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The effect of DDT upon the metabolism of estradiol in coho salmon (Oncorhynchus kisutch) Harvey, Brian John 1972

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r ° . y : , \ 1 THE EFFECT OF DDT UPON THE METABOLISM OF ESTRADIOL IN COHO SALMON (ONCORHYNCHUS KISUTCH) by B r i a n John Harvey B . S c , U n i v e r s i t y o f V i c t o r i a , 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Zoology We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1972 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e -quirements f o r an advanced degree at t h e U n i v e r s i t y o f B r i -t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t per-m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by t h e Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t copying or pub-l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be a l -lowed without my w r i t t e n p e r m i s s i o n . Department of Zoology The U n i v e r s i t y o f B r i t i s h Columbia Vancouver &t Canada i i ABSTRACT In the f i r s t experiment, s e x u a l l y immature male and f e -male coho salmon were exposed f o r 21 days t o d i e t a r y DDT at a l e v e l o f 10 or 100 p a r t s per m i l l i o n (ppm)}, or to methoxy-c h l o r at a' l e v e l of 100 ppm. Exposure t o 100 ppm DDT was found to i n c r e a s e the l e v e l o f l i v e r microsomal Cytochrome P-450 from a c o n t r o l l e v e l o f 1 . 3 2 + . 0 4 nmoles/1000 mg l i v e r to a l e -v e l of 1 . 9 # ± . 0 4 nmoles/1000 mg l i v e r , a s t a t i s t i c a l l y s i g n i f -i c a n t d i f f e r e n c e (P<,001). None o f the treatments were found to a f f e c t the hepato-somatic index. In the second experiment, l i v e r s l i c e s from s e x u a l l y ma-t u r i n g male and female coho salmon f e d 100 ppm DDT f o r 21 days or a c o n t r o l d i e t were incubated with 4 - C ^ - e s t r a d i o l - 1 7 c 3 i n v i t r o . M e t a b o l i t e s produced were e x t r a c t e d w i t h dichlorometh-ane, separated by t h i n - l a y e r chromatography and assayed u s i n g s c i n t i l l a t i o n c o u n t i n g t e c h n i q u e s . Prodxaced i n the i n c u b a t i o n were es t r o n e , e s t r i o l and one o t h e r u n i d e n t i f i e d p o l a r meta-b o l i t e . DDT treatment was found t o s i g n i f i c a n t l y i n c r e a s e the amount o f e s t r i o l and u n i d e n t i f i e d m e t a b o l i t e produced (P< .001). In the t h i r d experiment, s e x u a l l y maturing male and f e -male coho salmon f e d 100 ppm DDT f o r 21 days or f o r 7 days or a c o n t r o l d i e t were i n j e c t e d with 625,500 dpm 4 - C - ^ - e s t r a d i o l -178 and p e r m i t t e d t o metabolize the hormone i n v i v o . S e r i a l blood samples were e x t r a c t e d , chromatographed and s u b j e c t e d to s c i n t i l l a t i o n c o unting techniques t o o b t a i n values f o r M e t a b o l i c Clearance Rate, H a l f - l i f e Time and Volumes o f D i s t r i b u t i o n o f the i n j e c t e d s t e r o i d . I t was found t h a t i n g e s t i o n o f DDT had no s i g n i f i c a n t e f f e c t upon any o f these parameters (P<.001). The i i i pattern of metabolites produced i n vivo c l o s e l y resembled that produced i n v i t r o . The evidence presented i n t h i s study suggests that en-hancement of the a c t i v i t y of the Mixed Function Oxidase syst i n coho salmon may occur upon ingestion of an organochlorine i n s e c t i c i d e , but that the phenomenon may have l i t t l e s i g n i f -icance i n vivo. i v TABLE OF CONTENTS Page I n t r o d u c t i o n 1 Methods and M a t e r i a l s 9 R e s u l t s 20 D i s c u s s i o n 32 Summary 42 B i b l i o g r a p h y 43 Appendix I 47 Appendix I I 48 V LIST OF TABLES Table Page I Hepato-somatic indexes f o r c o n t r o l and 21 i n s e c t i c i d e - t r e a t e d f i s h . I I Cytochrome P -450 content i n c o n t r o l and 24 i n s e c t i c i d e - t r e a t e d f i s h . I I I In v i v o metabolism of ^ - C ^ - e s t r a d i o l - 31 17{3 VI LIST OF FIGURES Figure Page 1 Sectioning of the TLC sheets f o r scin- 14 t i l l a t i o n counting. 2 Resolution of the two exponential com- 16* ponents of the clearance curve f o r es-t r a d i o l . 3 Carbon monoxide difference spectra i n 23 control f i s h . 4 Metabolites produced by l i v e r s l i c e s i n - 25 cubated with 4-d4~estradiol-17 p . 5 Autoradiogram made a f t e r TLC separation 26 of metabolites produced i n incubations. 6 Autoradiograms from the 60-rainute i n j e c - 28 t i o n s e r i e s . 7 Disappearance curves f o r t o t a l plasma r a - 30 d i o a c t i v i t y , dichloromethane-extractable r a d i o a c t i v i t y , and 4-C 1 / ('-estradiol-17p . 8 The biosynthesis of key sex steroids. 35 9 Some in t e r r e l a t i o n s h i p s of estrogen me- 36 tabolism. 10 Two-compartment model describing the me- 39 tabolism and transport of e s t r a d i o l . v i i ACKNOWLEDGMENT I wish to thank ray s u p e r v i s o r s , Dr. E.M. Donaldson and Dr. W.S. Hoar, f o r t h e i r a s s i s t a n c e i n completing t h i s work. T e c h n i c a l help from Helen Dye i s a l s o acknowledged. 1 INTRODUCTION In 1961, Rachel Carson produced S i l e n t S p r i n g , a book t h a t i n r e t r o s p e c t has proven a landmark i n the b r i e f h i s t o r y of 'environmental r e s e a r c h ' . Heated p r o f e s s i o n a l and p o l i t i -c a l c r i t i c i s m o f the book f a i l e d t o d e t e r i n c r e a s i n g numbers of s c i e n t i s t s from t u r n i n g toward what has now become a co-h e s i v e and c o n c e r t e d e f f o r t t o a s c e r t a i n the impact of man-made p o l l u t a n t s upon l i v i n g systems from s i n g l e c e l l s t o pop-u l a t i o n s . Organochlorine i n s e c t i c i d e s (see Appendix I f o r r e -p r e s e n t a t i v e s t r u c t u r a l formulae and p r o p e r t i e s ) , by v i r t u e o f t h e i r e x t r a o r d i n a r y p e r s i s t a n c e i n both a b i o t i c and b i o t i c com-ponents o f t h e environment, soon occupied a l a r g e share o f s c i e n t i s t s ' a t t e n t i o n , and i t was not l o n g before a s e r i e s of d i s q u i e t i n g r e p o r t s made 'DDT' a household word. In 1 9 6 3 , a s i g n i f i c a n t a c c i d e n t a l d i s c o v e r y was made i n the o b s e r v a t i o n t h a t the s p r a y i n g o f animal rooms with the o r -g a n o c h l o r i n e i n s e c t i c i d e chlordane shortened the d u r a t i o n of b a r b i t u r a t e - i n d u c e d s l e e p i n the r a t (Hart and Fouts, 1 9 6 3 ) . F u r t h e r i n v e s t i g a t i o n showed that the o r a l a d m i n i s t r a t i o n o f chlordane i n c r e a s e d the b i o t r a n s f o r m a t i o n o f the b a r b i t u r a t e by i s o l a t e d l i v e r microsomes, and t h a t such i n s e c t i c i d e - c y t o -plasmic i n t e r a c t i o n c o u l d occur with a wide v a r i e t y o f organo-c h l o r i n e i n s e c t i c i d e s and i n a wide v a r i e t y of s p e c i e s . Ex-amples i n c l u d e d r a t s t r e a t e d with DDT (Hart and Fouts, I 9 6 5 ) , w i t h DDD ( G i l l e t t e et a l . , 1 9 6 6 ) , with l i n d a n e (Creaven et a l . , 1 9 6 6 ) , with d i e l d r i n (Ghazal et a l . , 1 9 6 4 ), with methoxychlor and with perthane (Hart et a l . , 1 9 6 5 ) , s q u i r r e l monkeys t r e a t e d 2 with DDT (Juchau et a l . , 1966) and chlordane (Cram et a l . , 1 9 6 5 ) , mice t r e a t e d w i t h DDT (Cram et a l . , 1967) and chlordane (Cram et a l . , 1 9 6 5 ) , and Japanese q u a i l t r e a t e d with DDT ( G i l -l e t t e et a l . , 1 9 6 6 ) . The phenomenon i s now known to be one o f enzyme i n d u c t i o n , and o r g a n o c h l o r i n e i n s e c t i c i d e s comprise but one o f a number of c l a s s e s o f compounds capable o f a c t i n g as inducers ( o t h e r s i n c l u d e p o l y c y c l i c hydrocarbons (Conney et a l . , 1 9 5 7 ) , drugs such as c h l o r c y c l y z i n e and chlorpromazine (Conney et a l . , 1961) and such environmental contaminants as a n t i o x i d a n t s (Kuntzman, 1969) and p o l y c h l o r i n a t e d b i p h e n y l s (Risebrough, I 9 6 & ), com-pounds b e a r i n g c o n s i d e r a b l e s t r u c t u r a l resemblance t o DDT). The enzymes a c t e d upon are members o f what has been termed the Mixed F u n c t i o n Oxidase system (MFO), a mono-oxygenase system r e s i d i n g i n the endoplasmic r e t i c u l u m o f the l i v e r . (Mono-ox-ygenases may be d e f i n e d as enzymes which, i n the presence o f an e l e c t r o n donor and m o l e c u l a r oxygen, c a t a l y z e the i n c o r p o r -a t i o n o f oxygen i n t o a l i p h a t i c and aromatic s u b s t r a t e s . See Ap-pendix I I f o r an o u t l i n e of the present c o n c e p t i o n o f the sys-tem). The system e x h i b i t s a s u r p r i s i n g l a c k o f s u b s t r a t e s p e c i -f i c i t y , appearing to e f f e c t the b i o t r a n s f o r m a t i o n ( i n a c t i v a -t i o n ) o f both ' f o r e i g n ' and endogenous compounds wi t h equal ease. S t u d i e s i n s e v e r a l l a b o r a t o r i e s have shown t h a t l i v e r mi-crosomes c o n t a i n NADPH-dependent enzyme systems which hydroxy-l a t e s t e r o i d s such as e s t r a d i o l , progesterone, t e s t o s t e r o n e , androsterone and the c o r t i c o i d s , thereby r e n d e r i n g them, by v i r t u e of t h e i r i n c r e a s e d p o l a r i t y , more r e a d i l y e x creted (Co-3 ney, 1967). Additional studies have demonstrated many s i m i l -a r i t i e s between the enzyme systems which hydroxylate steroids and those which oxidize drugs, and these observations sug-gested that steroid hormones are naturally-occurring substrates f o r drug-metabolizing enzymes i n l i v e r microsomes (Kuntzman et a l . , 1 9 6 4 ) . This l a t t e r conclusion was arrived at i n 1964, and i t was thus only three years a f t e r the publication of Miss Car-son's book that a possible l i n k between insecticide-mediated induction of the MFO system and the position of steroid hormones as normal substrates f o r that enzyme system suggested i t s e l f . The question asked upon the recognition of t h i s t i e - i n was the following: does an animal, upon exposure to an organo-chlorine i n s e c t i c i d e , become capable of s i g n i f i c a n t l y a c c e l -erated biotransformation of endogenous steroids? Numerous stu-dies addressed to t h i s question soon appeared, embracing a wide variety of species, compounds and dosages. Representative ex-amples include: two-fold stimulation by chlordane of the l i v e r microsomal metabolism of estradiol-17^ in rats to a mixture of hydroxylated metabolites more polar than the substrate (Conney et a l . , 1 9 6 7 ) , enhanced hepatic metabolism of testosterone and estradiol-17t3 in chickens pretreated with DDT or Aroclor 1 2 5 4 , a polychlorinated biphenyl (Nowicki and Norman, 1971), stimu-l a t i o n of metabolism of C o r t i s o l in man to 6-p-OH-cortisol following administration of o,p DDD, an analog of DDT (South-ren et a l . , 1 9 6 6 ), and stimulation of testosterone and pro-gesterone metabolism in l i v e r microsomes from pigeons fed DDT (10 ppm) or d i e l d r i n (2 ppm) f o r one week (Peakall, 1 9 6 7 ) . The l a t t e r example may serve to emphasize the v a l i d i t y of the 4 problem as a r e s e a r c h t o p i c - d i e t a r y l e v e l s o f 2-10 ppm o f an o r g a n o c h l o r i n e i n s e c t i c i d e are not without precedent i n the food o f man. By f a r the g r e a t e r part o f the i n f o r m a t i o n gathered on i n s e c t i c i d e - a l t e r e d s t e r o i d metabolism has been obtained u-t i l i z i n g i n v i t r o t e c h n i q u e s , u s u a l l y i n v o l v i n g the i n c u b a t i o n of the s u b s t r a t e w i t h l i v e r microsomal suspensions, whole l i -v e r homogenates, or l i v e r s l i c e s from animals t r e a t e d with the compound under i n v e s t i g a t i o n . T h i s i s the case i n a l l the ex-amples j u s t c i t e d . In v i v o evidence, when i t appears, i s i n -d i r e c t , and e f f e c t s on metabolism are g e n e r a l l y deduced from an a l t e r a t i o n i n b i o l o g i c a l response to an ad m i n i s t e r e d com-pound. Examples i n c l u d e a decreased a n e s t h e t i c a c t i o n o f pro-gesterone i n r a t s f o l l o w i n g a d m i n i s t r a t i o n o f DDT (Conney et a l . , 1966), and a di m i n i s h e d u t e r o t r o p i c e f f e c t o f ad m i n i s t e r e d e s t r a d i o l i n female r a t s f o l l o w i n g treatment with chlordane (Conney et a l . , 1967). The e f f e c t s o f o r g a n o c h l o r i n e i n s e c -t i c i d e s on the metabolism o f t r u l y endogenous s t e r o i d s remain to be e l u c i d a t e d , as does the importance of endocrine feedback loops i n c o u n t e r a c t i n g any hormonal imbalance which c o u l d the-o r e t i c a l l y a r i s e . Contamination o f the hydrosphere, whether from m i s a p p l i -c a t i o n o f i n s e d t i c i d e f o r m u l a t i o n s or through gradual l e a c h i n g and e r o s i o n o f the chemicals from farmlands i n t o s u r f a c e run-o f f waters, has not escaped a t t e n t i o n as a source o f danger f o r a q u a t i c organisms of a l l t y p e s . The e f f e c t s o f o r g a n o c h l o r i n e i n s e c t i c i d e s on f i s h have, i n p a r t i c u l a r , been v i g o r o u s l y i n -v e s t i g a t e d , and c o n s i d e r a b l e i n f o r m a t i o n has been gathered con-5 c e r n i n g such t o p i c s as route o f uptake (Macek and Korn, 1970), e f f e c t s on behaviour (Anderson, 1971), e f f e c t s on growth and r e p r o d u c t i o n (Macek, 1963), and g e n e t i c and e c o l o g i c a l con-sequences o f exposure t o o r g a n o c h l o r i n e i n s e c t i c i d e s (Cope, 1971). C u r i o u s l y absent from the l i t e r a t u r e , however, are r e -p o r t s concerning the i n d u c t i v e e f f e c t o f these compounds on mi-crosomal enzymes i n f i s h , a t o p i c which, as we have seen, has r e c e i v e d c o n s i d e r a b l e a t t e n t i o n with r e s p e c t t o t e r r e s t r i a l o r -ganisms . The e x p l a n a t i o n f o r t h i s d i s c r e p a n c y l i e s not i n any b e l i e f t h a t f i s h are not prone t o such e n d o c r i n o l o g i c a l imbal-ances as a r e , say, r a t s and pigeons, but r a t h e r i n an uncer-t a i n t y w i t h i n the s c i e n t i f i c community with r e s p e c t t o the de-gree o f b i o c h e m i c a l p a r a l l e l i s m between the dru g - m e t a b o l i z i n g c a p a b i l i t i e s o f f i s h and those o f t e r r e s t r i a l v e r t e b r a t e s . In an ext e n s i v e study on the comparative metabolism o f f o r e i g n compounds, c a r r i e d out i n 1962, evidence was presented t o support the co n t e n t i o n t h a t f i s h d i d not possess microsomal mixed-function oxidase systems as d i d t e r r e s t r i a l s p e c i e s , and t h a t d i s p o s a l o f many l i p i d - s o l u b l e f o r e i g n compounds was e f -f e c t e d through gill-membrane d i a l y s i s i n t o the surrounding wa-t e r (Brodie and M a i c k e l , 1962). However, recent s t u d i e s have shown t h a t t h i s i s not the case, and t h a t indeed t h e r e e x i s t s a s u r p r i s i n g s i m i l a r i t y between the b i o c h e m i c a l s t r a t e g i e s em-ployed by t e r r e s t r i a l organisms and f i s h i n the d i s p o s i t i o n o f f o r e i g n compounds (Buhler and Rasmusson, 1963; Adamson, 1967); numerous t r a n s f o r m a t i o n s ( h y d r o x y l a t i o n , demethylation, d e a l -k y l a t i o n , to name but a few) are e f f e c t e d by both groups, o n l y d i f f e r e n t optimal c o n d i t i o n s w i t h r e s p e c t t o temperature, pH, 6 s u b s t r a t e c o n c e n t r a t i o n , a c t i v a t o r s and i n h i b i t o r s (Ludke et a l . , 1971). Although p o s s e s s i o n o f microsomal MFO systems by f i s h has been amply demonstrated, i n d u c t i o n o f the enzymes by organo-c h l o r i n e i n s e c t i c i d e s has not. One recent r e p o r t has i n f e r r e d i n d u c t i o n from the o b s e r v a t i o n t h a t a d m i n i s t e r e d DDT reduces the amount o f s t o r e d d i e l d r i n i n rainbow t r o u t (Mayer and S t r e e t , 1970). However, r i g o r o u s b i o c h e m i c a l demonstration o f t h e phe-nomenon, such as has been made i n the case o f many t e r r e s t r i a l v e r t e b r a t e s , has not yet been attempted. With the f o r e g o i n g i n f o r m a t i o n i n mind, the f o l l o w i n g two qu e s t i o n s suggested themselves, and i t i s the attempt t o shed some l i g h t upon these q u e s t i o n s which forms the major part o f t h i s t h e s i s . Stated i n purposely g e n e r a l terms, they are: (1) can exposure t o an o r g a n o c h l o r i n e i n s e c t i c i d e exert any meas-u r a b l e b i o c h e m i c a l e f f e c t upon the MFO system o f f i s h ? and (2) i f the answer t o the f i r s t q u e s t i o n i s a f f i r m a t i v e , can t h i s e f f e c t have p h y s i o l o g i c a l s i g n i f i c a n c e f o r the animal? Question (1) lends i t s e l f most r e a d i l y t o examination by i n v i t r o t e c h -niques, q u e s t i o n (2) by i n v i v o methods. As has a l r e a d y been mentioned, i n f o r m a t i o n concerning the f i r s t q u e s t i o n i s scant and i n c o n c l u s i v e ; i t f o l l o w s that i n f o r m a t i o n p e r t a i n i n g t o the second i s even more so. I t should a l s o be mentioned t h a t , i n o r -der t o draw c o n c l u s i o n s concerning q u e s t i o n ( 1 ) , the assumption must be made t h a t , as i s the case i n t e r r e s t r i a l v e r t e b r a t e s , endogenous s t e r o i d hormones are acted on by the MFO system. In the l i g h t o f the s t r i k i n g b i o c h e m i c a l p a r a l e l l i s m between the systems i n a q u a t i c and t e r r e s t r i a l v e r t e b r a t e s , t h i s seems a 7 reasonable assumption. S p e c i f i c ways of a t t a c k i n g these very g e n e r a l q u e s t i o n s presented themselves. Coho salmon were s e l e c t e d as the exper-im e n t a l organisms, and DDT as the ad m i n i s t e r e d compound. T h i s l a t t e r c h o i ce was made f o r the f o l l o w i n g reasons: (1) p h y s i c a l , chemical and b i o l o g i c a l p r o p e r t i e s of DDT have come t o be r e - , garded as roughly r e p r e s e n t a t i v e o f o r g a n o c h l o r i n e i n s e c t i c i d e s as a group, (2) a l a r g e p r o p o r t i o n o f the pre v i o u s r e s e a r c h i n the area o f enzyme i n d u c t i o n by o r g a n o c h l o r i n e s has i n c l u d e d DDT among the compounds i n v e s t i g a t e d , and (3) worldwide usage of DDT, i n p a r t i c u l a r i n l e s s developed areas, i s s t i l l con-s i d e r a b l e . C h r o n i c , r a t h e r than acute a d m i n i s t r a t i o n o f the i n -s e c t i c i d e was undertaken i n the b e l i e f t h a t t h i s r e p r e s e n t s a more e c o l o g i c a l l y r e a l i s t i c regime o f a q u i s i t i o n . A r e p r o d u c t i v e s t e r o i d , estradiol - 1 7 @, was chosen as sub-s t r a t e f o r the MFO system. T h i s c h o i c e was made i n the l i g h t o f , f i r s t , the l a r g e amount o f i n f o r m a t i o n from t e r r e s t r i a l s p e c i e s concerning the metabolism o f t h i s s t e r o i d under normal c o n d i -t i o n s as w e l l as under those r e s u l t i n g from DDT exposure and, second, the r e c o g n i t i o n o f t h e p o s i t i o n o f e s t r a d i o l as an es-trogen o f major importance i n the o v a r i a n t i s s u e and plasma of salmonids (Cedard et a l . , 1961; B o t t i c e l l i and Hisaw, 1964). As has a l r e a d y been mentioned, i n v i t r o t echniques were employed i n the attempt t o answer the f i r s t g e n e r a l q u e s t i o n . S p e c i f i c a l l y , two experiments were undertaken. In the f i r s t , use was made of the o b s e r v a t i o n t h a t , i n t e r r e s t r i a l v e r t e -b r a t e s and b i r d s , i n d u c t i o n o f the MFO system i s accompanied by, and may v a l i d l y be estimated by, and i n c r e a s e i n the amount 8 of Cytochrome P-450, a hemoprotein which f u n c t i o n s as the t e r -minal oxidase i n the system ( G i l l e t t e et a l . , 1972) and has been i d e n t i f i e d i n f i s h (Chan et a l . , 1967; Fukami et a l . , 1969). The l e v e l o f l i v e r microsomal Cytochrome P-450 was t h e r e f o r e used as an index of MFO a c t i v i t y . The second exper-iment invoved i n c u b a t i o n o f the s t e r o i d s u b s t r a t e e s t r a d i o l - 1 7 f3 with components o f the MFO system and subsequent e x t r a c t i o n and i d e n t i f i c a t i o n o f the m e t a b o l i t e s , an approach modelled upon t h a t used so e x t e n s i v e l y i n the corresponding work on t e r r e s t r i a l v e r t e b r a t e s , although t a k i n g i n t o account the d i f f e r -ences i n o p t i mal c o n d i t i o n s f o r enzymatic a c t i v i t y a l r e a d y men t i o n e d . To i n v e s t i g a t e the second g e n e r a l problem, an attempt was made to f o l l o w , with as much p h y s i o l o g i c a l v a l i d i t y as p o s s i b l the a c t i v i t y o f the MFO system upon an endogenous s t e r o i d . A c l o s e approximation t o t h i s s i t u a t i o n was made by the i n j e c -t i o n o f a t r a c e amount o f r a d i o a c t i v e e s t r a d i o l - 1 7 - § i n t o the l i v i n g a nimal, m o n i t o r i n g o f the plasma l e v e l o f the s u b s t r a t e and the subsequent performance of c a l c u l a t i o n s y i e l d i n g a math e m a t i c a l d e s c r i p t i o n of i t s metabolic f a t e . 9 METHODS AND MATERIALS Fish and aquaria A l l experiments were performed i n the laboratories of the Fish e r i e s Research Board of Canada, West Vancouver, B r i t i s h Columbia. Male and female coho salmon (Oncorhynchus kisutch), hatched i n March, 1970 and maturing i n the f a l l of 1 9 7 2 , were maintained i n sea water i n large outdoor Fiberglass stock tanks, and transferred to i n d i v i d u a l , 570 l i t e r oval Fiberglass aquar-i a , provided with running sea water at 8-12 degrees Centigrade and aeration, f o r administration of the experimental d i e t s . Four f i s h were placed i n each tank; an acclimation period of one week was always allowed between transfer from the stock tanks and administration of the experimental d i e t s . A l l exper-iments were performed on f i s h from the same o r i g i n a l stock. Experiment I . Determination of Cytochrome P-450 This experiment was car r i e d out during the summer of 1 9 7 1 , using sexually immature coho salmon whose approximate average body weight was 60 grams. No attempt was made to include sex of the f i s h as an experimental variable. Pure p, p' DDT ( 2 , 2 - b i s (p-chlorophenyl)-l,l,l-trichloroethane; Aldrich Chemical Co., Milwaukee, Wis.) and technical grade methoxychlor ( 2 , 2 - b i s (p-methoxyphenyl)-l,l,l-trichloroethane; Du Pont Chemical Co., Montreal, P.Q.) were dissolved i n corn o i l and incorporated i n -to the diet at l e v e l s of 10 and 100 ppm f o r the former and 100 ppm for the l a t t e r ; control f i s h received the corn o i l vehicle alone. In a l l cases the food was read i l y accepted. Fish were fed ad libitum twice d a i l y , 7 days a week fo r 21 days; mortal-i t y during the feeding period was 5 . 0 $ . A t o t a l of 36' f i s h (9 10 f o r each o f the f o u r treatments) was used i n the experiment. At the t e r m i n a t i o n of the f e e d i n g p e r i o d , f i s h were k i l l e d by a blow t o the head, weighed, and moved to a c o l d room at 2°C, where the l i v e r s were r a p i d l y e x c i s e d and weighed. De-t e r m i n a t i o n o f Cytochrome P-4-50 was c a r r i e d out a c c o r d i n g t o the method o f Omura and Sato ( I 9 6 4 ) ; a l l o p e r a t i o n s p r i o r to the f i n a l spectrophotometric measurements were c a r r i e d out at 0-2°C. L i v e r s were homogenized f o r 60 seconds i n 6 volumes o f 1.15% KC1, u s i n g a g l a s s homogenizes equipped with a T e f l o n pes-t l e . An a l i q u o t o f the homogenate was c e n t r i f u g e d f o r 30 min-utes at 12,000 x g i n a S o r v a l l r e f r i g e r a t e d c e n t r i f u g e , and the p e l l e t d i s c a r d e d . Microsomes were sedimented from the su-pernatant f l u i d by c e n t r i f u g a t i o n f o r 90 minutes at 73,000 x g i n a Spinco p r e p a r a t i v e u l t r a c e n t r i f u g e . The r e s u l t i n g mi-crosomal p e l l e t was resuspended by homogenization i n 1.15$ KC1 and r e c e n t r i f u g e d as above. The washed microsomes were then r e -suspended i n 2 ml of 0.1M sodium phosphate b u f f e r ; suspensions from t h r e e f i s h were combined to a t o t a l volume o f 6 ml. Carbon monoxide d i f f e r e n c e s p e c t r a were determined i n a Cary Model 15 dual-beam r e c o r d i n g spectrophotometer u s i n g s t a n -dard s i z e quartz c u v e t t e s o f 1 cm o p t i c a l path. Suspensions were p a r t i t i o n e d i n t o sample and r e f e r e n c e c u v e t t e s , and chem-i c a l l y reduced by the a d d i t i o n o f 2 mg s o l i d sodium d i t h i o n i t e ( N a 2 S 2 0 J i f ) . Carbon monoxide (99.5$ pure; Matheson Chemical Co., Vancouver, B.C.) was then bubbled through the sample c u v e t t e f o r 20 seconds, and the d i f f e r e n c e i n l i g h t absorbancy between the sample and r e f e r e n c e c u v e t t e s o b t a i n e d as a f u n c t i o n o f wavelength, between 520 and 390 mp. 11 Experiment I I . Incubation of 4-C~^-estradiol-17f3 w i t h l i v e r s l i c e s T his experiment was c a r r i e d out during the summer of 1972, u s i n g s e x u a l l y maturing male and female coho salmon of 500 grams approximate average body weight. Pure p,p' DDT (100 ppm) was fed as before f o r 21 days; c o n t r o l f i s h r eceived the corn o i l v e h i c l e . There was no m o r t a l i t y during the course of the experiment. F i v e f i s h were used f o r each treatment, two male and three female i n each case. At the t e r m i n a t i o n of the feeding p e r i o d , f i s h were k i l l e d by a blow to the head, weighed, and the l i v e r s r a p i d l y e x c i s e d , weighed and placed on i c e . Incubation of l i v e r s l i c e s with 4-C^-estradiol-17r3 was c a r r i e d out according t o the meth-od of Buhler and Rasmusson (1968), w i t h m o d i f i c a t i o n s as to op-t i m a l c o n d i t i o n s of p r o t e i n c o n c e n t r a t i o n and shaking speed suggested by Fouts (1970). L i v e r s l i c e s of 0 . 5 mm t h i c k n e s s were prepared using a Stadie-Riggs t i s s u e s l i c e r ; 3 0 0 mg l i v e r were combined i n 25 ml Erlenmyer f l a s k s w i t h 2 ml .05M MgCl 2, 0 . 3 ml .01M NADPH (Sigma Chemical Co., S t . L o u i s , Mo.), 6 2 5 , 5 0 0 dpm 4-C 1 / 4--estradiol-17p (estr-1,3 , 5( 10)-triene-3,17 - d i o l ; spe-c i f i c a c t i v i t y 5 8 . 0 mCi/mmole; New England Nuclear, Boston, Mass.) i n 6% s a l i n e / e t h a n o l 9 : 1 , andOO.lM Tris(hydroxymethyl) aminomethane (TRIS) b u f f e r {99*9% pure; F i s h e r S c i e n t i f i c Co., F a i r l a w n , N . J . ) , pH 7.4, t o a f i n a l volume of 5 . 0 ml. Radio-l a b e l l e d e s t r a d i o l was checked p e r i o d i c a l l y f o r p u r i t y , using t h i n - l a y e r chromatographic techniques as described i n the f o l -lowing paragraph. Incubations were c a r r i e d out at 25°C f o r 60 minutes i n the presence of f l o w i n g oxygen i n c a Grant Model 12 SSB4 Metabolic Shaking Incubator at a r a t e of 160 complete os-c i l l a t i o n s per minute. Oxygenation of samples was achieved through the use of a gassing hood f i t t e d t o the incubator. Upon t e r m i n a t i o n of the in c u b a t i o n p e r i o d , contents of the f l a s k s were e x t r a c t e d twice w i t h 10 volumes of r e - d i s t i l l e d dichloromethane and c e n t r i f u g e d f o r 5 minutes at 600 x g i n a P h i l l i p s - D r u c k e r c e n t r i f u g e . The f i n a l aqueous phase was r e -moved by a s p i r a t i o n and 5 ml of the organic phase placed i n a 10 ml c o n i c a l c e n t r i f u g e tube. Two drops of a s o l u t i o n of au-t h e n t i c e s t r a d i o l , estrone ( e s t r - l , 3 , 5 ( 1 0 ) - t r i e n e - 3 - o l - 1 7 - o n e ) and e s t r i o l ( e s t r - 1 , 3 , 5 ( 1 0 ) - t r i e n e - 3 - l 6 ,17 - t r i o l ; Calbiochem, La J o l l a , C a l i f . ) i n methanol (1 mg/ml) were added to each tube and the s o l u t i o n evaporated t o dryness under f l o w i n g c e r t i f i e d n i t r o g e n . The d r i e d s t e r o i d s were then r e d i s s o l v e d i n 25 \fl r e -d i s t i l l e d methanol, d e l i v e r e d c a r e f u l l y down the side s of the tube to ensure complete s o l u t i o n . Using a 25 ^1 Hamilton s y r i n g e , a t o t a l of 10 p i of t h i s f i n i s h e d e x t r a c t was spotted on d u p l i -cate Thin Layer Chromatography sheets (Eastman Kodak Type 6060 s i l i c a g e l , with f l u o r e s c e n t i n d i c a t o r ) and subjected t o ascend-i n g chromatography i n a mixture of benzene/ethanol 9:1 (Rander-a t h , 1963). Upon completion of the run the sheets were developed by exposure t o i o d i n e vapor and the p o s i t i o n s of the marker s t e r o i d s i d e n t i f i e d by a l i g h t l y p e n c i l l e d c i r c l e . One member of the p a i r of d u p l i c a t e TLC sheets was used to produce an autoradiogram by p l a c i n g i t i n contact w i t h Kodak No-Screen X-Ray F i l m (Eastman Kodak Co., Rochester, N.Y.) w i t h i n a l i g h t - t i g h t c a s s e t t e f o r a period of 10 days. Films were de-> veloped f o r 5 minutes at 20°C i n Kodak L i q u i d X-Ray Developer 1 3 and f i x e d f o r 3 minutes i n Kodak L i q u i d X-Ray F i x e r . A l l darkroom operations were c a r r i e d out under i l l u m i n a t i o n from a Kodak Model A 2-way Safelamp, equipped with a 6B f i l t e r . A contact p r i n t was made from each developed autoradiogram as a permanent record. The second TLC sheet was cut i n t o s e c t i o n s as o u t l i n e d i n Figure 1; each s e c t i o n was placed i n a s c i n t i l l a t i o n v i a l w i t h 10 ml Aquasol (New England Nuclear, Boston, Mass.) and assayed f o r C 1^ a c t i v i t y f o r 5 0 minutes i n a Packard Tri-Carb Model 3 3 7 5 l i q u i d s c i n t i l l a t i o n counter oper a t i n g at an o v e r a l l count-i n g e f f i c i e n c y of 7 3 $ . Values i n dpm/ml were then p l o t t e d as a f u n c t i o n of dis t a n c e from the o r i g i n . Experiment I I I . In v i v o metabolism of 4-C' L^-estradiol-17ff T h i s experiment was c a r r i e d out during the summer of 1972, using s e x u a l l y maturing male and female coho salmon o f 5 0 0 grams approximate average body weight. Pure p,p f DDT (100 ppm) was fed as before f o r 21 days or f o r 7 days; i n a d d i t i o n , a c o n t r o l treatment of r e g u l a r d i e t l e s s the corn o i l v e h i c l e was i n c l u -ded. There was no m o r t a l i t y during the course of the experiment. T h i r t y f i s h were used. I n j e c t i o n o f 4-C"^-estradiol-17@ and e x t r a c t i o n of the me-t a b o l i c products were c a r r i e d out according t o the method of Donaldson and Fagerlund (1963). A l l i n j e c t i o n s were performed at the same time of day, between 9 and 10 A.M., i n order t o el i m i n a t e any p o s s i b l e e f f e c t of d a i l y rhythms. T h i r t y minutes p r i o r to i n j e c t i o n , the aquarium water supply was shut o f f and 4 . 5 grams of t r i c a i n e methane sulphonate (Kent Chemicals, Van-couver, B.C.) added t o give a con c e n t r a t i o n i n the water of 14 S O L V E N T F R O N T O E S T R O N E O E S T R A D I O L o E S T R I O L DWO o o « * o e O R I G I N F i g u r e 1. S e c t i o n i n g of the TLC sheets f o r s c i n t i l l a t i o n c o u n t i n g . S e c t i o n s 1 and 5 s u b d i v i d e d i n t o 1 cm l e n g t h s and counted as such. 15 1:26,000. Although t h i s c o n c e n t r a t i o n caused a l o s s o f e q u i l -i b r i u m , the f i s h r e t a i n e d the a b i l i t y t o swim and c o u l d s u r -v i v e immersion i n the s o l u t i o n f o r over 7 hours, e x h i b i t i n g ap-p a r e n t l y complete r e c o v e r y upon removal to c l e a n sea water. Af-t e r 45 minutes i n the a n e s t h e t i c the salmon was wrapped i n a wet paper towel and t r a n s f e r r e d to a 'V c r o s s - s e c t i o n f i s h h o i * der, where 625,500 dpra of 4-C 1 / 4--estradiol-17p , d i s s o l v e d i n 0.25 ml 6% s a l i n e / e t h a n o l 9:1, were i n j e c t e d i n t o the d o r s a l a-o r t a i n the r o o f o f the mouth from a 1 ml d i s p o s a b l e s y r i n g e by way o f a 25 gauge, V $ i n c h hypodermic needle. The salmon was then r e t u r n e d t o the a n e s t h e t i c bath. The f i s h was removed from the aquarium 10 minutes a f t e r i n -j e c t i o n and p l a c e d v e n t r a l s u r f a c e uppermost i n the f i s h h o l -der. A 0.5 ml sample o f blood was removed from the t a i l r e g i o n u s i n g a 2 ml h e p a r i n i z e d V a c u t a i n e r and a 20 gauge, 1^ i n c h n e e d l e . The f i s h was then r e t u r n e d to the aquarium and the blood sample placed on crushed i c e . F u r t h e r blood samples were taken i n the same manner at 20, 40, 80, 160, 210 and 270 minutes a f -t e r i n j e c t i o n . The f i s h was then k i l l e d by a blow t o the head. Whole blood was c e n t r i f u g e d at 5000 x g at 2°C and the c e l l f r a c t i o n d i s c a r d e d . T o t a l r a d i o a c t i v i t y was determined by c o u n t i n g 0.1 ml plasma i n 10 ml Aquasol i n the manner a l r e a d y d e s c r i b e d . 0.35 ml o f the remaining plasma was shaken f o r 2 minutes wi t h 10 ml r e d i s t i l l e d dichloromethane i n a 10 ml stop-pered g l a s s c e n t r i f u g e tube. A f t e r c e n t r i f u g a t i o n at 600 x g, a 5.0 ml a l i q u o t o f the o r g a n i c phase was t r a n s f e r r e d t o a se-cond 10 ml c e n t r i f u g e tube and, a f t e r the a d d i t i o n o f a u t h e n t i c e s t r a d i o l as marker, evaporated t o dryness under n i t r o g e n . The 16 d r i e d s t e r o i d s were then r e d i s s o l v e d i n 25 \sl r e d i s t i l l e d me-t h a n o l . T o t a l d i c h l o r o m e t h a n e - e x t r a c t a b l e r a d i o a c t i v i t y was de-termined by e v a p o r a t i n g t o dryness 5 . 0 u l of the methanol ex-t r a c t and counting f o r 50 minutes i n Aquasol. Another 5 . 0 j J l was a p p l i e d t o a TLC sheet and chromatographed i n the system benzene/ethanol 9 : 1 , developed i n i o d i n e vapor and t h e area c o r r e s p o n d i n g t o e s t r a d i o l marked as b e f o r e . T h i s zone o f the chromatogram was then cut out and counted i n 10 ml Aquasol, and r e p r e s e n t s the amount of u n a l t e r e d e s t r a d i o l i n the p a r t i c u l a r plasma sample. To enable a p r e l i m i n a r y i d e n t i f i c a t i o n o f the m e t a b o l i t e s produced i n v i v o , 4 female salmon (2 f e d DDT f o r 21 days, 2 f e d the c o n t r o l d i e t c o n t a i n i n g the corn o i l v e h i c l e ) were i n -j e c t e d as b e f o r e , and a s i n g l e blood sample taken from each a f -t e r 60 minutes. T h i s sample was s u b j e c t e d t o the e x t r a c t i o n pro-cedures a l r e a d y d e s c r i b e d , with the e x c e p t i o n t h a t e s t r a d i o l , estrone and e s t r i o l were a l l added as markers p r i o r t o evapor-a t i o n . Each e x t r a c t was then chromatographed i n t r i p l i c a t e , u s i n g the f o l l o w i n g t h r e e systems: benzene/ethanol 9 : 1 , c h l o r -oform/acetone 9 : 1 and c h l o r o f o r m (Randerath, 1 9 6 3 ) . A f t e r d e v e l -opment i n i o d i n e vapor and marking o f the r e f e r e n c e s t e r o i d s , each chromatogram was placed i n contact with X-Ray f i l m as de-s c r i b e d e a r l i e r . Contact p r i n t s o f the developed autoradiograms were agai n made. A disappearance curve f o r e s t r a d i o l f o r each f i s h was con-s t r u c t e d by p l o t t i n g the number o f dpm/ml plasma a s c r i b e d t o es-t r a d i o l a g a i n s t time on s e m i l o g a r i t h m i c paper. I t was apparent t h a t , i n a l l f i s h , r a d i o a c t i v i t y f e l l o f f r a p i d l y f o r between 17 40 and 60 minutes a f t e r i n j e c t i o n , and t h a t , a f t e r that p o i n t , the s l o p e o f the disappearance curves became l e s s steep. From t h i s i t was concluded t h a t the i n j e c t e d 4 - C ^ - e s t r a d i o l - 1 7 ^ was d i s t r i b u t e d i n more than one compartment. Procedures f o r the mathematical e v a l u a t i o n of s t e r o i d dynamics i n a two-com-partment system have been provided by T a i t et a l . (1962) and T a i t ( I 9 6 3 ) . In accordance with these authors the disappearance curves f o r i n j e c t e d 4 - C ^ - e s t r a d i o l - 1 7 r 3 were c o n s i d e r e d i n two p a r t s - an ' i n i t i a l ' segment between 10 and 40 minutes and a ' t e r m i n a l ' segment between 160 and 270 minutes. A s t r a i g h t l i n e of i n t e r c e p t B and s l o p e $ was f i t t e d t o the t e r m i n a l p a r t of the curve by g r a p h i c e s t i m a t i o n , and the values at 1 0 , 20 and 40 minutes on t h i s ' t e r m i n a l ' l i n e , e x t r a p o l a t e d t o zero time, were s u b t r a c t e d from the r e s p e c t i v e v a l u e s on the ' i n i t i a l ' p a r t o f the curve. Using the p o i n t s thus obtained, a second l i n e of i n t e r c e p t A and slope cc was obtained by graphic e s t i -mation ( F i g u r e 2 ) . The composite curve represented by the two l i n e s may be d e s c r i b e d by the formula: C = Ae_<cT+Be~*T where C i s the number o f d i s i n t e g r a t i o n s per minute a s c r i b e d t o e s t r a d i o l i n 1 . 0 ml plasma at time T. The r a t e constants K]_ f o r the interchange between the i n n e r and outer pools and Kg f o r s e c r e t i o n from the i n n e r pool were obtained from the f o r -mulae : Ki _ A*+ BB A + B K o_d ^ ( A 4 - B) ~ A^4- B«. 18 Figure 2. Resolution of the two exponential components of the clearance curve f o r e s t r a d i o l . 19 The h a l f - l i f e o f 4-C J- Z 4'-estradiol-17^ i n the i n n e r pool was c a l c u l a t e d from the formula: T i _ 0.693 and o u t e r pools r e s p e c t i v e l y were c a l c u l a t e d , f o l l o w i n g the me-thod o f Wagner and Northam (1967), from the formulae: where D i s the t o t a l amount o f i n j e c t e d s t e r o i d i n dpm. The metabolic c l e a r a n c e r a t e (MCR), which has been d e f i n e d as the volume o f blood c l e a r e d completely and i r r e v e r s i b l y o f s t e r o i d i n u n i t time ( T a i t , 1963), was c a l c u l a t e d on the b a s i s of 1000 grams of f i s h , u s i n g the formula: V 1 = D A+ B MCR = V 1 x K 2 20 RESULTS Experiment I . Determination of Cytochrome P - 4 5 0 Hepato-somatic indexes (HSI) f o r the experimental f i s h are r e p o r t e d i n Table I . At the dosages f e d , DDT and methoxy-c h l o r do not appear to a f f e c t t h i s parameter. The reduced form o f Cytochrome P - 4 5 0 binds with carbon monoxide t o form a complex a b s o r b i n g l i g h t at 450 my. A peak at 450 mp was observed i n a l l f i s h s t u d i e d , i n a d d i t i o n t o a sec-ond, s m a l l e r one at 420 my. T h i s l a t t e r peak was seen t o i n c r e a s e i n height a t t h e expense o f t h a t at 450 mu when samples were l e f t u n d i s t u r b e d i n the spectrophotometer; the phenomenon i s r e p r e s e n t e d i n F i g u r e 3 . O v e r a l l shape of the d i f f e r e n c e spec-t r a was unchanged i n i n s e c t i c i d e - t r e a t e d f i s h . Using a molar e x t i n c t i o n c o e f f i c i e n t of 91 x 10-* cm^/mmole (Omura and Sato, 1964) andAO.D. between 450 and 490 mu, the content o f Cyto-chrome P-450 was c a l c u l a t e d f o r the f o u r treatments. The r e s u l t s are summarized i n Table I I . Experiment I I . I n c u b a t i o n o f 4 - C 1 ^ - e s t r a d i o l - 1 7 £ 3 with l i v e r s l i c e s R a d i o a c t i v i t y on the t h i n - l a y e r chromatograms, s u b d i v i d e d and counted as d e s c r i b e d e a r l i e r , i s presented i n F i g u r e 4 . Each s i n g l e bar o f the histograms r e p r e s e n t s the t o t a l number o f d i s i n t e g r a t i o n s per minute per ml i n c u b a t i o n medium i n one segment o f the chromatogram. Assignment of m e t a b o l i t e s to each segment i s made on the b a s i s o f a u t o r a d i o g r a p h i c evidence. A c o n t a c t p r i n t made from one of the autoradiograms i s i n -cluded as F i g u r e 5 . E s t r a d i o l , estrone and e s t r i o l are i d e n t i -f i e d on the b a s i s o f the chromatographic m o b i l i t i e s o f a u t h e n t i c 21 Table I . Hepato-somatic indexes f o r c o n t r o l and i n s e c t i c i d e -t r e a t e d f i s h . Treatment No. f i s h Mean body weight HSI i n grams C o n t r o l 9 49.9 1.6 ± .2 DDT 10 ppm 9 53.2 1.8 ± .2 DDT 100 ppm 9 60.1 1.7 i .3 Methoxychlor 100 ppm 9 62.2 1.6 ± .2 expressed as meant standard d e v i a t i o n D i f f e r e n c e s i n mean HSI are s t a t i s t i c a l l y n o n - s i g n i f i c a n t ( s i n g l e c l a s s i f i c a t i o n a n a l y s i s of v a r i a n c e ; P<.001). 22 Figure 3. Carbon monoxide d i f f e r e n c e spectra i n c o n t r o l f i s h . (A) i n i t i a l reading (B) a f t e r 15 minutes (C) a f t e r 55 minutes (D) a f t e r 95 minutes (E) a f t e r 135 minutes 2 3 0.05--0.05 390 420 450 480 WAVELENGTH (mu ) 24 Table I I . Cytochrome P - 4 5 0 content i n c o n t r o l and i n s e c t i -c i d e t r e a t e d f i s h . Treatment No. f i s h Cytochrome P - 4 5 0 c o n t e n t 3 C o n t r o l 9 1 . 3 2 ± . 0 4 DDT 10 ppm 9 1 . 2 6 ± . 0 3 DDT 100 ppm 9 1 . 9 8 ± . 0 4 " * * Methoxychlor 100 ppm 9 1 .35 ± . 0 4 a mean v a l u e , expressed as nraoles/ 1 0 0 0 mg l i v e r ± standard d e v i a t i o n S i n g l e c l a s s i f i c a t i o n a n a l y s i s o f v a r i a n c e i n d i c a t e s t h a t 100 ppm DDT i n t h e d i e t s i g n i f i c a n t l y i n c r e a s e s the l e v e l o f l i v e r microsomal Cytochrome P-450 (P<.001). 25 F i g u r e 4. M e t a b o l i t e s produced by l i v e r s l i c e s incubated w i t h 4_Cl4-estradiol-17£ . Student's ' t ' t e s t s i n d i c a t e s i g n i f i c a n t (P<.001) i n c r e a s e s i n r a d i o a c t i v i t y i n the ' U n i d e n t i f i e d P o l a r M e t a b o l i t e ' and ' E s t r i o l plus U n i d e n t i f i e d M e t a b o l i t e s ' segments of the chrO' matograms. D P M / M I N / M L INCUBATION M E D I U M o estrone I-unaltered unidentified polar metabolite(s) estr iol plus unidentified metabolite(s) !1H estrone unaltered estradiol unidentif ied polar metabolite(s) estriol plus unidentified metabol ite(s) 26 • • • o o F i g u r e 5 . Autoradiogram made a f t e r TLC s e p a r a t i o n of met b o l i t e s produced i n l i v e r s l i c e i n c u b a t i o n s . C t r o l f i s h . 27 marker s t e r o i d s . Experiment I I I . In vivo metabolism of / f - C ^ - e s t r a d i o l - 1 7 3 Representative autoradiograms from the 60-minute i n j e c -t i o n s e r i e s are included as Figure 6. I d e n t i f i c a t i o n of metab-o l i t e s i s made on the b a s i s of the r e l a t i v e m o b i l i t i e s of au-t h e n t i c e s t r a d i o l , estrone and e s t r i o l i n the three chromato-graphic systems employed. Disappearance curves f o r t o t a l plasma r a d i o a c t i v i t y , d i -chloromethane-extractable r a d i o a c t i v i t y and un a l t e r e d e s t r a d i o l are presented i n Figure 7. The p a r t i c u l a r curves shown repre-sent mean values f o r the ten c o n t r o l f i s h f ed the i n s e c t i c i d e v e h i c l e ; curves f o r the remaining three treatments are v i r t u -a l l y i d e n t i c a l . C a l c u l a t e d values f o r T i , V±, V 2 and MCR f o r the f o u r treatments are presented i n Table I I I . P r e l i m i n a r y s t a t i s t i c a l a n a l y s i s i n d i c a t e d that p o o l i n g of values f o r male and female f i s h was permissable ( s i n g l e c l a s s i f i c a t i o n a n a l y s i s of var-i a n c e , P <.001). 28 (a) (b) (c) Figure 6. Autoradiograms from the 60-minute i n j e c t i o n s e r i e s . Control f i s h . (a) benzene/ethanol 9:1 (b) chloroform/acetone 9:1 (c) chloroform 2 9 F i g u r e 7. Disappearance curves f o r t o t a l plasma r a d i o a c t i v -i t y , d i c h l o r o m e t h a n e - e x t r a c t a b l e r a d i o a c t i v i t y , and 4-C l z»-estradiol-17^ f o r c o n t r o l f i s h . 30 < CO < i a. O 600-500-1 • total plasma radioactivity dichloromethane- extractable radioactivity estradiol 400-300-4 200-i 100-90 -80-70-60 -50-40-i 30-20-\ \ . \ • \ • \ 20 40 60 80 I 100 i 120 140 "Ho" 180 — I 200 220 240 T I M E I N M I N U T E S 31 Table I I I . In viv o metabolism of 4-C" 1"^-estradiol-17^ . Treatment No. f i s h T ia V - ^ V 2C MCRd C o n t r o le 10 45.8 ± 3.8 1 5 . 7 ± . 7 5 7 . 9 ± 4 . 8 1 4 3 . 0 * - 1 4 . 6 C o n t r o lf 5 4 6 . 0 ± 5 . 6 1 5 . 0 ± . 5 5 7 . 0 ± 4 . 7 1 3 7 . 3 ± 14 .3 DDT 21 days 10 4 7 . 7 ^ 2 . 1 1 5 . 2 ± . 4 5 5 . 9 ± 1 . 7 139.Oi 1 8 . 0 DDT 7 days 5 4 7 . 6 + 0 . 8 15 .4 ± . 5 5 7 . 2 + 4 . 3 1 3 7 . 0 + 1 0 . 9 ^ expressed i n minutes + standard d e v i a t i o n expressed as % body weight ± standard d e v i a t i o n ^ expressed as $ body weight ± standard d e v i a t i o n expressed as ml/kg body weight/hr ± standard d e v i a t i o n ® fed the corn o i l v e h i c l e corn o i l v e h i c l e omitted from d i e t S i n g l e c l a s s i f i c a t i o n a n a l y s i s of variance demonstrated no s i g n i f i c a n t added variance component among the four t r e a t -ments (P <.001). 3 2 DISCUSSION Among the salmonids, coho salmon e x h i b i t a s u r p r i s i n g r e -s i s t a n c e t o d i e t a r y o r g a n o c h l o r i n e i n s e c t i c i d e s . C u t t h r o a t t r o u t , f o r example, have been found t o be 13.5 times as sus-c e p t i b l e t o DDT i n terms o f m o r t a l i t y (Post and Schroeder, 1971). The r e s u l t s o f the present study would c e r t a i n l y appear to i n d i c a t e t h a t coho salmon can withstand exposure t o 100 ppm DDT or methoxychlor w i t h l i t t l e or no apparent gross p h y s i o l -o g i c a l upset; the s t a b i l i t y o f the hepato-somatic index through-out the f e e d i n g p e r i o d i s o f f e r e d as evidence o f t h i s , as are ; the low m o r t a l i t i e s observed. The f a c t t h a t , i n the e n t i r e course o f the study, o n l y one i n s e c t i c i d e - t r e a t e d f i s h d i e d i s p a r t i c u l a r l y impressive; i t i s a l s o i n t e r e s t i n g t o note t h a t the lone c a s u a l t y was an immature f i s h . I t has been amply dem-o n s t r a t e d t h a t f i s h s i z e g r e a t l y i n f l u e n c e s t o x i c i t y ; not onl y are s m a l l e r , younger f i s h more s u s c e p t i b l e t o a given d i e t than l a r g e r , o l d e r f i s h , but Median S u r v i v a l Time i s d i r e c t l y pro-p o r t i o n a l t o body weight i n young coho o f the same age (Buhler and Shanks, 1970). The s t a b i l i t y o f the hepato-somatic index i s best viewed i n the l i g h t o f the often-made o b s e r v a t i o n t h a t , i n mammals at l e a s t , a d m i n i s t r a t i o n o f o r g a n o c h l o r i n e i n s e c t i c i d e s i n t he d i e t b r i n g s about an i n c r e a s e i n l i v e r weight (Remmer and Merker, 1964; Hart and Fouts, 1965), although i t should be mentioned t h a t Buhler et al.(1969) r e p o r t e d a s i g n i f i c a n t reduc-t i o n i n l i v e r s i z e i n coho f i n g e r l i n g s f o l l o w i n g 60 days expos-ure t o d i e t a r y DDT at a l e v e l o f 100 ppm. The carbon monoxide d i f f e r e n c e s p e c t r a obtained are i n every way t y p i c a l o f those r e p o r t e d f o r mammals and i n s e c t s 33 (Fukami et a l . , 1969), and s t r o n g l y suggest the presence of Cytochrome P-450 i n coho l i v e r microsomes. Although the a b i l i t y o f f i s h to perform numerous b i o t r a n s f o r m a t i o n s o f f o r e i g n com-pounds would appear t o imply i t s presence, t h e r e e x i s t s but one demonstration o f t h i s , and that i n rainbow t r o u t l i v e r (Chan et a l . , 1966). In t h i s i n s t a n c e the amounts r e p o r t e d are about twice those found i n the present study; i t should be r e c a l l e d , how-ever, t h a t the l a t t e r study u t i l i z e d immature f i s h and t h a t , i n r a t s , the microsomal content o f Cytochrome P-450 has been shown t o i n c r e a s e w i t h the onset o f sexual m a t u r i t y ( G i l l e t t e e t a l . , 1972). The change i n the r e l a t i v e h e i g h t s o f the absorbance peaks at 450 and 420 mp may be i n t e r p r e t e d as a c o n t i n u i n g con-v e r s i o n of Cytochrome P - 4 5 0 t o Cytochrome P-420, i t s i n a c t i v e form ( M i t a n i et a l . , 1971). Of more s i g n i f i c a n c e , however, i s the o b s e r v a t i o n t h a t exposure t o 100 ppm DDT i n the d i e t f o r 21 days s i g n i f i c a n t l y i n c r e a s e d the l e v e l o f l i v e r microsomal Cytochrome P - 4 5 0 i n j u -v e n i l e coho. Here, then, i s a measurable b i o c h e m i c a l e f f e c t of an o r g a n o c h l o r i n e i n s e c t i c i d e upon the Mixed F u n c t i o n Oxidase system of a f i s h - a p a r t i a l answer t o the f i r s t g e neral ques-t i o n posed i n the I n t r o d u c t i o n . C o n c l u s i o n s as t o the p h y s i o l -o g i c a l importance o f t h i s i n c r e a s e remain t o be drawn i n the l i g h t o f i n v i v o evidence t o be d i s c u s s e d l a t e r . The l a c k o f an e f f e c t o f methoxychlor upon the content o f Cytochrome P-450 does, however, bear immediate c o n s i d e r a t i o n , f o r i t i s t h i s com-pound which has assumed re c e n t importance as one o f a number of 'replacements' f o r DDT (Kapoor and M e t c a l f , 1970), a d e s i g -n a t i o n prompted p r i m a r i l y by i t s lower t o x i c i t y t o mammals (Ed-34 wards and G l a s s , 1 9 7 1 ) . The apparent i n a b i l i t y o f methoxy-c h l o r , d e s p i t e i t s c l o s e s t r u c t u r a l s i m i l a r i t y to DDT, t o induce Cytochrome P -450 may c o n s t i t u t e a f u r t h e r , more p a r t i c u l a r r e -commendation f o r t h a t compound. A d i s c u s s i o n o f the remainder o f the r e s u l t s c o n cerning the e f f e c t o f DDT upon the metabolism of e s t r a d i o l must now be d e f e r r e d i n f a v o r o f a b r i e f o u t l i n e of the v a r i o u s pathways of b i o s y n t h e s i s and metabolism o f t h a t hormone. The mammalian pat-t e r n of estrogen b i o s y n t h e s i s , from c h o l e s t e r o l v i a t e s t o s t e r -one and progesterone, i s presented i n F i g u r e The a p p l i c a b i l -i t y o f such a scheme to salmonids remains t o be r i g o r o u s l y dem-o n s t r a t e d , although the g e n e r a l p a t t e r n o f b i o s y n t h e s i s i s now f e l t t o be s i m i l a r i n f i s h and h i g h e r v e r t e b r a t e s (Breuer and Ozon, 1 9 6 5 ) . I n f o r m a t i o n concerning the metabolism o f estrogens i n f i s h i s , however, much l e s s complete - F i g u r e 9 , which again r e p r e s e n t s the mammalian p a t t e r n , i s i n c l u d e d simply as an i n d i -c a t i o n of the d i v e r s i t y of pathways i n v o l v e d , pathways which have been but l i t t l e i n v e s t i g a t e d i n f i s h . As a f u r t h e r compli-c a t i o n , i t should be r e c a l l e d t h a t l i v e r metabolism o f s t e r o i d hormones may i n v o l v e c o n j u g a t i o n with g l u c u r o n i c and s u l f u r i c a c i d s ; p r e l i m i n a r y experiments undertaken i n the present study i n d i c a t e d c o n s i d e r a b l e involvement of one o r both of these com-pounds i n the metabolism o f 4-C*'"^-estradiol-17(3 both i n v i t r o and i n v i v o . With t h i s i n f o r m a t i o n i n mind, i t i s i n t e r e s t i n g t o note t h a t , i n the present study and w i t h i n the l i m i t s o f chromato-graphic evidence, both e s t r i o l and estrone appear to occur as metabolic products o f e s t r a d i o l ; t h a t the l i v e r i s the main s i t e T e s t o s t e r o n e Androstenedione 17cC-Hydroxy-E s t r i o l Estradiol-17(3 E s t r o n e F i g u r e #. The b i o s y n t h e s i s of key sex s t e r o i d s . From Yamamoto" (I969). 6oC-Hydroxyestrone 6<c-Hydroxyestradiol-17p ^ 6-Ketoestradiol - 1 7 p ;, * 6-Ketoeestrone 6$-Hydroxyestradiol-17p 7<t-Hydroxy e s t r a d i o l 15ot-Hydroxyestradiol 1 5 @ - H y d r o x y e s t r a d i o l * — x E s t r a d i o l - 1 7 ^ * z z l 6^-Hydroxyestrone 15<t-Hydroxyestrone 158-Hydroxyestrone E s t r i o l Estrone 2 - H y d r o x y e s t r i o l I 2-Methoxyestriol 2-Hydroxyestradiol 2-Methoxyestradiol l6«t-Hydroxyestrone *• 2-Hydroxyestrone ^16-Ketoestrone i I * 2-Methoxyestrone 1 6 - E p i e s t r i o l Figure 9. Some i n t e r r e l a t i o n s h i p s of estrogen metabolism. From White et a l . , (1968). 37 of metabolic alteration i s suggested by the similarity between the chromatographic patterns of metabolites produced in vitro  a n (^ in vivo. Furthermore, clear demonstration has been made of the presence of at least one further unidentified polar metab-ol i t e whose chromatographic mobility is approximately midway between that of estradiol and e s t r i o l . More sophisticated sep-aration and identification techniques may indeed demonstrate, to a much greater extent than has been done here, a considerable similarity between the metabolic transformations of estrogens performed by fish and those outlined in Figure 9. Returning now to the effect of dietary DDT upon the meta-bolism of estrogens in coho, we find that, in maturing male and female fish in vitro, significant increases occur in the pro-duction of es t r i o l and the as yet unidentified polar metabol-ites from estradiol-17@ . As was the case in the Cytochrome P-450 experiments, these results bear considerable similarity to those obtained in mammals and birds, and appear to substanti-ate the suspicion of an insecticide-mediated disturbance of the normal metabolic process, effected through an increase in activ-ity of the microsomal Mixed Function Oxidase system. We turn now to a consideration of the results obtained from the experiments performed in vivo. A possible model for the metabolism and transport of a steroid consists of an inner pool (which, for steroids weakly bound to plasma proteins other than albumin, likely includes the plasma, extracellular volumes and, assuming a relatively rapid transport from plasma to meta-bolic c e l l s , the liver) and an outer pool. The whole of the in-ner pool i s assumed to be in rapid equilibrium with the plasma, 38 while t r a n s p o r t o f the s t e r o i d i n t o the out e r pool i s r e l a -t i v e l y slow. T a i t et a l . (1962) s t r e s s t h a t the two pools need not n e c e s s a r i l y be w e l l - d e f i n e d a n a t o m i c a l l y . F i g u r e 10 r e -pr e s e n t s the two-compartment s i t u a t i o n , where i s a r a t e con-stant d e s c r i b i n g t r a n s p o r t between the i n n e r p o o l o f volume and the o u t e r p o o l o f volume V g . The o v e r a l l metabolic a c t i v -i t y o f the l a t t e r i s d e s c r i b e d by the r a t e constant K^. The disappearance curves o b t a i n e d , then, r e f l e c t a combination o f the e f f e c t s o f metabolism and t r a n s p o r t o f the s t e r o i d . I t w i l l be r e c a l l e d t h a t the M e t a b o l i c Clearance Rate r e p r e s e n t s the volume of blood i r r e v e r s i b l y c l e a r e d o f s t e r o i d per u n i t o f time. With these r e l a t i o n s h i p s i n mind, then, i t i s i n t e r e s t i n g t o c o n s i d e r the r e s u l t s o f the i n j e c t i o n experiments. As has a l -ready been mentioned, two-compartment k i n e t i c s were a p p l i e d t o the data as a r e s u l t o f the c l e a r a n c e curves d i s p l a y i n g a two-ex p o n e n t i a l form. Examination o f the data i n d i c a t e s t h a t the i n j e c t e d 4 - C ^ - e s t r a d i o l - 1 7 ( 3 i s r a p i d l y d i s t r i b u t e d i n t o a v o l -ume of about 150 ml and t h a t , a f t e r t h i s i n i t i a l d i s t r i b u t i o n , the r a d i o a c t i v i t y appears to move more slowly i n t o a t o t a l c a l -c u l a t e d volume o f about 65O ml. Donaldson and Fagerlund (1968), who performed s i m i l a r experiments u s i n g r a d i o l a b e l l e d C o r t i s o l and Oncorhynchus nerka, obtained volumes of d i s t r i b u t i o n f o r th a t s t e r o i d o f about 50 and 350 ml r e s p e c t i v e l y ; MCR's f o r C o r t i s o l were approximately one f i f t h those obtained i n t h e pre-sent study. S i m i l a r d i s c r e p a n c i e s between C o r t i s o l c l e a r a n c e and a l d o s t e r o n e c l e a r a n c e i n man have r e p e a t e d l y been noted ( T a i t , 1963; T a i t et a l . , 1962) , and have been a t t r i b u t e d t o the 39 INJECTED ESTRADIOL OUTER POOL VOLUME = V2 TRANSPORT INNER POOL VOLUME = V, K-METABOLISM F i g u r e 10. Two-compartment model d e s c r i b i n g the metabolism and t r a n s p o r t of e s t r a d i o l - 1 7 6 . From T a i t et a l . (1961). 40 s t r o n g b i n d i n g of C o r t i s o l to plasma t r a n s c o r t i n , a s i t u a t i o n which p r e c l u d e s i t s r a p i d deployment throughout the two body compartments and subsequent r a p i d metabolism. That an analo-gous e x p l a n a t i o n may be advanced i n the case o f e s t r a d i o l i s i n d i c a t e d by the o b s e r v a t i o n t h a t , i n the r a t a t any r a t e , plasma estrogens are but weakly bound to p r o t e i n s o t h e r than albumin (de Hertogh et a l . , 1970). F u r t h e r experimentation i s necessary to e s t a b l i s h whether, as i s the case with a l d o s t e r -one i n man, the a p p a r e n t l y r a p i d and e f f i c i e n t metabolism o f e s t r a d i o l i n d i c a t e s an i d e n t i t y between MCR and h e p a t i c blood flow ( T a i t , 1963). E f f i c i e n c y o f metabolism must, however, be c o n s i d e r e d i n a r e l a t i v e sense. Although the present r e s u l t s would appear t o i n d i c a t e a much more r a p i d metabolism o f e s t r a -d i o l than C o r t i s o l i n salmonids, the metabolism of e s t r a d i o l i n salmonids, expressed per u n i t o f body weight, i s 6-8 times l o -wer than the c o r r e s p o n d i n g v a l u e s i n r a t s and humans (de Her-togh et a l . , 1970). That t h i s should be the case i s not sur-p r i s i n g ; although f i s h l i v e r microsomes have indeed been shown t o c a r r y out many o f the m e t a b o l i c t r a n s f o r m a t i o n s f i r s t ob-served i n mammals, a b s o l u t e metabolic a c t i v i t y i s o f t e n lower i n the former. N-demethylation o f aminopyrine, f o r i n s t a n c e , c a r -r i e d out under o p t i m a l c o n d i t i o n s i n both r a t and t r o u t l i v e r microsomes, i s e f f e c t e d about 15 times as r e a d i l y i n the f o r -mer (Dewaide and Henderson, 1968). Perhaps the most s t r i k i n g f e a t u r e o f the c l e a r a n c e r e s u l t s i s , however, the v i r t u a l i d e n t i t y between the v a l u e s f o r c o n t r o l and i n s e c t i c i d e - t r e a t e d f i s h . Although the i n v i t r o r e s u l t s c l e a r l y p o i n t e d to an i n c r e a s e i n e s t r a d i o l - m e t a b o l i z i n g a c t i v -41 i t y i n l i v e r microsomes, no such e f f e c t i s apparent when t h i s h y p o t h e s i s i s t e s t e d i n v i v o . Although t h e r e i s s t i l l every reason t o b e l i e v e t h a t the MFO system i s i n some way a l t e r e d as a r e s u l t o f exposure to d i e t a r y DDT, i t i s p l a i n t h a t meta-bo l i s m of e s t r a d i o l i n the normal s t a t e i s so r a p i d as t o r e -main u n a f f e c t e d by any changes produced. Measurable a l t e r a t i o n s i n the amounts o f m e t a b o l i t e s w i l l thus show up o n l y when the amount o f s u b s t r a t e i s the l i m i t i n g f a c t o r , a s i t u a t i o n d e l i b -e r a t e l y c r e a t e d i n the t i s s u e i n c u b a t i o n experiments. These c o n c l u s i o n s c o n t a i n an o b j e c t l e s s o n concerning the p h y s i o l o g i c a l a p p l i c a b i l i t y of r e s u l t s o b t a i n e d i n v i t r o . The expected e f f e c t o f DDT upon the microsomal MFO system was i n -deed found; with a l i t t l e j u d i c i o u s j u g g l i n g o f the i n c u b a t i o n c o n d i t i o n s and c o f a c t o r s , i t c o u l d l i k e l y have been c o n s i d e r -a b l y 'magnified*. Under more r e a l i s t i c i n v i v o c o n d i t i o n s , how-ever, the phenomenon appears to be of l i t t l e r e a l importance. C e r t a i n l y i n t h i s i n s t a n c e , and perhaps i n many o t h e r s , the whole animal remains unimpressed by t e s t - t u b e b i o l o g y . 42 S U M M A R Y The t h e s i s attempts t o shed some l i g h t upon the f o l l o w -i n g two general questions: (1) can exposure to an organochlor-ine i n s e c t i c i d e exert any measurable biochemical e f f e c t upon the Mixed Function Oxidase system of a f i s h ? and (2) i f the an-swer t o the f i r s t question i s a f f i r m a t i v e , can t h i s e f f e c t have p h y s i o l o g i c a l s i g n i f i c a n c e f o r the animal? Information concerning the f i r s t question was obtained through measurement of the l i v e r microsomal l e v e l of Cytochrome P-450, a component of the MFO system, and through i n c u b a t i o n of a normal substrate f o r that system, estradiol-17@ , w i t h l i v e r s l i c e s i n v i t r o . Evidence obtained from these experiments i n d i -cated that the answer to the f i r s t question was a f f i r m a t i v e -s t a t i s t i c a l l y s i g n i f i c a n t increases were noted i n a l l parameters measured. Information concerning the second question was obtained i n v i v o , through measurement of the clearance of t r a c e amounts of i n j e c t e d e s t r a d i o l - 1 7 ^ . i n the i n t a c t animal. Evidence obtained from these experiments i n d i c a t e d t h a t , i n coho salmon, enhance-ment of MFO a c t i v i t y i n v i t r o i s not r e f l e c t e d i n an enhanced clearance of e s t r a d i o l i n v i v o . The i n v i t r o events woxild ap-pear t o have l i t t l e p h y s i o l o g i c a l s i g n i f i c a n c e . 43 BIBLIOGRAPHY Adamson, R.H. 1 9 6 7 . Drug metabolism i n marine v e r t e b r a t e s . Fed. Proc. 2 6 : 1 0 4 7 - 1 0 5 5 . Anderson, J.M. 1971. Assessment of the e f f e c t s of p o l l u t a n t s on physiology and behaviour. 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Some i n v i t r o assay conditions that af f e c t detection and quantitation of phenobarbitol-induced increases in hepatic microsomal drug-metabolizing a c t i v i t y . Tox. App. Pharmacol. 16: 48-65. Fukami, J . I . , T. Shishido and J.E. Casida. 1969. Oxidative me-tabolism of rotenene i n f i s h , mammals and insects and i t s r e l a t i o n to selective t o x i c i t y . J . Ag. and Food Chem. 17: 1217-1226. Ghazal, A., W. Koranski and T. Po r t i g . 1964. Acceleration of det o x i f i c a t i o n reactions i n various i n s e c t i c i d e s . Arch. Pharmakol. Exptl. Pathol. 249: 1-10. 45 G i l l e t t e , J.W., T.M. Chan and L.C. T e r r i e r e . 1966. Interaction between DDT analogs and microsomal epoxidase systems. J. Ag. and Food Chem. 14: 540-555. G i l l e t t e , J.W., D.C. Davis and H.A. Sasame. 1972. Cytochrome P-450 and i t s role i n drug metabolism. Ann. Rev. Pharmacol. 12: 57-84. Hart, L.G. and J.R. Fouts. 1963. Ef f e c t s of acute and chronic DDT administration on hepatic microsomal drug metabolism i n the r a t . Proc. Soc. Exptl. B i o l . Med. 114: 388-392. Hart, L.G. 1965. Further studies on the stimulation of hepatic microsomal drug-metabolizing enzymes by DDT and i t s analogs. Arch. Pharmakol. Exptl. Pathol. 249: 486 -500. Juchau, M.R., T.E. Gram and M.R. Fouts. 1966. Stimulation of he-patic microsomal drug-metabolizing enzyme systems i n primates by DDT. Gastroenterol. 51: 213-218. Kapoor, D. and M. Metcalf. 1970. Comparative metabolism of me-thoxychlor, methiochlor and DDT i n mouse and insects i n a mo-del ecosystem. J . Ag. and Food Chem. 18: 1145-1152. Kuntzman, R., M. Jacobson, K. Sceidman and A. Conney. I964. S i m i l a r i t i e s between oxidative drug-metabolizing enzymes and steroid hydroxylases i n l i v e r microsomes. J . Pharmacol. Exp. Ther. 146: 280-2&5. Kuntzman, R. I969. Drugs and enzyme induction. Ann. Rev. Phar-macol. 9: 21-36. Ludke, J.L. C.R. Gibson and J.R. 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Steroids 19: 85-99. 46 Omura, T. and R. Sato. 1964. The carbon monoxide-binding pig-ment of l i v e r microsomes. J . B i o l . Chem. 239: 2370-2385. Peakall, D.B. 1967. Pesticide-induced enzyme breakdown of ster-oids i n birds . Nature 216: 505-506. Post, G. and T. Schroeder. 1971. The t o x i c i t y of four insec-t i c i d e s to four salmonids. B u l l . Envir. Contain. Tox i c o l . 6: 144-155. Randerath, K. 1963. Thin-layer chromatography. Academic Press, N.Y. 248p. Remmer, H. and K.J. Merker. 1965. Effect of drugs on the f o r -mation of smooth endoplasmic reticulum and drug-metabolizing enzymes. Ann. N.Y. Acad. S c i . 123: 79-97. Risebrough, R.W. 1968. PCB's in the global ecosystem. Nature 220: 1098-1105. Sokal, R. and F. Rohlf. 1969. Biometry. W.H. Freeman, S.F. 776p. Southren, A.L., Tochimoto, S., K. I s u r i g i and G.G. Gordon. 1966. Effect of DDD on the metabolism of infused C o r t i s o l . Steroids 7: 11-29. T a i t , J.F., S.A. T a i t , B. L i t t l e and R. Laumas. 1961. The d i s -appearance of aldosterone i n the plasma of normal subjects. J . C l i n . Invest. 40: 72-80. T a i t , J.F., B. L i t t l e , A.S. Tait and J . Flood. 1962. The meta-bol i c clearance rate of aldosterone i n pregnant and non-pregnant subjects estimated by both s i n g l e - i n j e c t i o n and constant infusion methods. J . C l i . Invest. 41: 2093-2100. T a i t , J.F. 1963. The use of isotopic steroids f o r the meas-urement of steroid production rates i n vivo. J . C l i n . Endocr. Metab. 23: 1285-1297. White, A., P. Handler and E. Smith. 1963. P r i n c i p l e s of bio-chemistry. McGraw-Hill, N.Y. Il87p. Yamamoto, T. 1969. Sex d i f f e r e n t i a t i o n . In Fish Physiology, v. I l l , ed. Hoar and Randall. AcademicTress, N.Y., 43,5p. 47 Appendix I . The o r g a n o c h l o r i n e i n s e c t i c i d e s The i n s e c t i c i d a l p r o p e r t i e s o f DDT were d i s c o v e r e d by M u l l e r i n 1 9 3 9 . The f i r s t commercially produced organochlor-ine i n s e c t i c i d e , i t i s s t i l l an important f a c t o r i n i n s e c t con-t r o l . Although DDT i s t e c h n i c a l l y a c h l o r i n a t e d hydrocarbon ( t h e r e i s no heteroatom i n i t s s t r u c t u r e ) , i t e x h i b i t s w i t h the remainder o f the o r g a n o c h l o r i n e i n s e c t i c i d e s the p r o p e r t i e s o f p e r s i s t a n c e and i n s o l u b i l i t y i n water which are p r e s e n t l y ma-k i n g t h e i r c o n t inued, l a r g e - s c a l e use q u e s t i o n a b l e . Subgroups w i t h i n the o r g a n o c h l o r i n e s i n c l u d e such s t r u c -t u r a l l y d i s s i m i l a r groups as DDT and i t s analogs, the c y c l o -dienes.and the hexachlorocyclohexanes. Compounds used i n the present study belong t o the f i r s t - m e n t i o n e d group; they a r e : CC\=> DDT; 2 , 2 - b i s ( p - c h l o r o p h e n y l ) - 1 , 1 , 1 - t r i c h l o r o e t h a n e O r a l LD 50 ( r a t ) : 250 mg/kg Methoxychlor; 2 , 2 - b i s ( p - m e t h o x y p h e n y l ) - l , 1 , 1 - t r i c h l o r o -ethane O r a l LD 50 ( r a t ) : 3000 mg/kg 4* Anrjendix I I . The Kixed Function Oxidase system Cytochrome P-450 s u b s t r a t e +5 NADPH-Cytochrome c ^ reductase Cytochrome P-450 • s u b s t r a t e s u b s t r a t e >+3 0-Cytocfrrome P-450 ^—^products r, n i r - ^ 1 ^ -u Cytochrome P-450 • product Cytochrome P - 4 5 0 - 0 * s u b s t r a t e J „ unknown donor 2H* Cytochrome P-450 - 0 « substrate The s u b s t r a t e i s b e l i e v e d t o combine f i r s t with the o x i d i z e d form of Cytochrome P - 4 5 0 , a carbon monoxide-sensitive hemo-p r o t e i n . The r e s u l t i n g complex i s then reduced by an e l e c t r o n from the f l a v o p r o t e i n , NADPH-Cytochrome c reductase; the r e -duced complex i n t u r n r e a c t s w i t h oxygen to form a s u b s t r a t e -P-45O-O2 complex. I t i s be l i e v e d t h a t a second e l e c t r o n then reduces t h i s complex t o form an a c t i v e oxygen intermediate which decomposes w i t h the formation of the product and o x i -d i z e d Cytochrome P -450 ( G i l l e t t e et a l . , 1 9 7 2 ) . 

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