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Studies in selenium toxicology with particular reference to chemotherapeutic means of treatment Derrick, Jack Bryan Devereux 1949

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STUDIES IN SELENIUM TOXICOLOGY with p a r t i c u l a r r e f e r e n c e to chemotherapeutic means of treatment by Jack Bryan Devereux D e r r i c k , B.A, A T h e s i s Submitted i n P a r t i a l F u l f i l m e n t of the Requirements f o r the Degree of Master of A r t s In the Department of B i o l o g y and Botany THE UNIVERSITY OF BRITISH COLUMBIA August, 1949. STUDIES IH SELENIUM TOXICOLOGY with p a r t i c u l a r r e f e r e n c e to chemotherapeutie means of treatment by Jack Bryan Devereux D e r r i c k , B.A, A B S T R A C T 1. The experimental evidence Of the present work i n selenium t o x i c o l o g y Is such as to cast grave doubts on the a u t h e n t i c i t y of some aspects of the work o f p r e v i o u s i n -v e s t i g a t o r s i n t h i s f i e l d . The response o f the l a b o r a t o r y animals, subjected t o tho v a r i o u s procedures p r a c t i s e d by these i n v e s t i g a t o r s i n attempts t o produce a c o n d i t i o n of c h r o n i c selenium p o i s o n i n g , suggests t h a t these animals a r e , In a d d i t i o n t o being the v i c t i m s o f selenium p o i s o n -i n g , t h e v i c t i m s of I n a n i t i o n and/or d e h y d r a t i o n . 2. The presence of up t o 15 p.p.m. sodium s e l e n i t e (6.9 p.p.m. Se) i n t h e d r i n k i n g water i s t o l e r a t e d without any d r a s t i c r e d u c t i o n i a food and water i n t a k e by l a b o r a t o r y v r a t e , p rovided t h a t t h i s c o n c e n t r a t i o n i s reached g r a d u a l l y . At t h i s l e v e l o f selenium intake %he animals w i l l remain f o r at l e a s t 25 weeks i n good c o n d i t i o n and w i l l e x h i b i t growth o n l y s l i g h t l y l e s s r a p i d t han t h a t of c o n t r o l animals. Any attempts t o r a i s e the selenium content above t h i s l e v e l r e s u l t s In a compensatory l o w e r i n g of the water i n t a k e and a r e s u l t a n t r e d u c t i o n i n food i n t a k e of the animals. 3. The r e s u l t s obtained i n the attempts t o induce Chronic selenium p o i s o n i n g by d i e t a r y methods were such as to cause these methods t o be abandoned due to t h e i r u n s u i t -a b l l l t y f o r the present p r o j e c t • A method i n v o l v i n g t h e subcutaneous i n j e c t i o n of s u b - l e t h a l doses o f sodium s e l e n i t e i n c o r p o r a t e d i n t o an a b s o r p t i o n d e l a y i n g v e h i c l e was evolved and, a f t e r t e s t i n g , was deemed s u i t a b l e f o r the purpose o f i n d u c i n g a sub-acute c o n d i t i o n o f selenium t o x i c i t y t o be used as a means of a s s e s s i n g the value of chemotherapy* 4. Experimental data on the u r i n a r y e x c r e t i o n l e v e l s of selenium was made a v a i l a b l e by the development of a new c o l o r I m e t r i c procedure f o r the q u a n t i t a t i v e d e t e r m i n a t i o n o f STUDIES IN SELENIUM TOXICOLOGY ABSTRACT - Cont. selenium i n b i o l o g i c a l samples. The data obtained by t h i s method r e v e a l e d t h a t the maximum q u a n t i t y of selenium ex-c r e t e d i n the u r i n e of r a t s over a p e r i o d as l o n g as t e n days a f t e r i n j e c t i o n was about one t h i r d o f the q u a n t i t y o r i g i n a l l y i n j e c t e d . No p a r t i c u l a r advantage i n the use of the a b s o r p t i o n d e l a y i n g v e h i c l e was d i s c e r n a b l e by t h i s method but s i n c e , under t h e l i m i t a t i o n s imposed by t h e q u a n t i t i e s of u r i n e a v a i l a b l e , the delay i n hours would be undetectable the v e h i c l e was r e t a i n e d i n the experiments a s s e s s i n g the v a l u e of BAL ( 2 , 3 - d i t h i o p r o p a n o l ) as a chemo-t h e r a p e u t i c agent. 5. The use of BAL as a chemotherapeutic agent f o r combatting selenium t o x i c i t y has a d e l e t e r i o u s e f f e c t on the i n t o x i c a t e d animals. The subcutaneous i n j e c t i o n o f q u a n t i t i e s of BAL i n peanut o i l at l e v e l s c o n sidered w e l l w i t h i n the l i m i t s of s a f e t y r e s u l t e d i n an i n t e n s i f i c a t i o n bf the d i s t r e s s of selenium i n j e c t e d r a t s . Deaths were more frequent than i n t h e case of non BAL-treated animals. The animals which s u r v i v e d the e f f e c t s o f both poison and treatment showed a s l i g h t l y h i g h e r and a more prolonged ex-c r e t i o n o f selenium i n t h e u r i n e . 6. Suggestions have been made f o r improved means of therapy f o r acute and sub-acute selenium p o i s o n i n g i n v o l v i n g the use of a l e s s t o x i c d i t h i o l compound and the a d d i t i o n of s u c c i n a t e t o c a r r y . t h e animal over the i n i t i a l shock t o the a f f e c t e d enzyme systems. ACKNOWLEDGEMENTS S i n c e r e expressions of g r a t i t u d e are ex-tended t o a l l of the people r e s p o n s i b l e f o r assistance, i n the completion of t h i s work. In p a r t i c u l a r , I would l i k e t o thank Dr. A.H. Hutchinson f o r h i s encouragement t o pursue the work and fori h i s p e r m i s s i o n to carry-i t out i n the L a b o r a t o r y of Animal N u t r i t i o n , and P r o f e s s o r H.M. K i n g f o r the courteous co-o p e r a t i o n I have always r e c e i v e d from the Department of Animal Husbandry. Drs. J.A. A l l a r d y o e and A . J . Wood, both of whom c o n t r i b u t e d much of t h e i r time, c o n s i d e r a -t i o n and c o n s t r u c t i v e c r i t i c i s m t o the d i r e c t i o n of t h i s work are the r e c i p i e n t s of my g r a t e f u l thanks. The g e n e r o s i t y of The C o n s o l i d a t e d M i n i n g and Smelting Company of Canada and the B r i t i s h Columbia Research C o u n c i l i n extending f i n a n c i a l a s s i s t a n c e to make t h i s p r o j e c t p o s s i b l e i s a l s o g r a t e f u l l y acknowledged. F i n a l l y my s i n c e r e s t thanks go to Miss Joan W i l l i a m s f o r p r e p a r i n g t h i s work f o r p r e s e n t a t i o n . TABLE OF CONTENTS Page FOREWORD 1 I . CHRONIC SELENIUM TOXICITY with p a r t i c u l a r r e f e r e n c e t o the i n d u c t i o n of a c o n d i t i o n of ch r o n i c selenium p o i s o n i n g i n l a b o r a t o r y animals.-. 5 ( i ) A Summary of Pre v i o u s Work and E x p e r i m e n t a t i o n . . . . . . . . . . . . . . . . j> ( i i ) Experiments i n the I n d u c t i o n of Chronic Selenium T o x i c i t y i n the White Rat 10 ( i i i ) Experiments i n the Ind u c t i o n of a C o n d i t i o n of Selenium T o x i c i t y i n the White Rat S u i t a b l e f o r the Purpose of As s e s s i n g the E f f e c t i v e n e s s of Chemotherapy 27 I I . A NEW COLORIMETRIC METHOD FOR THE QUANTITATIVE DETERMINATION OF SELENIUM IN SMALL BIOLOGICAL SAMPLES...... 48 H I . CHEMOTHERAPEUTICS, METAL TOXICOLOGY AND THE SELENIUM PROBLEM. 64 IV. EXPERIMENTAL CHEMOTHERAPY: The use of BAL as a means of t r e a t i n g sub-acute selenium p o i s o n i n g i n the r a t . . . 86 V. SUMMARY AND CONCLUSIONS 94 APPENDICES Pa&e APPENDIX I . Data. Experiment A 99 APPENDIX I I . A Summary of the Attempts to E s t a b l i s h the Q u a n t i t y of Selenium E x c r e t e d Through the Lungs of A c u t e l y S e l e n i -zed Animals ................. 103 APPENDIX I I I . F u r t h e r Data on the L.D.50 of Sodium S e l e n i t e , v i a the I n t r a p e r i t o n e a l I n j e o t i o n Route, f o r V a r i o u s Species of Animals... 107 APPENDIX 17. A Summary of the Gross Pathology Observed i n Animals of the V a r i o u s Experimental Groups.......... 115 TABLE OF REFERENCES 117 FOREWORD The p r e l i m i n a r y work on the problem of selenium t o x i c o l o g y ( D e r r i c k , 1948) c a r r i e d out d u r i n g 1947 and 1948 i n the Animal N u t r i t i o n L a b o r a t o r y appeared to v a l i d a t e , at l e a s t p a r t i a l l y , the hypothesis set forward with r e g a r d t o the mechanism of selenium p o i s o n i n g . Chemotherapeutic t r e a t -ment, ad m i n i s t e r e d on the s u p p o s i t i o n that the mode of a c t i o n of selenium was as p o s t u l a t e d , showed evidence of value i n the r e c o v e r y of White Wistar r a t s which had been f e d on a s e l e n i z e d d i e t . I t became i n c r e a s i n g l y obvious d u r i n g the course of experimentation, however, t h a t many of the techniques employed by previous workers (Moxon, 1937)(Smith, 1939) and u t i l i z e d i n these p r e l i m i n a r y i n v e s t i g a t i o n s were not ade-quate and were indeed adding to the gen e r a l bewilderment e x i s t i n g i n the f i e l d . When i t was r e s o l v e d to continue the work on selenium t o x i c o l o g y , t h e r e f o r e , i t was a l s o decided that i t was necessary to employ ne.w techniques f o r the f o l l o w i n g phases of the work. 1. The i n d u c t i o n of a s t a t e of c h r o n i c selenium p o i s o n i n g i n l a b o r a t o r y animals. 2. The measurement of the in t a k e and e x c r e t i o n of selenium. S i n c e i t was necessary to produce a l a r g e number of selenium poisoned animals i n the chr o n i c c o n d i t i o n i n order to v e r i f y the e f f e c t i v e n e s s of v a r i o u s types of therapy, 2 the primary purpose of the new work, i t was a l s o necessary that a r e l a t i v e l y simple method o f i n d u c i n g the t o x i c c o n d i -t i o n should be employed. The work of Smith et a l . (1940) o f f e r e d what appeared to be the most promising means of pro-ducing a c o n d i t i o n of selenium p o i s o n i n g , s u i t a b l e f o r the a p p l i c a t i o n of therapy, i n the White Wistar r a t . T h i s method was t h e r e f o r e adopted as a means of g e t t i n g the animals i n t o the t o x i c c o n d i t i o n r e q u i r e d f o r the new s e r i e s of i n -v e s t i g a t i o n s . The r e s u l t s which attended t h i s d e c i s i o n * may be looked upon as both unfortunate and f o r t u n a t e ; u n f o r t u n a t e i n that much of the t h e r a p e u t i c work which was p r i m a r i l y intended to be c a r r i e d out i n these i n v e s t i g a t i o n s has not reached f r u i t i o n and, i n a d d i t i o n , t h a t i t i s the w r i t e r ' s unpleasant task t o p o i n t out t h a t much of the previous work o f other i n v e s t i g a t o r s of selenium p o i s o n i n g has been based on a f a l s e premise; f o r t u n a t e i n t h a t the f a i l u r e s attendant on the experiment served to p o i n t out some s i g n i f i c a n t a spects of the selenium p o i s o n i n g problem which might not otherwise have come to the f o r e . The outcome of the repeated f a i l u r e s t o develop true selenium p o i s o n i n g , uncomplicated by extraneous f a c t o r s , by d i e t a r y means has been the development i n t h i s work of what i s b e l i e v e d t o be a new technique i n metal t o x i c o l o g y ? * While i t i s obvious t h a t t h i s new technique a l s o has i t s shortcomings i t i s the s i n c e r e o p i n i o n of the writer that, ? See p. 6. s s S e e p. 48. e x p e r i m e n t a l l y , the p r i n c i p l e o f the method i s more sound than the r e l a t i v e l y haphazard means of i n d u c i n g the t o x i c s t a t e u t i l i z e d i n selenium t o x i c o l o g y work h e r e t o f o r e . The development of a new method of q u a n t i t a t i v e a n a l y s i s f o r selenium i n b i o l o g i c a l samples, during the course of t h i s work, has s i m p l i f i e d the problem of determining the e x c r e t i o n o f selenium by the experimental animals. P r e v i o u s l y e x i s t i n g methods were, i n the main, f a r too ponderous, time consuming, and expensive f o r the s m a l l l a b o r a t o r y with l i m i t e d personnel and thus were i m p r a c t i c a l f o r use i n t h i s study. The present method has made i t p o s s i b l e t o measure the r e t e n t i o n of selenium, and i t s e l i m i n a t i o n under v a r i o u s c i r -cumstances, with comparative ease. In a d d i t i o n , i t o f f e r s the p o s s i b i l i t y o f c a r r y i n g out analyses i n numbers s i g n i f i -cant enough to be u t i l i z e d as dependable data. The e a r l i e r study ( D e r r i c k , 1948) o f f e r e d a f a i r l y complete review of the a v a i l a b l e l i t e r a t u r e on selenium p o i s o n i n g up to the time of w r i t i n g . Much of the work d i s -cussed had been approached from the a p p l i e d r a t h e r than the fundamental poi n t of view. I t was f e l t , upon completion of the former work, that the sub j e c t should be approached from a more fundamental point of view and, with t h i s i n mind, an i n t e n s i v e review of the sub j e c t of chemotherapeutics was c a r r i e d out. The r e s u l t s of t h i s review only serve to con-f i r m the previous p o s t u l a t i o n s made wit h r e f e r e n c e to the 4 mechanism of>true, or perhaps one should say t h e o r e t i c a l , selenium p o i s o n i n g . U n f o r t u n a t e l y , however, i n view of some of the f i n d i n g s of t h i s present i n v e s t i g a t i o n i t i s not at a l l c e r t a i n t h a t chemotherapeutic measures of the "BAL n type, which should t h e o r e t i c a l l y be of b e n e f i t i n cases o f selenium p o i s o n i n g t w o u l d be o f value i n the case of animals s u f f e r i n g from the c o n d i t i o n concomitant w i t h the presence of selenium b e a r i n g m a t e r i a l s i n the d i e t . Thus the work o f f e r e d here i s a c o n s i d e r a b l y m o d i f i e d p r e s e n t a t i o n of the i n v e s t i g a t i o n s o r i g i n a l l y planned. I t i s f e l t , however, t h a t the e l i m i n a t i o n of many of the d i f f i -c u l t i e s f o r m e r l y impeding progress i n the f i e l d of selenium t o x i c o l o g y has alone made the p r o j e c t w e l l worth w h i l e . In a d d i t i o n the evolvement of new techniques and the r e s u l t s of t h e i r a p p l i c a t i o n have served t o add new knowledge to the problem. 5 I. CHRONIC SELENIUM TOXICITY  with p a r t i c u l a r r e f e r e n c e to the i n d u c t i o n  of a c o n d i t i o n of c h r o n i c selenium po i s o n - ing i n l a b o r a t o r y animals ( i ) A Summary of Previous Work and P r e l i m i n a r y Experimenta- t i o n The i n d u c t i o n of a s t a t e of c h r o n i c metal p o i s o n i n g may be accomplished by s e v e r a l means which have become standard procedures of t o x i c o l o g y . (a) I n g e s t i o n of food i n t o which t o x i c m a t e r i a l has been i n c o r p o r a t e d (Moxon, 1937 )(Smith, 1939 ) . (b) I m b i b i t i o n of a water s o l u t i o n of the t o x i c m a t e r i a l (Smith et a l . , 1940). (c) I n h a l a t i o n of v o l a t i l i z e d t o x i c m a t e r i a l (Chem. Warfare R e p o r t s ) . (d) I n j e c t i o n of t o x i c m a t e r i a l subcutaneously ( i n a s u i t a b l e vehicle)(Cameron, 1947 ) . An attempt was made to induce c h r o n i c selenium p o i s o n i n g i n White Wistar r a t s , by means of the poisonous food i n g e s t i o n method, i n a s e r i e s of e a r l i e r i n v e s t i g a t i o n s c a r r i e d out i n t h i s l a b o r a t o r y ( D e r r i c k , 1948) . The animals were o f f e r e d s e l e n i z e d food of two types: (a) A n u t r i t i o n a l l y adequate d i e t c o n t a i n i n g n a t u r a l l y s e l e n i f e r o u s wheat and having a f i n a l c o n c e n t r a t i o n l e v e l of 12 p.p.m. selenium. (b) A stock r a t i o n d i e t of P u r i n a fox chow t r e a t e d w i t h sodium s e l e n i t e and having a f i n a l c o n c e n t r a t i o n l e v e l of 20 p.p.m. selenium. The two groups (male and female r a t s , one month old) given the s e l e n i f e r o u s wheat d i e t r e f u s e d to i n g e s t more than an extremely s m a l l q u a n t i t y of the food. The experiment was 6 abandoned when i t became obvioua t h a t a s t a t e of s t a r v a t i o n was being superimposed on the selenium t o x i c i t y . S i m i l a r groupings of r a t s given the s e l e n i z e d stock r a t i o n consumed the food at a s l i g h t l y more s a t i s f a c t o r y l e v e l and were maintained on the d i e t (the selenium content of which was r a i s e d to 40 p.p.m. a f t e r two weeks) u n t i l they were i n a c o n d i t i o n d e s c r i b e d by Moxon (1937) and by Smith, L i l l i e , Stohlman and W e s t f a l l (1940) as being t y p i c a l of sub-acute selenium p o i s o n i n g . T h i s c o n d i t i o n was reached i n about JO days. However, at no time d u r i n g the i n v e s t i g a t i o n was the food i n t a k e of these animals s u f f i c i e n t l y high to s a t i s f y the b i o e n e r g e t i c requirements of maintenance and growth. I t was t h e r e f o r e obvious that the question of super-i m p o s i t i o n of extraneous symptoms of s t a r v a t i o n on those o r i g i n a t i n g from selenium p o i s o n i n g would have to be s o l v e d before the problems of the mechanism of selenium p o i s o n i n g and i t s treatment could be approached. Smith et a l . (1940) r e p o r t comparison of the m o r t a l i t y r a t e of a group of r a t s from an i n b r e d colony of the White Wistar s t r a i n , r e c e i v i n g 15 parts per m i l l i o n selenium,as sodium s e l e n i t e , i n the d r i n k i n g water,with that of a s i m i l a r group of r a t s r e c e i v i n g 16 p a r t s per m i l l i o n selenium as i t occurs n a t u r a l l y i n wheat. Continuous records of the water and food consumption i n d i c a t e d t hat the l e v e l of selenium 7 i n t a k e i n the two sets of animals was very n e a r l y the same, namely from 1 to 1.5 mgm. of selenium per k i l o g r a m of body weight per day f o r animals over a weight range of from 70 to 104 grams. At such a l e v e l o f i n t a k e a 100 gram r a t on the water experiment consumed from 6.8 to 10 cc. of water per day, r e p r e s e n t i n g an a c t u a l consumption of 100 to 150 gamma of selenium. Animals r e c e i v i n g t h i s l e v e l of selenium showed 100/. m o r t a l i t y i n 80 days and 50% m o r t a l i t y i n 50 days. In an i n i t i a l attempt to reproduce the work of Smith et a l , as a means of i n d u c i n g c h r o n i c s e l e n o s i s i n r a t s , p r e p a r a t o r y to the use of v a r i o u s t h e r a p e u t i c s i n t h i s l a b o r a -t o r y , the f o l l o w i n g o b s e r v a t i o n s were made. Two groups of White Wistar r a t s from an i n b r e d colony, 6 females and 6 males, of an age (39 days old) and weight range (79 to 111 grams) s i m i l a r to the animals used by Smith et a l . (1940) were o f f e r e d 15 p a r t s per m i l l i o n s e l e n -ium as sodium s e l e n i t e i n t h e i r d r i n k i n g water. During the f i r s t 24 hours of the experiment the animals consumed from 4 to 6 cc. of s e l e n i z e d water per r a t while a s i m i l a r group of female c o n t r o l r a t s consumed 19 c c . of water per r a t . In the succeeding t e n day p e r i o d the female experimental r a t s consumed an average of 4.0 c c . of s e l e n i z e d water per r a t per day, the male experimental r a t s consumed an average of 6.1 cc. of s e l e n i z e d water per r a t per day and the female c o n t r o l r a t s 8 consumed an average of 20 . 8 cc. of water per r a t per day. During t h i s p e r i o d the experimental r a t s l o s t an average of from 10 to 1? grams body weight per r a t (female and male r e s p e c t i v e l y ) while the c o n t r o l animals gained 39.4 grams body weight per r a t . The average food consumption was from 4.4 grams ( f e -males) to 6.9 grams (males) per r a t per day f o r the e x p e r i -mental r a t s as compared to a consumption of 13 grams per r a t per day f o r the female c o n t r o l animals. I t became i n c r e a s i n g l y obvious, dur i n g the course of the ten day o b s e r v a t i o n p e r i o d , t h a t the experimental r a t s were f a i l i n g to consume water at an adequate l e v e l as adjudged by the water i n t a k e of the l i t t e r - m a t e c o n t r o l s . When the experimental animals were o f f e r e d selenium f r e e water t h e i r ' consumption re t u r n e d t o a normal l e v e l f o l -lowing an i n i t i a l abnormally l a r g e consumption. The p r e l i m i n a r y experiment i n i t s o r i g i n a l form was t h e r e f o r e terminated s i n c e i t appeared q u i t e evident t h a t the e f f e c t s of water d e p r i v a t i o n were being superimposed on those of the t o x i c metal. F o l l o w i n g a s i x day r e c o v e r y p e r i o d , d u r i n g which water and feed consumption f i g u r e s and weight gains of the experimental r a t s c l o s e l y approximated those of the c o n t r o l animals f o r the p r e c e d i n g week, they were again s u b j e c t e d to experimental procedure. The animals were o f f e r e d s e l e n i z e d water But the l e v e l of selenium was g r a d u a l l y i n c r e a s e d over a 21 day p e r i o d from an i n i t i a l c o n c e n t r a t i o n of 1.5 p a r t s per m i l l i o n Na 2Se0j ( i . e . 0.69 p.p.m. Se) to a f i n a l c o n c e n t r a t i o n of 11 p.p.m. _JJ a 2 S e O j ( i . e . 5.0 p.p.m. S e ) . In a d d i t i o n a l l d r i n k i n g water b o t t l e s were s c a l d e d d a i l y t o r i d them of an unpleasant odor concomitant with standing s e l e n i z e d water and the d i l u t i o n s were made f r e s h d a i l y . The animals were maintained at t h i s f i n a l c o n c e n t r a t i o n of selenium s i n c e i t appeared t o be the high e s t l e v e l at which the element could be ad m i n i s t e r e d without imposing other d e f i c i e n c y symptoms on the selenium t o x i c i t y p i c t u r e . A f t e r three weeks on the 11 p.p.m. Na2Se0j.concentra-t i o n the water consumption of the experimental animals was from 14 to 16 cc. per r a t per day i n the female and male groups r e s p e c t i v e l y as compared to 24 c c . per r a t per day i n the female c o n t r o l group. Food consumption i n experimental groups was 10.2 to 12 grams per r a t per day as compared to 13 grams per r a t per day i n the c o n t r o l group. Weight gains over a 7 day p e r i o d were 4.4 to 5 gms. per r a t i n the female and male experimental groups r e s p e c t i v e l y and i n the female c o n t r o l group 4.8 grams per r a t . The occurrence of a death i n the female experimental group at 8 days and i n the male experimental group at 12 days a f t e r r e a c h i n g the maximum c o n c e n t r a t i o n l e v e l of 5 p.p.m. 10 selenium i n the d r i n k i n g water, followed by other deaths i n the male group at 27 days and at 39 days suggested the p o s s i -b i l i t y t h a t selenium might w e l l be t o x i c at a d a i l y intake l e v e l c o n s i d e r a b l y lower than the 0.3 mgm. per k i l o g r a m of body weight per day given by Smith et a l . The marked s i m i l a r -i t y between the si g n s observed i n the s i c k animals and the d e s c r i p t i o n s g i v e n of selenium p o i s o n i n g i n the r a t by Smith et a l . (1940), Moxon (1937 )' and other i n v e s t i g a t o r s a l s o served to emphasize t h i s p o s s i b i l i t y . Since the d e f i n i t e establishment of the l e v e l of selenium i n t a k e r e q u i r e d to induce a c h r o n i c t o x i c c o n d i t i o n appeared to be necessary f o r a more comprehensive concept of the selenium i n t o x i c a t i o n p i c t u r e a l a r g e s c a l e experiment was set up using the r e s u l t s obtained i n the above mentioned p i l o t experiment as a guide. T h i s was done not o n l y that t h i s problem might be solved but a l s o that a c o n s i d e r a b l e number of c h r o n i c a l l y poisoned r a t s might l a t e r be a v a i l a b l e f o r experimental t h e r a p e u t i c s . An o u t l i n e of the experiment f o l l o w s : ( i i ) Experiments i n the I n d u c t i o n of Chronic Selenium  T o x i c i t y i n the White Rat* Experiment A Procedure F i f t y female and f o r t y - t h r e e male White W i s t a r r a t s aged 30 days and weighing an average of 76.1 and 87.6 grams r e s p e c t i v e l y , were p l a c e d i n wire cages i n groups of 3 or 6 r a t s per cage. 11 The animals were given a stock r a t i o n d i e t of P u r i n a Fox Chow and s e l e n i z e d water. The c o n c e n t r a t i o n of selenium was g r a d u a l l y i n c r e a s e d from an i n i t i a l 0.69 pa r t s per m i l -l i o n selenium ( i . e . 1.5 p.p.m. Na 2Se0j) to 5.0 p a r t s per m i l -l i o n selenium (11 p.p.m. Na 2Se0j) over a p e r i o d of 9 days. S i g h t male and e i g h t female l i t t e r - m a t e s of the experimental animals were r e t a i n e d as c o n t r o l animals. With the exception t h a t they r e c e i v e d normal water i n place of s e l e n i z e d water these c o n t r o l r a t s were maintained under s i m i l a r c o n d i t i o n s t o those of the experimental r a t s . Complete records of the d a i l y water and food i n t a k e and the weekly weight changes of a l l animals were maintained f o r the d u r a t i o n of the experiment. A f t e r 8 weeks i t was found p o s s i b l e to r a i s e the selenium c o n c e n t r a t i o n o f the d r i n k i n g water s l i g h t l y higher to a l e v e l of 6.9 p.p.m. selenium ( i . e . 15 p.p.m. Na 2Se0j) without any l a r g e r e d u c t i o n of water consumption becoming apparent. However, h i g h e r l e v e l s i n a " t e s t case" were r e s i s t e d by the f a m i l i a r method of c u t t i n g down water i n t a k e . At 11 weeks a f t e r the i n c e p t i o n o f the experiment, when i t had become obvious t h a t a c o n s i d e r a b l y longer time would be necessary to induce a ch r o n i c s t a t e of s e l e n o s i s i n the animals, the number of experimental r a t s was reduced by h a l f . The animals were used f o r f u r t h e r L . D . ^ Q 3 1 d a t a . ^Appendix I I I . 1 2 At 1? weeks the number of experimental animals was again halved w i t h s i m i l a r u t i l i z a t i o n of the r a t s removed. The remainder of the animals were c a r r i e d on u n t i l the 26th week a f t e r the i n c e p t i o n of the experiment at which time they were s a c r i f i c e d and post mortem examinations were c a r r i e d out. R e s u l t s The f i n d i n g s w i t h r e f e r e n c e to the average weight changes and the average water and food i n t a k e per k i l o g r a m of body weight are presented i n t a b u l a r form i n Appendix I. and i n graphic form on pages 1J 16 . The graphs are s e l f -e xplanatory and w i l l be d e a l t w i t h under the d i s c u s s i o n of r e s u l t s . In summary i t may be s t a t e d that selenium a d m i n i s t e r e d i n the d r i n k i n g water at the l e v e l s i n d i c a t e d does not appear to be dangerously t o x i c t o White Wistar r a t s even when the p e r i o d of a d m i n i s t r a t i o n i s extended to s i x months. In a d d i t i o n i t would a l s o appear t h a t the experimental animals l i m i t t h e i r i n t a k e " o f water to allow a more or l e s s constant, s u b - t o x i c intake of selenium. There i s no doubt that the s e l e n i z e d d r i n k i n g water method of i n d u c i n g selenium i n t o x i c a t i o n i n r a t s i s h i g h l y u n s a t i s f a c t o r y . A D i s c u s s i o n of the Attempts to Produce a S t a t e of Chronic  Selenium P o i s o n i n g by the A d m i n i s t r a t i o n of Selenium i n  the D i e t The o r i g i n a l purpose of t h i s experiment was to o b t a i n a l a r g e number of c h r o n i c a l l y selenium poisoned r a t s f o r the 17 demonstration of the e f f e c t i v e n e s s of v a r i o u s t h e r a p e u t i c measures. These measures, as may be a n t i c i p a t e d from the d i s c u s s i o n s presented elsewhere i n t h i s paper, were to be chemotherapeutic i n nature. In a d d i t i o n to o f f e r i n g a u s e f u l means of t r e a t i n g the t o x i c c o n d i t i o n the t h e r a p i e s would, by t h e i r e f f e c t i v e n e s s , c o nfirm or deny the present t h e o r y of the mechanism of selenium p o i s o n i n g . The deaths observed i n the p i l o t experiment r e s u l t e d i n the erroneous c o n c l u s i o n that selenium administered i n the d r i n k i n g water was a s a t i s f a c t o r y means of i n d u c i n g the c h r o n i c selenium poisoned s t a t e . In view of the remarkable p a r a l l e l observed between the symptomologies of the e x p e r i -mental r a t s i n the p i l o t experiment and the animals d e s c r i b e d by Smith et a l . (1940) such a c o n c l u s i o n was e n t i r e l y v a l i d . To r e i t e r a t e these symptoms b r i e f l y , they are as follows*, s i g n i f i c a n t l o s s e s i n weight, i n t e r m i t t e n t a n o r e x i a f o l l o w e d by a s c i t e s , p l e u r a l e f f u s i o n , l i v e r damage and more or l e s s severe anemia. These symptoms have been observed i n experiments i n the present study under both f e e d i n g and d r i n k i n g types of selenium a d m i n i s t r a t i o n . However, i t must a l s o be mentioned that they have not occurred u n l e s s accompanied by the inanition and/or water d e p r i v a t i o n which occur a u t o m a t i c a l l y when the r a t i s o f f e r e d water or food c o n t a i n i n g selenium at the l e v e l s of c o n c e n t r a t i o n c i t e d by Smith et a l . (1940). 18 O b s e r v a t i o n of the symptoms of the experimental animals i n the c o n d i t i o n induced by the experimental methods of Smith et a l . (1940), Moxon (1937),and t h i s l a b o r a t o r y i n the present experiments;show them t o bear a marked resemb-lance to the g e n e r a l e f f e c t s of i n a n i t i o n or c h r o n i c p a r t i a l s t a r v a t i o n . For example, l o s s of weight and s t u n t i n g may be a t t r i b u t e d not o n l y to chronic selenium p o i s o n i n g but a l s o to c h r o n i c p a r t i a l s t a r v a t i o n and dehydration; anemia may a l s o be a secondary r e s u l t of i n a n i t i o n , c h r o n i c p o i s o n i n g or a u t o i n t o x i c a t i o n ; p l e u r a l e f f u s i o n and a s c i t e s may w e l l be a consequence of the d e p l e t i o n of plasma p r o t e i n concomitant with c h r o n i c p a r t i a l s t a r v a t i o n and r e s u l t a n t changes i n osmotic p r e s s u r e . L i v e r damage and l o s s of i n t e r n a l f a t may a d d i t i o n a l l y be c o r r e l a t e d to the changes i n metabolism accompanying c h r o n i c p a r t i a l s t a r v a t i o n and/or d e h y d r a t i o n . In e a r l i e r s t u d i e s of the selenium problem ( D e r r i c k , 1948) the hypothesis was put forward t h a t the r o l e of selenium i s t h a t of an enzyme i n h i b i t o r y agent. The en-zymes systems i n h i b i t e d are b e l i e v e d t o be mainly those concerned with the carbohydrate metabolism energy c y c l e . I t may w e l l be argued t h a t the a n o r e x i a , and consequent i n a n i t i o n ob-served i n the animals subjected to selenium p o i s o n i n g "per-os" are simply a r e s u l t of the law of mass a c t i o n e x e r t -in g i t s e f f e c t as a r e s u l t of the accumulation of products at the p a r t i c u l a r l y a f f e c t e d point or p o i n t s i n the m e t a b o l i c 1? c y c l e . However, t h i s argument i s r e f u t e d by the response of the s o - c a l l e d " s e l e n i z e d a n i m a l s n when o f f e r e d food or water which does not c o n t a i n selenium at a time when t h e i r r e f u s a l to consume selenium t r e a t e d feed or water i s both obvious and c o n s i s t e n t . O f f e r e d as a p a r t i c u l a r l y good example i s the response of the " c o n t r o l s e l e n i z e d animals" to normal d i e t i n an e a r l i e r s e r i e s of experiments conducted by the author ( D e r r i c k , 1948). A graphic comparison of the changes i n body weight observed i n the animals of the p i l o t experiment d u r i n g the i n i t i a l t e n day p e r i o d of 15 p.p.m. selenium i n water admini-s t r a t i o n , and i n c o n t r o l groups of animals maintained f o r a s i m i l a r l e n g t h of time on n o n - s e l e n i z e d water and food i n amounts r e s t r i c t e d to the l e v e l of the experimental animals, i s g i v e n on page 20. The evidence t h a t the experimental animals were indeed the v i c t i m s of i n a n i t i o n and dehydration as w e l l as of s e l e n i -um p o i s o n i n g i s c o n v i n c i n g l y p ortrayed i n t h i s graph. There i s every reason to b e l i e v e t h a t , c a r r i e d f u r t h e r , the c o n t r o l experiment would have r e s u l t e d i n the p r o d u c t i o n of the extreme symptoms of m a l n u t r i t i o n . The s i m i l a r response of both groups of animals to the removal of the r e s p e c t i v e types of r e s t r i c t i o n s on food and water i n t a k e serves as a d d i t i o n a l evidence that the anorexia and consequent i n a n i t i o n of the experimental animals i s , at 21 l e a s t i n p a r t , the r e s u l t of the presenoe of selenium i n the d i e t r a t h e r than the r e s u l t of selenium p o i s o n i n g . A comparison of the d a i l y c a l o r i c i n take of the c o n t r o l (normal) r a t s with that of the experimental r a t s i n the p i l o t experiment f u r t h e r serves to emphasize that these animals, fed on d i e t s i n t o which selenium has been i n c o r p o r a t e d e i t h e r i n the food or i n the water at doses above the t o l e r a n c e l e v e l , s u f f e r from extreme m a l n u t r i t i o n i n a d d i t i o n t o selenium p o i s o n i n g . For example, under the c o n d i t i o n s of the a d m i n i s t r a t i o n of 15 p.p.m. selenium i n the d r i n k i n g water i n the p i l o t experiment an experimental 100 gram female animal would have a d a i l y c a l o r i c i n t a k e of 16.6 c a l o r i e s as com-pared to an int a k e of 37.4 c a l o r i e s d a i l y by a 100 gram con-t r o l female r a t ( c a l c u l a t e d on the b a s i s o f 75$ d i g e s t i b i l i t y and 4 c a l o r i e s per gram of t o t a l d i g e s t i b l e n u t r i e n t s (T.D.N.) (Brody,(1945)). When i t i s considered that Hays et a l . (1947) demonstrated t h a t normal r a t s p l a c e d on 50$ of normal c a l o r i c i n t ake showed a 50/» r e d u c t i o n i n body weight i n 6 weeks i t i 3 not s u r p r i s i n g t h a t the animals subjected to a d i e t c o n t a i n i n g selenium ( e i t h e r i n the food or the water), at l e v e l s r e s u l t -i n g i n v o l u n t a r y r e s t r i c t i o n of t h e i r c a l o r i c i n t a k e to one-h a l f of the normal i n t a k e , should e x h i b i t the l a c k of growth, l o s s of body weight and other symptoms of i n a n i t i o n u s u a l l y r e f e r r e d to as a part of the selenium i n t o x i c a t i o n p i c t u r e . With the f o r e g o i n g thoughts i n mind i t i s of p a r t i c u l a r i n t e r e s t t o note the statements made by Moxon (1937)(194?) and Smith and L i l l i e (1940) with r e f e r e n c e to the much gr e a t e r s u s c e p t i b i l i t y of young (4 to 3 weeks old) r a t s to selenium p o i s o n i n g . Moxon (1937) s t a t e s t h a t r a t s placed on s e l e n i f e r o u s d i e t s at 21 days of age l o s t weight and were a l l dead i n 20 days while most of the animals not r e c e i v i n g s e l e n i f e r o u s d i e t s u n t i l they were over 42 days of age l i v e d f o r more than 200 days. An examination of the work of Harte, Travers and S a r i c h (1948) r e v e a l s that i n r a t s the f o u r t h and f i f t h weeks of l i f e mark a maximum c a l o r i c i n take of from 21 to 23 c a l -o r i e s per square decimeter and t h a t from t h i s p e r i o d u n t i l the t w e l f t h week there i s a g r a d u a l d e c l i n e to a constant value of 12.3 to 13 c a l o r i e s per square decimeter. S i m i l a r l y Brody (1943) s t a t e s t h a t the f i f t h week of l i f e i s the p e r i o d of g r e a t e s t energy metabolism per u n i t of body s u r f a c e i n the r a t . The c o n c l u s i o n s which may be drawn from these observa-t i o n s and the f a c t t h a t young r a t s , when placed on a s e l e n i -ferous d i e t having a selenium content above t o l e r a n c e l e v e l * cut down t h e i r c a l o r i c i n t a k e by h a l f , (Moxon, 1937; 1943), are obvious. F u r t h e r , a p e r u s a l of the a v a i l a b l e l i t e r a t u r e on selenium p o i s o n i n g r e v e a l s t h a t experimentation i n v o l v i n g the The " t o l e r a n c e l e v e l " appears to vary with age; i n l i g h t of present work i t would appear to be between 5 and 6.9 p.p.m. f o r the r a t . 23 use of mature l a b o r a t o r y animals seldom r e s u l t s i n the pro-d u c t i o n of s p e c i f i c s i g n s and symptoms t h a t might be c o n s i -dered pathognomonic of selenium p o i s o n i n g even under c o n d i -t i o n s of extremely long term d i e t a r y a d m i n i s t r a t i o n . Such f a c t s are considered i n d i c a t i v e of the complete inadequacy of the d i e t a r y methods of selenium a d m i n i s t r a t i o n (Moxon, 1937)(Smith, 1940) as a means of i n d u c i n g c h r o n i c selenium p o i s o n i n g . In a l l f a i r n e s s i t must be admitted t h a t the p o s s i -b i l i t y of a low c a l o r i c i n t ake e x e r t i n g some i n f l u e n c e on the symptomatology of c h r o n i c selenium t o x i c i t y d i d not escape Moxon (Moxon, 194-3) • However, i t i s tempting to go much f u r t h e r and t o suggest that the chronic c o n d i t i o n induced by the use of such methods as the above i s not selenium p o i s o n i n g at a l l . Rather i t i s more probably a s t a t e of i n a n i t i o n and/or dehydration with, p o s s i b l y , a d d i t i o n a l com-p l i c a t i o n s r e s u l t i n g from a continuous i n g e s t i o n of a low l e v e l of selenium i n the d i e t . Such symptoms as a s c i t e s , and some of the p a t h o l o g i c a l changes o c c u r r i n g i n the l i v e r , are f a r more probably a r e s u l t of the selenium p o i s o n i n g than of the i n a n i t i o n . On the other hand, how much l e s s s e r i o u s might these, and other, symptoms be i n the case of a w e l l nourished animal r e c e i v i n g a s i m i l a r q u a n t i t y of selenium i n the d i e t . For example, i t i s w e l l known that the symptom of p r o g r e s s i v e anemia, so c h a r a c t e r i s t i c of c h r o n i c selenium po i s o n i n g in.young r a t s , i s absent i n the case of selenium 24 poisoned c a t s . Smith and h i s co-workers (1940) a t t r i b u t e the d i f f e r e n c e as probably due to the selenium to p r o t e i n r a t i o of the d i e t . While the w r i t e r concurs with t h i s view i t i s f e l t that these i n v e s t i g a t i o n s might have gone much f u r t h e r and suggested t h a t the d i f f e r e n c e i s probably due to the h i g h b i o l o g i c a l value of the meat p r o t e i n of the cat d i e t i n r e l a t i o n to the blood supply. With these f a c t s t o hand i t at l e a s t must be admitted that animals i n such a c o n d i t i o n are e n t i r e l y u n s u i t e d f o r s t u d i e s i n v o l v i n g the t o x i c mechanism of selenium p o i s o n i n g or i t s treatment by chemotherapeutic means. The r e s u l t s of the long term experiment (Experiment A) i n v o l v i n g the a d m i n i s t r a t i o n of selenium at l e v e l s which would not r e s u l t i n the simultaneous i n d u c t i o n of a s t a t e of c h r o n i c m a l n u t r i t i o n are of such a nature (see graphs, p J 3 - l 6 ) (Appendix I. ) as to r e q u i r e very l i t t l e d i s c u s s i o n . I t may be observed that while the water i n t a k e of the experimental r a t s , even at 5 and 6 . 9 p.p.m. Se c o n c e n t r a t i o n s , was con-s i s t e n t l y lower than that of the c o n t r o l animals the food i n t a k e and weight i n c r e a s e s of both groups compare f a v o r a b l y throughout the experiment. T h i s occurred i n c o n j u n c t i o n with an average d a i l y i n t a k e of 0.465 mgm. of selenium per k i l o -gram f o r female r a t s and 0 . 5 3 2 mgm. of selenium per k i l o g r a m f o r male r a t s during the e n t i r e 1 8 week experimental p e r i o d . These r e s u l t s amply confirm e a r l i e r o b s e r v a t i o n s by Smith HNote: In the case of two groups of 6 r a t s of each sex the p e r i o d was extended to 2 5 weeks. 2 5 et a l . (1940) that doses of selenium up to 0.5 mgm. per k i l o -gram may be t o l e r a t e d f o r long p e r i o d s without causing s e r i o u s symptoms or pronounced t i s s u e damage. The r e s u l t s of autopsies on some of the animals (Appendix IV.) a l s o c o nfirm Smith's o b s e r v a t i o n with r e f e r e n c e to the p o s s i b i l i t y t h a t the a d m i n i s t r a t i o n of t h i s l e v e l of selenium i n the d i e t , and even lower l e v e l s , may cause minor symptoms of systemic p o i s o n i n g . Whether these symptoms are t r u l y the r e s u l t of the presence of selenium p o i s o n i n g at very low l e v e l s or are the r e s u l t of a s l i g h t l y d i s t u r b e d metabolism due t o a c o n s i s t e n t l y lower water intake cannot be confirmed or denied. I t i s f e l t , however, on the b a s i s of t h i s work and the e a r l i e r d i s c u s s i o n of the probable reasons f o r the " t o x i c i t y " of selenium to l a b o r a t o r y animals at higher l e v e l s t h a t Smith's c o n c l u s i o n s with r e f e r e n c e to the probable safe l i m i t s of selenium intake (Smith, 1940) must be m o d i f i e d . Since the d a i l y i n take of amounts of selenium of 1 mg. per ki l o g r a m or more, the s o - c a l l e d "danger l e v e l " of Smith, i n v o l v e s the presence of at l e a s t 15 p.p.m. selenium i n the d i e t which i n t u r n would appear to i n v o l v e a u t o m a t i c a l l y the i n d u c t i o n of a s t a t e of i n a n i t i o n and d ehydration i n the t e s t animal i t i s obvious that no d e f i n i t e danger l e v e l of intake can be c i t e d on t h i s b a s i s . The c a l o r i c i n t a k e of the e x p e r i -mental r a t s of Experiment A ( i . e . of r a t s with an i n t a k e of 0.5 mg. of selenium (or l e s s ) per k i l o g r a m per day) i s 26 s u f f i c i e n t l y h i g h to al l o w f o r growth as w e l l as maintenance which accounts f o r the a p p a r e n t l y innocuous e f f e c t s of t h i s dose l e v e l . The c a l o r i c i n t a k e of r a t s subjected to the presence of 15 p.p.m. or more selenium i n the d i e t ( i . e . , an intake of 1.0 mg. or more selenium per ki l o g r a m per day) i s s u f f i c i e n t only f o r maintenance, (Brody, 1945) s i n c e t h e i r a c t u a l i n t a k e i s s l i g h t l y l e s s than one-half that of t h e i r c o n t r o l c o u n t e r p a r t s . U n t i l such time as a more s u i t a b l e means of a d m i n i s t e r i n g selenium i n the d i e t of experimental animals can be evolved, which e l i m i n a t e s the co m p l i c a t i o n s concomitant with l a c k of p a l a t a b i l i t y and r e s u l t a n t v o l u n t a r y r e s t r i c t i o n of the c a l o r i c i n t a k e t o the danger p o i n t of i n a n i t i o n , there w i l l be no r e l i a b l e i n f o r m a t i o n on the symptomatology of selenium p o i s o n i n g , i t s mechanism i n the l i v i n g animal, the danger l e v e l of i n t a k e , or means of therapy. It was with these thoughts i n mind that the w r i t e r abandoned t h i s l i n e of i n v e s t i g a t i o n of the selenium problem and embarked on a study of the t h i r d means of i n d u c i n g the c h r o n i c s t a t e of t o x i c i t y , namely by the i n j e o t i o n r o u t e . 27 ( i i i ) Experiments i n the I n d u c t i o n of a C o n d i t i o n of  Selenium T o x i c i t y i n the White Rat S u i t a b l e f o r  the purpose of A s s e s s i n g the E f f e c t i v e n e s s of  Chemotherapy. Experiment B Experiments i n the i n d u c t i o n of c h r o n i c selenium i n t o x i c a t i o n by use of the i n j e c t i o n route had been c a r r i e d out by Cameron i n 1 9 4 0 (Cameron, 1 9 4 7 ) and were a v a i l a b l e p r i o r to the commencement of the present work. In view of the statements made i n previous d i s c u s s i o n s i n t h i s paper, i t i s f e l t t h a t some of the o b s e r v a t i o n s of Cameron bear r e p e t i -t i o n . T h i s i n v e s t i g a t o r determined the acute L.D. v i a the po subcutaneous i n j e c t i o n route as being 3.5 mg. Se/Kg. body weight f o r h i s s t r a i n of white r a t s . He induced a s t a t e of chronic t o x i c i t y by means of subcutaneous i n j e c t i o n s of a 0 . 1 percent aqueous s o l u t i o n of sodium s e l e n i t e t o mature young r a t s , twice weekly, over a 1 4 0 day p e r i o d . The i n -j e c t i o n s were g r a d u a l l y i n c r e a s e d from an i n i t i a l dosage of 1.6 mg. Se/Kg. body weight to f i n a l dosages of 1 2 mg. Se/Kg. body weight. Hence i t i s evident t h a t the animals r e c e i v i n g bi-weekly doses were s u r v i v i n g on dosage l e v e l s approximately three times as high as the acute L . D . ^ Q . During the e n t i r e experimental p e r i o d the r a t s ate v o r a c i o u s l y although t h e i r body weight descended to a much lower l e v e l (128 grams) than "the i n i t i a l l e v e l " ( 1 8 5 grams). Of the o r i g i n a l e ighteen animals twelve s u r v i v e d to the end of the experimental p e r i o d . H a l f of these s u r v i v o r s were allowed to l i v e f o r 60 days 28 f o l l o w i n g the experimental p e r i o d and, with the c e s s a t i o n of i n j e c t i o n s , they recovered and gained s t e a d i l y i n weight (190 grams). Cameron f u r t h e r r e p o r t s t h a t i n view of the enormous a p p e t i t e of the i n j e c t e d animals l o s s of weight must be a t t r i b u t e d to some metabolic d i s t u r b a n c e r a t h e r than t o s t a r v a t i o n . In a d d i t i o n many of the symptoms commonly a s s o c i a t e d with selenium p o i s o n i n g as i t i s observed i n v a r y i n g degrees i n the " a l k a l i d i s e a s e " of animals i n s e l e n i -ferous areas were d e s c r i b e d . S k i n d i s t u r b a n c e s with dryness, s c a l i n e s s and f a l l i n g out of h a i r were prominent; l a r g e patches of s k i n became b a l d as the i n t o x i c a t i o n developed and a y e l l o w i s h brown pigmentation of such areas was noted. A s c i t e s did not develop but p l e u r a l e f f u s i o n occurred i n f a t a l cases. L i v e r damage was ex t e n s i v e and i s d i s c u s s e d f u l l y by the i n v e s t i g a t o r . While i t i s not proposed to enter i n t o a d i s c u s s i o n of the h i s t o l o g i c a l p i c t u r e of the l i v e r damage i t i s i n t e r e s t i n g to note that Cameron co n s i d e r e d t h a t the v a r i o u s f i n d i n g s are of a type d i f f e r i n g from t h a t u s u a l l y a s s o c i a t e d with i n a n i t i o n . In a d d i t i o n t h e r e are fundamental d i f f e r e n c e s between the l i v e r damage observed i n Cameron's experiment and the p a t h o l o g i c a l f i n d i n g s i n the f e e d i n g experi-ments of L i l l i e and Smith (1940), Smith et a l . (1937, 1940), Moxon (1943 l i t . rev.) and o t h e r s . I t i s p o s s i b l e that there i s a s e r i e s of changes r e s u l t i n g from the chemical a c t i o n of 29 the selenium s a l t and t h a t t h i s a c t i o n i s m o d i f i e d t o some extent by the route of a d m i n i s t r a t i o n and r e s u l t a n t d i s t r i b u -t i o n o f selenium i n the body t i s s u e s . The i n f o r m a t i o n given by Cameron was extremely u s e f u l as a guide t o s e t t i n g up experiments f o r the purpose of t e s t -i n g the e f f e c t i v e n e s s of v a r i o u s means of therapy and study-in g the mechanism of the p o i s o n i n g . In a d d i t i o n i t was f e l t t h a t the obser v a t i o n s he recorded served to confirm e a r l i e r work c a r r i e d out by the w r i t e r i n 1948.* The avoidance of the v o l u n t a r y r e s t r i c t i o n of c a l o r i c i n t a k e encountered i n f e e d -ing experiments was an obvious advantage. On the other hand the e x t e n s i v e l i v e r damage induced by t h i s method of selenium p o i s o n i n g was not a d e s i r a b l e f e a t u r e i n the case of animals to be s u b j e c t e d t o d i t h i o l (BAL) treatment. I t may w e l l be argued at t h i s p o i n t t h a t s i n c e Cameron had shown that animals recovered and gained weight when a d m i n i s t r a t i o n of selenium ceased, removal from the source of selenium i s o b v i o u s l y the most e f f e c t i v e means of th e r a p y . However, when i t i s considered that such c o n d i t i o n s as " b l i n d staggers" (Beath et a l . , 1934, 1935) and the more acute forms of selenium p o i s o n i n g r e s u l t i n the death of the a f f l i c t e d animal before such a therapy can be e f f e c t i v e the advantages of a treatment which w i l l cause the r a p i d m o b i l i z a t i o n and e x c r e t i o n of the t o x i c metal are apparent. Such acute and xNot with r e f e r e n c e t o the L.D,,.n» (See d i s c u s s i o n s , p. 42.) 3 0 sub-acute t o x i c c o n d i t i o n s a l s o a r i s e too q u i c k l y t o produce the type of l i v e r damage seen i n Cameron's experiments. I t must be admitted, however, t h a t Cameron's work was i n f l u e n t i a l i n the c o n f i r m a t i o n of the f e a r s expressed p r e v i o u s l y i n 1948 by the author, that BAL would be an i n e f f e c t i v e means of therapy where p o i s o n i n g had r e s u l t e d i n severe l i v e r damage. Thus, w h i l e the experimental procedures of Cameron were u s e f u l as a guide t o the development of the techniques u l t i m a t e l y employed i n t h i s work they could not be a p p r o p r i a t e d f o r use without c o n s i d e r a b l e m o d i f i c a t i o n . The c o n d i t i o n r e q u i r e d t o t e s t the e f f e c t i v e n e s s of t h i o l s i n m o b i l i z i n g selenium was one of a sub-acute nature i n which i t i s suspected t h a t selenium p o i s o n i n g i s e s s e n t i a l l y a problem of mass i n h i b i t i o n of the s u l f h y d r y l groups of c e r -t a i n enzyme systems. Thus the dosage r e q u i r e d to b r i n g about t h i s c o n d i t i o n must be on the verge of l e t h a l i t y but not so heavy as t o succeed i n producing the pulmonary m a n i f e s t a t i o n s which are a part of the acute, l e t h a l c o n d i t i o n . The e a r l i e r i n t r a p e r i t o n e a l determinations of the L.D.^ 0 of sodium s e l e n i t e i n the r a t ( D e r r i c k , 1948) were e s s e n t i a l f o r the establishment of the dosage l e v e l which would produce t h i s c o n d i t i o n by subcutaneous a d m i n i s t r a t i o n . However, i n order t o assure t h a t the low dose of selenium should not be e x c r e t e d too q u i c k l y and to permit an e f f e c t i v e combination with the s u l f h y d r y l groups of the v a r i o u s t i s s u e 3 1 and enzyme systems, i t was considered that the r e q u i r e d dosage should be i n j e c t e d i n combination with some type of a b s o r p t i o n d e l a y i n g v e h i c l e . I t was f e l t t h a t the use of such a v e h i c l e would permit the maintenance of e f f e c t i v e c o n c e n t r a t i o n s i n the blood over a longer p e r i o d of time. A 27» s o l u t i o n of aluminum-monostearate i n peanut o i l had been proved s u c c e s s f u l i n d e l a y i n g the a b s o r p t i o n and m a i n t a i n i n g the blood c o n c e n t r a t i o n of water s o l u b l e p e n i c i l l i n compounds by v a r i o u s i n v e s t i g a t o r s ( S u l l i v a n et a l . , 1948) and i t was considered, t h e r e f o r e , that i t would probably be s u i t a b l e as a means of accomplishing s i m i l a r e f f e c t s i n the case of sodium s e l e n i t e . Procedure Two groups of animals c o n s i s t i n g of 4 male r a t s and 4 female r a t s of s i m i l a r age (mature, young animals) were placed i n metabolism cages f o r a p e r i o d of 24 hours or more p r i o r to i n j e c t i o n . Urine was c o l l e c t e d and placed i n the r e f r i g e r a t o r f o r f u t u r e use as a c o n t r o l f o r the d e t e r m i n a t i o n of selenium e x c r e t i o n . The animals were weighed and marked f o r i d e n t i t y p r i o r t o the i n j e c t i o n . Sodium s e l e n i t e was i n j e c t e d i n q u a n t i t i e s concomitant with t h e i r body weight and the dosage l e v e l per k i l o g r a m . The i n j e c t i o n s were made as deeply subcutaneous as p o s s i b l e , i n the lumbar area, s i n c e i t i s d i f f i c u l t t o c a r r y out i n t r a m u s c u l a r i n j e c t i o n , i n the r a t , of the q u a n t i t y of 32 s o l u t i o n r e q u i r e d . The s o l u t i o n s f o r i n j e c t i o n were f r e s h l y prepared and were of the f o l l o w i n g t ypes: (a) An aqueous s o l u t i o n of a selenium concentra-t i o n which would a l l o w approximately 0.25 ml. of the s o l u t i o n f o r i n j e c t i o n i n t o a 200 gram r a t . (b) An aqueous s o l u t i o n of a selenium concentra-t i o n which, when mixed 1:1 with a 2% s o l u t i o n of aluminum-monostearate i n peanut o i l , would a l s o a l l o w approximately 0.25 ml, of the mix-tu r e f o r i n j e c t i o n i n t o a 200 gram r a t . (c) A 1:3 mixture of the aqueous selenium s o l u t i o n of (b) and 2% aluminum-monostearate to al l o w approximately 0.5 ml. of the mixture f o r i n -j e c t i o n of a 200 gram r a t . S t r i c t a s e p s i s was s u c c e s s f u l l y maintained. Following i n j e c t i o n the animals were placed i n the metabolism cages with feed and water a v a i l a b l e at a l l t i m e s . Urine c o l l e c t i o n s were made twice d a i l y and weight f l u c t u a t i o n s and v a r i o u s other o b s e r v a t i o n s were c a r e f u l l y recorded d u r i n g the e x p e r i -mental p e r i o d f o l l o w i n g i n j e c t i o n . A l l u r i n e s were kept i n the r e f r i g e r a t o r u n t i l they were analyzed f o r selenium. The maximum time these u r i n e s were kept was 3 days d u r i n g which p e r i o d l o s s e s of selenium by v o l a t i l i z a t i o n were proved by experimentation to be i n s i g n i f i c a n t . The metabolism cages were thoroughly washed and rinsed d a i l y d u r i n g the experimental p e r i o d . The i n d i v i d u a l experiments and t h e i r r e s u l t s f o l l o w . Expt. B-^  Since the L.D.^ of an aqueous s o l u t i o n of sodium s e l e n i t e f o r white male r a t s had been determined (Derrick, 1948) 33 as 14.3 mgm. Na 2SeOj/Kg. body weight (6.6 mg. Se/Kg.) by the i n t r a p e r i t o n e a l route i t was considered that a subcutaneous i n j e c t i o n of about 8 mg. Na 2Se0-j/Kg, (3.7 mg» Se/Kg.) as a mixture of aqueous s o l u t i o n and 2% aluminum-monostearate i n peanut o i l might serve to induce the more or l e s s sub-acute selenium t o x i c i t y i n r a t s as o u t l i n e d above. The mixture was prepared by e m u l s i f y i n g the aqueous s o l u t i o n with the aluminum-monostearate i n peanut o i l i n a Waring Blendor. Experience showed t h a t t h i s was most s u c c e s s f u l l y accomplished by c h i l l i n g the two s o l u t i o n s and the Blendor previous to the mixing p r o c e s s . The v a r i o u s amounts r e q u i r e d were c a l c u l a t e d : (a) At a dose l e v e l of 8 mg. Na 2Se0j/Kg. a 200 gram r a t r e q u i r e s 1.6 mgms. Na 2 S e 0 j . (b) Since the f i n a l amount f o r i n j e c t i o n i s r e q u i r e d to be approximately 1/4 ml. and the mixture of aqueous s o l u t i o n and " d e l a y i n g v e h i c l e " i s r e q u i r e d t o be 1:1 then 1.6 mgms. Na 2Se0j must be contained i n 1/8 ml. of aqueous s o l u t i o n . There-f o r e , 1.6 x 1 = 12.8 mgms./ml. = concen-178" t r a t i o n of aqueous s o l u t i o n . (c) 50 ml. of 12.8 mgms./ml. s o l u t i o n p l u s 50 ml. of 2$ Al-monostearate i n peanut o i l gives an e m u l s i f i e d mixture c o n t a i n -i n g 6.4 mgms. Na 2Se0j per ml. (d) 1/4 ml. of a 6.4 mgm. Na^eO^/ml. emuls-io n i s the q u a n t i t y s u f f i c i e n t f o r a 200 gram r a t . T h e r e f o r e , ( l / 4 x ^ )ml. i s q u a n t i t y s u f f i c i e n t f o r a r a t of weight x. 34 A group of 4 male r a t s aged 9 months, and another group of 4 s i m i l a r l y aged female r a t s were used i n t h i s p r e l i m i n a r y experiment. These animals were given subcutaneous i n j e c t i o n s and then su b j e c t e d t o the g e n e r a l experimental procedure o u t l i n e d p r e v i o u s l y f o r Experiment B. R e s u l t s W ithin 10 to 15 minutes a f t e r the i n j e c t i o n the usual symptomatology of selenium p o i s o n i n g by the i n j e c t i o n route was apparent (Moxon, 19 4 3 ) ( D e r r i c k , 1948)(Cameron, 1948). The male animals were very i l l f o r 3 days f o l l o w i n g the i n j e c t i o n and i n f a c t showed very l i t t l e a c t i v i t y even up to the f i f t h day. On t h i s day a l i t t l e green feed was eaten but no food p e l l e t s were consumed u n t i l the seventh day a f t e r i n j e c t i o n . The animals were k i l l e d on the e i g h t h day and a u t o p s i e d . No p a t h o l o g i c a l evidence was apparent. The female animals were a f f e c t e d much more s e v e r e l y by the s i m i l a r dose l e v e l . Two of them died w i t h i n 24 hours and a t h i r d animal was dead w i t h i n 48 hours. The remaining animal s u r v i v e d and e x h i b i t e d about the same r e c o v e r y progress as t h a t o u t l i n e d f o r the males. Weight Changes — Male Female Average wt. at i n j e c t i o n time 311 gms. 226 gms. Average wt. 3 days a f t e r i n j e c t i o n time 276 gms. Average wt. 7 days a f t e r i n j e c t i o n time 270 gms. U r i n a r y E x c r e t i o n of Selenium No r e l i a b l e f i g u r e s can be given f o r the u r i n a r y 35 e x c r e t i o n of selenium i n t h i s experiment s i n c e i n t e r f e r e n c e by copper and i r o n o r i g i n a t i n g from the metabolism cages i n use at t h i s time was r e s p o n s i b l e f o r a s e r i e s of f a l s e values,(See method, p, 5 9 ) These cages were r e p l a c e d by improved u n i t s of Monel metal and s t a i n l e s s s t e e l wire, f a b r i c a t e d u s i n g s i l v e r s o l d e r f o r a l l j o i n t s , Expt, B 9 Four male r a t s aged 9 months and a s i m i l a r l y aged group of 4 female r a t s were p l a c e d i n the Monel metal meta-b o l i s m cages and u r i n e c o l l e c t i o n s were made twice d a i l y over a p e r i o d of 3 days. These u r i n e s when sub j e c t e d to a n a l y s i s f o r selenium gave no evidence of i n t e r f e r e n c e with the t e s t by extraneous m e t a l l i c ions and were, t h e r e f o r e , r e t a i n e d f o r use as "reagent blanks" dur i n g the experimental p e r i o d . The male animals were given subcutaneous i n j e c t i o n s of 8 mgm, Na 2SeOj/Kg. body weight as p r e v i o u s l y d e s c r i b e d i n Expt, B]_, The female animals were given i n j e c t i o n s of 6 mgm. Na2Se0j/Kg. body weight. As before the i n j e c t i o n m a t e r i a l was a 1:1 e m u l s i f i e d mixture of aqueous sodium s e l e n i t e s o l u t i o n and Z% aluminum-monostearate i n peanut o i l . These animals were then s u b j e c t e d t o the g e n e r a l experimental procedure o u t l i n e d f o r Expt. B. R e s u l t s The u s u a l symptoms were observed about 10 minutes a f t e r i n j e c t i o n i n both groups. "Selenium breath" was n o t i c e a b l e and the animals were p r o s t r a t e and s u f f e r i n g from 36 r e s p i r a t o r y d i s t r e s s . Two of the male r a t s died w i t h i n 24 hours and the two s u r v i v o r s g r a d u a l l y recovered over a p e r i o d of 8 days. During t h i s time the animals were very i n a c t i v e and d o c i l e . T h e i r food i n t a k e was very s m a l l and they con-t i n u e d t o l o s e weight u n t i l a f t e r the 7th day. At the time they were k i l l e d , on the morning of the 10th day a f t e r i n j e o t i o n , they were j u s t commencing t o r e g a i n the l o s t weight. The female r a t s a l l s u r v i v e d and recovered i n a s i m i l a r man-ner to that of the males. Weight Changes Male Female Ave. wt. of) s u r v i v o r s )at i n j e c t i o n time 34-9 gms. 230 gms. " 11 3rd day a f t e r i n j e c t i o n time 331 gms. 210 gms. '» n 6th day a f t e r i n j e c t i o n time 322 gms. 206 gms. " " 10th day a f t e r i n j e c t i o n time 326 gms. 215 gms. Ur i n a r y E x c r e t i o n of Selenium The u r i n a r y e x c r e t i o n of selenium was c a l c u l a t e d as i n the f o l l o w i n g example. (a) The t o t a l u r i n e e x c r e t i o n over 24 hour p e r i o d . was 23 ml. b) A 10 ml. a l i q u o t was d i g e s t e d . c) The d i g e s t a t e waB d i l u t e d to 30 ml. and an a l i q u o t was taken f o r c o l o r development. (d) Galvanometer r e a d i n g = 74.5 which on the standard curve i s the e q u i v a l e n t of 6.7 gamma of selenium. T h e r e f o r e , one m i l l i l i t e r of u r i n e contained 6.7 gamma of selenium. (e) Since the o r i g i n a l 24 hour e x c r e t i o n was . 23 ml. then 23 x 6.7 - 154- gamma of selenium or 336 gamma Na2Se0j excreted i n 24 hours. (f ) T h e r e f o r e 536 x 1000 = 365 gamma of Na2Se05/<(, 922 ^ were excreted i n 24 hours, where 922 gms. was the o r i g i n a l combined wts. of the animals i n j e c t e d . KSee p. 52 f o r method. 37 T h i s system of c a l c u l a t i o n i s complicated by some in a c c u r a c y when animals d i e during the 24 hour p e r i o d . The on l y course which could be adopted was t h a t of c o n s i d e r i n g these animals as c o n t r i b u t i n g toward the t o t a l selenium e x c r e t i o n f o r the e n t i r e 24 hour p e r i o d . Since animals r a r e l y d i e a f t e r the f i r s t 48 hours of the post i n j e c t i o n p e r i o d t h i s d i s c r e p a n c y has not been too disadvantageous, however, f o r reasons which w i l l be apparent i n the d i s c u s s i o n of t h i s work. U r i n a r y Selenium E x c r e t i o n Male Se per Kg. of su r -v i v o r s T o t a l equiv. excretion per Kg. or survivors NazSeOj j Female Se per v i v o r s Kg. of Sur- )dur i n g ) a f t e r 1st 24 h r s . i n j ect ion 171 Y 167 v it n d u r i n g a f t e r 2nd 24 h r s . i n j e c t i o n 168 162 Y it n d u r i n g a f t e r 3rd 24 h r s . i n j e c t i o n 92 105 V tt » d u r i n g a f t e r 4th 24 h r s . i n j e c t i o n 73 Y 98 r ii u during a f t e r 5th 24 h r s . i n j e c t i o n 50 Y 61 Y n ii d u r i n g a f t e r 6th 24 h r s . i n j e c t i o n 60 Y 132 Y it II d u r i n g a f t e r 7th 24 h r s . i n j e c t i o n 58 Y 75* ti it during a f t e r 8th 24 h r s . i n j e c t i o n 64 If 98* n tt d u r i n g a f t e r 9th 24 h r s . i n j ect ion 0 0 T o t a l e x c r e t i o n of) during e n t i r e p e r i o d 736 Y 898 Y 1.6 mgm. 2.0 mgm, 3 8 Expt. B,, Two groups of r a t s , 4 males and 4 females aged 100 days were s u b j e c t e d to the general experimental procedure as given f o r Experiment B. Dosage l e v e l s of i n j e c t i o n were 6 mgm. Na 2Se0j/Kg. body weight f o r males and 5 mgm. Na2Se0j/Kg. body weight f o r females. The r a t i o of aqueous sodium s e l e n i t e s o l u t i o n to 2% aluminum-monostearate i n peanut o i l was 3 * 1 i n the e m u l s i f i e d i n j e c t i o n mixture. R e s u l t s A l l the animals i n both groups s u r v i v e d although the u s u a l symptoms seen a f t e r i n j e c t i o n were q u i t e severe. Recovery i n both groups proceeded along s i m i l a r l i n e s i n that both food i n t a k e and a c t i v i t y were very r e s t r i c t e d f o r about 5 days a f t e r the i n j e c t i o n . F o l l o w i n g t h i s p e r i o d the animals appeared to be r e c o v e r i n g r a p i d l y u n t i l the 11th day,when they were k i l l e d and a u t o p s i e d . The general autopsy p i c t u r e was one of apparent n o r m a l i t y . Male Female Ave. wt. of) s u r v i v o r s )at i n j e c t i o n t ime 219 gms o 1 7 0 gms. tt tt 1 day i a f t e r i n j e c t . 2 1 3 gms. 1 6 5 gms. tt tt 2 days tt it 2 0 5 gms. 1 6 5 gms. tt it 3 days n » 2 0 2 gms. 1 6 3 gms. n tt 4 days it tt 1 9 7 gms. 1 5 6 gms. it it 5 days it it 198 gms. 154 gms. tt ii 6 days it it 1 9 3 gms. 1 4 6 gms. ti tt 8 days ti II 2 0 3 gms. 1 5 6 gms. « n 1 0 days it it 2 0 8 gms • l 6 l gms. n ti 1 1 days it it 2 0 9 gms. 1 6 2 gms. 39 U r i n a r y Selenium E x c r e t i o n U r i n a r y E x c r e t i o n of) Se per Kg. of Sur- ) d u r i n g 1st 5 h r s . v i v o r s ) a f t e r i n j e c t i o n d u r i n g 5th to 24th hrs, a f t e r i n j e c t i o n Male 160 Y 378 v Female 88 x 221 v du r i n g a f t e r d u r i n g a f t e r d u r i n g a f t e r d u r i n g a f t e r d u r i n g a f t e r d u r i n g a f t e r d u r i n g a f t e r d u r i n g a f t e r d u r i n g a f t e r d u r i n g a f t e r 1st 24 h r s . i n j e c t i o n 2nd 24 h r s . i n j e c t i o n 3rd 24 h r s . i n j ect i o n 4th 24 h r s . i n j e c t i o n 5th 24 h r s . i n j e c t i o n 6th 24 h r s . i n j e c t i o n 7th 24 h r s . i n j e c t i o n 8th 24 h r s . i n j e c t i o n ?th 24 h r s . i n j e c t i o n 10th 24 hrs, i n j e c t i o n T o t a l e x c r e t i o n of) Se per Kg. of sur-) v i v o r s ) T o t a l e q u i v a l e n t ) e x c r e t i o n of Na2Se02) per Kg. of survivors ) du r i n g e n t i r e p e r i o d 538 Y 19?Y 102 Y 34 * 0 6 0 * 49 Y 982Y 309 Y 144 Y 0* 0 453Y 2.2 mgm. 1.0 mgm. 40 Expt. Procedure Two groups of r a t s , 4 males and 4 females, aged 111 days were s u b j e c t e d to the g e n e r a l experimental procedure f o r Experiment B. An aqueous s o l u t i o n of sodium s e l e n i t e was used f o r the i n j e c t i o n of 6 mgm, Na 2Se0j/Kg. body v/eight i n t o the male r a t s and of 5 mgm, Na 2SeOj/Kg. body weight i n t o the female r a t s . R e s u l t s Pronounced symptoms of acute selenium p o i s o n i n g were apparent about 10 minutes a f t e r the i n j e c t i o n . In the male group one animal d i e d at 28 hours a f t e r the i n j e c t i o n . A s m a l l amount of food was consumed on the t h i r d p o s t - i n j e c t i o n day but the animals were s t i l l very i n a c t i v e . By the f i f t h day the t h r e e s u r v i v o r s appeared q u i t e w e l l and were e a t i n g at a l e v e l - o n l y s l i g h t l y below normal. The seventh day a f t e r i n j e c t i o n found the animals w e l l on the way t o complete r e c o v e r y . A l l the female r a t s s u r v i v e d the i n j e c t i o n but recovery of a normal a p p e t i t e was slower than i n the case of the males. Only a s m a l l amount of food was consumed even to t h e seventh day a f t e r i n j e c t i o n . By the n i n t h day, however, the a p p e t i t e was c l o s e to normal and the animals were r e c o v e r i n g q u i c k l y . A c t i v i t y , as i n the case of the males, was r e s t r i c t e d f o r about three days a f t e r i n j e c t i o n . Weight Changes Ave. wt. of) s u r v i v o r s )at i n j e c t i o n time " " 1 day a f t e r i n j e c t . 2 days " " 3 days 5 days 6 days 7 days 8 days 9 days 10 days 41 tt ii it n n it it it tt n u ti it n II it tt it it ti it it tt it u tt t» Male 25? 248 24? 244 S24? s242 XK239 X3E236 244 gms < gms, gms, gms, gms, gms, gms, gms, gms, gms < Female 162 154 14? 145 14 3 141 143 143 144 14? gms. gms. gms. gms. gms. gms. gms. gms. gms. gms. K0ne animal was r e g a i n i n g weight on these days. H HTwo animals were r e g a i n i n g weight while the t h i r d continued t o l o s e weight (40 gms. l o s t i n 8 da y s ) . U r i n a r y Selenium E x c r e t i o n Male Female U r i n a r y E x c r e t i o n of) Se per v i v o r s Kg. of Sur- ) d u r i n g ) a f t e r 1st 5 h r s . i n j e c t i o n 125 v 95 i d u r i n g a f t e r 5th to 24th his i n j e c t i o n 412 Y 214 % it II d u r i n g a f t e r 1st 24 h r s . i n j e c t i o n 537 Y 309v » n during a f t e r 2nd 24 h r s . i n j ect i o n 144 Y 245 V tt it during a f t e r 3rd 24 h r s . i n j e c t i o n 76 Y 0 it tt during a f t e r 4th 24 h r s . i n j e c t i o n 0 0 ti tt d u r i n g a f t e r 5th 24 h r s . i n j e c t i o n 0 0 n tt d u r i n g a f t e r 6th 24 h r s . i n j e c t i o n 0 0 « tt d u r i n g a f t e r 7th 24 h r s . i n j e c t i o n 0 0 ti tt d u r i n g a f t e r 8th 24 h r s . i n j e c t i o n 0 0 II it d u r i n g a f t e r 9th 24 h r s . i n j e c t i o n 0 0 ti «t T o t a l e x c r e t i o n per Kg. of survivors d u r i n g a f t e r of Se) . y d u r i n g 10th 24 h r s . i n j e c t i o n e n t i r e p e r i o d 0 757t 0 554Y 4 2 A D i s c u s s i o n of the R e s u l t s of Subcutaneous I n j e c t i o n of  S u b - l e t h a l Doses of Sodium S e l e n i t e P a i n t e r (1941) has observed that c o n s i d e r a b l e d i s -agreement e x i s t s with r e f e r e n c e to minimum l e t h a l d-ose of selenium f o r v a r i o u s animals. Moxon (1943) f e e l s t h a t the d i f f e r e n t r e s u l t s r e p o r t e d are due to s p e c i e s d i f f e r e n c e s i n s u s c e p t i b i l i t y , mode of a d m i n i s t r a t i o n and p o s s i b l y to i m p u r i t i e s i n compounds used. He found,in h i s review of the l i t e r a t u r e on selenium t o x i c o l o g y (which i n c l u d e d much of the work emanating from h i s own l a b o r a t o r y ^ t h a t the l e a s t amount of selenium as sodium s e l e n i t e that i s r e p o r t e d f a t a l to the r a t by subcutaneous, i n t r a p e r i t o n e a l or intravenous i n j e c t i o n i s from 3.0 to 3 .7 mgm. Se per Kilogram of body weight. E q u a l l y u n c e r t a i n were the v a r i o u s " f a t a l doses" r e p o r t e d f o r v a r i o u s other s p e c i e s . The c o n f u s i o n engendered by the use of the term, " f a t a l dose" was considered by the w r i t e r to be the o r i g i n of much of the disagreement with respect to the acute t o x i -c i t y data. As a r e s u l t , a s e r i e s of experiments were con-ducted ( D e r r i c k , 1948) to determine the 50% l e t h a l dose ( L . D . J J O ) °f sodium s e l e n i t e f o r v a r i o u s s p e c i e s by 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 r o u t e . The method of Reed and Muench (1938) was used f o r c a l c u l a t i o n of the L . D O ^ Q . Among the r e s u l t s of t h i s work was the establishment of an L . D . ^ Q of 14.3 mgm. l ^ S e O ^ or 6.6 mgm. Se per Kg. of body weight f o r mature male r a t s and of 10.5 mgm. Na2Se0j 43 or 4.3 mgm. Se per Kg. of body weight f o r mature female r a t s . The d i f f e r e n c e observed between these r e s u l t s and the L * D c 5 0 value f o r the t o x i c i t y of selenium as sodium s e l e n i t e i n the r a t given by Cameron (1947)(although i t was not c l e a r l y s t a t e d with r e f e r e n c e t o the age and sex of the animals) was s t a r t l i n g . A check made with the supply house on the chemi-c a l a n a l y s i s of the sodium s e l e n i t e used i n the determina-t i o n s by the w r i t e r r e v e a l e d t h a t the compound was q u i t e o l d . Thus the v o l a t i l i z a t i o n of some of the selenium i n t h i s com-pound could have been r e s p o n s i b l e , at l e a s t i n p a r t , f o r the observed d i f f e r e n c e s . Not to be overlooked was the a d d i t i o n a l p o s s i b i l i t y of v a r i a t i o n s i n s u s c e p t i b i l i t y of p a r t i c u l a r s t r a i n s of r a t s . However, the approximate agreement e x i s t i n g between the r e s u l t s of Experiment B^,which was c a r r i e d out u s i n g sodium s e l e n i t e known to be f r e s h and of d i f f e r e n t o r i g i n , and the t o x i c i t y l e v e l s determined e a r l i e r tends to v a l i d a t e these values at l e a s t with r e f e r e n c e to the r a t s used i n t h i s l a b o r a t o r y . The p r e l i m i n a r y nature of Experiments B;j_ and B 2 o b v i a t e s the n e c e s s i t y of d i s c u s s i n g t h e i r outcome i n d e t a i l . The deaths of two of the eight male animals i n j e c t e d with 8 mgm. Na 2Se0j/Kg. body weight and of a l l four of the female animals r e c e i v i n g a s i m i l a r i n j e c t i o n were approximately what might be expected as a p o s s i b l e r e s u l t of the use of the 44 a b s o r p t i o n d e l a y i n g v e h i c l e . The s u r v i v a l of a l l f o u r of the female animals i n j e c t e d at the 6 mgm. Na 2SeOj/Kg. body weight l e v e l p ointed to the probable e x i s t e n c e of a narrow margin between s u r v i v a l and death under the c o n d i t i o n s of i n j e c t i o n i n c o n j u n c t i o n with the 2f» aluminum-monostearate i n peanut o i l . Experiments Bj and B4, however, d i d l i t t l e t o prove whether or not the t h e o r i z e d r e s u l t of the i n c o r p o r a t i o n of the sodium s e l e n i t e i n t o the a b s o r p t i o n d e l a y i n g emulsion f o r i n j e c t i o n o c c u r r e d . The appearance of acute symptoms of pois o n i n g was e q u a l l y prompt i n both cases. The r e c o v e r y of a p p e t i t e and normal a c t i v i t y appeared to occur a l i t t l e more r a p i d l y i n the animals i n j e c t e d with the aqueous s o l u t i o n (B^) than i n the animals r e c e i v i n g the emulsion i n j e c t i o n ( B j ) . Changes i n weight observed i n the two experiments were very s i m i l a r , however, with a l l groups, i n both sexes, showing l o s s e s i n weight u n t i l the s i x t h day a f t e r i n j e c t i o n and the commencement of a tendency t o r e g a i n weight on the seventh day. Although the female r a t s o f Expt. Bj showed a lower t o t a l u r i n a r y e x c r e t i o n of selenium than d i d the female animals i n Expt. B4 any s i g n i f i c a n c e which may be a t t r i b u t e d to t h i s o b s e r v a t i o n i s o f f s e t by the op p o s i t e r e s u l t s ob-served i n the case of the two male groups. The remarkable agreement e x i s t i n g between the u r i n a r y selenium v a l u e s f o r the 1st 24 hours a f t e r i n j e c t i o n i n both experiments may or 45 may not be s i g n i f i c a n t s i n c e the a c t i o n of the a b s o r p t i o n d e l a y i n g v e h i c l e does not extend beyond t h i s p e r i o d . Attempts to break down the e x c r e t i o n data to s h o r t e r time l e v e l s were l i m i t e d by the q u a n t i t i e s of u r i n e a v a i l a b l e . The s m a l l q u a n t i t i e s of selenium excreted by the males of Expt. on the 7th and 8th days a f t e r i n j e c t i o n are unaccountable. The two experiments d i d s o l v e the problems of the dosage l e v e l of selenium and the r e q u i r e d techniques which could be used t o induce a c o n d i t i o n of selenium p o i s o n i n g i n r a t s s u i t a b l e f o r the assessment of chemotherapeutic means of treatment. The procedure of Expt. B j was, t h e r e f o r e , adopted f o r t h i s purpose i n Experiment C (p.86 ). The reasons f o r r e t a i n i n g the a b s o r p t i o n d e l a y i n g v e h i c l e i n the i n j e c t i o n mixture are d i s c u s s e d under that heading. The f o r e g o i n g experiments are by no means regarded as complete. Had time permitted i t would have been d e s i r a b l e t o determine the blood l e v e l s of selenium at v a r i o u s time i n t e r v a l s a f t e r i n j e c t i o n . T h i s procedure would s o l v e the problem of whether or not the i n c o r p o r a t i o n of selenium i n t o the emulsion s u c c e s s f u l l y d e l a y s i t s a b s o r p t i o n and maintains an e f f e c t i v e c o n c e n t r a t i o n i n the blood f o r a longer p e r i o d of time. In a d d i t i o n i t i s f e l t t h a t s e v e r a l more e x p e r i -ments should be c a r r i e d out to e s t a b l i s h a u r i n a r y selenium e x c r e t i o n curve f o r t h i s l e v e l of i n j e c t i o n . Another problem, as yet unsolved i s the e x c r e t i o n of selenium through the 46 lungs* Although, the amount excreted by t h i s route i s pro-bably not l a r g e (Appendix JI.pJ.05) i t i s d e s i r a b l e to have more i n f o r m a t i o n on the q u a n t i t y l o s t by t h i s means. The problem of the l o s s of selenium from the u r i n e by v o l a t i l i z a t i o n under the c o n d i t i o n s of these experiments has been shown to be n o n - e x i s t e n t . On the other hand the l o s s of u r i n e by a b s o r p t i o n i n t o the feed and feces s c a t t e r e d by the animals i n t o the f u n n e l of the metabolism u n i t and a d d i t i o n a l l o s s e s which might seem by evaporation from t h i s s u r f a c e can-not be s o l v e d . Measurements c a r r i e d out by the author sug-gest t h i s l o s s of u r i n e i s of the order of from 15 to 25%. Such a l o s s i s more or l e s s constant, however, and as a r e s u l t would not i n t e r f e r e too g r e a t l y with the assessment of the e f f i c i e n c y of chemotherapeutic agents i n promoting the e x c r e t i o n of selenium. In view of the r e p o r t s of Cameron (1947) with r e f e r -ence t o the v o r a c i o u s a p p e t i t e s of h i s experimental animals while r e c e i v i n g twice weekly i n j e c t i o n s of sodium s e l e n i t e , the anorexia e x h i b i t e d by the animals i n the present s e r i e s of experiments i n response to one i n j e c t i o n i s i n t e r e s t i n g . The only p o s s i b l e c o n c l u s i o n which can be drawn i s t h a t con-t i n u e d i n j e c t i o n s r e s u l t i n a c o n s i d e r a b l y i n c r e a s e d t o l e r a n c e as suggested by Cameron and a l s o , p o s s i b l y , i n changing the paths of metabolism. I n t e r e s t i n g , a l s o , i s the problem of the f a t e of the 4 7 selenium which was not e x c r e t e d by the i n j e c t e d animal d u r i n g the p e r i o d of o b s e r v a t i o n . Even a l l o w i n g f o r a 25% l o s s of u r i n e only about J>Q% of the amount of selenium i n j e c t e d can be accounted f o r by u r i n a r y e x c r e t i o n . I t i s almost c e r t a i n t h a t only a small percentage of the remainder i s e x c r e t e d as a v o l a t i l e compound through the lungs. Thus a s t r o n g p o s s i -b i l i t y e x i s t s t h a t the " i n vi-v.o." mechanism of the p o i s o n i n g i s one of formation of a q u i t e s t a b l e complex probably with the s u l f h y d r y l groups of v a r i o u s t i s s u e p r o t e i n s and enzymes r a t h e r than an o x i d a t i o n r e a c t i o n of the type d e s c r i b e d by P a i n t e r (see p.80 )• Profound changes must take plac e i n the v a r i o u s metabolic pathways and i n a l l p r o b a b i l i t y the r e s t o r a t i o n to a normal s t a t e i s concerned with the dynamic nature of the p r o t e i n s and the r e s u l t a n t gradual e x c r e t i o n of extremely s m a l l , non-detectable q u a n t i t i e s of selenium. 48 II. A NEW GOLORIMETRIC METHOD FOR TEE QUANTITATIVE  DETERMINATION OE SELENIUM IN  SMALL BIOLOGICAL SAMPLES The development of a quick, r e l a t i v e l y simple method of determining selenium quantitatively, i n the minute amounts i n which i t i s usually present i n b i o l o g i c a l samples, i s es s e n t i a l to the success of studies i n selenium toxicology. The available methods have proved to be time consuming, costly, and often so cumbersome as to render the performance of s i g n i f i c a n t numbers of analyses Impossible under the ordinary conditions of a small laboratory. On the other hand, unless the r e s u l t s of problems involving plant and animal tissue analysis, excretion studies et a l . are pre-sented i n quantity they must, by virtue of the considerable fluctuation of values encountered i n work of t h i s kind, be regarded with a considerable amount of doubt. In addition to the accepted, standard method of quantitative selenium analysis (Methods of Anal. A.O.A.C. 6th Ed.) at least two other methods (Gortner, R.A., J r . and Lewis, H.B., 1?40) (Smith, M.I., R.D. L i l l i e , E.E. Stohlman and B.B. Westfall, 1940) had been considered before work on the present method was i n i t i a t e d . The standard method was abandoned as being too cumbersome for exis t i n g laboratory f a c i l i t i e s and too time consuming for u t i l i z a t i o n by a 4? s i n g l e i n v e s t i g a t o r . The other two methods were only s l i g h t l y l e s s time-consuming but c o n s i d e r a b l y more i n a c c u r a t e and were, t h e r e f o r e , a l s o d i s c a r d e d . Other methods of selenium a n a l y s i s have s i n c e appeared i n the l i t e r a t u r e (McNulty, 1947)(Lobanov, and Goldina, 1947) and, w h ile they may have been s u i t a b l e f o r a d a p t a t i o n i n the present study, they have not been i n v e s t i g a t e d s i n c e the new method presented here has proved to be q u i t e s a t i s f a c t o r y . In a search f o r a r e a c t i o n s u i t a b l e f o r a d a p t a t i o n as a c o l o r i m e t r i c procedure the v a r i o u s q u a l i t a t i v e spot t e s t s of F e i g l (1?46) were examined. The t e s t i n v o l v i n g the con-v e r s i o n of compounds of selenium to s e l e n i o u s a c i d which i n t u r n i s reduced to selenium by s i m u l t a n e o u s l y o x i d i z i n g asymmetric d i p h e n y l h y d r a z i n e to v i o l e t q u i n o n e a n i l d i p h e n y l -hydrazone was chosen as the one o f f e r i n g the c o n d i t i o n s most s u i t a b l e f o r a d a p t a t i o n as a c o l o r i m e t r i c q u a n t i t a t i v e method of a n a l y s i s . Numerous d i f f i c u l t i e s were encountered i n the develop-ment of t h i s r e a c t i o n i n t o a q u a n t i t a t i v e a n a l y t i c a l pro-cedure. C h i e f among them were: 1. The d i g e s t i o n of proteinaoeous m a t e r i a l without the l o s s of selenium by v o l a t i l i z a t i o n . 2. The e l i m i n a t i o n of i n t e r f e r e n c e i n the c o l o r r e a c t i o n by o x i d i z i n g agents employed i n the d i g e s t i o n p r o c e s s . 3. The adjustment of q u a n t i t i e s of reagents and m a t e r i a l s f o r a n a l y s i s to a s u i t a b l e volume f o r measurement by a p h o t o - e l e c t r i c c o l o r i m e t e r or spectrophotometer while s t i l l r e t a i n i n g the conditions of pH, reagent r a t i o , etc* r e q u i s i t e for the color reaction to occur. The problem of digestion of proteinaceous material without the accompanying v o l a t i l i z a t i o n of selenium was l a r g e l y solved with the help of a review of the l i t e r a t u r e on the use of selenium c a t a l y s i s i n the Kjeldahl method for nitrogen determination (Seebold, R.E., 1948). Study of the reactions between selenium and concentrated s u l f u r i o acid has determined that i n a solution of hot concentrated s u l -f u r i c acid the only stable form of selenium i s selenious acid (H 2SeOj). I f selenic acid (H^SeO^) i s added to t h i s hot solution i t i s reduced to the selenious acid* This i s true regardless of the o r i g i n a l form of the selenium ( i . e . Se metal, selenium dioxide, e t c . ) . If mercuric oxide i s added to a selenium digest of organic material the selenium i s oxidized to selenic acid* After the organic matter i s digested the selenium i s pre-sent as selenic aoid i f mercuric oxide i s s t i l l present and as selenious aoid i f the mercuric oxide i s spent owing to the formation of mercury-ammonia complexes. This informa-t i o n was adapted to s a t i s f y the demands of the F e i g l reaction by allowing the mercuric oxide to become spent during the digestion process. To further insure against the loss of selenium a system of p a r t i a l digestion of the organic material i n the cold was worked out involving the addition 51 of concentrated n i t r i c a c i d and over n i g h t s t a n d i n g . Since the only I n t e r f e r i n g o x i d i z i n g agents added i n « the o u t l i n e d d i g e s t i o n process were n i t r i c a c i d , which could be v o l a t i l i z e d even w i t h low temperature completion of d i g e s t i o n , and mercuric oxide, which i s spent d u r i n g the d i g e s t i o n p r o c e s s , the second d i f f i c u l t y was a l s o overcome. The f i n a l problem of r e t e n t i o n of the c o n d i t i o n s r e q u i s i t e f o r the c o l o r r e a c t i o n t o occur was s o l v e d o n l y by the performance of numerous t e s t r u ns. I t was found u l t i m a t e l y t h a t the i n t r o d u c t i o n of s u l f u r i c a c i d i n t o the method was causing i n t e r f e r e n c e with the c o l o r r e a c t i o n , probably due t o i t s a t t r a c t i o n f o r water. The q u a n t i t y o f water present appears to be q u i t e c r i t i c a l f o r c o l o r development to occur* The procedure which f o l l o w s has proved t o be a reasonably a c c u r a t e method (plus or minus f i v e percent) of determining the q u a n t i t y of selenium present i n u r i n e * P r e l i m i n a r y t r i a l s have i n d i c a t e d t h a t p l a n t and animal t i s s u e analyses can be c a r r i e d out with s i m i l a r d i s p a t c h . THE COLORIMETRIC DETERMINATION O J SELENIUM IN URINE  Reagents Concentrated s u l f u r i c a c i d C,P. Concentrated n i t r i c a c i d Y e llow m e r c u r i c oxide (Merck) Saturated aqueous s o l u t i o n of o x a l i c a c i d (10?.) 6 N h y d r o c h l o r i c a c i d C P . 0*17. s o l u t i o n of asymmetric d i p h e n y l h y d r a z i n e (Eastman) i n concentrated g l a c i a l a c e t i c a c i d C P . 52 Procedure (a) 10 ml. of urine (or a known aliquot) are placed in a macro Kjeldahl digestion flask (J00 ml. size used i n th i s laboratory). (b) To the fl a s k are added 5 ml. concentrated HgSO^, 5 ml. concentrated HNOj and approximately 0.1 to 0.2 grams of yellow HgO. The f l a s k i s rotated following the addition of each reagent and capped after adding the mercuric oxide. (o) The flasks are placed i n a fume cupboard and allowed to stand overnight. (Note: It has been found con-venient to oarry out t h i s portion of the procedure i n the late afternoon.) (d) Following the period of standing the flasks are heated at low temperature (120°-125°C. measured) on a Kjeldahl digestion rack to expel the n i t r i c acid and the excess water and to complete digestion. The appearance of dense s u l f u r i c acid fumes marks the point at which the expulsion of n i t r i c acid and water are almost complete. Continuation of the heating (temperature now approximately 240°C.) for at least twelve minutes a f t e r the appearance of these fumes i s considered desirable to ensure that the digest is free of n i t r i c a c i d . The urine w i l l usually be completely digested by t h i s time and the general appearance of the digest w i l l be clear and colorless with the formation of a c r y s t a l l i n e deposit of what i s probably mercuric sulfate o c c u r r i n g at the neck of the f l a s k . Should the d i g e s t i o n be incomplete at t h i s time ( i . e . a s l i g h t c o l o r a t i o n p e r s i s t e n c e ) t e s t s have shown that the m a t e r i a l may be heated f o r as long as twenty minutes a f t e r the appearance of fumes without any a p p r e c i a b l e change i n the selenium c o n t e n t . Although i t has not been t e s t e d i t i s c o n s i d e r e d t h a t i n view o f the f a c t that the selenium i s , under the c o n d i t i o n s of the d i g e s t i o n , i n i t s most s t a b l e form, f u r t h e r h e a t i n g of the d i g e s t should not endanger the selenium content too g r e a t l y . However, s i n c e at t h i s stage the m ercuric oxide and the n i t r i c a c i d are spent I t would be wise i n the case of incomplete d i g e s t i o n to repeat the a n a l y s i s u s i n g l a r g e r q u a n t i t i e s of these two r e a g e n t s . The problem has not been encountered during the present i n v e s t i g a t i o n s however, even when one gram samples of p l a n t or animal t i s s u e are d i g e s t e d u s i n g the above q u a n t i t i e s of r e a g e n t s . In the event t h a t a l a r g e volume of u r i n e i s passed by experimental animals, n e c e s s i t a -t i n g an o r i g i n a l a l i q u o t g r e a t e r than 10 ml., s a t i s f a c t o r y r e s u l t s may be achieved by adding 10 ml. of H N O j ( c o n c ) f o r each a d d i t i o n a l 10 ml. of u r i n e . (e) F o l l o w i n g completion of d i g e s t i o n the f l a s k s are allowed to c o o l , p r e f e r a b l y being removed from the d i g e s t i o n r a c k . (f) Upon c o o l i n g the contents of the f l a s k are d i l u t e d to e x a c t l y 30 ml. by the a d d i t i o n of d i s t i l l e d water with care being taken to r i n s e the o r i g i n a l c o n t a i n e r as 54 thoroughly as p o s s i b l e . (g) A 6 ml. a l i q u o t of the d i l u t i o n i s p i p e t t e d i n t o a l a r g e t e s t tube. 2 ml. of the s a t u r a t e d aqueous s o l u t i o n of o x a l i c a o i d are added and the s o l u t i o n s are mixed and allowed to stand f o r approximately f i v e minutes. 2 ml. of 6 N HC1 a c i d are added to the mixture and the contents are again mixed and allowed to stand f o r approximately t e n minutes* (h) The appearance of very minute amounts of a p r e c i p i -t a t e may occur at t h i s p o i n t . To e l i m i n a t e the p o s s i b i l i t y Of nephelometric i n t e r f e r e n c e or c o l o r a d s o r p t i o n onto t h i s s l i g h t p r e c i p i t a t e f i l t r a t i o n i s c a r r i e d out r o u t i n e l y . The s o l u t i o n i s f i l t e r e d u s i ng o r d i n a r y No. 1 q u a l i t a t i v e f i l t e r paper. ( i ) A 5 ml. a l i q u o t of the f i l t r a t e i s p l a c e d i n a cuvette and 1 ml. of the 0,1% s o l u t i o n of asymmetric d i p h e n y l -hydrazine i n g l a c i a l a c e t i c a c i d i s added wi t h r o t a t i o n of the tube to i n s u r e thorough mixing* ( j ) The f i n a l mixture i s allowed t o stand i n a dark cupboard f o r t h r e e hours, and then p l a c e d i n the s p e c t r o -photometer to be measured f o r the a b s o r p t i o n of l i g h t i n the 510 m i l l i m i c r o n wave band. (k) A b r i e f mathematical check of the v a r i o u s a l i q u o t s and d i l u t i o n s quoted above r e v e a l s t h a t the f i n a l a l i q u o t of the mixture i s e q u i v a l e n t to 1 ml. of u r i n e . Since the 55 standard curve i s set up to cover c o n c e n t r a t i o n s of from l»5*to 15* comparison of the galvanometer r e a d i n g f o r the sample wi t h the values given on the curve g i v e s the concen-t r a t i o n of selenium i n gamma per m i l l i l i t e r , or p a r t s per m i l l i o n , of u r i n e . PREPARATION OJ? A STANDARD CURVE FOR THE SELENIUM-ASYMMETRIC  PIPHENYLHYDRAZINE REACTION The p r e p a r a t i o n of a standard curve f o r any c o l o r i -m e t r i c r e a c t i o n r e s o l v e s i t s e l f i n t o two problems: 1. The d e t e r m i n a t i o n of the most s u i t a b l e wavelength to be used f o r a b s o r p t i o n ( u s u a l l y , but not neces-s a r i l y the p o i n t of maximum ab s o r p t i o n ) of l i g h t by the s o l u t i o n . 2. The s e t t i n g up of a s e r i e s of known standards of the m a t e r i a l t o be determined and the s u b j e c t i o n of t h i s s e r i e s t o the procedure of a n a l y s i s con-templated w i t h the attainment of r e p e a t a b l e , constant readings over a number of d e t e r m i n a t i o n s . The a b s o r p t i o n measurements on the Coleman S p e c t r o -photometer showed t h a t f o r the v i o l e t q u i n o n e a n i l d i p h e n y l -hydrazone dye produced i n the r e a c t i o n the p o i n t of maximum ' l i g h t a b s o r p t i o n and l e a s t i n t e r f e r e n c e was at 510 m i l l i -microns (See graph, p.56 ). To o b t a i n a s e r i e s of standards an aqueous s o l u t i o n of sodium s e l e n i t e c o n t a i n i n g 150 gamma of selenium (as sodium s e l e n i t e ) per m i l l i l i t e r was prepared. The a d d i t i o n of t h i s s o l u t i o n to normal human u r i n e ( i n K j e l d a h l f l a s k s ) was made i n amounts v a r y i n g from 0.1 ml. (equiv. 15*Se) to 1 ml. (equiv. 150 * Se) with the q u a n t i t y of u r i n e v a r y i n g from 9.9 ml. to 9 ml. r e s p e c t i v e l y t o make iii iiii! iii 111 111 ill Hi iii iiiiiiii iii iiii ii! H I iiiiiiiii iiiiiiiii ::::: iiiiiiii mmwmm iiii! ::: JBBB :::: :::: •••••••••a ii :: • i BSHKBSSSS: ::::! Ik liiii ES ss:: 1 • • i i a I III i i : iiii \ Mi I ::»:::»: _ _ _ _ _ • • • • I B I H iM'4aK*>l :....::::::::..::::::=! ii! iaa ::: B:B mmm li Hi IIIIBI :::! •••• • • • • • ik-.«» iii»iiiu :::::: iii • • • • :::: ii Iliilii Illllliililiiiiiiillliiiiii ii mm ii urn ••••••• iiiiiii iiii ::::::: • B B B B B B iiiiiii I:::::: :::::::::::::::::::::: :::: iiiiiii ::::::: ::::::: :::::::::::::::::::: Si:::»:: • • • • :::: IBI ::::::: ::::: :::: "SSnaS {{[[{jjjjjjjjjlj:: [jljijijjjjililll Hill Iiiiiiiii ii iiiiiiiiiiiiiiiiiiiiii iiii iiii ::::::::::::: I iiiiiiiii iiii : : . : :s: . : : : : : .s: : . : :s: ..::::::::::;::::.:::: • • • • • • • • • • • • • • • • • • B B B B B B • • • • • B B B B B aaaaaaaaaaaaaaaaiaaa :::::: •818 B I B ! •aaaaaiaaaaa aaaaa illliiiiiiiiiiili!!; HH! ia aaaaa at l a a a a a i i a laaaaaaaaa•• :::::: maiaaaaaaaaaaaaaa • mmimnaaaaa aaaaa f r i i i ' i i i iiii iiii ;:: •aai ••••• is::: SSSSSBBH! iiii iii iii iii i l l liiii! ::sss: ;::::: 600 650 700 750 WAVE LENGTH MILLIMICRONS COLEMAN SPECTROPHOTOMETER Model No l i f t . Serial No 57 a t o t a l of 10 ml. of l i q u i d m ixture. Thus a range of the equ i v a l e n t of 1.5xto 15* Se per m i l l i l i t e r of u r i n e was ob-t a i n e d . A reagent blank was a l s o prepared u s i n g 10 ml. of normal u r i n e . These standards were then s u b j e c t e d t o the a n a l y t i c a l procedure d e s c r i b e d above. The standard curve obtained by t h i s prooedure (see p.58 ) compared v e r y f a v o r a b l y with a curve obtained u s i n g aqueous sodium s e l e n i t e s o l u t i o n and the r e s p e c t i v e reagents without c a r r y i n g through the d i g e s t i o n procedure (see p.52). A DISCUSSION OF THE METHOD The method, i n common with most c o l o r i m e t r i c methods of a n a l y s e s , s u f f e r s from some shortcomings. C h i e f among these i s the i n t e r f e r e n c e with the r e a c t i o n due to the p r e -sence of other o x i d i z i n g agents which may a l s o cause forma-t i o n of the d y e s t u f f . The p o s s i b i l i t y of i n t e r f e r e n c e of o x i d i z i n g oxy-acids has been e l i m i n a t e d from the procedure, however, by the use of the d i g e s t i o n process o u t l i n e d above. The d i g e s t i o n problem was perhaps the most d i f f i c u l t one t o overcome during the development of the method. For some time there appeared t o be no a l t e r n a t i v e s other than those of l o s s of Belenium d u r i n g long d i g e s t i o n processes i n v o l v i n g the use of n o n - i n t e r f e r i n g reagents on one hand or i n t e r f e r e n c e from stronger o x i d i z i n g reagents on the other hand. I t i s d i f f i -c u l t t o p l a c e the o r e d i t f o r the process f i n a l l y evolved s i n c e i t i s a composite t h i n g r e s u l t i n g from the study of 5? d i g e s t i o n methods used i n numerous d i f f e r e n t types of a n a l y t i -c a l procedures. Tungstates, molybdates, F e ( I I I ) and Cu(II) s a l t s are a l s o mentioned by F e i g l as being sources of i n t e r f e r e n c e in. the c o l o r r e a c t i o n . He s t a t e s t h a t i f any of these are p r e -sent o x a l i c a c i d should be added to the h y d r o c h l o r i c a c i d and complex o x a l a t e s are formed which do not r e a c t w i t h asymmetric d i p h e n y l h y d r a z i n e . The w r i t e r had ocoasion to doubt t h a t t h i s statement was a p p l i c a b l e i n the present method duri n g a s e r i e s of t e s t s i n v o l v i n g the use of meta-bolism u n i t s which had f u n n e l s f o r u r i n e c o l l e c t i o n made of' g a l v a n i z e d i r o n s h e e t i n g and copper t u b i n g . Test runs i n v o l v i n g the i n c o r p o r a t i o n of f e r r i c sulphate and c u p r i c sulphate i n t o reagent mixtures s i m i l a r to those employed i n the method r e v e a l e d t h a t the two metals were indeed i n t e r -f e r i n g to the f o l l o w i n g e x t e n t . -Amts. of Cu or Fe per ml. of Urine Galvanometer Reading Cu Se E q u i -v a l e n t of Read-i n g Galvanometer Reading Fe Se E q u i -v a l e n t of Read-ing 25 */ml. 7 8 . 5 6 . 0 jr/ml. 87 4 . 8 50 tf/ml. 66 8 . 1 j/ml. 82 5 . 6 r/ml. 7 5 r/ml. 54 1 0 . 5 J/ml. 70 7 . 5 '/ml. lOOr/ml. 4 8 . 5 1 1 . ? v/ml. 53 1 0 . 8 v/ml. I t may Well be t h a t the s o l u b i l i t y of the complex ions i s a f f e c t e d by the presence of a c o n s i d e r a b l e excess of oxalate ions and, i f such i s the case, i t would be d i f f i c u l t 6 0 to e l i m i n a t e i n t e r f e r e n c e by t h i s means. On the other hand i t may a l s o be that the c o n d i t i o n s of pH, e t c . imposed by the reagents of the q u a n t i t a t i v e procedure preclude the formation of the complex i o n . For the present, however, the o x a l a t e has been r e t a i n e d i n the method s i n c e i t does appear to enhance the c o l o r r e a c t i o n . V a r i o u s m o d i f i c a t i o n s of the method were attempted to e l i m i n a t e the i n t e r f e r e n c e encountered when i r o n and copper are present i n a p p r e c i a b l e amounts. Among them were the i n c o r p o r a t i o n i n t o the procedure of the v a r i o u s p r e c i p i t a t i o n r e a c t i o n s , such as the formation and removal of hydroxides, e t c . However a l l the approaches attempted i n t e r f e r e d with the o p t i m a l c o n d i t i o n s of the c o l o r r e a c t i o n . The i n t e r -ference was no doubt due to such t h i n g s as changes i n hydrogen i o n c o n c e n t r a t i o n , the i n t r o d u c t i o n of f o r e i g n i o n s , e t c . and the r e s u l t was t h a t c o l o r p r o d u c t i o n under these c o n d i t i o n s was u n r e l i a b l e . The presence of the two metals under normal concentra-t i o n c o n d i t i o n s such as are u s u a l l y found i n b i o l o g i c a l samples o f f e r s v e r y l i t t l e hazard to the method, however. Samples of l i v e r , f o r example, have not shown an excess of c o l o r p r o d u c t i o n too great t o be taken care of by the i n -c l u s i o n of the normal t i s s u e reagent "blank". The removal of i r o n by p r e c i p i t a t i o n u s i n g c u p f e r r o n . or neo-cupferron ( S a n d e l l , 1?44) i s suggested as a procedure 6 1 which may w e l l be s u i t a b l e f o r i n c o r p o r a t i o n i n t o the method in cases where c o n c e n t r a t i o n s of the metal cause c o n s i d e r a b l e i n t e r f e r e n c e . The f a c t t h a t t h i s p r e c i p i t a t i o n occurs under a c i d medium c o n d i t i o n s would appear q u i t e promising. The same author o f f e r s a means of e x t r a c t i n g copper from an a c i d medium by shaking with a s o l u t i o n of d i t h i z o n e i n an o r g a n i c s o l v e n t . It i s r e i t e r a t e d , however, that such procedures should not be necessary under normal c o n d i t i o n s of experimentation. Suoh d i f f i c u l t i e s as were encountered d u r i n g the use of the meta-bol i s m u n i t s of sheet i r o n and copper t u b i n g may a l s o be over-come i n other ways. In the present study the o f f e n d i n g u n i t s were coated with p a r a f f i n wax which s u c c e s s f u l l y e l i m i n a t e d the i n t e r f e r e n c e . L a t e r these cages were r e p l a c e d with u n i t s of Monel metal which appears to be the most s a t i s f a c t o r y means of a v o i d i n g i n t e r f e r e n c e . Tungstates and molybdates, mentioned e a r l i e r as p o s s i b l e i n t e r f e r i n g agents i n the t e s t , could h a r d l y be regarded as hazards to the method i n most cases where the m a t e r i a l s being examined are of b i o l o g i c a l o r i g i n . In the case of plan t t i s s u e a n a l y s i s , however, the p o s s i b i l i t y of t h e i r presence should not go unchecked. Many of the minor d i f f i c u l t i e s , such as incomplete d i g e s t i o n of v a r i o u s types of samples, may be s o l v e d by the simple expedient of a d j u s t i n g the v a r i o u s q u a n t i t i e s of r e a g e n t s . I t must be emphasized, however, t h a t the c o l o r r e a c t i o n depends on a r a t h e r c r i t i c a l balance of reagents and the e f f o r t should be made to preserve a f i n a l reagent r a t i o c l o s e l y corresponding to the one presented above. The time f o r c o l o r development has been e m p i r i c a l l y d e f i n e d as three hours. T h i s p r e c i s e d e f i n i t i o n i s necessary s i n c e the c o l o r change i s a p r o g r e s s i v e t h i n g and i s not o n l y a f u n c t i o n of c o n c e n t r a t i o n but a l s o of time. The use of instruments other than the Coleman Spectrophotometer may r e q u i r e t h a t the time be lengthened or shortened, a c c o r d i n g to the s e n s i t i v i t y of the instrument. L i g h t w i l l o x i d i z e the asymmetric d i p h e n y l h y d r a z i n e s l o w l y and f o r t h i s reason the tubes c o n t a i n i n g the f i n a l mixture must be placed i n a dark cupboard d u r i n g the p e r i o d of c o l o r development. S i m i l a r l y the reagent s o l u t i o n of asymmetric d i p h e n y l h y d r a z i n e i n g l a c i a l a c e t i c a c i d should be kept i n a dark b o t t l e and may be used only up to a p e r i o d of about one week a f t e r being made up. In t h i s l a b o r a t o r y i t has been found convenient t o make up about 50 ml. of the reagent s o l u t i o n at a time. Observation of the standard curve r e v e a l s t h a t Beer's Law does not h o l d too w e l l under c o n d i t i o n s of low concentra-t i o n . I t i s suggested t h a t , where i t i s p o s s i b l e , the o r i g i n a l a l i q u o t of samples f a i l i n g w i t h i n t h i s low concentra-t i o n range should be i n c r e a s e d t o a l l o w the f i n a l r e a d i n g t o f a l l on the p o r t i o n of the curve which adheres s t r i c t l y to Beer's Law. T h i s m o d i f i c a t i o n w i l l a p p r e c i a b l y i n f l u e n c e the 63 accuracy of the method,, In cases where the c o n c e n t r a t i o n i s too h i g h to f a l l on the curve the di g e s t may be d i l u t e d with the "reagent blank" d i g e s t m a t e r i a l before being again submitted to the c o l o r development procedure or a s m a l l e r o r i g i n a l a l i q u o t may be used,, 6 4 I I I . CHEMOTHERAPEUTICS. . METAL TOXICOLOGY AND THE  SELENIUM PROBLEM The g e n e r a l l e t h a l a c t i o n of low c o n c e n t r a t i o n s of c e r t a i n m e t a l l i c s a l t s on l i v i n g c e l l s was considered so remarkable when i t was f i r s t d i s c o v e r e d that i t was given the s p e c i a l name, "oligodynamic a c t i o n " . Many e l a b o r a t e t h e o r i e s were proposed to e x p l a i n the nature of the l e t h a l e f f e c t (Buchanan and Fulmer, 1928 ) and, although i t remained f o r l a t e r i n v e s t i g a t o r s to o f f e r more v a l i d c o n c l u s i o n s w i t h r e f e r e n c e t o the mechanisms i n v o l v e d the ob s e r v a t i o n s made by the e a r l i e r s c i e n t i s t s such as Heubel, Koch, E h r l i c h and others (Work and Work, 1948) were the foundation of the scien c e of chemotherapy. E h r l i c h , u s u a l l y considered the f a t h e r of the sc i e n c e of chemotherapy, b e l i e v e d " t h a t the ways and means by which drugs are d i s t r i b u t e d over the body must be of gre a t e s t importance i n the r a t i o n a l development of t h e r a -p e u t i c s " ( E h r l i c h and Hata, 1911). As a means of stud y i n g t h i s d i s t r i b u t i o n he i n i t i a t e d h i s well-known experiments on v i t a l s t a i n i n g i n the f i n a l years of the l a s t century ( c i r c a 1885). E h r l i c h , on k i l l i n g an animal which had been 65 i n j e c t e d with methylene blue some time p r e v i o u s , found t h a t the o n l y t i s s u e s dyed were those of the nervous system. F u r t h e r experimentation showed him t h a t c e r t a i n dyes s t a i n e d s p e c i f i c a l l y c e r t a i n organs or types of c e l l s while others were f a i r l y g e n e r a l i n t h e i r a c t i o n . Many b a s i c dyes such as Bismarck brown, n e u t r a l r e d , f l a v a n i l i n or methylene b l u e , were found by E h r l i c h t o s t a i n nerve t i s s u e only while o n l y one a c i d i c dye, a l i z a r i n had t h i s p r o p e r t y . These dyes l o s t t h e i r " n e u r o t r o p i c " p r o p e r t y on co n v e r s i o n t o s u l p h o n i c a c i d d e r i v a t i v e s . E h r l i c h assumed t h a t a c i d i c dyes are bound i n the blood by the a l k a l i present while b a s i c dyes are not h e l d i n blood by any chemical a f f i n i t i e s and are thus f r e e t o d i f f u s e i n t o surrounding t i s s u e s . In a d d i t i o n he emphasized t h a t s i m i l a r d i f f e r e n c e s i n the d i s t r i b u t i o n o f c o l o r l e s s substances i n the body are l i k e l y to occur. " V i t a l s t a i n i n g " ( K n a y s i , 1944) assumed a p o s i t i o n of primary importance i n E h r l i c h ' s s t u d i e s and he continued with such problems as the r e l a t i v e oxygen combining powers of va r i o u s t i s s u e s as evidenced by the r e d u c t i o n of the dyes indophenol or a l i z a r i n blue t o the leuco product. He con-cluded that i n c e l l protoplasm t h e r e e x i s t e d s i d e chains whose f u n c t i o n was oxygen f i x a t i o n and that t h e i r a f f i n i t y f o r oxygen v a r i e d i n d i f f e r e n t organs. I t was d u r i n g t h i s s e r i e s of s t u d i e s that the idea s were conceived upon which E h r l i c h ' s l a t e r chemotherapeutic t h e o r i e s were based. 66 From work with animals E h r l i c h turned h i s a t t e n t i o n to the s t a i n i n g of b a c t e r i a w i t h a n i l i n e dyes and was succes s -f u l i n developing many new te c h n i q u e s . In 1891 he performed h i s f i r s t chemotherapy experiment i n c o l l a b o r a t i o n w i t h Guttman u t i l i z i n g much of h i s own work and the work of others such as Rozsahegyi (I887) who had demonstrated the b a c t e r i -c i d a l a c t i o n of dyes and S t i l l i n g (1890) who had c a r r i e d the work f u r t h e r and was recommending a mixture of blue dyes f o r the treatment of minor s u r g i c a l i n f e c t i o n s . E h r l i c h found t h a t methylene blue, which s t a i n e d m a l a r i a l p a r a s i t e s v e r y e f f e c t i v e l y , when administered t o m a l a r i a l p a t i e n t s was a f a i r l y s u c c e s s f u l cure f o r cases of t e r t i a n m a l a r i a although not s u f f i c i e n t l y so t o warrant i t s s u b s t i t u t i o n f o r q u i n i n e . E h r l i c h continued to be a l e a d e r i n chemotherapy developments i n a d d i t i o n t o p l a y i n g a major r o l e i n the development of immunology. He c o n t i n u a l l y emphasized the chemical s i d e of immunology and h i s fundamental t h e o r i e s of immunity and of chemotherapy were c l o s e l y a l l i e d . The bases of both these t h e o r i e s were the " r e c e p t o r u n i t s " or s i d e chains which E h r l i c h p o s t u l a t e d to be present i n a l l p r o t o -plasm. Such r e c e p t o r s were considered by E h r l i c h t o be u t i l i z e d i n normal c e l l metabolism as a means of c a t c h i n g or anchoring a v a r i e t y of m e t a b o l i t e s p r i o r t o t h e i r incorpora-t i o n i n t o the c e l l substance. T o x i c substances were thought to be capable of d i v e r t i n g the r e c e p t o r s from t h e i r normal 67 f u n c t i o n . In the case of immunological r e a c t i o n s the c e l l was b e l i e v e d to be s t i m u l a t e d to produce not onl y r e c e p t o r s to r e p l a c e the i n a c t i v a t e d ones but a l s o an excess of re c e p t o r s which entered the blood stream and c o n s t i t u t e d the s p e c i f i c a n t i t o x i n or ant i b o d y . The chemotherapy theory of E h r l i c h resembled h i s immunology theory i n essence s i n c e i t accounted f o r the a c t i o n o f a drug by i t s combination with a s p e c i f i c c e l l r e c e p t o r . R e s i s t a n c e of an organism to a p a r t i c u l a r drug was regarded as due to the r e d u c t i o n of a f f i n i t y of a r e c e p t o r f o r one drug without a change i n com-b i n i n g powers f o r other drugs. These t h e o r i e s , now known to have many f a i l i n g s , h a d the m e r i t t h a t they kept t o the f o r e the important concept that chemical s p e c i f i c i t y i s an e s s e n t i a l f e a t u r e of immunology and chemotherapy. It would be u n f a i r t o give c r e d i t t o E h r l i c h alone f o r the enormous advances made i n the f i e l d s o f immunology and chemotherapy i n the l a t e n i n e t e e n t h and e a r l y t w e n t i e t h c e n t u r i e s . Such i n v e s t i g a t o r s as Meyer (1889) and Overton (1901),who c o n t r i b u t e d the the o r y that chemical compounds may be d i v i d e d i n t o d i f f e r e n t groups by t h e i r r a p i d i t y of d i f f u s i o n i n t o the c e l l protoplasm as a r e s u l t of the c o e f f i c i e n t s of d i s t r i b u t i o n between f a t and water,were h e l p -in g t o e s t a b l i s h a sound s c i e n t i f i c b a s i s f o r chemotherapy. Many others were a l s o c o n t r i b u t i n g to the f i e l d and yet i t remained f o r E h r l i c h t o o f f e r the most s u c c e s s f u l c o n t r i b u -t i o n of the decade when he i n i t i a t e d the work i n the f i e l d of 68 o r g a n o - a r s e n i c a l s . T h i s work culminated i n the p r o d u c t i o n of such drugs as a t o x y l (p-aminophenyl a r s o n a t e ) , which was used by Koch to cure the human form of trypanosomiasis ( s l e e p i n g s i c k n e s s ) , and i t s d e r i v a t i v e s , p-aminophenylarsenoxide, and p-aminophenyl arsenobenzene, the l a t t e r of which was found to be l e s s t o x i c to the c e l l s of the host animal. A t o x y l and i t s d e r i v a t i v e s were a l s o found to be e f f e c t i v e a g a i n s t s p i r o -chaetes. I t was from these type a r s e n i c a l compounds t h a t compound 606 (the h y d r o c h l o r i d e of dihydroxydiaminoarseno-benzene) known as s a l v a r s a n , which was s u c c e s s f u l i n c u r i n g r e l a p s i n g f e v e r , s y p h i l i s and t r y p a n o s o m i a s i s , was f i n a l l y e volved. The more s o l u b l e n e o s a l v a r s a n (a condensation pro-duct of s a l v a r s a n with sodium formaldehyde sulphoxide) was e v e n t u a l l y placed on the open market, a f t e r c a r e f u l c l i n i c a l t r i a l s , as an a n t i s y p h i l i t i c drug and was used i n a d d i t i o n as a h i g h l y s u c c e s s f u l means of t r e a t i n g another human s p i r o -c h a e t a l i n f e c t i o n , namely, framboesia, (yaws). The develop-ment of n e o s a l v a r s a n marked the g r e a t e s t achievement of chemotherapy up to the d i s c o v e r y of the sulphonamides and i t was indeed as a r e s u l t of the e a r l i e r work on b a c t e r i o s t a t i c dyes that sulphonamides were p e r f e c t e d . From these beginnings the s c i e n c e of chemotherapy has expanded i n t o the more s p e c i a l i z e d branches of c e l l p h y s i o -logy, t o x i c o l o g y , enzymology and a host of other approaches to the fundamental f a c t s i n v o l v e d i n the metabolic processes of the l i v i n g c e l l . 6 9 Most of E h r l i c h ' s chemotherapeutio s t u d i e s were con-f i n e d to diseases of p r o t o z o a l or s p i r o c h a e t a l o r i g i n . He d i d , however, c a r r y out some work on the chemotherapeutic treatment of b a c t e r i a l i n f e c t i o n s which was of c o n s i d e r a b l e t h e o r e t i c a l importance. For example, i n an i n v e s t i g a t i o n of the g e r m i c i d a l powers of s u b s t i t u t e d phenols, Bechhold and E h r l i c h (1906)(1909) found a number of compounds which f a r exceeded a l l p r e v i o u s l y known phenols i n t h e i r b a c t e r i c i d a l a c t i o n " i n v i t r o " i n n u t r i t i v e b r o t h . T h e r a p e u t i c e x p e r i -ments on i n f e c t e d animals, however, proved u n s u c c e s s f u l and the f a i l u r e was shown to be due to the f a c t t h at these d i s -i n f e c t a n t s combined with serum p r o t e i n s to such an extent that t h e i r g e r m i c i d a l power was depressed. Other i n v e s t i -gators (Morgenroth and Levy, 1 9 1 1 ) ( C h u r c h m a n , 1912) of chemotherapeutic b a c t e r i c i d a l t e c h n i q u e s , w h i l e they met with l i t t l e success i n e f f e c t i n g cures of b a c t e r i a l i n f e c t i o n s , were c o n t r i b u t i n g a l a r g e volume of i n f o r m a t i o n and p o s t u l a -t i o n to the f i e l d of chemotherapy. In 1914 Simon and Wood showed only b a s i c dyes had an i n h i b i t o r y a c t i o n . In a d d i t i o n these authors d e s c r i b e d the phenomenon of the development of drug r e s i s t a n t s t r a i n s of organisms,although the o b s e r v a t i o n s were not e n t i r e l y o r i g i n a l s i n c e drug r e s i s t a n c e development had been noted i n the n i n e t e e n t h century. The p u b l i c a t i o n s of Simon and Wood were most not a b l e , however, f o r the presented theory of drug a c t i o n and r e s i s t a n c e which d i f f e r e d 70 somewhat from E h r l i c h ' s theory and, i n f a c t , bears a marked resemblance t o some of the modern t h e o r i e s * These authors a s c r i b e d , as d i d E h r l i c h , the i n h i b i t o r y a c t i o n of dyes to the e x i s t e n c e of r e c e p t o r s i n the c e l l to which the dye was anchored with the r e c e p t o r s probably c o n s i s t i n g of a c i d i c groups which r e a c t e d with the b a s i c dyes. These s o - c a l l e d " n u t r i c e p t o r e " were thought to be r e s p o n s i b l e f o r c a r r y i n g on the metabolism of the c e l l and when they were blocked by combination with a dye the c e l l d i e d . T h i s occurred not n e c e s s a r i l y because the c e l l had been poisoned, but because a s u f f i c i e n t number of the n u t r i c e p t o r s had been e f f e c t i v e l y blocked and the c e l l c o u l d no longer m etabolise and m u l t i p l y . The a c q u i r e d a d a p t a t i o n t o i n h i b i t o r y amounts of the dye was a t t r i b u t e d to the e x i s t e n c e or development of other r e c e p t o r s by means of which the c e l l c o u l d c a r r y out i t s n u t r i t i o n and r e p r o d u c t i o n . While no suggestion was made as to the nature of these r e c e p t o r s i t was noted t h a t " s i n c e i n t r a c e l l u l a r metabolism i s i n t i m a t e l y connected with the a c t i o n of enzymes, the qu e s t i o n has n a t u r a l l y suggested i t s e l f whether the d e l e t e r i o u s a c t i o n of the dyes may not i n p a r t be r e f e r a b l e to i n t e r f e r e n c e with the a c t i v i t y of these components". In g e n e r a l , s y n t h e t i c chemotherapy, however, remained c h i e f l y concerned with o r g a n i c and o r g a n o - m e t a l l i c trypano-c i d a l and a n t i s p i r o c h a e t a l drugs and has advanced to i t s present s t a t e r a t h e r s u c c e s s f u l l y on what may appear to be a somewhat e m p i r i c a l b a s i s . N e v e r t h e l e s s some workers were emphasizing the importance of the a c t i o n , d i s t r i b u t i o n and e x c r e t i o n of chemotherapeutic drugs i n the body s i n c e they f e l t i t was t h i s s i d e of chemotherapy which would determine to a great extent whether or not a drug would be as a c t i v e " i n v i v o " as " i n v i t r o " . One of these i n v e s t i g a t o r s , ( V o e g t l i n , 1925) attempted t o e x p l a i n the d i f f e r e n t i a l a c t i o n of drugs i n p o i s o n i n g p a r a s i t e s r a t h e r than t h e i r hosts (a f a c t simply accepted by E h r l i c h ) by suggesting t h a t e i t h e r the c a p a c i t y of the host c e l l s to convert the t o x i c form of a drug i n t o a non - t o x i c form was g r e a t e r than that of the p a r a s i t e s , or that p a r a s i t e s had a g r e a t e r p e r m e a b i l i t y f o r the drug than the host c e l l s . Voegtlin.was a pioneer i n the f i e l d of metal t o x i c -ology. He p r o v i d e d the f i r s t p r a c t i c a l demonstration of E h r l i c h ' s theory t h a t the pentavalent form of an a r s e n i c a l drug i s converted by the host i n t o the a c t i v e t r i v a l e n t oxide form and, i n a d d i t i o n , he showed that arsenobenzene d e r i v a t i v e s are not a c t i v e as such but r a t h e r are t r a n s -formed i n the host t o the a c t i v e arsenoxide form. This p o i n t seems t o have escaped E h r l i c h i n h i s development of s a l v a r s a n ( V o e g t l i n , Dyer and Leonard, 1923). V o e g t l i n et a l . found t h a t when s a l v a r s a n or other arsenobenzene d e r i v a t i v e s were i n j e c t e d i n t o r a t s i n f e c t e d w i t h trypanosomes the d e s t r u c t i o n of the p a r a s i t e s was preceded by a l a t e n t p e r i o d of s e v e r a l hours. However, the t r i v a l e n t oxide form of s a l v a r s a n had an immediate t r y p a n o c i d a l a c t i o n . The t o x i c e f f e c t of arsenoxides was shown to be due to t h e i r a c t i o n on s u l f h y d r y l groups i n the host t i s s u e s s i n c e g l u t a t h i o n e , a s u l f h y d r y l c o n t a i n i n g p e p t i d e , a f f o r d e d p r o t e c t i o n t o animals i n j e c t e d with i t immediately f o l l o w i n g a l e t h a l dose of arsenoxide. With t h i s work V o e g t l i n i n t r o d u c e d a new concept i n chemotherapy. E s s e n t i a l l y he was showing t h a t i t was pos-s i b l e t o e f f e c t i v e l y denature the p r o t e i n s of l i v i n g p r o t o -plasm by means of simple chemical combination. T h i s concept ap p a r e n t l y f a i l e d t o take r o o t , however, s i n c e the a c t i o n o.f drugs was a subject of s p e c u l a t i o n f o r a number of years f o l l o w i n g . F i l d e s i n 1929 and Knight and F i l d e s i n 1930 presen t -ed evidence that anaerobic b a c t e r i a would not grow i f the o x i d a t i o n - r e d u c t i o n p o t e n t i a l of the medium was maintained p o s i t i v e to a c e r t a i n l e v e l - — t h u s i m p l y i n g that growth occurs only when a c e r t a i n degree of reducing i n t e n s i t y i s present i n the medium. Diminution of t h i s r e d u c i n g i n t e n s i t y by oxygen or by o x i d i z i n g agents such as f e r r i c y a n i d e , t h i o n i n e , or methylene blue caused i n h i b i t i o n of growth u n t i l the agent has been reduced. Anaerobic metabolism was shown to i n v o l v e the presence of reduced substances i n the n u t r i e n t s or at l e a s t freedom from o x i d i z i n g substances. A s i m i l a r 73 i n h i b i t i n g e f f e c t may be observed with aerobes when d i f f u s i b l e dyes such as t h i o n i n e or methylene blue are present i n s u f -f i c i e n t c o n c e n t r a t i o n to impose a e r o b i c c o n d i t i o n s w i t h i n the c e l l . In 1937 F i l d e s observed that the i n h i b i t i o n o c c u r r e d o n l y with d i f f u s i b l e dyes and not with n o n - d i f f u s i b l e dyes of s i m i l a r o x i d i z i n g c a p a c i t y and concluded t h a t the i n h i b i t i o n was due to o x i d a t i o n of some substance w i t h i n the c e l l which was r e q u i r e d by c e l l metabolism to be reduced. A more recent review of t h i s work ( F i l d e s , 1940) phrases the observa-t i o n as f o l l o w s , " I t , ( i n h i b i t i o n ) was due to an i n t e r f e r e n c e by o x i d a t i o n with an e s s e n t i a l m e t a b o l i t e which r e q u i r e d to be reduced i n order that the enzyme a s s o c i a t e d with i t might f u n c t i o n normally as i n growth." F i l d e s d u r i n g t h i s p e r i o d had been a l e a d i n g worker i n the i s o l a t i o n of e s s e n t i a l m e t a b o l i t e s and, i n p a r t i c u l a r , had shown that s u l f h y d r y l groups were necessary i n the meta-bol i s m of s t a p h y l o c o c c i ( F i l d e s and Richardson, 1937)* V o l -kansky, (1932) had a l s o p r e v i o u s l y shown the -SH group t o be e s s e n t i a l i n the metabolism of moulds. The c o n s i d e r a t i o n , by F i l d e s , o f the p o s s i b i l i t y t h a t the a n t i b a c t e r i a l a c t i o n of mercury was due to the combination of th a t metal with e s s e n t i a l s u l f h y d r y l groups w i t h i n the c e l l r e s u l t e d i n a s e r i e s of experiments i n which he showed t h a t mercury combined 74 with -SH i n the t e s t tube i n the proper molecular p r o p o r t i o n s to form a s o l u b l e compound devoid of -SH. T h i s compound was not i t s e l f i n h i b i t o r y s i n c e i n h i b i t i o n was only observed when mercury i n a mixture of mercury and -SH was i n excess of the proper molecular p r o p o r t i o n s . I t was concluded t h a t the a n t i b a c t e r i a l a c t i o n of mercury was due to the entrance of the d i f f u s i b l e s a l t i n t o the c e l l and consequent d e p r i v a t i o n of an e s s e n t i a l m e t a b o l i t e . An excess of the e s s e n t i a l m e t a b o l i t e allowed normal c e l l growth. It w i l l be remembered t h a t V o e g t l i n had concluded t h a t a r s e n i c i n the t r i v a l e n t form i s a s p e c i f i c p o i s o n a f f e c t i n g the -SH groups of the protoplasm ( V o e g t l i n , Dyer and Leonard, 1923). F i l d e s by h i s work i n 1940 showed t h a t t h i s mode of a c t i o n was not co n f i n e d to a r s e n i c but was a l s o common to mercury. R e c e n t l y i t has been found t h a t both the trypano-c i d a l a c t i o n and the systemic t o x i c i t y f o r animals of t r i -v a l e n t a n t i m o n i a l s such as t a r t a r emetic and antimony t h i o -g l y c o l l a t e are antagonized by c y s t e i n e . The t o x i c i t y of pentavalent a n t i m o n i a l s i s not a f f e c t e d (Chen, G e r l i n g and MacHatton, 1945). I t may be considered as f i r m l y e s t a b l i s h e d , then, that the t o x i c e f f e c t of metal complexes upon l i v i n g c e l l s i s antagonized by n a t u r a l l y o c c u r r i n g s u l f h y d r y l compounds (Work and Work, 1948). The F i l d e s t h e o r y ( F i l d e s , 1940) i n essence suggests t h a t o r g a n o m e t a l l i c compounds are t o x i c 75 because they deprive c e l l s of t h e i r e s s e n t i a l s u l f h y d r y l -c o n t a i n i n g m e t a b o l i t e s . Work and Work (1948) have another e x p l a n a t i o n f o r the t o x i c i t y of o r g a n o - m e t a l l i c compounds. These authors suggest t h a t i t i s u n l i k e l y t h at the low con-c e n t r a t i o n of such compounds " i n v i v o " would deprive a c e l l of any l a r g e percentage of the a v a i l a b l e e s s e n t i a l m e t a b o l i t e s such as g l u t a t h i o n e and c y s t e i n e . However, some enzymes known as "SH enzymes" have been shown to possess, i n the undenatured s t a t e , s e v e r a l exposed s u l f h y d r y l groups which are a b s o l u t e l y e s s e n t i a l f o r enzyme a c t i v i t y . I t i s suggested that the " s u l f h y d r y l enzymes" of which about t h i r t y are now recogn i z e d (Barron and S i n g e r , 194-3) may be i n a c t i v a t e d by the chemical combination of s u l f h y d r y l and m e t a l l i c r a d i c l e s . T h i s e x c e p t i o n to F i l d e s * t h e o r y i s p o o r l y taken s i n c e he s t a t e s c l e a r l y that the c e l l i s deprived of e s s e n t i a l meta-b o l i t e s , an a c t i o n e q u a l l y a p p l i c a b l e i n the case of enzyme i n h i b i t i o n . N e i t h e r the concept of enzyme i n h i b i t i o n or the con-cept of the s i t e of a t t a c k by the drugs on the enzymeB i s new (Jacoby, 1916)(Rona et a l . , 1920-23) but the proof of the s i t e of a t t a c k awaited the work on c e l l metabolism o r i g i n a t i n g l a r g e l y with Quastel's experiments on the e f f e c t o f a n t i -s e p t i c s on b a c t e r i a (Quastel et al.(1927) (1931)(1932)). T h i s work was extended by enzyme chemists such as Barron (1943) and P e t e r s (1945). 76 The work of Stamp (1939) and l a t e r of F i l d e s (1940) on the mode of a c t i o n of s u l f a n i l a m i d e , a b a c t e r i o s t a t i c drug which previous to t h i s time had been developed on a more or l e s s e m p i r i c a l b a s i s , showed i t to p a r a l l e l the a c t i o n of o r g a n o m e t a l l i c compounds i n t h a t fundamentally i t s a c t i o n was to deprive the c e l l of an e s s e n t i a l m e t a b o l i t e . Para-aminobenzoic a c i d was shown to antagonize e f f e c t i v e l y the a c t i o n of s u l f a n i l a m i d e ( K i t t s , 1949)* I t i s p l a u s i b l e t h a t the antagonism i s the r e s u l t of c o mpetition between the s u l f a n i l a m i d e and the paraaminobenzoic a c i d f o r an enzyme of which the l a t t e r i s the n a t u r a l s u b s t r a t e . It i s t h i s concept of competition between substances f o r the occupancy of s p e c i f i c p o r t i o n s of the a c t i v e s u r f a c e s of the c e l l which has become the f o u n d a t i o n of the theory of modern t o x i c o l o g y and chemotherapeutics. Such a theory i s as e q u a l l y a p p l i c a b l e to the pheno-menon of selenium p o i s o n i n g as i t i s to .sulphonamide therapy and p o s s i b l y even to a n t i b i o t i c a c t i o n (Work and Work, 1948) In b r i e f the s c i e n c e of chemotherapy i s the s c i e n c e of the fundamental r e a c t i o n s of l i v i n g protoplasm. The s o l u t i o n to any problem i n t h i s f i e l d i s t h e r e f o r e i n t i m a t e l y bound up with s o l u t i o n of many other problems i n c e l l p h y s i o l o g y . The problem of the p o i s o n i n g of c a t t l e by s e l e n i f e r o u s p l a n t s i s e s s e n t i a l l y the same t h i n g as the problem of the p o i s o n i n g of trypanosomes by the a r s e n i c a l d e r i v a t i v e s . Chemotherapy 7 ? i s t o x i c o l o g y i n r e v e r s e . T h i s statement i s emphasized by the work of Pe t e r s i n a r s e n i c t o x i c o l o g y and t h e r a p e u t i c s ( P e t e r s , Stocken and Thompson, 1945) which was adequately .reviewed i n the e a r l i e r work on selenium t o x i c o l o g y ( D e r r i c k , 1948). To r e i t e r a t e b r i e f l y , P e t e r s * work was based on the assumption t h a t the r a i s e d blood pyruvate l e v e l s observed i n a r s e n i c a l p o i s o n i n g were the r e s u l t of the i n h i b i t i o n of the enzyme pyruvate oxidase. I t was a l s o known that -SH groups were e s s e n t i a l f o r the a c t i v i t y of the pyruvate oxidase system and t h a t s u l f h y d r y l compounds such as g l u t a t h i o n e a f f o r d e d some degree of p r o t e c t i o n a g a i n s t a r s e n i c a l s . The sum t o t a l of evidence t h e r e f o r e p o i n t e d to the p o s s i b i l i t y t h a t a r s e n i c a l s s e l e c t i v e l y i n h i b i t e d the pyruvate oxidase system by i n a c t i v a -t i o n of i t s s u l f h y d r y l groups. With t h i s p o s s i b i l i t y i n mind P e t e r s proceeded t o t e s t the a b i l i t y of v a r i o u s t h i o l compounds to compete a c t i v e l y with the -SH groups of the enzyme f o r the a r s e n i c . 1:2-Di-t h i o l s were shown to be much more e f f e c t i v e than monothiols i n r e v e r s i n g the i n h i b i t o r y a c t i o n of a r s e n i c . The work u l t i m a t e l y r e s u l t e d i n the pr o d u c t i o n o f BAL, or B r i t i s h A n t i - L e w i s i t e ( i . e . , 2-3 dimercapto-propanol) which i s a powerful a n t i d o t e t o p o i s o n i n g by a r s e n i c and some other metals (McDonald, 1946)(Ryder, Cholak and Kehoe, 1947). 78 The. d e t o x i c a t i n g e f f e c t i s due to speedy formation of a r a p i d l y excreted c y c l i c compound, by r e a c t i o n of the v i c i n a l -SH groups of the d i t h i o l w i t h the t o x i c m e tal. Thus again the concept of competition i s i n t r o d u c e d but i n t h i s case the antagonism i s between the enzyme s u r f a c e and the d i t h i o l f o r p o s s e s s i o n of the t o x i c group. T h i s new development does not f a l l w i t h i n the o l d e r , g e n e r a l l y accepted connotation of chemotherapy f o r the reason t h a t i t i s not d i r e c t e d a g a i n s t an i n f e c t i o n . However, i t i s o f f e r e d as a model of the type of neat and c o n c i s e r e a s o n i n g which i s a r a d i c a l departure from the e m p i r i c a l methods of the past, when chemotherapy i n v o l v e d the t r i a l of endless v a r i a n t s of a chemical type p r e v i o u s l y found by chance t o possess some t h e r a p e u t i c powers. The d i s c o v e r y of the t o x i c e f f e c t of selenium on the enzyme su c c i n o x i d a s e ( C o l l e t , Rheinberger and L i t t l e , 1 9 3 3 )> and the subsequent work of P o t t e r and Elvehjem), ( 1 9 3 6 ) , B e r s i n , ( 1 9 3 5 ) and Wright (1940) p o i n t i n g to the e l i m i n a t i o n of an e s s e n t i a l m e t a b o l i t e i n a reduced form and the i n h i b i t i o n of enzymes known t o r e l y on s u l f h y d r y l groups f o r t h e i r a c t i v i t y (Barron and S i n g e r , 1 9 4 3 ) ,was d i s c u s s e d i n the previous work of the w r i t e r on selenium t o x i c o l o g y ( D e r r i c k , 1948). A l s o d i s c u s s e d was the work of Barron (Barron and K a l n i t s k y , 1947) on the t o x i c i t y of c e r t a i n metals ( i n c l u d i n g selenium) to the s u c c i n o x i d a s e system and the r e a c t i v a t i o n of 7? the system by d i t h i o l s . T h i s l a t t e r work r e v e a l e d by the method of " i n v i t r o 1 1 experimentation t h a t many metals i n h i b i t the enzymatic a c t i v i t y of s u c c i n o x i d a s e . That t h i s i n h i b i -t i o n was almost c e r t a i n l y due to a r e a c t i o n with the -SH groups of the p r o t e i n moiety was shown by the r e v e r s a l of the i n h i b i t i o n secured i n v a r y i n g degrees by the a d d i t i o n of v a r i o u s d i t h i o l s ( m o d i f i c a t i o n s of the o r i g i n a l BAL of P e t e r s ) . Selenium i n h i b i t e d s u c c i n o x i d a s e was not r e a c t i v a -t e d by the d i t h i o l s e i t h e r as r a p i d l y or with the same degree of e f f i c i e n c y as was the case when the enzyme was i n h i b i t e d by heavy metals. The r a t i o of d i t h i o l to selenium r e q u i r e d to produce even 50% r e a c t i v a t i o n was 7 to 1 as compared t o much lower r a t i o s with a f a r higher percentage of r e a c t i v a -t i o n i n the case of the h e a v i e r metals. T h i s phenomenon i n d i c a t e d t h a t the i n h i b i t o r y e f f e c t of selenium on the succ i n o x i d a s e system was q u i t e d i f f e r e n t to t h a t of the hea v i e r metals. Since i t was considered t h a t the response of the h e a v i e r metals to d i t h i o l treatment c o n s t i t u t e d i n d i r e c t evidence of combination with the s u l f h y d r y l groups of the p r o t e i n moiety t o form a metal c y c l i c compound (as e a r l i e r suggested by Pet e r s (1?45) f o r a r s e n i c i n h i b i t o r y a c t i o n ) i t was a l s o c onsidered, due to the l a c k of response to d i t h i o l treatment, that selenium i n h i b i t i o n must occur by some other means. 80 I t was suggested i n the d i s c u s s i o n of t h i s work that the l a c k of success encountered i n the r e a c t i v a t i o n of selenium poisoned succinoxidase by d i t h i o l s might be account-ed f o r by the p o s s i b i l i t y t h a t s e l e n i t e a c t s as an o x i d i z i n g agent. Thus, ra t h e r than combination with the -SH groups of the p r o t e i n moiety, i t might be that any one of the f o l l o w i n g types of o x i d a t i o n takes place as shown by P a i n t e r (1941): 0 1. H-SH + Se0 2 • RS-Se-SR + H 20 2. 4RSH + Se0 2 > RS-Se-SR + RS-SR + 2H 20 3. 4RSH + Se0 2 -> 2RS-SR + Se + 2H g0 In such a case the p a r t i a l r e a c t i v a t i o n encountered could occur as the r e s u l t of the reducing power of the d i -t h i o l s . I t i s h i g h l y probable that hexavalent selenium i s converted to the t e t r a v a l e n t form i n much the same manner as the pentavalent to t r i v a l e n t conversion of a r s e n i c noted e a r l i e r i n t h i s d i s c u s s i o n . The lower t o x i c i t y of the s e l e n -ates ( D e r r i c k , 1948) could be explained by the e x c r e t i o n of some of the higher valence form before the w l n v i v o " valence change i s completed. Such o x i d a t i o n r e a c t i o n s could, i n view of the discovery of an amino a c i d of the form H00G-0_NH2-CH2-S-Se-S-CHg-OHNHg-GOOH (selenium d i c y s t e i n e ) by P a i n t e r (1941), be considered as w e l l w i t h i n the bounds of p r o b a b i l i t y since i t not only conforms wi t h the type 2 r e a c t i o n but a l s o allows f o r p a r t i a l r e a c t i v a t i o n by a d i t h i o l . 8 1 Another h y p o t h e s i s , advanced by the w r i t e r , was based on the suggestion of Barron (Barron and S i n g e r , 1 9 4 3 ) t h a t the p o s i t i o n of the -SH groups i n the p r o t e i n moiety-might be a f a c t o r i n the s p e c i f i c i t y encountered between c e r -t a i n enzymes and p a r t i c u l a r i n h i b i t o r y agents. I t was f e l t t h a t s i n c e the d i s t a n c e apart of the -SH groups appears to be a f a c t o r i n the r e a c t i o n of the p r o t e i n s u l f h y d r y l groups with a p a r t i c u l a r i n h i b i t o r y agent i t i s e n t i r e l y p o s s i b l e t h a t a s i m i l a r type r e l a t i o n s h i p must a l s o e x i s t between the i n h i b i t o r y agent and the s u l f h y d r y l groups of a t h i o l com-pound i f r e a c t i v a t i o n was to occur. In f a c t , the circum-stances r e q u i r e d f o r chemical combination t o occur between the i n h i b i t o r y agent and the t h i o l compound must be more f a v o r a b l e than those encountered i n the p r o t e i n - i n h i b i t o r y agent r e l a t i o n s h i p (e.g., the formation of the more s t a b l e c y c l i c compound i n the As-BAL r e a c t i o n ) . Such a th e o r y c a l l s f o r the use of a s p e c i f i c t h i o l compound f o r a s p e c i f i c i n h i b i t o r y agent. Whether the l a t e r work of Barron (Barron and K a l n i t s k y , 1 9 4 7 ) on the " i n v i t r o " e f f i c i e n c y of v a r i o u s d i t h i o l s as r e a c t i v a t i n g agents f o r s u c c i n o x i d a s e poisoned by a number of d i f f e r e n t i n h i b i t o r y metals was based on the f o r e g o i n g theory, or was simply an e m p i r i c a l a t t a c k on the problem, i s not known. However, the d i f f e r e n c e s i n the i n -h i b i t o r y powers of v a r i o u s metals and i n the r e s u l t s obtained by the use of v a r i o u s d i t h i o l s i n Barron's i n v e s t i g a t i o n s do tend to l e n d credence to the t h e o r y . 8 2 Such an approach to the design of new chemotherapeutic agents holds the key not only to means of therapy but also to the mechanism of the poisoning by the p a r t i c u l a r i n h i b i t o r y agent. Knowledge of the chemical configuration of a success-f u l r e a c t i v a t i n g agent would shed further l i g h t on the "mode of attack" of the i n h i b i t i n g agent. In the case of the selenium problem now under i n v e s t i -gation, the s l i g h t promise exhibited by e a r l i e r therapeutic administration of BAL (Derrick, 1948) was, i n the l i g h t of Barron's work (Barron and Kalnitsky, 1947) considered possibly to be due to the variations often observed between " i n vitro'* and " i n vivo" work. It was f e l t that BAL might, under " i n vivo" conditions, be s u f f i c i e n t l y s p e c i f i c to reactivate, at least p a r t i a l l y , the inhibited enzymes of selenium poisoned animals. In addition the promising results obtained i n the " i n vivo" r e a c t i v a t i o n by BAL of "sulfhydryl enzyme" systems inh i b i t e d by a wide var i e t y of agents such as mercury salt s (Stocken, 1947), gold s a l t s (Swanson et a l . , 1947), copper (MacDonald, 1946), coumarin and protoanemonin (in plants)(Thimann and Bonner, 1949) indicated that the speci-f i c i t y of the t h i o l compound might be more e l a s t i c i n l i v i n g animals or plants. The t o x i c i t y of BAL has afforded considerable d i f f i c u l t y i n investigations of i t s effectiveness as a thera-py for " s u l f h y d r y l enzyme" i n h i b i t i o n poisoning " i n v i t r o " . 85 Graham and Hood i n 1948 conducted a survey of the e f f e c t s of the t h i o l compound and found i t to have an L.D #^ 0 o f 1 0 0 mgm. per Kg. when 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 i n mice. Pet e r s i n a p e r s o n a l communication gave the L.D._rt f o r r a t s as 110 mgm. per Kg. by the same ro u t e . In mice poisoned with a r s e n i c , mercury, antimony, or chromium BAL. exerts a p r o t e c t i v e e f f e c t whereas i n mice poisoned with gold? bismuth or l e a d i t e x e r t s a d e l e t e r i o u s e f f e c t (Graham and Hood, 1948). I t would appear that what may be an optimum t h e r a p e u t i c dosage of BAL i n one type of p o i s o n i n g may be e i t h e r an i n e f f e c t i v e or d e l e t e r i o u s dosage i n another type of p o i s o n i n g . The formation of BAL-metal complexes i n the d e t o x i f y i n g mech-anism of BAL i s thought to produce anomalies i n which the complexes may be absorbed more q u i c k l y , or be more t o x i c than the m e t a l l i c s a l t s themselves. The t o x i c p r o p e r t i e s of BAL, as w e l l as the v a r i a -b i l i t y of e f f e c t s , suggest t h a t some other r e l a t e d compound such as the BAL«glucoside of - D a n i e l l i and D a n i e l l i (194?) would be t h e r a p e u t i c a l l y p r e f e r a b l e . However, at the time of the present i n v e s t i g a t i o n t h i s compound was not a v a i l a b l e . As a p o s s i b l e a l t e r n a t i v e to BAL i t was suggested by A l l a r d y c e , during the course of d i s c u s s i o n s p r i o r t o the i n i t i a t i o n of t h i s second study, t h a t the t h e r a p e u t i c admini-s t r a t i o n of the h y d r o l y s a t e s of p r o t e i n s h i g h i n s u l f h y d r y l c o n t a i n i n g amino a c i d s might be of value i n selenium sNo e x p l a n a t i o n can be given f o r the apparent disagreement e x i s t i n g between the statements of Graham and Hood (1948) and Swanson et a l . (1947). 34 p o i s o n i n g . A subsequent study of the l i t e r a t u r e r e v e a l e d t h a t somewhat s i m i l a r t h e r a p e u t i c measures i n v o l v i n g the a d m i n i s t r a t i o n of g l u t a t h i o n e , c y s t e i n e , e t c . had been a t -tempted i n cases of selenium and other m e t a l l i c poisonings but had l a c k e d success probably because the presence of these s u l f h y d r y l compounds d i d not r e s u l t i n the formation of a more s t a b l e , more r a p i d l y e x c r e t e d compound of the As-BAL, « type. In view of subsequent o b s e r v a t i o n s with r e f e r e n c e to the i n a n i t i o n c o m p l i c a t i o n s of chronic selenium p o i s o n i n g , however, i t i s unfortunate that the work has not yet reached the p o i n t where the p r o t e i n h y d r o l y s a t e therapy might be a p p l i e d since, i n a l l p r o b a b i l i t y , s u c h treatment might be of v a l u e . The a d m i n i s t r a t i o n of the p r o t e i n h y d r o l y s a t e would most c e r t a i n l y speed the recovery from i n a n i t i o n and, i n a d d i t i o n , the h i g h i n c i d e n c e of s u l f h y d r y l amino a c i d s might w e l l serve as a means of replacement f o r the selenium i n h i b i -t e d amino a c i d s of the p r o t e i n moiety of the a f f e c t e d enzyme systems, i f such seleno-amino a c i d complexes a c t u a l l y e x i s t . A f u r t h e r a p p l i c a t i o n of chemotherapy to the selenium problem suggested by Wood, and c o n s i d e r e d to h o l d promise, i n v o l v e d a new approach to the a r s e n i c therapy of Moxon (1941, 1943, 1945). Moxon showed t h a t the i n c l u s i o n of 3 parts per m i l l i o n of a r s e n i c i n the d r i n k i n g water of dogs fed r a t i o n s c o n t a i n i n g as much as 1 3 p.p.m. selenium i n the n a t u r a l form was e f f e c t i v e i n c o u n t e r a c t i n g or p r e v e n t i n g the 85 symptoms of c h r o n i c selenium p o i s o n i n g . In experiments on r a t s the a r s e n i c was of l i t t l e value i n t r e a t i n g animals which had been f e d selenium f o r more than t h i r t y days but was an e f f e c t i v e therapy f o r animals which had been fed selenium f o r not more than 20 days be f o r e treatment s t a r t e d . V a r i o u s t h e o r i e s f o r the e f f e c t i v e n e s s of the a r s e n i c were put forward by Moxon i n h i s d i s c u s s i o n of the work, the most notable being that of the i n h i b i t i o n of the a b s o r p t i o n of selenium from the g a s t r o - i n t e s t i n a l t r a c t . However, on the b a s i s of the a p p l i c a t i o n of the newer aspects of chemotherapy to the problem i t i s f e l t that the probable mechanism i s e i t h e r one of gradual replacement of the bound selenium by a r s e n i c which i n t u r n may be more e a s i l y d i s p l a c e d d u r i n g the c o n t i n u a l r e o r g a n i z a t i o n r e s u l t i n g from the dynamic nature of p r o t e i n or a simple case of the formation of a s e l e n i u m - a r s e n i c complex which may be f a i r l y r a p i d l y e x c r e t e d . Whatever the mechanism may be t h e r e i s no doubt t h a t the known e f f e c t i v e n e s s of BAL as a means of r e l i e v i n g a r s e n i c t o x i c i t y o f f e r s the p o s s i b i l i t y of a new chemotherapeutic approach t o the selenium problem. I t may w e l l be that a r s e n i c could be used i n doses h e r e t o f o r e p r o h i b i t i v e to r e l i e v e the t i s s u e s of selenium and t h a t BAL could, i n t u r n , be used to r e l i e v e the t i s s u e s of a r s e n i c . 86 IY. EXPERIMENTAL CHEMOTHERAPY The Use of BAL as a Means of T r e a t i n g Sub- acute Selenium P o i s o n i n g i n the Rat. Experiment C Procedure (General) The experimental procedure was e s s e n t i a l l y the same . as f o r Experiment B. An e m u l s i f i e d 1:3 mixture of aqueous sodium s e l e n i t e s o l u t i o n and 2% aluminum-monostearate i n peanut o i l was used,as i n Expt. B j , s i n c e i t was f e l t t h a t any p o s s i b l e delay i n the a b s o r p t i o n of the selenium would be of value i n view of the r e l a t i v e l y slow a b s o r p t i o n of BAL i n peanut o i l . The dosage l e v e l s of sodium s e l e n i t e were a l s o as f o r Expt. B^; 6 mgm. N a 2 S e 0 j / K g . body weight f o r male r a t s and 3 mgm. N a 2 S e 0 j / K g . body weight f o r female r a t s . BAL i n peanut o i l , d i l u t e d from the 10% commercial p r e p a r a t i o n (BAL(2,3-dithiopropanol) 10% Benzyl Benzoate 20% i n Peanut O i l . The Hynson, Westcott and Dunning Labora-t o r i e s , B a l timore) t o a 0.37» s o l u t i o n with a d d i t i o n a l peanut o i l was i n j e c t e d i n t o the animals subcutaneously (as deep as p o s s i b l e ) at the f o l l o w i n g times and dosage l e v e l s : Immediately f o l l o w i n g the ... 3 mgm. BAL/Kg. body i n j e c t i o n of the sodium weight s e l e n i t e 3 hours a f t e r the f i r s t ..... 2.5" mgm. BAL/Kg. body i n j e c t i o n weight 6 hours a f t e r the f i r s t 2.5 mgm. BAL/Kg. body i n j e c t i o n weight 8 7 Expt. 0^ Procedure Four male r a t s and 4 female r a t s aged 128 days were i n j e c t e d with sodium s e l e n i t e and . BAL , i n o i l a c c o r d i n g to the general procedure d e s c r i b e d above. The animals were then p l a c e d i n metabolism u n i t s and su b j e c t e d t o the ge n e r a l pro-cedure of Experiment B. R e s u l t s Symptoms of acute selenium t o x i c i t y were apparent i n both groups but a few exceptions t o the ge n e r a l p i c t u r e were noted. There appeared to be c o n s i d e r a b l y l e s s odor of " s e l -enium breath" emanating from the animals than i s u s u a l l y the case. In a d d i t i o n the animals, although o b v i o u s l y unwell, were l e s s s e r i o u s l y a f f e c t e d by the u s u a l symptoms of p r o s t r a -t i o n , r e s p i r a t o r y d i s t r e s s and o c c a s i o n a l cases of d i a r r h o e a w i t h i n 3 or 4 hours a f t e r the selenium i n j e c t i o n than they were about 6 t o 8 hours a f t e r the i n j e c t i o n . At t h i s time, however, the symptoms were i n t e n s i f i e d and a l l the animals i n both groups were very i l l . In the male group two of the animals died w i t h i n 21 hours of the selenium i n j e c t i o n ; another animal died at 29 hours and the f i n a l death occurred w i t h i n 46 hours of the i n j e c t i o n . In the female group one animal d i e d 29 hours a f t e r the selenium i n j e c t i o n and another animal died at 47 hours. The two s u r v i v o r s showed a very gradual improvement i n c o n d i t i o n s t a r t i n g on the t h i r d day but very l i t t l e i n c l i n a -t i o n toward e a t i n g or a c t i v i t y of any k i n d was noted u n t i l the 7th day a f t e r i n j e c t i o n . On t h i s day they were destroyed. Weight Changes Ave. wt. of) s u r v i v o r s )at time of i n j e c t i o n " 11 1 day a f t e r i n j e c t i o n " 2 days a f t e r i n j e c t i o n " » 3 days » « n 4 d a y s ti " n 5 days " »» it 6 days " n i> , 7 days " Female 18? 180 17 3 167 161 157 156 161 gms . gms. gms. gms. gms. gms. gms. gms. U r i n a r y Selenium E x c r e t i o n U r i n a r y E x c r e t i o n of) Se per Kg. of Sur- ) d u r i n g 1st 24 hours v i v o r s ) a f t e r i n j e c t i o n " 11 during 2nd 24 hours a f t e r i n j e c t i o n T o t a l e x c r e t i o n of) Se per Kg. of s u r - j v i v o r s T o t a l e q u i v a l e n t I e x c r e t i o n of ) Na 2Se0j per Kg. of) s u r v i v o r s ) during a f t e r d u r i n g a f t e r d uring a f t e r d u r i n g a f t e r d uring a f t e r 3rd 24 hours i n j e c t i o n 4th 24 hours i n j e c t i o n 5th 24 hours i n j e c t i o n 6th 24 hours i n j e c t i o n 7th 24 hours i n j e c t i o n Male 392 Y d u r i n g e n t i r e p e r i o d Female 455 y 274 jr 144 x 198 1 259Y 0 0 1330>-2.9 mgm. Expt. Cp Four male r a t s and 4 female r a t s aged 137 days were subjected to a procedure s i m i l a r i n a l l r e s p e c t s to that of Expt. c 1 . 8 9 R e s u l t s In both groups the symptomatology of the animals-resembled e n t i r e l y the c o n d i t i o n of the animals of Expt. C Three of the male r a t s d i e d w i t h i n 22 hours and the f o u r t h animal died w i t h i n 48 hours of the o r i g i n a l i n j e c t i o n . In the female group three of the animals a l s o died w i t h i n 22 hours. The f o u r t h animal was extremely i l l f o r 3 days a f t e r the o r i g i n a l i n j e c t i o n but had recovered s u f f i c i e n t l y by the 5th day to eat a l i t t l e food. By the 7th day the animal was i n q u i t e good c o n d i t i o n . On t h i s day i t was destroyed. Weight Changes Wt. of Female) S u r v i v o r )at time of i n j e c t i o n 205 gms. " 2 days a f t e r i n j e c t i o n 185 gms. " 2 days a f t e r i n j e c t i o n 175 gms. " 4 days a f t e r i n j e c t i o n 170 gms. " 5 days a f t e r i n j e c t i o n 175 gms. " 7 days a f t e r i n j e c t i o n 180 gms. U r i n a r y Selenium E x c r e t i o n U r i n a r y E x c r e t i o n of) K Se per Kg. of Sur- )during 1st 24 hours 307 Y v i v o r s ) " « d u r i n g 2nd 24 hours K260 v « « d u r i n g 3rd 24 hours 183 Y" M " d u r i n g 4th 24 hours 204v « « d u r i n g 5th 24 hours 146 Y " " d u r i n g 6th 24 hours 73v " " d u r i n g 7th 24 hours 73r T o t a l e x c r e t i o n of) during e n t i r e p e r i o d 1246 v Se per Kg. of sur-) v i v o r ) T o t a l e q u i v a l e n t ) w tt " 2«7 mgm. e x c r e t i o n of Na2Se0j) per Kg. of s u r v i v o r ) • i n a c c u r a c y here i s probably high due t o l a r g e number of deaths i n f i r s t 24 hours and to an u n w i l l i n g n e s s to subject the s u r v i v o r to moving t o a c l e a n cage f o r the second 24 hours. A D i s c u s s i o n of the Use of BAL as a Means of T r e a t i n g Sub- acute Selenium P o i s o n i n g i n the Rat The two experiments have e s t a b l i s h e d beyond doubt that the a d m i n i s t r a t i o n of BAL to ratB s u f f e r i n g from acute or sub-acute selenium p o i s o n i n g i s dangerously d e l e t e r -ious t o the c o n d i t i o n of the animals. Whether t h i s e f f e c t i s the r e s u l t of the formation Of a more t o x i c Se-BAL complex of the type suggested by Graham and Hood (1948), or whether i t i s the r e s u l t of the f a c t that BAL i s i t s e l f i n h i b i t o r y t o the s u c c i n o x i d a s e system and thus may exert an a d d i t i o n a l t o x i c a c t i o n on an 91 a l r e a d y s e r i o u s l y i n c a p a c i t a t e d system, would be d i f f i c u l t t o determine. In view of the apparent tendency of BAL to promote the e x c r e t i o n of selenium (see graph, p. 92) f o r a longer p e r i o d of time the former p o s s i b i l i t y i s probably more l i k e l y than the l a t t e r . I t would be expected i n the case of simultaneous i n j e c t i o n s of selenium and BAL t h a t the for m a t i o n of a t h e o r i z e d more r a p i d l y excreted s t a b l e complex would r e s u l t i n a more r a p i d e x c r e t i o n of selenium than i s the case when selenium i s administered alone. That such a c t i o n was not observed tends t o obvi a t e t h i s p o s s i b i l i t y and r a t h e r f a v o r s the idea of the form a t i o n of a more e a s i l y absorbed Se-BAL complex which i s h e l d w i t h i n the t i s s u e s f o r a lo n g e r p e r i o d of time and i s t h e r e f o r e r e l e a s e d over a longer p e r i o d . The f a c t t h a t BAL i n c r e a s e d the t o t a l amount of selenium excreted may or may not be s i g n i f i c a n t under these c o n d i t i o n s . The Se-BAL complex, w h i l e i t may be more t o x i c immediately, may a l s o be more e a s i l y r e l e a s e d from the t i s s u e s of an animal a b l e t o withstand i t s t o x i c i t y than i s the selenium i t s e l f . On the other hand i t may be t h a t m o b i l i -z a t i o n of the selenium from the t i s s u e s by the BAL s t i l l a v a i l a b l e a f t e r the i n i t i a l t o x i c i t y has been overcome may be delayed f o r some reason at present unaccountable. In such an event the f u r t h e r a d m i n i s t r a t i o n of BAL l a t e r on would appear to be necessary. Since the experimentation has y i e l d e d some q u i t e 95 p o s i t i v e r e s u l t s , however, i t i s f e l t that the present s i t u a -t i o n c a l l s f o r a c t i o n r a t h e r than hypothesis and i t i s s i n -c e r e l y hoped t h a t the techniques developed i n t h i s work w i l l be u t i l i z e d i n f u r t h e r i n v e s t i g a t i o n s . The problem may be sol v e d to a c o n s i d e r a b l e degree by the a d m i n i s t r a t i o n of BAL-Intrav., (the BAL g l u c o s i d e of D a n i e l l i et a l . , 1947) i n p l a c e of BAL. In a d d i t i o n the simultaneous a d m i n i s t r a -t i o n of s u c c i n a t e which has proved t o be of p r o t e c t i v e value i n " i n v i t r o " experiments with a r s e n i t e , t e l l u r i t e , s e l e n i t e and t a r t a r emetic (Bergstermann and Mangier, 1948) with BAL or BAL-Intrav. might be of v a l u e . The f u n c t i o n of the s u c c i n a t e administered i n c o n j u n c t i o n with the t h i o l com-pound would be that of a "pump primer" t o c a r r y through the metabolic c y c l e d u r i n g the p e r i o d of i n c a p a c i t a t i o n of the enzyme system. The a d m i n i s t r a t i o n of the s u c c i n a t e might w e l l be a s u c c e s s f u l treatment of i t s e l f s i n c e i t could probably serve t o c a r r y a su b - a c u t e l y poisoned animal over the c r i t i c a l p e r i o d of t o x i c i t y . On the other hand t h i s compound co u l d not be considered as capable of m o b i l i z i n g selenium. For t h i s reason i t i s considered t h a t a combination of the mode of a c t i o n of the s u c c i n a t e and that of the t h i o l might w e l l r e -s u l t i n the evolvement of an e f f i c i e n t therapy not only of selenium p o i s o n i n g but of other s i m i l a r t o x i c i t i e s as w e l l . 94 Y . SUMMARY AND CONCLUSIONS 1 . In the f i e l d of t o x i c o l o g y i t i s customary to r e c o g n i z e f o r the purposes of c l a s s i f i c a t i o n , s e v e r a l t o x i c s t a t e s 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 of poisonous m a t e r i a l s t o animals. The acute s t a t e of i n t o x i c a t i o n r e f e r s to a complete breakdown of normal p h y s i o l o g i c a l processes r e s u l t i n g , u s u a l l y , i n the death of the animal. Such a c o n d i t i o n i s b e l i e v e d t o a r i s e , i n the case of m e t a l l i c and s i m i l a r l y a c t i n g poisons, as a r e s u l t of mass blockage of s e v e r a l enzyme systems. Acute p o i s o n i n g i s nor m a l l y caused by s i n g l e l a r g e doses of the t o x i c m a t e r i a l . The dosage of selenium r e q u i r e d to produce the a c u t e l y t o x i c s t a t e i n v a r i o u s s p e c i e s ( i . e . L.D.^ Q or gre a t e r ) i s presented i n Appendix I I I . The symptomatology of t h i s s t a t e has been d e s c r i b e d i n d e t a i l i n an e a r l i e r r e p o r t ( D e r r i c k , 1 9 4 8 ) . I t s u f f i c e s here to repeat that the a c u t e l y s e l e n i z e d animal, immediately a f t e r i n j e c t i o n and on post mortem examination, shows a l l the s i g n s of shook t h a t would be expected to occur as a r e s u l t of mass enzyme i n h i b i t i o n . The sub-aoute s t a t e of i n t o x i c a t i o n i s , as i n f e r r e d by the e x p r e s s i o n , a s l i g h t l y l e s s severe c o n d i t i o n than the acute s t a t e and one from which the animal normally Just r e c o v e r s i f no secondary involvement a r i s e s . I t i s sug-g e s t i v e of a p a r t i a l blockage of one or more enzyme systems, 95 a blockage which, disappears on removal of the e x c i t i n g cause. Sub-acute s e l e n o s i s has been produced i n animals i n the present work. The c h r o n i c s t a t e i s the l e a s t r e a d i l y r e c o g n i z a b l e s t a t e of i n t o x i c a t i o n . I t r e f e r s t o a c o n d i t i o n of impaired p h y s i o l o g i c a l a o t i v i t y induoed by the p o i s o n . However, the symptoms noted may, or may not, be d i r e c t l y a t t r i b u t a b l e to the s p e c i f i c e f f e c t s of the t o x i c m a t e r i a l on metabolic systems. The chronic c o n d i t i o n i s u s u a l l y achieved by the a d m i n i s t r a t i o n of low l e v e l s of a poison f o r long p e r i o d s of time. When the means of a d m i n i s t r a t i o n i s d i e t a r y v o l -u n t a ry r e s t r i c t i o n of food and water intake may occur. In such casesthe r o l e of i n a n i t i o n i n producing p a t h o l o g i c a l changes i n the exposed animals o f t e n goes unrecognized. The experimental evidence of the present work i n selenium t o x i c o l o g y i s such as to cast grave doubts on the a u t h e n t i c i t y of some aspects of the work of previous i n v e s t i -gators i n t h i s f i e l d . The response of the l a b o r a t o r y animals, s u b j e c t e d t o the v a r i o u s procedures p r a c t i s e d by these i n v e s t i g a t o r s i n attempts t o produce a c o n d i t i o n o f c h r o n i c selenium p o i s o n i n g , suggests t h a t these animals are, i n a d d i t i o n to being the v i c t i m s of selenium p o i s o n i n g , the v i c t i m s of i n a n i t i o n and/or d e h y d r a t i o n . While i t has been p r e v i o u s l y admitted t h a t the low food i n t a k e of such animals was i n part r e s p o n s i b l e f o r t h e i r c o n d i t i o n i t i s f e l t t h a t the anor e x i a e x h i b i t e d by them 96 was too f r e e l y a s c r i b e d t o the e f f e c t s of the poison r a t h e r than to the presence of the poison i n the d i e t . T h i s l a t t e r reason i s made more v a l i d by the immediate response of animals p r e v i o u s l y e x h i b i t i n g a n o r e x i a , while s u b j e c t e d t o a d i e t c o n t a i n i n g s e l e n i z e d food or water, when a selenium f r e e d i e t i s made a v a i l a b l e * The a v e r s i o n to a s e l e n i f e r o u s d i e t ( e i t h e r n a t u r a l l y or a r t i f i c i a l l y s e l e n i z e d ) i s obvious i n l a b o r a t o r y r a t s from the f i r s t day of e x p e r i m e n t a t i o n . In view of these o b s e r v a t i o n s the a p p a r e n t l y normal a p p e t i t e of dogs r e c e i v i n g s e l e n i f e r o u s food and, s i m u l -t a n e o u s l y , 5 p.p.m. a r s e n i c i n the d r i n k i n g water (Moxon, 1943, 1945) i s i n e x p l i c a b l e s i n c e i t would appear t h a t most experimental animals are a b l e , i n some manner, t o sense the t o x i c i t y of the food. 2. The presence of up to 15 p.p.m. sodium s e l e n i t e (6,9 p.p.m. Se) i n the d r i n k i n g water i s t o l e r a t e d without any d r a s t i c r e d u c t i o n i n food and water i n t a k e by l a b o r a t o r y r a t s , pro-v i d i n g t h i s c o n c e n t r a t i o n i s reached g r a d u a l l y . At t h i s l e v e l of selenium intake the animals w i l l remain f o r at l e a s t 25 weeks i n good c o n d i t i o n and w i l l e x h i b i t growth onl y s l i g h t l y l e s s r a p i d than t h a t of c o n t r o l animals. Any attempts t o r a i s e the selenium content above t h i s l e v e l r e s u l t i n a compensatory lo w e r i n g of the water intake and a r e s u l t a n t r e d u c t i o n i n food i n t a k e of the animals. 97 5. The r e s u l t s obtained i n the attempts to induce c h r o n i c selenium p o i s o n i n g by d i e t a r y methods were such as t o cause these methods t o be abandoned due t o t h e i r u n s u i t a b i l i t y f o r the present p r o j e c t . A method i n v o l v i n g the subcutaneous i n j e c t i o n of s u b - l e t h a l doses of sodium s e l e n i t e i n c o r p o r a t e d i n t o an a b s o r p t i o n d e l a y i n g v e h i c l e was evolved and, a f t e r t e s t i n g , was deemed s u i t a b l e f o r the purpose of i n d u c i n g a sub-acute c o n d i t i o n of selenium t o x i c i t y to be used as a means of a s s e s s i n g the value of chemotherapy. 4. Experimental data on the u r i n a r y e x c r e t i o n l e v e l s of selenium were made a v a i l a b l e by the development of a new c o l o r i m e t r i c procedure f o r the q u a n t i t a t i v e d e t e r m i n a t i o n of selenium i n b i o l o g i c a l samples. The data obtained by t h i s method r e v e a l e d t h a t the maximum q u a n t i t y of selenium ex-cret e d i n the u r i n e or r a t s over a p e r i o d as l o n g as ten days a f t e r i n j e c t i o n was about o n e - t h i r d of the q u a n t i t y o r i g i -n a l l y i n j e c t e d . No p a r t i c u l a r advantage i n the use of the a b s o r p t i o n d e l a y i n g v e h i o l e was d i s c e r n i b l e by t h i s method but s i n c e , under the l i m i t a t i o n s imposed by the q u a n t i t i e s of u r i n e a v a i l a b l e , the delay i n hours would be undetectable the v e h i c l e was r e t a i n e d i n the experiments a s s e s s i n g the value of BAL (2 , 3 - d i t h i o p r o p a n o l ) as a chemotherapeutic agent. 5. The use of BAL as a chemotherapeutic agent f o r combatting selenium t o x i c i t y has a d e l e t e r i o u s e f f e c t on the i n t o x i c a t e d 98 animals. The subcutaneous i n j e c t i o n of q u a n t i t i e s o f BAL i n peanut o i l at l e v e l s considered w e l l w i t h i n the l i m i t s of s a f e t y r e s u l t e d i n an i n t e n s i f i c a t i o n of the d i s t r e s s of selenium i n j e c t e d r a t s . Deaths were more frequent than i n the case of non BAL-treated animals. The animals which s u r v i v e d the e f f e c t s of both poison and treatment showed a s l i g h t l y h i g h e r and a more prolonged e x c r e t i o n of selenium i n the u r i n e . 6. Suggestions have been made f o r improved means of therapy f o r acute and sub-acute selenium p o i s o n i n g , i n v o l v i n g the use of a l e s s t o x i c d i t h i o l compound and the a d d i t i o n of s u c c i n a t e to c a r r y the animal over the i n i t i a l shock t o the a f f e c t e d enzyme systems. 99 APPENDIX I. ( i ) Data Experiment A Table 1 C o n t r o l r a t s - Male Age - 30 days 8 animals Average Wt. 'Water Intake 1 Food Intake ' Week of Weekly Ob- D a i l y Average D a i l y Average Experiment s e r v a t i o n s f o r one week f o r one week — . , , . . i n grams ml. per ml. per gm. per gm. per r a t Kg. r a t Kg.  86.3 - - — - 0 116.2 21.8 187.5 14.5 125.0 1 151.0 24.2 160.0 18.0 119.2 2 178.0 26.2 147.0 I6.3 91.6 3 204.0 27.1 136.0 21.6 106.0 4 233.0 28.5 122.2 19.4 83.3 5 236 ;o 28.5 120.8 18.0 76.4 6 245.0 30.3 123.8 18.0 73.5 7 254.0 29.8 117.3 16.0 63.0 8 257.0 28.0 109.0 18.0 70.0 9 263.0 26.2 99.6 15.6 59.4 10 267.0 27.5 103.0 16.6 62.3 11 267.0 26.4 99.0 16.7 62.5 12 263.0 26.0 ' 98.8 17.0 64.7 13 266.0 25.8 97.0 15.4 58.0 14 268.0 26.0 97.0 14.3 53.4 15 267.O 24.2 90.7 13.5 50.5 16 269.0 24.4 90.7 17 100 ( i i ) Data Experiment A Table 2 C o n t r o l r a t s - Female Age - JO days 8 animals Average Wt, Weekly Ob-se r v a t i o n s Water Intake D a i l y Average f o r one week Food Intake D a i l y Average f o r one week Week of Experiment i n grams m l . p e r ml. per gm. per gm. per ra t Kg. ra t Kg. • 79,? _ _ a . -* 0 104 .0 19.0 182.7 12.5 120.0 1 121.0 20.4 168.5 14 .1 116.5 2 135.0 •20.8 154 .0 15.0 111.0 3 147.4 20.4 138.3 16.0 108;5 4 156.3 20.5 131.5 20.6 131.5 5 165.4 23*4 141 .5 16.9 102.2 6 170.6 23.4 137.0 17.7 103.3 7 176.6 23.9 135.0 17.0 96.7 8 179.6 23.9 133.0 15.9 88.7 9 I69.6 24 .0 133.5 17.5 97.5 10 184*6 23.4 127.0 I6.3 88.3 11 185.7 23.3 125 .3 17.4 93.7 12 185.0 22.8 123.3 15.5 83.9 13 186.7 20.7 111.0 15.9 85.3 14 188.5 24.2 128.5 14.4 76.5 15 192.4 21.1 109.5 13.8 71.8 16 194.4 21.3 109.5 13.6 70.8 17 101 ( i i i ) Data Experiment A Table 3 Experimental r a t s - Male Age - 30 days 43 animals Water Intake Pood Intake'" Na2Se0jIn-D a i l y Average D a i l y Average take Ave. Average Wt. Weekly Ob-Week of E x p e r i -servat ions f o r i Dne week f o r < Dne week f o r one wk. ment i n grams ml. ml. gm. gm. micro- micro-per per per per gram gram rat Kg. r a t Kg. per per r a t Kg. 87.6 - - - _ - - 0 118.6 18.5 125.0 15.3 129.0 99.4 840 1 150.5 19.0 , 126.3 17.3 115.0 209.8 1390 2 176.4 19.2 109.0 16.3 92.5 210.8 1200 3 1??.2 19.6 98.5 18.6 93.5 216.0 1083 4 219,4 20.6 94.0 18.4 84.0 227.0 1035 5 " 233.6 21.5 92.2 . 18.0 77.2 236.3 1012 6 240,6 19.3 . 8o.o 16.9 70.3 212.0 885 7 244.6 19.0 . 77.7 15.7 64.0 209 .0 855 8 24?,1 18.2 73.2 17.5 .70.3 273.0 1095 9 255.0 18.3 72.0 17.3 68.0 '274.0 1077 10 258.6 17.9 69.3 17.9 69.0 268.0 1040 11 260.7 20.0 76.7 18.1 69.3 300.0 1150 12 249.0 20.5 82.4 17.6 70.7 308.0 1210 13 258.0 23.1 89.5 19.3 74.8 347.0 1345 14 257..0 21.1 . 82.3 19.3 75.0 316.0 1233 15 259.0 21.5 83.0 17.5 67.7 323.0 1245 16 260,3 20.3 78.0 14.3 55.0 305.0 1170 17 272.0 19.5 71.7 16.? 62.3 293.0 1075 18 1 0 2 ( i v ) Data Experiment A Table 4 Experimental r a t s - Female Age- JO days 50 animals Water Intake Food Intake N a 2 S e 0 j i n -D a i l y Average D a i l y Average take Ave. f o r one week f o r one week f o r one wk. Average Wt. Weekly Ob-s e r v a t i o n s Week of E x p e r i -ment i n grams ml. ml. gm. gm. micro- micro-per per per per gram gram r a t Kg. r a t Kg. per r a t per K S. 7 6 . 1 *• _ mm — 0 9 6 . 5 1 5 . 9 165 . 5 1 3 . 5 140 . 0 8 4 . 3 875 1 1 1 1 . 2 1 3 . 3 1 2 0 . 0 14 . 5 120 ..3 147 ..0 1 3 2 0 2 1 2 6 . 1 1 3 . 7 1 1 0 . 0 1 5 . 5 122. .8 1 5 1 . 5 1200 3 134.? 1 2 . 9 9 6 . 0 1 5 . 5 1 1 5 . 0 142 . 5 1 0 5 5 4 140 . 5 1 2 . 7 9 1 . 0 14 . 1 1 0 0 . 5 140.5 1000 5 1 5 0 . 1 1 3 . 3 8 9 . 0 1 5 . 0 1 0 0 . 0 146 . 7 ? 8 0 6 1 5 6 . 1 1 2 . 3 7 9 . 2 15 . 3 9 8 . 0 1 3 6 . 0 8 7 0 7 1 5 9 . 7 1 2 . 5 7 8 . 6 14 . 4 9 0 . 7 1 3 8 . 0 865 8 1 5 9 . 7 1 1 . 2 7 0 . 6 1 5 . 8 9 9 . 0 1 6 9 . 0 1 0 6 0 9 1 6 3 . 5 1 1 . 6 7 1 . 2 1 5 . 2 9 3 . 0 1 7 4 . 5 1070 10 1 6 3 . 7 1 1 . 6 7 1 . 3 1 6 . 3 9 8 . 0 1 7 5 . 0 1070 11 1 6 6 . 0 1 2 . 8 7 7 . 0 14 . 0 8 4 . 0 1 9 2 . 0 1155 12 1 6 7 . 0 1 3 . 3 7 9 . 6 1 3 . 6 8 0 . 5 1 9 9 . 5 1 1 9 5 13 169 . 0 1 3 . 2 7 8 . 2 1 2 . 7 8 0 . 6 1 9 8 . 0 1 1 7 0 14 1 7 0 . 0 1 3 . 0 7 6 . 5 1 3 . 0 7 6 . 5 1 9 5 . 0 1 1 5 0 15 1 7 3 . 0 1 3 . 2 7 6 . 4 1 2 . 6 7 8 . 0 1 9 8 . 0 1145 16 174 . 0 1 2 . 9 7 4 . 5 1 2 . 0 69.O 1 9 3 . 5 1 1 1 0 17 103 APPENDIX I I . A Summary of the Attempts to E s t a b l i s h the  Quan t i t y of Selenium E x c r e t e d through the  Lungs of A c u t e l y S e l e n i z e d Animals, Apparatus The apparatus c o n s i s t e d of a Wet Test Gas Meter ( P r e c i s i o n S c i e n t i f i c Co., Chicago) through which a i r was drawn i n t o a 5 l i t e r vacuum d e s i c c a t o r which served as a chamber. Attached by gum rubber t u b i n g i n s e r i e s w i t h the "chamber" were two a b s o r p t i o n towers f i l l e d with g l a s s beads and absorbing s o l u t i o n . A i r was drawn from the chamber and through the a b s o r p t i o n towers by means of a s u c t i o n pump attached t o a'water f a u c e t . The r a t e of flow of a i r was recorded by the wet t e s t gas meter. Procedure 1 Four male r a t s aged 85 days were i n j e c t e d i n t r a -p e r i t o n e a l l y at a dosage l e v e l of 7 mgm. NagSeOj per Kg. of body weight. The animals were immediately p l a c e d i n the chamber and s u c t i o n was a p p l i e d t o the apparatus to draw a i r through at a r a t e of approximately 90 l i t e r s per hour. The a i r temperature v a r i e d from 24°C. to 26°C. durin g the e x p e r i -mental p e r i o d . The t o t a l a i r displacement was 600 l i t e r s . The absorbing s o l u t i o n was 757* of 16.77« H 2 S 0 4 (cone.) 257° of 50y. HC1 ( c o n e ) . T h i s s o l u t i o n i s a d u p l i c a t i o n of the f i n a l mixture obtained when the procedure f o r the c o l o r i -m e t r i c d e t e r m i n a t i o n of selenium i n ur i n e i s f o l l o w e d . * HSee p. t h i s paper. 104 Each of the a b s o r p t i o n towers contained JO ml. of s o l u t i o n . At the end of one hour 10 ml. of f r e s h absorbing s o l u t i o n were added to each c o n t a i n e r and the contents were thoroughly mixed. 10 ml. of t h i s mixture were removed f o r a d e t e r m i n a t i o n of selenium content. This procedure was repeated at the end of each hour f o r s i x hours at which time the experiment was t e r m i n a t e d . A 4 c c . a l i q u o t of each sample ( l / l 6 of o r i g i n a l sample) was p l a c e d i n a cuvette and to i t were added 1 ml. of 10$. o x a l i c a c i d (aqueous) and 1 ml. of 0.1°/. a s - d i p h e n y l h y d r a z i n e i n g l a c i a l a c e t i c a c i d . The contents of the cuvette were mixed, stoppered and placed i n a dark cupboard f o r three hours. The samples were then measured f o r c o l o r a b s o r p t i o n i n a Coleman Spectrophotometer at wavelength 510 m i l l i m i c r o n s . R e s u l t s and Conclusions (a) No selenium-asymmet. d i p h e n y l h y d r a z i n e was observed i n -d i c a t i n g at l e a s t two p o s s i b i l i t i e s ! ( i ) There was not a l a r g e enough q u a n t i t y of selenium excreted to be d e t e c t a b l e i n the d i l u t i o n s used. ( i i ) The selenium compound excreted was not absorbed i n t o the absorbing s o l u t i o n . (b) A l l f o u r animals s u r v i v e d the i n j e c t i o n and were k i l l e d a f t e r 48 hours. Procedure 2 Four male r a t s aged 105 days were given 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 of sodium s e l e n i t e at a dosage l e v e l of 6 mgm./Kg. body weight and p l a c e d i n the chamber of the 105 e x h a l a t i o n apparatus immediately. A i r was drawn through the apparatus as f o r the previous procedure. The t o t a l a i r d i s -placement was 976 l i t e r s and the a i r temperature was 25°C. The absorbing s o l u t i o n was 6MI01. Each a b s o r p t i o n tower con-t a i n e d 15 ml. of t h i s s o l u t i o n . At the end of 4 hours the two a b s o r p t i o n towers were r e p l a c e d with f r e s h towers again c o n t a i n i n g 15 ml. 6N HC1 each. At the end of the second 4 hour p e r i o d these towers were a l s o removed and the experiment terminated. 3 ml. a l i q u o t s of 'the contents of each tower were placed i n c u v e t t e s . To each were added 1 ml. of water, 1 ml. of 10% o x a l i c a c i d and 1 ml. of 0.17° asymmetric d i -phenylhydrazine s o l u t i o n . The samples were allowed to stand f o r 3 hours i n a dark place and measured f o r c o l o r a b s o r p t i o n as i n the previous procedure. R e s u l t s and Conclusions (a) Comparison of the galvanometer readings obtained with the samples with those of a standard curve assembled f o r the purpose gave v a l u e s f o r the selenium content of the a b s o r p t i o n towers as f o l l o w s : $1 and $2 (1st 4 hours) 21 micrograms of selenium i n each tower #3 and $4 (2nd 4 hours) 22.5 micrograms of selenium i n each tower The r e s u l t s showed that selenium was again not being e f f i c i e n t l y absorbed by the absorbing s o l u t i o n . The s i m i l a r -i t y of the values f o r the two towers i n each case made t h i s c o n c l u s i o n obvious. 106 (b) A l l four of the animals s u r v i v e d the i n j e c t i o n s and were k i l l e d a f t e r 48 hours. Procedure 3 A s i m i l a r l y aged group of male r a t s was subjected to a procedure i d e n t i c a l i n a l l r e s p e c t s to Procedure 2 with the exception that the absorbing f l u i d was r e p l a c e d with 15 ml. of concentrated HgSO^. The r e s u l t s were e q u a l l y n e g a t i v e . A l l f o u r animals s u r v i v e d the i n j e c t i o n s and were k i l l e d a f t e r 48 hours. D i s c u s s i o n The d i f f i c u l t y w ith the a b s o r p t i o n medium was not a n t i c i p a t e d when the experiment was set up. Were i t not f o r the f a c t t h at the second a b s o r p t i o n tower showed s i m i l a r values i n Procedure 2 i t might have been considered t h a t the e x c r e t i o n of selenium through the lungs i s extremely low. The course of f u t u r e i n v e s t i g a t i o n i s obvious. Before f u r t h e r experiments u t i l i z i n g animals are c a r r i e d out the e f f i c i e n c y of v a r i o u s s o l u t i o n s i n absorbing H 2Se, a gaseous form of selenium, should be determined. P r e l i m i n a r y i n v e s t i -g ations suggest that none of the above mentioned s o l u t i o n s are s u i t a b l e f o r t h i s purpose. A p r i v a t e communication from Pittman (1949) suggests bubbling the exhaled gases through a weak aqueous s p l u t i o n of Se0 2 t h u s . o b t a i n i n g a c o l l o i d a l suspension of selenium which could be estimated c o l o r i m e t r i c -a l l y . 107 APPENDIX I I I . Further Data on the L . D . ^ of Sodium Selenite. v i a the Intraperitoneal Injection Route, for Various Species of Animals 8 Bioassays have been carried out throughout the period of investigation of the selenium problem. Whenever suf-f i c i e n t animals of any species were available an attempt was made to further the establishment of the L.D.^Q l e v e l for a pa r t i c u l a r species. This work i s of fundamental importance to the problem as a whole since i t serves to make possible the investigation of such related problems as acquired tolerance, and species s u s c e p t i b i l i t y , t o the poison i n addition to establishing the various dosage l e v e l s at which selenium may be administered to produce a certain condition of t o x i c i t y . In the e a r l i e r work the author disoussed the sali e n t points with reference to the procedure employed to determine the L.D.^Q, the means of cal c u l a t i o n (Reed & Muench, 1938), and the sign i f i c a n c e of the various r e s u l t s . It i s proposed therefore to record the various additional r e s u l t s obtained and to incorporate them into a table representing the r e s u l t s of the L.D,^ G determinations on selenium obtained by t h i s investogator up to the present time. A l l the determinations were made using aqueous (,001 N HC1) sodium selenite solutions given by the intraperitoneal i n j e c t i o n route, and i n the new series the ^Reference, Derrick, 1?48, 1 0 8 animals were i n j e c t e d and maintained under c o n d i t i o n s ap-proximating those of previous determinations as c l o s e l y as p o s s i b l e * The new data are as f o l l o w s : 1 . The White Mouse (a) L.D*^0 f o r the young (35-42 day old) female white mouse  Dose of Percent Na 2Se0j i n L i v e d Died Lived Died M o r t a l i t y mgm*/Kg. 1 1 mgm. 6 0 12 0 0 1 2 mgm. 4 2 6 2 2 5 1 3 mgm. 1 5 2 7 7 8 14 mgm* 1 5 1 12 9 2 1 2 . 5 mgm . Na 2Se0j/Kg. body wt. (b) L.D* 5 0 f o r the young (35-42 day old) male white mouse Dose of Percent Na 2Se0j i n L i v e d Died Lived Died M o r t a l i t y mgm./Kg. 1 1 mgm. 4 2 15 2 1 1 . 8 12 mgm. 6 0 1 1 2 15.4 1 3 mgm. 3 3 5 5 5 0 . 0 14 mgm. 2 4 2 9 8 2 . 0 L.D.^Q s 1 3 mgm. Na 2Se0j/Kg* body wt. 109 ( c ) L . D . ^ Q for the mature (3 months female white mouse old) Determination 1 Dose of Na2Se0j mgm./Kg. in Lived Died Lived Died Peroent Mortality 9 mgm. 10 mgm. 11 mgm. 12 mgm. 5 6 6 3 1 20 1 0 15 1 0 ? l 3 3 4 4.8 6.3 10.0 57.0 L.D»£0 = 11.8 mgm. Na2Se0j/Kg. body • wt. Determination 2 Dose of. Na9Se0, in Lived Died Lived Died Percent Mortality 10 mgm. 11 mgm. 12 mgm. 5 5 3 1 13 1 3 8 4 3 3 7 7.1 33.4 70.0 L.D. 5 0 = 11.45 mgm. Na2Se0j/Kg. body wt. (d) L.D.5Q for the mature (3 months male white mouse old) Determination 1 Dose of NagSeOz mgm./Kg. in Lived Died Lived Died Percent Mortality 10 mgm. 11 mgm• 12 mgm. 13 mgm. 4 3 2 1 2 10 2 3 6 5 4 3 ? 5 1 14 16.7 45.5 75.0 93.0 L , D , 5 0 " 11.2 mgm. Na2Se0^/Kg. body wt. 110 Determination 2 Dose of Peroent Na 2Se03 i n L i v e d Died L i v e d Died M o r t a l i t y mgm./Kg. 10 mgm. 6 0 11 mgm. 6 0 12 mgm. 6 0 L*P«50 cannot be determined. • Note: No e x p l a n a t i o n can be made f o r t h i s phenomenon s i n c e the same s o l u t i o n was used f o r male and female i n each d e t e r -m i n ation and t h e r e i s an o b v i o u s l y good check i n the agree-ment obtained i n the ad u l t female white mouse L . D . ^ Q v a l u e s . 2. The Guinea P i g (a) L.D.JJQ f o r the young a d u l t (approx. 6 months old) male guinea p i g s Dose of Na 2Se03 i n mgm./Kg. L i v e d Died L i v e d Peroent Died M o r t a l i t y 1.2 mgm. 3 0 8 0 0 1.7 mgm. 4 0 3 0 0 2.2 mgm. 1 3 1 3 75 L » D » 5 0 r 2.0 mgm . Na 2SeO j/Kg. body wt. I l l (b) L . D . ^ Q for the young adult (approx. 6 months old) female guinea pig Dose of Na 2SeOj i n mgm*/Kg. Lived Died Lived Died Percent Mor t a l i t y 1 . 2 mgm. 3 0 6 0 0 1.7 mgm. 3 1 3 1 25 2.2 mgm. 0 4 0 5 100 L.D.^o " 1»85 mgm. Na 2Se0j/Kg. body wt. (c) L .D .JJQ for young (approx. 78 days old) female guinea pigs Dose of Na 2Se0j i n mgm./Kg. Lived Died Lived Died Percent Mortality 1 .9 mgm. 2 . 1 mgm. 3 1 5 2 2 . 2 1 3 1 6 . 7 6 0 . 0 L.D. JJQ s 2 . 0 7 mgm. Na 2Se0j/Kg. body wt. (d) L.D • ^ 0 f o r yo^ng (approx. 78 days old) male guinea pigs -Dose of Na 2Se0j i n mgm./Kg. Lived Died Lived Died Percent Mortality 1 .9 mgm. 2 . 1 mgm. - 4 0 7 3 1 1 0 3 0 7 5 L.D. 5 0 = 2.0 mgm. Na 2Se0j/Kg. body wt • 112 3 . The L.D,go f o r Rats (aged 15 weeks) P r e v i o u s l y Administered  Small Q u a n t i t i e s of Selenium as Sodium S e l e n i t e i n the  D r i n k i n g Water (5 t o 6 , 9 p.p.m.) f o r a P e r i o d of 11 Weeks Male Rats Dose of Percent Na 2Se0j i n L i v e d Died L i v e d Died M o r t a l i t y mgm./Kg. 10 mgm. 6 0 12 0 0 12 mgm. 3 3 6 3 33 14 mgm. 2 4 3 7 7 0 16 mgm. 1 5 1 12 92 L.D. 50 = 1 2 . 9 mgm. Na 2 SeOj/Kg. body wt. Female Rats Dose of Percent Na 2SeOj i n L i v e d Died L i v e d Died M o r t a l i t y mgm./Kg. 10 mgm. 6 0 14 0 0 12 mgm. 5 1 8 1 11 14 mgm. 1 5 . . . 3 6 66 16 mgm. 2 4 2 10 8 3 L . D . ^ Q = 13,4 mgm. Na 2Se0j/Kg. body wt. 113 4. The L.D.go f o r Rats (aged 22 weeks) P r e v i o u s l y Administered  Small Q u a n t i t i e s of Selenium as Sodium S e l e n i t e i n the Drink i n g Water (5 to 6.9 p.p.m.) f o r a Pe r i o d of 18 Weeks. Male Rats Dose of Per0ent Na 2Se0j i n Lived Died L i v e d Died M o r t a l i t y mgm./Kg. 12 mgm. 2 4 3 4 57 13 mgm. 1 5 1 ? 90 L . D # 5 0 » 11.8 mgm. Na 2Se0 5/Kg. body wt. Female Rats Dose of Percent Na 2SeOj i n Li v e d Died Li v e d Died M o r t a l i t y mgm./Kg. 12 mgm. 4 2 10 2 17 13 mgm. 4 2 6 4 40 14 mgm. 2 4 2 8 80 L.D,^0 = 13.2 mgm. Na 2Se0j/Kg. body wt. 114 A Table of the L . D . ^ Q of Sodium Selenite Injected I n t r a p e r i -toneally as an Aqueous (.001 N HOI) Solution. (Time of death within 48 hrs.) ! Species of Animal Age Sex Other Information L^D.^Q Na2Se0j i : mgm./Kg. Rat 5 mos. Male 14 . 5 Rat ft n Female 10*5 Rat 4 " Male Receiving 5-6.?p»p>m. Se i n drinking wat-er for 11 weeks 12.9 Rat 4 • Female n n 13.4 Rat ft M Male Receiving 5-6.9p.p.m. Se i n drinking wat-er for 18 weeks 11 .8 Rat ft" Female N It 13.2 Mouse 5 - 6 wks. Male • 13.0 Mouse 5 - 6 wks. Female 12 .5 Mouse 3 mos. Male Determination #1 11.2 Mouse 3 mos* Male Determination $2 Could not be deter-mined. Mouse 3 mos. Female Determination #1 10 .7 Mouse 3 mos. Female Determination #2 11 .8 Mouse 3 mos. Female Determination $ 3 • 11 . 5 Guinea pig Approx. 1 yr. Male Tentative value 1 to 2 Guinea pig Approx. 6 mos. Male 2.0 Guinea pig Approx. 6 mos. Female 1 .9 Guinea pig 3 mos. Male 2.0 Guinea pig 3 mo s • Female 2.1 Rabbit 8 mos* to 1 yr. Male Tentative value 3 to 4 115 APPENDIX IV. A Summary of the Gross Pathology Observed i n  Animals of the V a r i o u s Experimental Groups ( i ) Rats dying w i t h i n 44 t o 91 days a f t e r the i n i t i a t i o n  of the p r e l i m i n a r y experiment u s i n g sodium s e l e n i t e  i n the d r i n k i n g water (p. l o l Lungs - p a l e , s l i g h t l y edematous, smal l amount of p l e u r a l e f f u s i o n . Heart - p a l e , f l a b b y , s l i g h t l y e n l a r g e d . Spleen - enlarged, p a l e , p i t t e d s u r f a c e i n some cases and smooth i n o t h e r s . L i v e r - a t r o p h i e d , q u i t e f a t t y i n appearance, p a l e , i n some cases evidences of s l i g h t adhesions. G e n i t a l s - s m a l l , p o o r l y developed. A s c i t e s was u s u a l l y , but not always, observed and the f l u i d was sometimes bloody and sometimes c l e a r . Anemia was o u t s t a n d i n g . Fat d e p o s i t i o n was poor. ( i i ) Rats i n j e c t e d with sodium s e l e n i t e a f t e r being subjected  to d r i n k i n g water c o n t a i n i n g 0.69 to 6.9 p.p.m. Se over. an 11 week p e r i o d . Expt. A. (p. 11) Only the l i v e r s of these animals were con-s i d e r e d important with r e s p e c t t o post mortem f i n d i n g s s i n c e other organs u s u a l l y showed.the e f f e c t s of the acute c o n d i t i o n . In a few cases there was p i t t i n g and mottling evident on the s u r f a c e of-the l i v e r . S l i g h t evidence of f a t t y degeneration of t h i s organ was observed o c c a s i o n -a l l y . In g e n e r a l , however, the l i v e r appeared q u i t e 116 normal. Fat d e p o s i t i o n was good i n a l l cases. ( i i i ) Rats s u b j e c t e d t o d r i n k i n g water c o n t a i n i n g from 0.69  to 6,9 p.p.m. Se over a p e r i o d of 25 weeks. Expt. A. The post mortem appearance of these animals v a r i e d very l i t t l e from normal. L i v e r damage was no more extensive than observed i n ( i i ) ; f a t d e p o s i t i o n was good although s l i g h t l y l e s s than i s u s u a l i n animals of t h i s age. In g e n e r a l , nothing of s i g n i f i -cance was apparent. 117 TABLE OF REFERENCES Barron, E.S.G., and S i n g e r , T.P., (1943). S u l f h y d r y l en-zymes i n carbohydrate metabolism. J . B i o l . Chem., 157. 221. Barron, E.S.G., and S i n g e r , T.P., (1943). S u l f h y d r y l en-zymes i n f a t and p r o t e i n metabolism. J . B i o l . 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