a n t l y l e s s than those of a l l o x a n c o n t r o l s , but were not s i g n i f i c a n t l y d i f f e r e n t from those of tumor c o n t r o l s (see TABLE I ) . Seventy-nine per cent of a l l o x a n i z e d tumor-bearing animals had normal or m i l d l y hyperglycemic blood sugar l e v e l s , and o n l y 17 per cent were s e v e r e l y d i a b e t i c . On the other hand, 74 per cent o f a l l o x a n i z e d c o n t r o l r a t s were moderately to seve r e l y d i a b e t i c (see TABLE I I I ) . 3. A l l o x a n administered to animals bearing sub-cutaneous tumor t r a n s p l a n t s . R e s u l t s w i t h tumor i n j e c t e d subcutaneously were not as c l e a r - c u t as when tumor was i n j e c t e d i n t r a p e r i t o n e a l l y . Some p r o t e c t i o n was obtained w i t h N o v i k o f f hepatoma i n j e c t e d e i t h e r 24 hours before or 24 hours a f t e r a l l o x a n . Walker 256 carcinoma, however, f a i l e d to give p r o t e c t i o n when i n j e c t e d subcutaneously 4 days p r i o r to a l l o x a n . FIGURE I I I . Comparison of blood sugar l e v e l s and tumor weights of rats transplanted i n t r a p e r i t o n e a l l y with Novikoff hepatoma 7 days a f t e r alloxanizing with those of alloxanized and tumor-bearing controls. FIGURE IV. Comparison of blood sugar levels and tumor weights of rats transplanted subcutaneously with Novikoff hepatoma 24 hours p r i o r to alloxanizing with those of alloxanized and tumor-bearing controls. FIGURE I I I . -22-A l l o x a n C o n t r o l s e t 3 4 . 9 5 T u m o r C o n t r o l 073 s e t I4.?f! A l l o x a n T u m o r 0*49 m s e i 1 4 4 8 1 1 1 1 1 % b o d y w t . IO 5 IOO 2 C O 3 0 0 m g % mean t u m o r mean f a s t i n g b l o o d g l u c o s e FIGURE IV. A l l o x a n C o n t r o l T u m o r C o n t r o l A l l o x a n T u m o r s e i 109 1_ s e t M7 I % b o d y w t . IO 5 m e a n tumor 14-64 s e i 1-83 s e t IO-64 J IOO 2 0 0 3 0 0 m g % mean f a s t i n g b l o o d g l u c o s e -23-Rats r e c e i v i n g a subcutaneous i n j e c t i o n o f N o v i k o f f hepatoma 24 hours before a diabetogenic dose of a l l o x a n had blood sugar l e v e l s averaging 173.1 mg. per cent (S.E. 10.64), as compared w i t h an average of 239.1 mg. per cent (S.E. 14.64) f o r c o n t r o l s r e c e i v i n g a l l o x a n o n l y , and 105«2 mg. per cent (S.E. 1.83) f o r c o n t r o l s r e c e i v i n g tumor o n l y (FIGURE I V ) . The blood sugar l e v e l s of tumor-bearing a l l o x a n i z e d animals were s i g n i f i c a n t l y d i f f e r e n t from both c o n t r o l groups (see TABLE I ) . Seventy-two per cent of a l l o x a n c o n t r o l r a t s were moderately or s e v e r e l y d i a b e t i c ; on the other hand, ofnthe r a t s r e c e i v i n g both tumor and a l l o x a n , 30 per cent had normal blood sugar l e v e l s , 40 per cent were m i l d l y d i a b e t i c , and the remaining 30 per cent were moderately or s e v e r e l y d i a b e t i c (see TABLE I V ) . Walker 256 carcinoma, on the other hand, f a i l e d to e x h i b i t any p r o t e c t i v e e f f e c t when i n j e c t e d subcutaneously 4 days p r i o r to a l l o x a n . A l l animals became ..severely hyper-glycemic, w i t h blood glucose l e v e l s averaging 263.7 mg. per cent (S.E. 0.84). This l e v e l i s higher than that of a l l o x a -n i z e d c o n t r o l s (225.2 mg. per cent, S.E. 28.92), but t h i s d i f f e r e n c e i s not s t a t i s t i c a l l y s i g n i f i c a n t . ( s e e TABLE I , and FIGURE V ) . Thus subcutaneous tumor implanted p r i o r to the i n j e c t i o n of a l l o x a n i s l e s s e f f e c t i v e i n preventing the development of diabetes than i s the same tumor implanted i n t r a p e r i t o n e a l l y ; -2.4-TABLE IV. S e v e r i t y o f diabetes i n r a t s a l l o x a n i z e d 24 hours a f t e r t r a n s p l a n t i n g subcutaneously w i t h N o v i k o f f hepatoma. Tumor A l l o x a n - A l l o x a n Blood Sugar L e v e l C o n t r o l s Tumor C o n t r o l s Normal 100% 30% 6% (under 130 mg. per cent) M i l d Hyperglycemia 40% 22% (131-190 mg. per cent) Moderate Hyperglycemia 15%' , . 17% (I9I-230 mg. per cent) Severe Hyperglycemia 15% 55% (over 250 mg. per cent) TABLE V. S e v e r i t y of diabetes i n r a t s a l l o x a n i z e d 24 hours p r i o r t o . t r a n s p l a n t i n g subcutaneously.with N o v i k o f f hepatoma. Tumor A l l o x a n - A l l o x a n Blood Sugar L e v e l c o n t r o l s Tumor Co n t r o l s Normal . 100% $5% (under 130 mg. per cent) M i l d Hyperglycemia 21% 13% (131-190 mg. per cent) Moderate Hyperglycemia 14% 39% (191-250 mg. per cent) Severe Hyperglycemia (over 250 mg. per cent) FIGURE V. Comparison of blood sugar l e v e l s and tumor weights of rats transplanted subcutaneously with Walker 2$G carcinoma .4 days p r i o r to allo x a n i z i n g with those of alloxanized and tumor-bearing controls. FIGURE 71.- Comparison of blood sugar l e v e l s and tumor weights of rats transplanted subcutaneously with Noyikoff hepatoma 24 hours aft e r alloxanizing with those of alloxanized and tumor-bearing controls. FIGURE V - 2 5 -A l l o x a n C o n t r o l T u m o r C o n t r o l A l l o x a n T u m o r % b o d y wt. IO 5 mean tumor _1_ s e t 0 - 8 4 J IOO 2 0 0 3 0 0 m g % mean f a s t i n g b l o o d g l u c o s e FIGURE V I . A l l o x a n C o n t r o l T u m o r C o n t r o l A l l o x a n T u m o r s e i I-08 % body w t . IO 5 mean t u m o r s e * II-40 IOO 2 0 0 3 0 0 m g % mean f a s t i n g b l o o d g l u c o s e -26-however some p r o t e c t i o n was achieved w i t h N o v i k o f f hepatoma homogenate i n j e c t e d 24 hours before a l l o x a n . • i ' • 4. A l l o x a n administered p r i o r to the subcutaneous i m p l a n t a t i o n o f tumors. N o v i k o f f hepatoma implanted subcutaneously 24 hours a f t e r the i n j e c t i o n o f a diabetogenic dose o f a l l o x a n prevented the appearance of diabetes i n 55 per cent of r a t s t r e a t e d i n t h i s way, and an a d d i t i o n a l 21 per cent were only m i l d l y d i a b e t i c . Controls r e c e i v i n g o n l y a l l o x a n were 87 per cent moderately to seve r e l y d i a b e t i c (TABLE V ) . Blood glucose l e v e l s o f alloxan-tumor animals averaged 128.8 mg. per cent (S.E. 13*50), and were s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s r e c e i v i n g a l l o x a n o n l y (255.0 mg. per cent, S.E. 11.40), but not from c o n t r o l s r e c e i v i n g tumor o n l y (108.4 mg. per cent, S.E. 4.20).(TABLE I , and FIGURE V I ) . Walker 256 carcinoma i n j e c t e d two weeks a f t e r a l l o x a n f a i l e d to reduce blood sugar l e v e l s below those of a l l o x a n i z e d . c o n t r o l s . The blood sugar l e v e l s o f the former averaged 273.0 mg. per cent (S.E. 7.02), w h i l e those o f the l a t t e r averaged 225.2 mg. per cent (S.E. 28.92); the d i f f e r e n c e i s not s i g n i -f i c a n t at the 5 per cent level.TABLE I , andJFIGURE V I I ) • FIGURE V I I . Comparison of blood sugar l e v e l s and tumor weights of rats transplanted subcutaneously with Walker 25& carcinoma 14 days aft e r alloxanizing with those of tumor-bearing controls. -27-FIGURE 711. T u m o r C o n t r o l A l l o x a n T u m o r s e t 7 0 2 _1 % body wt IO 5 mean tumor IOO 2 0 0 3 0 0 m g 7 0 mean f a s t i n g b l o o d g l u c o s e -28-B. E f f e c t o f a l l o x a n i z a t i o n on tumor growth i n r a t s  t r a n s p l a n t e d w i t h N o v i k o f f hepatoma and Walker 256  carcinoma. Diabetes induced by a l l o x a n reduced the s i z e o f i n t r a p e r i t o n e a l tumors as compared w i t h non-diabetic c o n t r o l s , but had a l e s s appreciable e f f e c t on the s i z e of subcutaneous tumors. Animals 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 w i t h N o v i k o f f hepatoma 24 hours before the a d m i n i s t r a t i o n o f a diabetogenic dose of a l l o x a n and s a c r i f i c e d one week l a t e r had tumors weighing on the average 3.03 per cent of t h e i r t o t a l body weight (S.E. 1.06). C o n t r o l r a t s r e c e i v i n g hepatoma but no al l o x a n had tumors averaging 7*33 per cent of body weight (S.E. 0.68). The d i f f e r e n c e i s s i g n i f i c a n t at the 1 per cent l e v e l (see TABLE V I , and FIGURE I I ) . Likewise a l l o x a n i z a t i o n 7 days before i n j e c t i n g w i t h n o v i k o f f hepatoma reduced tumor s i z e at one week to 2.89 per cent of body weight (S.E. 0.48), as compared w i t h 7.93 per cent (S.E. 0.78) i n c o n t r o l r a t s r e c e i v i n g tumor but no a l l o x a n . This d i f f e r e n c e was again s i g n i f i c a n t at p 0.01 (see TABLE I I , and FIGURE I I I ) . A l l o x a n i z a t i o n 4 days p r i o r to 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 Walker 256 carcinoma tended to reduce tumor weights, but the d i f f e r e n c e between experimental and c o n t r o l TABLE V I . D i f f e r e n c e between tumor weights o f a l l o x a n i z e d and non-alloxanized r a t s t r a n s p l a n t e d with. N o v i k o f f hepatoma and Walker 256 carcinoma. Tumor S i t e Substance Mean Tumor Weight I n j e c t e d Time (as % o f body wt.) F i r s t I n t e r v a l C o n t r o l s A l l o x a n i z e d S i g n i f i c a n c e o f D i f f e r e n c e N o v i k o f f i n t r a p e r i t a l l o x a n 24 hours 7.531 0.681 3.031 1.06 p=< 0.01 N o v i k o f f i n t r a p e r i t a l l o x a n 7 days 7 .93± 0.78 2 .89± 0.48 p=< 0.01 N o v i k o f f subcut a l l o x a n 24 hours 4.84i 1.08 6.671 1.31 p=> 0.05 N o v i k o f f subcut tumor 24 hours 4.711 1.09 3.12:1 1.17 p=> 0.05 N o v i k o f f i n t r a p e r i t tumor 24 hours 5.491 3.34 0.372 -Walker i n t r a p e r i t tumor 4 days 4.081 0.54 3.561 O.65 p=> 0.05 Walker • subcut tumor 4 days 8.56I 1.95 4.321 1.61 p=> 0.05 Walker subcut a l l o x a n 14 days 8.561 1.95 5.101 2.09 p^> 0.05 -^Standard e r r o r of the mean o One animal - 3 0 -at the 5 per cent l e v e l . However the number was s m a l l due to the h i g h m o r t a l i t y . Animals r e c e i v i n g both a l l o x a n and tumor i n j e c -t i o n s had tumors weighing 3•56 per cent o f body weight (S.E. 0.65), as compared w i t h tumors weighing 4 .08 per cent i n r a t s r e c e i v i n g i n j e c t i o n s o f tumor alone (S.E. 0.54) (FIGURE I ) . The e f f e c t o f a l l o x a n d i a b e t e s on the s i z e o f sub-cutaneous N o v i k o f f hepatoma was not as pronounced as was i t s e f f e c t on i n t r a p e r i t o n e a l tumors (FIGURES IV, and V I ) . In no case was the tumor s i z e o f a l l o x a n i z e d animals s i g n i f i c a n t l y d i f f e r e n t from that i n n o n - d i a b e t i c tumor-bearing c o n t r o l s . Rats r e c e i v i n g subcutaneous i n j e c t i o n s o f Walker 256 carcinoma 4 days and 2 weeks a f t e r a l l o x a n i z a t i o n had tumors weighing 4 .32 per cent o f body weight (S.E. 1.61) and 5.10 per cent o f body weight (S.E. 2 . 0 9 ) , r e s p e c t i v e l y . C o n t r o l s r e c e i v i n g o n l y carcinoma had tumors weighing 8.56 per cent o f body weight (S.E. 1.95) (FIGURES V and V I I , and TABLE V I ) . Tumor ?/eights i n a l l three groups were extremely v a r i a b l e , and t h i s , together w i t h s m a l l sample s i z e , may be r e s p o n s i b l e f o r the l a c k o f s i g n i f i c a n c e . C. E f f e c t of alloxanization on tumor incidence i n r a t s  transplanted with Novikoff hepatoma. The incidence of.Novikoff hepatomatransplanted. . i n t r a p e r i t o n e a l l y was reduced i n alloxanized animals, "but subcutaneous tumors showed no change in.incidence. Only 62 per cent of rats receiving a diabetogenic dose of alloxan either 24 hours or 7 days before the i n t r a p e r i t o n e a l transplantation of Novikoff hepatoma developed tumors. Non-alloxanized controls developed tumors i n 92 to 95 per cent of cases. Only 29 per cent of rats transplanted with tumor i n t r a -p e r i t o n e a l l y 24 hours before alloxan developed tumors (see TABLE V I I ) . Animals injected either 2.4 hours before or 24 hours af t e r the subcutaneous transplantation of Novikoff hepatoma, however, f a i l e d to have any e f f e c t on tumor incidence. Both tumor-bearing controls and animals receiving both tumor and alloxan developed tumors i n 93 to 95 per cent of cases. -32-TABLE V I I . Tumor incidence i n a l l o x a n i z e d and n o n - a l l o x a n i z e d r a t s t r a n s p l a n t e d w i t h N o v i k o f f hepatoma. Substance I n j e c t e d Time Tumor c o n t r o l s Tumor-alloxan S i t e F i r s t I n t e r v a l A B C A B C i n t r a p e r i t a l l o x a n 24 hours 40 37 92?. 40 25 62% i n t r a p e r i t a l l o x a n 7 days 20 19 95% 13 8 62% subcut a l l o x a n 24 hours 22 21 95f. 15 14 95% subcut tumor 2:4 hours 18 17 94% 17 16 93% i n t r a p e r i t tumor 24 hours 10 8 80% 7 2 29% A T o t a l number o f animals. B Number of animals developing tumor. C Tumor in c i d e n c e . -33-T). H i s t o l o g y o f the pancreas. 1. The i s l e t s o f Langerhans i n the pancreas o f the normal r a t . In the pancreas o f normal animals which had not been t r e a t e d e i t h e r w i t h tumor or w i t h a l l o x a n , i s l e t s o f d i f f e r e n t s i z e s , r a nging from 2 c e l l s to over 100 c e l l s were observed (FIGURES V I I I , IX, and X ) . The b e t a - c e l l s c o n s t i t u t e d about 80 per cent o f the t o t a l number o f c e l l s , and formed the c e n t r a l core o f the i s l e t . The a l p h a - c e l l s , c o n s t i t u t i n g the remaining 20 per cent o f the i s l e t , were l o c a t e d a t the p e r i p h e r y . A l l the b e t a - c e l l s were w e l l g r a n u l a t e d . Under phase c o n t r a s t t h e i r n u c l e i appeared w e l l rounded and s l i g h t l y l a r g e r i n s i z e than those o f the a l p h a - c e l l s , were g r a n u l a r i n appearance, and had prominent n u c l e o l i (FIGURE X I ) . 2. The i s l e t s o f Langerhans i n a l l o x a n i z e d r a t s . Animals o f t h i s group r e c e i v e d a l l o x a n i n t r a p e r i -t o n e a l l y 7 and 9 days p r i o r to s a c r i f i c i n g i n a dose o f 173 mg. per kg. o f body weight. The i s l e t s o f Langerhans were fewer i n number than i n normal pancreas, and t h e i r s i z e was markedly s m a l l e r . P u r p l e s t a i n e d g r a n u l e s were completely absent i n the i s l e t c e l l s o f animals t r e a t e d w i t h a l l o x a n 9 days p r i o r to s a c r i f i c e (FIGURE X I I ) . In animals a l l o x a n i z e d ' 7 days b e f o r e s a c r i f i c e , most o f the i s l e t s showed no s t a i n e d g r a n u l e s . However, i n one medium-sized i s l e t , a seemingly i n t a c t b e t a - c e l l w ith a h i g h c o n c e n t r a t i o n o f p u r p l e s t a i n e d g r a n u l e s was found FIGURE VIII. Medium-sized i s l e t of Langerhans from the pancreas of a normal r a t . B r i g h t f i e l d i l l u m i n a t ion. X550. FIGURE IX. Large-sized i s l e t of Langerhans from the pancreas. of a normal r a t . B r i g h t f i e l d illumination. Xj?j?0 FIGURE X. Medium-sized i s l e t o f Langerhans i n the pancreas o f a normal r a t , B r i g h t f i e l d i l l u m i n a t i o n . Xj5j>0. FIGURE X I . Same i s l e t as sho?/n i n FIGURE X. Phase c o n t r a s t . X550 FIGURE XI. FIGURE 211• I s l e t of Langerhans i n the pancreas of a r a t i n j e c t e d w ith a diabetogenic dose of a l l o x a n 9 days p r i o r to s a c r i f i c e . Blood sugar l e v e l 293 mg, per cent. B r i g h t f i e l d i l l u m i n a t i o n , X550. FIGURE X I I I * The same i s l e t as shown i n FIGURE X I I (above). Phase c o n t r a s t , X55°* -37-l o c a t e d i n the p e r i p h e r y o f the i s l e t . Most o f the s m a l l e r i s l e t s were composed mai n l y o f alpha-type c e l l s w i t h normal appearing n u c l e i (FIGURE X I I I ) . In l a r g e r i s l e t s the number o f p e r i p h e r a l a l p h a - c e l l s was i n c r e a s e d i n comparison w i t h those o f normal animals. In the c e n t r a l p o r t i o n s o f the i s l e t s some b e t a -c e l l s w i t h degranulated cytoplasm and p y c n o t i c n u c l e i were pre s e n t , but the m a j o r i t y o f c e l l s were degenerated beyond r e c o g n i t i o n . In s e v e r a l i n s t a n c e s the damage to the i s l e t s was severe, w i t h o n l y a few p e r i p h e r a l a l p h a - c e l l s p e r s i s t i n g , the r e s t o f the i s l e t being completely plasmolysed. 3. the i s l e t s o f Langerhans i n tumor-bearing r a t s . In animals t r a n s p l a n t e d e i t h e r w i t h Walker 256 c a r -cinoma subcutaneously f o r 7 days, or N o v i k o f f hepatoma i n t r a -p e r i t o n e a l l y f o r 10 days, the appearance o f the i s l e t s o f Langerhans was i d e n t i c a l w i t h that i n normal animals. The i s l e t s were numerous. T h e i r s i z e ranged from s m a l l i s l e t s c o n t a i n i n g o n l y 2 b e t a - c e l l s to v e r y l a r g e ones wi t h over 100 b e t a - c e l l s . Intense g r a n u l a t i o n , i n d i c a t e d by t h e i r deep p u r p l e s t a i n i n g p r o p e r t i e s , c h a r a c t e r i z e d the b e t a - c e l l s . T h e i r n u c l e i were o f normal appearance. The p r o p o r t i o n o f b e t a - c e l l s v a r i e d from 75 to 90 per cent o f the t o t a l number o f c e l l s i n the i s l e t s (FIGURES XIV, and XV). FIGURE XIV. Large i s l e t o f Langerhans i n the pancreas o f a r a t t r a n s p l a n t e d subcutaneously w i t h walker 256 carcinoma 7 days b e f o r e s a c r i f i c e . Blood sugar l e v e l 93 mg. p e r cent. B r i g h t f i e l d i l l u m i n a t i o n . X550. FIGURE XV. Small i s l e t o f Langerhans i n the pancreas o f a r a t t r a n s p l a n t e d subcutaneously w i t h Walker 256 carcinoma 7 days b e f o r e s a c r i f i c e . Blood sugar l e v e l 93 mg. per cent. B r i g h t f i e l d i l l u m i n a t i o n . X550. FIGURE XIV. -38-FIGURE XVI* I s l e t o f Langerhans i n the pancreas o f a r a t a l l o x a n i z e d 2 weeks p r i o r to the subcutaneous t r a n s p l a n t a t i o n o f 7falker 2J?6 carcinoma. Blood sugar l e v e l • „., 280 mg. per cent. B r i g h t f i e l d i l l u m i n a t i o n . X550. FIGURE XVII. The same i s l e t shown i n FIGURE XVI (above). Phase c o n t r a s t . Xj?50. -40-4. The i s l e t s o f Langerhans i n r a t s a l l o x a n i z e d 2 weeks p r i o r to t r a n s p l a n t i n g w i t h Walker 256 carcinoma subcutaneously. Animals i n t h i s group r e c e i v e d 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 a l l o x a n i n a dose o f 175 mg. per kg. o f body weight. Two weeks l a t e r 0.5 ml. o f a 40 per cent suspension o f Walker 256 carcinoma c e l l s was i n j e c t e d . Most i s l e t s were o f medium s i z e . In the m a j o r i t y there were no b e t a - c e l l s w i t h s t a i n e d g r a n u l e s p r e s e n t . In many o f them there was a c e n t r a l n e c r o t i c a r e a w i t h a p e r i p h e r a l r i n g o f i n t a c t a l p h a - c e l l s . A few o f the s m a l l e r i s l e t s appeared to be composed o f a l p h a - c e l l s o n l y . S e v e r a l s m a l l i s l e t s were noted i n which there were one to three b e t a - c e l l s w i t h deeply s t a i n e d g r a n u l e s p r e s e n t (FIGURE X V I ) . The g r a n u l e s were p r e s e n t In the same c o n c e n t r a t i o n as i n the b e t a - c e l l s o f normal animals, but the n u c l e i i n the a l l o x a n i z e d tumor-bearing animals had a s l i g h t l y p y c n o t i c appearance.(FIGURE X V I I ) . 5» The i s l e t s o f Langerhans i n r a t s t r a n s p l a n t e d subcutaneously w i t h Walker 256 carcinoma 4 days p r i o r to a l l o x a n i z i n g . Animals o f t h i s group were i n j e c t e d subcutaneously w i t h 0.5 ml. o f 40 per cent Walker carcinoma tumor homogenate. Four days l a t e r they were i n j e c t e d i n t r a p e r i t o n e a l l y w i t h 175 mg. per kg. o f body weight o f a l l o x a n . Seven days l a t e r theyi were s a c r i f i c e d . FIGURE X V I I I . Medium-sized i s l e t o f Langerhans i n the pancreas o f a r a t t r a n s -p l a n t e d subcutaneously with Walker 2j?6 carcinoma 4 days before a l l o x a n ! z i n g . Blood sugar l e v e l 28.0 mg. per cent. B r i g h t f i e l d i l l u m i n a t i o n . Xj?j?0. FIGURE XIX. The same i s l e t as shown i n FIGURE XVIII (above). Phase c o n t r a s t . X550. -41-FIGURE X V I I I . FIGURE XIX FIGURE XX. Medium-sized i s l e t o f Langerhans i n the pancreas o f a r a t t r a n s -p l a n t e d subcutaneously with Walker 2^ 6 carcinoma 4 days before a l l o x a n i z i n g . Blood sugar l e v e l 267 mg. per cent. B r i g h t f i e l d i l l u m i n a t i o n . X550. FIGURE XXI. The same i s l e t as shown i n FIGURE XX. (above). Phase c o n t r a s t . X550. - 4 3 -The appearance o f the i s l e t s was v e r y s i m i l a r to those i n the p a n c r e a t i c t i s s u e s o f a l l o x a n i z e d c o n t r o l s . The i s l e t s were s m a l l e r i n s i z e than i n normal pancreas, and fewer i n number, and were without g r a n u l a t e d b e t a - c e l l s . In some there was a c e n t r a l plasmolysed n e c r o t i c a r e a . A r i n g o f normal appreaing a l p h a - c e l l s was p r e s e n t i n a l l i s l e t s examined. In s e v e r a l i s l e t s there were one or two w e l l - g r a n u l a t e d b e t a - c e l l s p r e s e n t (FIGURES XVTII, XIX, XX, and X X I ) . 6. The i s l e t s o f Langerhans i n r a t s a l l o x a n i z e d one day p r i o r to t r a n s p l a n t i n g i n t r a p e r i t o n e a l l y w i t h N o v i k o f f hepatoma. Animals i n t h i s group r e c e i v e d 175 mg. per kg. o f body weight o f a l l o x a n i n t r a p e r i t o n e a l l y 24 hours p r i o r to 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 o f 0.5 ml. o f a 50 per cent suspension o f N o v i k o f f hepatoma. Tumor was found to be p r o l i f e r a t i n g i n the pancreas. The i s l e t s were as numerous as those o f normal animals. They ranged i n s i z e from s m a l l to medium; l a r g e i s l e t s were absent. W e l l - g r a n u l a t e d b e t a - c e l l s w i t h n u c l e i o f normal appearance c o n s t i t u t e d 65 to 80 per cent o f the l o t a l number o f i s l e t c e l l s . FIGURE XXII. pancreas o f r a t t r a n s p l a n t e d i n t r a p e r i t o n e a l l y with walker 2^ 6 carcinoma 4 days "before a l l o x a -n i z i n g . The l i g h t - c o l o u r e d t i s s u e occupying the l e f t h a l f o f the photograph i s tumor, i n v a d i n g the gray a c i n a r t i s s u e on the r i g h t . S e v e r a l i s l e t s are present i n the a c i n a r t i s s u e . Blood sugar l e v e l 106 mg. per cent. B r i g h t f i e l d i l l u m i n a t i o n . X150. FIGURE X X I I I . I s l e t o f Langerhans i n the pancreas o f a r a t t r a n s p l a n t e d i n t r a -p e r i t o n e a l l y w i t h Walker 256 carcinoma 4 days before a l l o x a -n i z i n g . T h i s i s l e t was l o c a t e d i n -a c i n a r t i s s u e remote from p r o l i f e r a t i n g tumor, and shows evidence o f n e c r o t i c degeneration. Blood sugar l e v e l 47 mg. per cent. B r i g h t f i e l d i l l u m i n a t i o n . X550. - 4 5 -7. The i s l e t s o f Langerhans i n r a t s t r a n s p l a n t e d i n t r a p e r i t o n e a l l y w i t h Walker 256 caroinoma 4 days p r i o r to a l l o x a n i z i n g . Animals i n t h i s group r e c e i v e d i n t r a p e r i t o n e a l t r a n s -p l a n t s o f Walker 256 carcinoma 4 days b e f o r e 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 o f a d i a b e t o g e n i c dose o f a l l o x a n . They were s a c r i f i c e d 8 days a f t e r the a d m i n i s t r a t i o n o f a l l o x a n . In most i n s t a n c e s tumor was found to be p r o l i f e r a t i n g i n the pancreas (FIGURE X X I I ) . The appearance o f the medium-s i z e d and l a r g e i s l e t s depended on t h e i r p r o x i m i t y to tumor t i s s u e . Those present i n the par.ts o f the pancreas d i s t a n t from tumor c o n t a i n e d o n l y v e r y few g r a n u l a t e d b e t a - c e l l s , and those t h a t were prese n t were p o s i t i o n e d i n the p e r i p h e r a l zone (FIGURE X X I I I ) . Degranulated b e t a - c e l l s i n the c e n t e r s o f these i s l e t s had p y c n o t i c n u c l e i and v a c u o l a t e d cytoplasm. The p e r i -p h e r a l a l p h a - c e l l s appeared normal. Large and medium-sized i s l e t s i n the p r o x i m i t y o f tumor c o n s i s t e d o f 50 to 65 per cent g r a n u l a t e d b e t a - c e l l s FIGURES XXIV, XXV, XXVI, XXVII, and 2 X V I I I ) . N u c l e i o f these, and o f the p e r i p h e r a l a l p h a - c e l l s appear normal. G r a n u l a t e d b e t a - c e l l s occupy the c e n t e r s o f the i s l e t s , and between them and the p e r i p h e r a l a l p h a - c e l l s are l o c a t e d degranulated b e t a -c e l l s w i t h p y c n o t i c n u c l e i , which are g e n e r a l l y s m a l l e r i n s i z e . A l a r g e number o f s m a l l i s l e t s , c o n t a i n i n g 90 per cent FIGURE XXIV. Medium-sized I s l e t o f Langerhans l o c a t e d c l o s e to p r o l i f e r a t i n g • tumor i n the pancreas o f a r a t t r a n s p l a n t e d i n t r a p e r i t o n e a l l y w i t h Walker 2j?6 carcinoma 4 days b e f o r e a l l o x a n i z i n g . Blood sugar 47 mg, per cent. B r i g h t f i e l d i l l u m i n a t i o n . Xj?50. FIGURE XXV. The same i s l e t shown i n FIGURE XXIV (above). Phase c o n t r a s t . X550» FIGURE XXVI. Medium-sized i s l e t o f Langerhans Located c l o s e to p r o l i f e r a t i n g tumor i n the pancreas o f a r a t t r a n s p l a n t e d i n t r a p e r i t o n e a l l y w i t h Walker 256 carcinoma 4 days before a l l o x a n i z i n g . Blood sugar l e v e l 47 mg. per cent. B r i g h t f i e l d i l l u m i n a t i o n . X550. FIGURE XXVII. The same i s l e t as shown i n FIGURE XXVI (above). Phase c o n t r a s t . X550* FIGURE XXVIII. Medium-sized i s l e t o f Langerhans l o c a t e d c l o s e to p r o l i f e r a t i n g tumor i n the pancreas o f a r a t t r a n s p l a n t e d i n t r a p e r i t o n e a l l y w i t h Walker 256 carcinoma 4 , days before a l l o x a n i z i n g . Blood sugar l e v e l 47 mg. per cen t . B r i g h t f i e l d i l l u m i n a t i o n . X550. FIGURE XXIX. Small i s l e t o f Langerhans • l o c a t e d c l o s e to p r o l i f e r a t i n g tumor i n the pancreas of a r a t t r a n s p l a n t e d i n t r a p e r i t o n e a l l y w i t h Y/alker 2% carcinoma 4 days before a l l o x a n i z i n g . Blood sugar l e v e l 106 mg. per cen t . B r i g h t f i e l d i l l u m i n a t i o n . X550. FIGURE XXX. Three s m a l l i s l e t s o f Langerhans c o n s i s t i n g o f b e t a - c e l l s o n l y i n a mass o f tumor p r o l i f e r a t i n g i n the pancreas o f a r a t t r a n s p l a n t e d i n t r a p e r i t o n e a l l y w i t h Walker 2J?6 carcinoma 4 days be f o r e a l l o x a n i z i n g . B lood sugar l e v e l 47 mg. per cent. B r i g h t f i e l d i l l u m i n a t i o n . X550. -49-FIGURE XXX. or more o f granulated b e t a - c e l l s was a l s o noted, mainly i n tne v i c i n i t y o f p r o l i f e r a t i n g tumor (FIGURES X X I I , and XXIX). Small I s l e t s composed only o f granulated "beta-cells were a l s o found i n a mass o f tumor c e l l s adjacent to tne pan-creas. There were no a c i n a r c e l l s bordering these i s l e t s (FIGURE XXX). E. Summary o f r e s u l t s . I n summary, tumor e s t a b l i s h e d i n t r a p e r i t o n e a l l y before the a d m i n i s t r a t i o n o f a s i n g l e diabetogenic dose of a l l o x a n modified i t s diabetogenic a c t i o n , w i t h blood sugar l e v e l s ranging from normal glycemia to severe hyperglycemia. I n t r a p e r i t o n e a l tumor t r a n s p l a n t e d 24 hours a f t e r a l l o x a n , o r subcutaneous tumor t r a n s p l a n t e d e i t h e r 24 hours before or 2:4 hours a f t e r a l l o x a n o f f e r e d p r o t e c t i o n against the development of diabetes i n some animals, and reduced i t s s e v e r i t y i n o t h e r s . However the e f f e c t o f subcutaneous tumor on the hyperglycemia induced by a l l o x a n was much l e s s s t r i k i n g than that o f i n t r a -p e r i t o n e a l tumor. H i s t o l o g i c a l examination o f the i s l e t s of Langerhans i n the pancreas showed that the high blood sugar l e v e l s f o l -lowing a l l o x a n i z a t i o n were accompanied by degranulatlon or n e c r o s i s o f the b e t a - c e l l s r e s p o n s i b l e f o r I n s u l i n p r o d u c t i o n . The presenco o f tumor t i s s u e invading the pancreas appeared -51-to have a p r o t e c t i v e or regenerative i n f l u e n c e on the b e t a - c e l l s o f a l l o x a n i z e d animals. The incidence and r a t e o f growth of i n t r a p e r i t o n e a l tumor t r a n s p l a n t s was reduced i n a l l o x a n i z e d animals as compared w i t h non-diabetic c o n t r o l s . However the e f f e c t o f a l l o x a n administered .before or a f t e r the subcutaneous i m p l a n t a t i o n of these tumors was much l e s s pronounced on tumor growth and had no e f f e c t on tumor i n c i d e n c e . Thus, a l l o x a n - d i a b e t e s and t r a n s p l a n t e d i n t r a p e r i t o n e a l tumor are mutually a n t a g o n i s t i c . The hyperglycemia c h a r a c t e r i s t i c o f a l l o x a n - diabetes i s r e l i e v e d by the presence of i n t r a p e r i -t o n e a l tumor, and the development and growth o f I n t r a p e r i t o n e a l tumors i s i n h i b i t e d by a l l o x a n - d i a b e t e s . IV. DISCUSSION. A. The I n h i b i t i o n o f a l l o x a n d i a b e t e s by tumor. The experimental r e s u l t s demonstrate t h a t the i n t r a -p e r i t o n e a l or subcutaneous i n j e c t i o n o f N o v i k o f f hepatoma e i t h e r p r i o r to o r a f t e r the i n j e c t i o n o f a d i a b e t o g e n i c dose o f a l l o x a n prevents the development o f d i a b e t e s i n many i n s t a n c e s and reduces i t s s e v e r i t y i n o t h e r s , although the e f f e c t o f subcutaneous t r a n s p l a n t s was l e s s pronounced. S i m i l a r r e s u l t s were ob t a i n e d w i t h Walker 256 carcinoma t r a n s p l a n t e d i n t r a p e r i -t o n e a l l y 4 days p r i o r to a i l l o x a n . These r e s u l t s are i n agreement w i t h those o b t a i n e d by Goranson, Botham and Willms (35)» who observed marked reduc-t i o n o f blood sugar l e v e l s i n a l l o x a n - d i a b e t i c r a t s w i t h i n t r a -p e r i t o n e a l t r a n s p l a n t s o f N o v i k o f f hepatoma. That t h i s r e d u c t i o n i n b l o o d sugar l e v e l s i s due to the p r o t e c t i v e or r e g e n e r a t i v e i n f l u e n c e o f tumor on-the b e t a -c e l l s o f the i s l e t s o f Langerhans was i n d i c a t e d by h i s t o l o g i c a l examination o f the pancreas. In a l l o x a n i z e d c o n t r o l animals d e g r a n u l a t i o n and c y t o l y s i s o f the b e t a - c e l l s , which are r e s p o n s i b l e f o r i n s u l i n p r o d u c t i o n , was observed. I s l e t s were reduced i n s i z e and number, and showed c e n t r a l areas o f n e c r o s i s . In a l l o x a n i z e d animals w i t h i n t r a p e r i t o n e a l tumors, Tumor t i s s u e was always present to some extent i n the p a n c r e a t i c r e g i o n . Tumor c e l l s were p r o l i f e r a t i n g between a c i n a r t i s s u e . Regenerating g r a n u l a t e d b e t a - c e l l s were pr e s e n t i n the c e n t e r s o f medium- to .Large-sized i s l e t s i n the p r o x i m i t y o f tumor t i s s u e , u s u a l l y w i t h a p e r i p h e r a l area o f degranulated c e l l s and an i n c r e a s e d number o f a l p h a - c e l l s . The presence o f l a r g e numbers o f "new" s m a l l i s l e t s composed o f g r a n u l a t e d b e t a - c e l l s was a l s o noted i n the p r o x i m i t y o f tumor. In two i n s t a n c e s s m a l l i s l e t s were found i n a mass of tumor c e l l s completely i s o l a t e d from normal a c i n a r t i s s u e . S e v e r a l l a r g e or medium-sized i s l e t s at a g r e a t e r d i s t a n c e from tumor were found to be completely devoid o f g r a n u l a t e d b e t a - c e l l s . The animals p r e s e n t i n g m t h i s h i s t o l o g i c a l p i c t u r e had normal b l o o d sugar l e v e l s . In a l l o x a n i z e d animals w i t h subcutaneous tumors no g r a n u l a t e d b e t a - c e l l s were found i n those animals which were examined h i s t o l o g i c a l l y . These animals had h i g h b l o o d sugar l e v e l s . T i s s u e s f o r h i s t o l o g i c a l examination were not o b t a i n e d f o r those animals i n which a l l e v i a t i o n o f d i a b e t e s was observed i n the presence o f subcutaneous tumor. These o b s e r v a t i o n s suggest that tumor does e x e r t a d i r e c t p r o t e c t i v e or r e g e n e r a t i v e i n f l u e n c e on the b e t a - c e l l s of the p a n c r e a t i c i s l e t s , and t h a t i t s c l o s e p r o x i m i t y to pan-creas i s e s s e n t i a l f o r t h i s a c t i o n . Conn, Hinerman and Buxton (16) r e p o r t e d on a human case i n which treatment o f i n s u l i n - p r o d u c i n g i s l e t c e l l adenoma ~54<-o f the .pancreas w i t h l a r g e doses o f a l l o x a n was attempted. S e v e r a l courses o f treatment w i t h a l l o x a n were g i v e n , from which the p a t i e n t r e c e i v e d o n l y temporary r e l i e f . F i n a l l y the tumor was removed by surgery, f o l l o w i n g which the p a t i e n t became d i a b e t i c . H i s t o l o g i c a l examination i n d i c a t e d t h a t , although the tumor co n t a i n e d i n t a c t b e t a - c e l l s r e s p o n s i b l e f o r the observed h y p e r i n s u l i n i s m , the i s l e t s o f the non-tumorous p o r t i o n s o f the pancreas had been destroyed by the a l l o x a n treatments. T h i s o b s e r v a t i o n supports the h y p o t h e s i s t h a t tumor t i s s u e e x e r t s a p r o t e c t i v e i n f l u e n c e on the b e t a - c e l l s a g a i n s t the s p e c i f i c t o x i c i t y o f a l l o x a n . C a r r i e and Ham (13) observed no e f f e c t on the hyper-glycemia induced by -alloxan i n W i s t a r r a t s w i t h sarcoma 57 t r a n s p l a n t s . I t i s o f i n t e r e s t to note t h a t the tumor i n t h i s i n s t a n c e was a subcutaneous t r a n s p l a n t , and t h a t i t was a sarcoma, or cancer d e r i v e d from c o n n e c t i v e t i s s u e , w h i l e both tumors o f f e r i n g p r o t e c t i o n (Walker 256 carcinoma and N o v i k o f f hepatoma) are o f e p i t h e l i a l o r i g i n , as are the b e t a - c e l l s o f the pancreas. Re c e n t l y , Vangerov and McKee (81) have confirmed the r e c i p r o c a l e f f e c t s o f i n t r a p e r i t o n e a l tumor t r a n s p l a n t ( E h r l i c h ' s mouse a c i t e s c e l l tumor) i n a m e l i o r a t i n g the d i a b e t e s i n a l l o x a n i z e d C57BL mice. B. P r e v e n t i o n and a l l e v i a t i o n o f a l l o x a n d i a b e t e s by  s u l p h y d r y l compounds. S e v e r a l authors have demonstrated the i n h i b i t i o n o f the d i a b e t o g e n i c a c t i o n o f a l l o x a n by s u l p h y d r y l compounds (3, 43 44, 45, 46, 47, 51, 52, 53, 54, 56, 58, 59, 65, and 7 0 ) . A c c o r d i n g to Lazarow, P a t t e r s o n , and Levy (56.) the p r o t e c t i v e a c t i o n o f s u l p h y d r y l compounds g i v e n p r i o r to o r immediately f o l l o w i n g the i n j e c t i o n o f a l l o x a n Is due to t h e i r forming a non-diabetogenic a d d i t i o n compound wi t h a l l o x a n . C y s t e i n e , g l u t a t h i o n e , and B r i t i s h A n t i - l e w i s i t e (2,3-dimercapto-propanol) were found to be e s p e c i a l l y e f f e c t i v e . The i n j e c t i o n o f a l l o x a n i n t o r a b b i t s (57) and r a t s (8) produced an immediate decrease i n the l e v e l of b l o o d g l u t a -t h i o n e , which was independent o f changes i n the blood g l u c o s e l e v e l s . T h i s suggests that g l u t a t h i o n e i s u t i l i z e d f o r a l l o x a n d e t o x i c a t i o n i . Ten minutes a f t e r the i n j e c t i o n o f a l l o x a n , a decrease i n h e p a t i c (17 per cent) and i n t e s t i n a l (42 per cent) g l u t a t h i o n e was observed ( 1 8 ) . A l s o , decreases i n the s u l p h y d r y l content o f l i v e r and kidney were observed 48 hours and 19 days a f t e r a l l o x a n : i n j e c t i o n (47). On the other hand, f a c t o r s which produce an i n c r e a s e i n the s u l p h y d r y l content o f the t i s s u e s a l s o prevent a l l o x a n d i a b e t e s (59). Houssay and M a r t i n e z (45) a l s o observed r e m i s s i o n o f a l l o x a n d i a b e t e s f o l l o w i n g treatment with t h i o u r a c i l , even though treatment s t a r t e d 16 days a f t e r a l l o x a n i z a t i o n . Sen and Bhattacharya (68) o b t a i n e d p r o t e c t i o n a g a i n s t a l l o x a n d i a b e t e s w i t h B r i t i s h A n t i - l e w i s i t e and c y s t e i n e , even though treatment s t a r t e d as much as h a l f an hour a f t e r the a d m i n i s t r a t i o n o f a l l o x a n , p r o v i d e d that subsequent i n j e c t i o n s were admi n i s t e r e d •§• hour and 1 hour l a t e r . The pancreas o f animals t r e a t e d i n t h i s way had normal i s l e t s . The authors conclude t h a t "the maintenance o f a h i g h c o n c e n t r a t i o n o f s u l p h y d r y l compounds i n the b l o o d f o r a f a i r l y long p e r i o d o f time was e s s e n t i a l f o r the r e v e r s a l o f the a c t i o n o f a l l o x a n " . , They p o s t u l a t e t h a t the a c t i o n o f a l l o x a n i s due to the o x i d a -t i o n o f e s s e n t i a l SH groups o f c e r t a i n s p e c i f i c b e t a - c e l l enzymes to S-S l i n k a g e s . T h i s o x i d a t i o n c o u l d be r e v e r s e d f o r a c e r t a i n p e r i o d o f time, but l a t e r becomes I r r e v e r s i b l e due to s u b s i d i a r y r e a c t i o n s . In view o f the p r o t e c t i v e a c t i o n of s u l p h y d r y l compounds f o r a l l o x a n d i a b e t e s o u t l i n e d above, and i n view o f the s i m i l a r p r o t e c t i v e a c t i o n e x e r t e d by tumors, as demonstrated by Goranson, Botham and Willms (35) and i n the present e x p e r i -ments, any m o d i f i c a t i o n o f s u l p h y d r y l metabolism i n tumor-b e a r i n g animals would be o f c o n s i d e r a b l e I n t e r e s t . A c c o r d i n g to Rapkine (66), changes o f SH groups to S-S groupg are one o f the key changes i n the p r o c e s s o f m i t o s i s . Mazia and Dan (60, 6l) have r e c e n t l y conducted a s e r i e s o f ~57~ experiments on the b a s i s o f which they p o s t u l a t e t h a t .the s p i n d l e f i b e r s and the whole m i t o t i c apparatus are formed by the p o l y m e r i z a t i o n o f r e l a t i v e l y s m a l l molecules r i c h i n SH groups through the formation o f S-S b r i d g e s . The s t i m u l a t i n g e f f e c t o f added g l u t a t h i o n e on growth and f i s s i o n i n c u l t u r e s o f Amoeba pr o t e u s ;was demonstrated by C h a l k l e y and V o e g t l i n (14). One o f the c h i e f c h a r a c t e r i s t i c s o f n e o p l a s t i c growth i s an i n c r e a s e i n the r a t e o f c e l l d i v i s i o n . S i n c e s u l p h y d r y l groups are o f g r e a t importance f o r t h i s p r o c e s s , and s i n c e they are s u p p l i e d to the body mainly i n the form o f amino a c i d s ( c y s t e i n e , c y s t i n e , and m e t h i o n i n e ) , r e d u c t i o n o f the i n c i d e n c e and growth o f tumors should accompany low d i e t a r y l e v e l s o f the s u l p h u r - c o n t a i n i n g amino a c i d s . T h i s has indeed been demon-s t r a t e d f o r s e v e r a l tumors. The i n c i d e n c e o f leukemia a f t e r p a i n t i n g w i t h methylcholanthrene was reduced to 10 per cent i n mice f e d on a low c y s t i n e d i e t , as compared wi t h 90 per cent i n mice on a f u l l d i e t or on the d e f i c i e n t d i e t supplemented by c y s t i n e (71, 72, 75, 76, and 77). M i l l e r e t a l (6?), H a r r i s et a l (42), and White and Edwards (73, 74) showed t h a t a low c y s t i n e d i e t reduced the i n c i d e n c e or prolonged the i n d u c t i o n p e r i o d o f hepatoma r e s u l t i n g from the a d m i n i s t r a t i o n o f p - d i -methylaminazobenzene. Brunschwig, A r n o l d , and Edgcomb (11) observed that a number o f d i f f e r e n t t h i o l compounds i n c r e a s e d the r a t e o f growth o f tumors. Walker 2jj6 carcinoma i n c r e a s e d * 5 8 -15 to 35 per cent i n animals t r e a t e d w i t h t h i o g l y c o l l a t e , methionine, and c y s t e i n e as compared with u n t r e a t e d c o n t r o l s . Shacter et a l (69) found an i n v e r s e r e l a t i o n s h i p between plasma s u l p h y d r y l l e v e l s and the r a t e o f growth o f Murphy r a t lympho-sarcoma. That m o d i f i c a t i o n o f s u l p h y d r y l metabolism does take place i n tumor was demonstrated by the work o f G r e e n s t e i n and Leuthardt (36, 37, and 38) on the c y s t i n e oxidase content o f normal and n e o p l a s t i c t i s s u e s . C y s t i n e oxidase c a t a l y s e s the d e g r a d a t i o n o f c y s t i n e or c y s t e i n e to p y r u v i c a c i d , hydrogen s u l p h i d e , and ammonia. C y s t i n e p e p t i d e s are a l s o a f f e c t e d by t h i s enzyme. G r e e n s t e i n demonstrated t h a t c y s t i n e oxidase was present i n descending amounts i n normal l i v e r , kidney, and pancreas. A l l o t h e r normal t i s s u e s , and a l l tumors s t u d i e d possess no a c t i v i t y . G r e e n s t e i n s t a t e s t h a t " c y s t i n e oxidase i s t h e r e f o r e the f i r s t system observed which, although present i n s e v e r a l normal t i s s u e s ( i n c l u d i n g l i v e r ) , i s completely absent i n a l l tumors s t u d i e d ( i n c l u d i n g hepatoma). These o b s e r v a t i o n s emphasize the importance o f the r o l e p l a y e d by s u l p h y d r y l groups i n n e o p l a s t i c growth, and i n d i c a t e t hat a t l e a s t one o f t h e i r major f u n c t i o n s i s l i n k e d to the process o f c e l l d i v i s i o n . -59-C. The i n f l u e n c e o f a l l o x a n d i a b e t e s on tumor growth  and i n c i d e n c e . The growth and i n c i d e n c e o f i n t r a p e r i t o n e a l tumors (both Walker 25& carcinoma and N o v i k o f f hepatoma), and the growth o f subcutaneous tumors was found to be reduced i n a l l o -x a n i z e d r a t s as compared wi t h n o n - d i a b e t i c tumor c o n t r o l s . I n h i b i t i o n o f the i n c i d e n c e and growth o f N o v i k o f f hepatoma was l i k e w i s e observed by Goranson, Botham and Willms (35)• S a l z b e r g and G r i f f i n (67) a l s o found t h a t the i n c i d e n c e o f hepatoma induced by f e e d i n g 3~methyl-4-dlmethylaminoazobenzene was markedly reduced i n a l l o x a n i z e d r a t s . The mechanism by which a l l o x a n i n h i b i t s the develop-ment and growth o f tumors remains unknown. Indeed, i t can not y e t be s t a t e d c a t e g o r i c a l l y whether the observed r e d u c t i o n i n tumor i n c i d e n c e i s due to f a i l u r e o f the tumor to develop, or to r e g r e s s i o n o f the tumor a f t e r i t has s t a r t e d to grow. However, the o b s e r v a t i o n that tumor i n c i d e n c e i s reduced even when tumor t r a n s p l a n t s are made 24 hours b e f o r e a l l o x a -n i z i n g suggests t h a t , i n some cases at l e a s t , r e g r e s s i o n o f tumors does o c c u r . A l s o , i t i s not known why the i n c i d e n c e o f subcutaneous tumors i s not reduced i n a l l o x a n i z e d r a t s , w h i l e i n t r a p e r i -t o n e a l tumor i n c i d e n c e i s d e c i d e d l y lower. V a s c u l a r i z a t i o n o f these tumors may be a c o n t r i b u t a r y f a c t o r , s i n c e subcuta-neous tumors were i n g e n e r a l we&l v a s c u l a r i z e d i n c o n t r a s t to the s i t u a t i o n w i t h r e s p e c t to i n t r a p e r i t o n e a l tumors which, i n the a l l o x a n i z e d animal, were o f t e n a v a s c u l a r . Goranson, Botham and Wlllms (35) suggest the p o s s i -b i l i t y t h a t the r e t a r d a t i o n i n tumor growth i n a l l o x a n i z e d r a t s may be due i n d i r e c t l y to the impairment i n l i v e r metabolism, so t h a t the supply o f e e r t a i n m e t a b o l i t e s e s s e n t i a l to the growing tumor i s r e s t r i c t e d . T h i s i s supported by the obser-v a t i o n o f P a l a y and Lazarow (65) who found t h a t a l l o x a n produced n e c r o s i s o f the l i v e r . I t i s i n t e r e s t i n g to note that t h i s h e p a t o t o x i c p r o p e r t y o f a l l o x a n i s enhanced by c y s t e i n e . S i n c e the i n h i b i t i o n o f tumor growth i s observed i n a l l o x a n - d i a b e t i c animals, i t might a l s o be expected t h a t normal growth too would be i n h i b i t e d . T h i s has i n f a c t been observed. C h e s l e r and T i s l o w i t z (15) i n j e c t e d young r a t s w i t h a l l o x a n and found marked r e t a r d a t i o n o f growth as compared w i t h normal animals. The degree o f i n h i b i t i o n was p r o p o r t i o n a l to the dose o f a l l o x a n i n j e c t e d . The work o f Young (79) and-of Milman, de Moor, and Lukens (80) i n d i c a t e s t h a t the.growth-promoting and p r o t e i n - a n a b o l i c e f f e c t o f growth hormone r e q u i r e s the supplementary a c t i o n o f i n s u l i n . The i n h i b i t o r y e f f e c t o f a l l o x a n i z a t i o n o f animals on the growth o f t r a n s p l a n t a b l e tumors may i n v o l v e metabolism o f the tumor c e l l d i r e c t l y , or i n d i r e c t l y through r e s t r i c t i o n i n the supply o f e s s e n t i a l m e t a b o l i t e s . The r e c i p r o c a l e f f e c t o f growing tumors i n a m e l i o r a t i n g d i a b e t e s due to p r e s e r v a t i o n o r r e g e n e r a t i o n of the b e t a - c e l l s o f the i s l e t s o f Langerhans i n the pancreas may c o n c e i v a b l y take p l a c e through i n t e r a c t i o n s i n v o l v i n g s u l p h y d r y l groups. The s i g n i f i c a n t work o f lazarow, Houssay, and o t h e r s on the s p e c i f i c i t y o f a c t i o n o f a l l o x a n on s u l p h y d r y l - c o n t a i n i n g enzymes suggests t h a t t h i s may be a worthwhile avenue f o r f u r t h e r i n v e s t i g a t i o n o f the p r o t e c t i v e e f f e c t o f tumor i n the a l l e v i a t i o n o f d i a b e t e s . -62** V. SUMMARY 1, P r e l i m i n a r y work by Goranson et a l (35) suggested that i n t r a p e r i t o n e a l N o v i k o f f hepatoma t r a n s p l a n t s and a l l o x a n - . diabetes were mutually a n t a g o n i s t i c . The present study was undertaken to extend t h i s work and to c o r r e l a t e i t w i t h the h i s t o l o g i c a l p i c t u r e i n the pancreas. 2. S e v e r a l s e r i e s o f experiments were performed i n which v i r g i n female W i s t a r r a t s were i n j e c t e d i n t r a p e r i t o n e a l l y (or o c c a s i o n a l l y subcutaneously) w i t h a s i n g l e diabetogenic dose o f a l l o x a n (175 mg. per kg. o f body weight) 14 days, 7 days, 4 days, and 24 hours p r i o r t o , and 24 hours and 4 days a f t e r t r a n s p l a n t i n g i n t r a p e r i t o n e a l l y or subcutaneously w i t h 0.5 ml. of 50 per cent tumor homogenate prepared from N o v i k o f f hepatoma o r Walker 256 carcinoma from stock tum o r - c a r r i e r animals. Animals were s a c r i f i c e d 5 to 7 days a f t e r t r a n s p l a n t i n g , at which time blood samples were taken f o r sugar a n a l y s i s , tumors were d i s s e c t e d out and weighed, and p a n c r e a t i c t i s s u e was preserved f o r s e c t i o n i n g . 5. A n a l y s i s of f a s t i n g blood sugar l e v e l s r e vealed t h a t i n a l l o x a n i z e d animals b e a r i n g i n t r a p e r i t o n e a l tumor t r a n s p l a n t s blood sugars were reduced to normal or a t l e a s t c o n s i d e r a b l y ameliorated as compared w i t h a l l o x a n - d i a b e t i c c o n t r o l s . The e f f e c t of subcutaneous tumors on sugar l e v e l s i n a l l o x a n i z e d -6? animals was much l e s s marked, although a l l e v i a t i o n was observed i n some in s t a n c e s . 4. The inoidenoe and growth of i n t r a p e r i t o n e a l tumors i n a l l o x a n i z e d animals was c o n s i d e r a b l y .less than t h a t i n non-d i a b e t i c c o n t r o l s . Although no changes i n tumor incidence o f suboutaneous t r a n s p l a n t s was observed, some r e t a r d a t i o n of tumor growth occurred. However, r e s u l t s w i t h subcutaneous tumor were f a r l e s s s t r i k i n g than those obtained w i t h i n t r a p e r i t o n e a l tumor* 5» High blood sugar l e v e l s i n a l l o x a n i z e d animals were cor-r e l a t e d w i t h degranulation o r n e c r o s i s o f the b e t a - c e l l s of the i s l e t s of Langerhans i n the pancreas. The i s l e t s were sma l l e r . i n s i z e and fewer i n number i n d i a b e t i c than i n normal animals. The a l p h a - c e l l s were not damaged. A l l o x a n i z e d animals bearing i n t r a p e r i t o n e a l tumor t r a n s p l a n t s , which had normal blood sugar l e v e l s , had i s l e t s i n which granulated b e t a - c e l l s were present, although f r e q u e n t l y reduced i n number. The c l o s e p r o x i m i t y o f invading tumor c e l l s i n the pancreas appeared to a f f o r d pro-t e c t i o n to or s t i m u l a t e regeneration o f the b e t a - c e l l s . 6* Recent l i t e r a t u r e r e l a t i n g the mechanism of a l l o x a n a c t i o n to i t s a f f i n i t y f o r s u l p h y d r y l groups, and on the importance of s u l p h y d r y l groups f o r m i t o s i s and tumor development and growth i s o u t l i n e d . The p o s s i b l e p a r t i c i p a t i o n of s u l p h y d r y l groups i n the m u t u a l l y a n t a g o n i s t i c a c t i o n o f a l l o x a n - d i a b e t e s and t r a n s p l a n t a b l e tumors i s suggested as worthy o f f u r t h e r i n v e s t i g a t i o n . -65-LITERATURE CITED 1. All e g r e t t i , N., and V. Fister. 1953. On the insulin poten-tiation of the diabetogenic action of alloxan. Arch, Internal Physiol. 6 l : 41^46. 2. Arnold, A. 1951* Beitrag zur quantitativen Histologic des Alloxandiabetes der Albinoratte: Die Kerngrossen des Inselzellen. Acta Anatomioa 1_2: 396-428. 3* Bailey, C.C, 1949. Alloxan Diabetes. Vitamins and Hormones 2: 565-582. 4. Bailey, C.C, and O.T. Bailey. 1943* The production of diabetes me11itus in rabbits with alloxan. A preliminary report. Jour* Amer. Med. Assoc. 122: Il65-ll6b. 5. Bailey, C.C., O.T. Bailey, and R.S. Leech. 1944. Alloxan diabetes with complications. New England Med. Jour., 230: 533-536. 6. Bailey, O.T., C.C. Bailey, and W.H. Hagan. 1944. Alloxan diabetes in the rabbit. A consideration of the morphologic and physiologic changes, Amer, Jour, Med. Sci., 203: 450-461. 7. Brachet, J. 1950. Chemical embryology. Interscience Publishers, Inc., New York. 533pp. 8. Bruckmann, G. and E. Wertheimer. 1947. Alloxan studies: The action of alloxan homologues and related compounds. Jour. Bi o l Chem. 168: 241-256. 9. Brunschwig, A., and J.G. Allen. 1944. Specific injurious action of alloxan upon pancreatic i s l e t cells and convoluted tubules of the kidney. Comparative study in the rabbit, dog and man. Attempted chemotherapy of insulin-producing i s l e t c e l l carcinomas i n man. Cancer Res., _4; 45-54. 10. Brunschwig, A., J.G. Allen, F.M. Owens, Jr., and T.F. Thornton. 1944. Alloxan in the treatment of insulin-pro-ducing i s l e t c e l l carcinoma of the pancreas. Jour. Amer. Med Assoc., 124: 212-216. 11. Brunschwig, A., J. Arnold, and J. Edgcomb. 1946. Stimulation and retardation of neoplastic growth by sulfhydryl compounds. Cancer Res., 6: 560-562. 12. Brunschwig, A., M.G. Goldner, J.G. Allen, and G. Gomori. 1943. Alloxan. Jour. Amer. Med. Assoc., 122: 966. -66-15. C a r r i e , A.W., and A.W. Ham, 1949. An experimental study o f the e f f e c t s o f malignancy and diabetes on each other . Cancer Res., j?: 629, 14, C h a l k l e y , H.W., and C, V o e g t l i n , 1940. The e f f e c t o f v a r i a -t i o n i n oxygen t e n s i o n ans s u l f h y d r y l c o n c e n t r a t i o n on nuc l e a r growth and f i s s i o n i n Amoeba proteus. J o u r , N a t l . Cancer I n s t . , 1: 63—75. 15, Chesler, A., and R. T i s l o w i t z . 1945. E f f e c t o f a l l o x a n diabetes i n the growing r a t . Science, 101; 468, 16, Conn, J.W.-,- D.L. Hinerman, and Buxton, R.W, 1947, E f f e c t s o f a l l o x a n upon the human pancreas. Jour, Lab, and C l i n , Med,, 32: 347-348. 17, Cruikshank, A.H. 1954. L i v e r n e c r o s i s and a l l o x a n hypo-glycemia. Jour, Path, and Baot. $Jj 227-236, 18, De Caro, L,, and E, Rovida, 1937.-Relazione t r a g l u t a t i o n e e vitamina C n e i t e s s u t i . I I I . Azlone dell»allossano e d e l l * a c i d o monoiodo a c e t i c o s u l contenuto i n g l u t a t i o n e e vitamina C d e g l i organ! d i oavie e d i r a t t i . B o l l , Soc, I t a l , B i o l . Sper,, 12: fill. C i t e d by Houssay,- A.B, 1950. Amer. Jour. Med. ScT7 219: 353-367. 19, Duff, G.L. 1945, The pathology o f the pancreas i n e x p e r i -mental diabetes m e l l i t u s . Amer. Jour. Med, S c i . , 210: 381-397. 20, Duff, G.L., and H. S t a r r . 1944. Experimental a l l o x a n diabetes i n hooded r a t s . Proo. Soc. E x p t l . B i o l , and Med., 57: 28O-282. 21, Dunn, J.S., E. Duffy, M.K. Gilmour, J . K i r k p a t r i c k , and N.G.B, McLetchie. 1-944, Jour. P h y s i o l , , 103: 233. 22, Dunn, J.S., J . K i r k p a t r i c k , N.G.B. McLetchie, and S.V. T e l f e r . 1943. N e c r o s i s o f the i s l e t s o f Langerhans produced e x p e r i m e n t a l l y . Jour, path, and Bact,, 55: 254-257. 23, Dunn, J.S,, and N.G.B. McLetchie. 1943. Production o f diabetes i n the r a t . Lancet 245: 384-387. 24, Dunn, J.S., H.L. Sheehan, and N.G.B. McLetchie. 1943. Necrosis of*, i s l e t s of Langerhans produced e x p e r i m e n t a l l y . Lancet 121: 484-487. -67-25. - Dunning, W.F., M.R. C u r t i s r and C. Friedgood. 1 9 4 9 . The incidence of benzpyrene-induced sarcomas i n diabetic and alloxan-refractive r a t s of three s t r a i n s . Cancer Res., £: 546. 26. Ferner, H., and W. Stoekenius, J r . 1951. Die Cytogenese des Inselsystems beim Menschen. Zeitschr. Z e l l f o r s c h . mikrosk. Ahat., 55: 147-175. 27. Fisher, R. A. 1948. S t a t i s t i c a l methods fo r research workers. 10th ed. O l i v e r and Boyd, Edinburgh. 354pp. 2:8. Goldner.. M.G., and G. Gomori. 1943. Alloxan diabetes i n the dog. Endocrinology, 33.: 297-308. . 29. Goldner, M.G., and G. Gomori. 1944. Mechanism of the diabeto-genic action of alloxan. Proc. Soc. E x p t l . B i o l , and Med., 55: 73-75. 30.. Goldner, M.G., and Gomori, G. 1944. Studies on* the mechanism of alloxan diabetes. Endocrinology, 35: 241-248. 31. Gomori, G. 1939. Studies on the c e l l s of the pancreatic i s l e t s . Anat. R e c , 74: 4 3 2-459. . [ 32. Gomori, G. 1950. Aldehyde-fuchsin: a new s t a i n f o r e l a s t i c t i ssue. Am e r ,« Jour. C l i n . Path., 20: 665-666, 35. Gomori, G»-» and M.G. Goldner. 1943. Production of diabetes mel l i t u s i n r a t s with alloxan. Proc. Soc. E x p t l . B i o l , and Med.,54: 287-290. 34. Gomori, G., and M.G. Goldner. 1945. Acute nature of alloxan damage. Proc. Soc. E x p t l . B i o l , and Med., 58: 232-835. 55. Goransqri,""E.S.", F. Botham,* and M. Wlllms. 1954. I n h i b i t i o n of growth of transplanted hepatomas i n alloxanized Wistar r a t s . Cancer Res., 14: 750-733. 3 6 . Greenstein, J.P. 1945. Degradation of cystine by normal l i v e r but not by transplanted hepatomas. Jour.' N a t l . Cancer Inst., j5: 491-494. 57.* Greenstein, J.P.,'and F.M. Leuthardt. 1945. Note on the cystine oxidase a c t i v i t y i n normal and neoplastic tissues of the mouse. Jour. N a t l . Cancer Inst., 5: 59. -68-38. Greenstein, J-.P., and F.M. Leuthardt. 1945. Degradation of oystine peptides by tissues. I. Exocystine desulfurase and debydropeptidase in rat l i v e r extracts. II. Distribution of exocystine desulfurase and debydropeptidase in tissue extracts of various species. III. Absence of exocystine desulfurase and dehydropeptidase- i n tumors. Jour. Natl. Cancer Inst., 3} 209-221; 223-225; 249. 39. Halml, N.S. 1952. Stain Technology, 2Jj 6 l . 40. Hard, W.L. 1944. The origin and differentiation of the alpha and beta cells in the pancreatic i s l e t s of the rat. Amer. Jour. Anat., -75: 369-403. -41. Hard, W.L., and C.J. Carr. 1944. Experimental diabetes produced by alloxan-. Proc. soc. Exptl. B i o l , and Med., 55: 214-216. 42. Harris, P.N., M.E. Krahl, and G.H.A. .Clowes. 1947. p-Dimethylaminoazobenzene carcinogenesis with purified diets varying in content of cysteine, cystine, l i v e r extract, protein, riboflavin and other factors. Cancer Bes., 7r 162. • 43. Houssay, B.A. 1950. Action of sulfur compounds on carbo-hydrate metabolism and on diabetes. Amer. Jour. Med. Sol., 219: 353-367. -44. Houssay, B.A., W.A. Lott, and C. Martinez. 1950. Accion de algunos compuestos con azufre sobre l a diabetes' aloxanica y pancreatica. Rev. Soc. Argentina Biol., 26: 335-344. Cited by Houssay, B.A. 1950. Amer. Jour. MecT. Sci., 219: 353-367. 45. Houssay, B.A., and C. Martinez. 1948. Prevention ou guerison du diabete- alloxanique par les thiouraclles ou l a cysteine. .C.R. Soc. Biol., Paris, 142: II67-II69. 46. Houssay, B.A., and C. Martinez. 1948. Prevencion y curacion de l a diabetes- aloxanica por tiouracilos o cisteina. Rev. Soc. Argentina Biol., 24: 63-92. Cited by Martinez, C. 1951* Acta Physiol. Latinoamer., 1: 135-162. 47'. Houssay, B.A.', C. Martinez, and R. Gaputtc. 1947. Influencia de l a diabetes y de l a adminlstracion de tlouraoilo sobre el oontenido' de SH de los tejidos. Rev. Soc. Argentina Bi o l . 23: 248. Cited by Houssay, B.A. 1950. Amer. Jour. Med. s*bT., 219: 353-367. 48. Hughes, H., L.L. Ware, and F.S. Young. 1944. Diabetogenic action of alloxan. Lancet 246: 148-150. -69-49. J e h l , J . , and, R.W. McKee. 1954. Influence o f a l l o x a n diabetes on s u r v i v a l time of mice i n o c u l a t e d w i t h E h r l i c h i a mouse a s c i t e s carcinoma c e l l s . Fed. P r o c , 13: 237. 50. Lackey, R.W., C.A. Bunde, A.J. G i l l , and L.C. H a r r i s . 1944. Glycogen i n a l l o x a n - t r e a t e d r a t s . Proc. Soc. E x p t l . B i o l , and Med., £7? 191-194. 51. Lazarow, .A. 1945. S u l f y d r y l groups and a l l o x a n diabetes.1945. Anat. R e c , 91: 286. 52. Lazarow, A. 1946. P r o t e c t i v e e f f e c t o f g l u t a t h i o n e and cy s t e i n e against a l l o x a n diabetes i n the r a t . Proc. Soc. E x p t l . B i o l , and Med., 6 l ; 441. 53. Lazarow, A. 1947. P r o t e c t i o n a g a i n s t a l l o x a n . d i a b e t e s . Anat. Rec , 97: 353. 54. Lazarow, A. .1947.-Further s t u d i e s . o f e f f e c t . o f s u l f u r com-pounds on production o f diabetes w i t h a l l o x a n . P r o c Soc. E x p t l . B i o l , and Med., 66: 4-7. 55. Lazarow,-A. 1949. F a c t o r s c o n t r o l l i n g the development and progression of diabetes. P h y s i o l . Revs., 29: 48-74. 56. Lazarow, A., J.Wf. P a t t e r s o n , and SLevy.. 1 9 4 8The mechanism o f c y s t e i n e and g l u t a t h i o n e p r o t e c t i o n a g a i n s t a l l o x a n diabetes. Science, 108: 308-309. 57. Leech, R.S., and C.C. B a i l e y . 1945. Blood a l l o x a n and blood g l u t a t h i o n e i n r a b b i t s i n j e c t e d w i t h a l l o x a n . Jour. B i o l . Chem., 157: 525-542. . . . * 58. Martinez, C. 1950. S u f h i d r i l o s y diabetes. Rev. A s s o c Med. Argentina, £ 4 : 127-129. 59. Martinez,.-C. 1951. The SH groups i n experimental d i a b e t e s . Acta P h y s i o l . Latinoamer., JL: 135-162. 60. Mazia, D. 1953. C e l l d i v i s i o n . S c e i n t i f i c Amer., 189: (2), 55-61. 61. Mazia, D., and Katsuma Dan. 1952. The i s o l a t i o n and b i o -chemical c h a r a c t e r i z a t i o n of t h e . m i t o t i c apparatus o f d i v i d i n g c e l l s . Proc. N a t l . Acad. S c i . , 38: 826-838. 62. McClung, C.E. ( e d i t o r ) . 1950. Handbook o f m i c r o s c o p i c a l technique.for workers i n both p l a n t and animal t i s s u e s . 3rd. ed. P.B. "Hoeber, Inc., New York. 790pp. -70-63. M i l l e r , E.C., J.A. M i l l e r , B.E. K l i n e , and H.P. Rusch. 1948. C o r r e l a t i o n of the l e v e l of hepatic r i b o f l a v i n w i t h the appearance of l i v e r tumors i n r a t s f e d aminoazo dyes. Jour. E x p t l . Med., J38: 89-98. 64. Nelson, N. 1944. Photometric adaptation of the Somogi method f o r the.determination.of glucose. Jour. B i o l . Chem., 153: 375-380. 65. Palay , S.A., and Lazarow, A. 1946. P r o t e c t i o n against d i a -betes and production o f hepa t i c n e c r o s i s by use of c y s t e i n e and a l l o x a n i n r a t s . Anat. R e c , 96: 55-76. 66. Rapkine, L. 1931. Ann. Physio. J_x 382. C i t e d by Brachet, J . Chemical Embryology. I n t e r s c i e n c e P u b l i s h e r s , Inc., New , York. 533pp. • 67. S a l z b e r g , D.A., and A.C. G r i f f i n . 1952. I n h i b i t i o n o f azo dye carcinogenesis i n the a l l o x a n - d i a b e t i c r a t . Cancer Res., 12_: 294. , 68. Sen, P.B., and G. Bhattacharya. 1952. Reversal o f the diabetogenic a c t i o n o f a l l o x a n by s u l f h y d r y l compounds. Science, 115: 41-43. 69. Shacter, B., C. Entenman, and M.B. Shimkin. 1952. Plasma s u l f h y d r y l l e v e l s during tumor growth. Cancer Res., 12: 295. • 70. Ueno, Y..1954. Studies on the r a b b i t a l l o x a n diabetes m e l l i t u s . I I . Preventive a c t i o n of a few drugs f o r -diabetogenic a c t i o n - o f a l l o x a n . F o l i a E n d o c r i n o l . Japonica, 29: 253-265. 71. White, J . , a n d H.B. Andervont. 1943. E f f e c t of d i e t r e l a -t i v e l y low i n c y s t i n e on the production of spontaneous , breast tumors i n - v i r g i n C 3 H mice. Cancer Res., j5: 129. 72. White, J . , and Andervont, H.B. 1943. E f f e c t of a d i e t r e l a t i v e l y low i n c y s t i n e on.the production of - spontaneous mammary gland tumors In s t r a i n C3H female mice. J o u r . N a t l . Cancer I n s t . .3: 449-451. 73. White, J . , and J.E. Edwards. 1942. E f f e c t of d i e t a r y c y s t i n e on the development of hepatic tumors i n r a t s f e d p-dimethyl-aminoazobenzene ( b u t t e r y e l l o w ) . Jour. N a t l . Cancer I n s t . , 2: 535-538.. 74. White, J . , and J.E. Edwards, 1942. E f f e c t of,supplementary methionine or c h o l i n e p l u s c y s t i n e on the incidence of p-dimethylaminoazobenzene-induced he p a t i c tumors i n the r a t . Jour. N a t l . Cancer I n s t . , 3: 43-59. - 7 1 -75. White, J . , G.B. Mider, and I . E . Heston. 1943. Note on the comparison o f dosage o f methylcholanthrene on the p r o d u c t i o n o f leukemia and s c l e r o t i c l e s i o n s 'in d i l u t e brown s t r a i n mice.on a r e s t r i c t e d c y s t i n e d i e t . J o u r . N a t l . Cancer I n s t . , 453-454. 76. White, J . , G.B. Mider, and W.E. Heston..1944. E f f e c t o f amino a c i d s on the i n d u c t i o n o f leukemia i n mice. J o u r . N a t l . Cancer I n s t . , _4: 409-411. 77. White, J . , F.R. White, and G.B. Mider. 1947. E f f e c t s o f d i e t s d e f i c i e n t i n c e r t a i n amino a c i d s on the i n d u c t i o n o f leukemia i n mice. Jo u r . N a t l . Cancer I n s t . , T} 199-202. 78. W i l d e r , R.M. 1946. P r o c . Amer. Diabetes Assoc., _6: 389-390. 79. Young, F.G. 1945. Growth and d i a b e t e s i n normal animals t r e a t e d w i t h - p i t u i t a r y , ( a n t e r i o r lobe) d i a b e t o g e n i c e x t r a c t . Biochem. J o u r . _39t 515-536. 8 0 . Milman, A.E., P. de Moor, and F.D.W. Lukens. 1951. R e l a t i o n o f p u r i f i e d p i t u i t a r y growth hormone and i n s u l i n i n r e g u l a t i o n o f n i t r o g e n b a l a n c e . Amer. Jour . P h y s i o l . , 166: 354-363. 81 . Vanergov, M., and R.W. MoKee. 1955. Metabolism o f g l u c o s e , l a c t a t e and a l a n i n e by E h r l i c h i a a s c i t e s carcinoma c e l l s from normal and a l l o x a n - d i a b e t i c mice. Fed. Proc., 14;:296. 

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