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Physiological and biochemical mechanisms of dieldrin-induced reproductive lesions in the SWV mouse Virgo, Bruce Barton 1974

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PHYSIOLOGICAL, AND BIOCHEMICAL MECHANISMS OF DIELDRIN-INDUCED REPRODUCTIVE LESIONS IN THE Slv'V MOUSE by BRUCE BARTON VIRGO B.Sc., (Honours) U n i v e r s i t y of B r i t i s h Columbia, 1965 M.Sc., U n i v e r s i t y of B r i t i s h Columbia, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the D i v i s i o n of Pharmacology and Toxi c o l o g y Department of Pharmaceutical Sciences F a c u l t y of Graduate Studies We accept t h i s t h e s i s as conforming to the re q u i r e d standard The U n i v e r s i t y of B r i t i s h Columbia October, 1973 In presenting 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 of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that 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 reference and study. I f u r t h e r agree that permission f o r extensive 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 the Head of 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 that copying or p u b 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 allowed without my w r i t t e n permission. Department of Pharmaceutical Sciences The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada D a t e November 29, 1973 1.1 ABSTRACT The e f f e c t s o\i d i e l d r i n on the h e p a t i c , mixed-function oxidase system and on the r e p r o d u c t i v e processes of SWV female mice were s t u d i e d . L i v e r parameters and the _in v i t r o microsomal metabolism of imipra-mine were determined at i n t e r v a l s i n mice fed d i e t s c o n t a i n i n g 5, 10, 15 or 20 ppm d i e l d r i n f o r 10 weeks. Mean values from c o n t r o l mice were: 57.9 mg of l i v e r / g of body weight; 15.3 mg of microsomal p r o t e i n / g of l i v e r ; 0.91 nmoles of cytochrome P-450/mg of microsomal p r o t e i n . D i e l d r i n caused do s e - r e l a t e d increases of up to 60% i n l i v e r mass and 230% i n P-450; microsomal p r o t e i n was increased by 30-357» at a l l doses. L i v e r mass and microsomal p r o t e i n reached plateau l e v e l s by 2 weeks of exposure; P-450 reached maximum l e v e l s by 3-4 weeks of exposure and then d e c l i n e d to p l a t e a u l e v e l s , 40% above normal, by 5-8 weeks of exposure. Imipramine hydroxyla-t i o n was increased by 307, a f t e r 8 days of exposure to 20 ppm; demethylation increased by 50% d u r i n g weeks 4-8 of exposure to 20 ppm; N - o x i d a t i o n was concomitantly decreased. S i m i l a r metabolic changes were caused by longer exposure to the lower doses. The in v i v o metabolism of p e n t o b a r b i t a l was increased by 430% i n mice fed e i t h e r 5, 15, or 25 ppm f o r 4 weeks. I t was concluded that the r e q u i s i t e c o n d i t i o n s f o r enhanced metabolis.m of the sex s t e r o i d s are met i n a l l SWV females fed 5 ppm, or more, f o r 2 weeks, or longer. Reproduction was s t u d i e d i n females which had weaned one l i t t e r and were then c o n t i n u o u s l y fed r a t i o n s c o n t a i n i n g 2.5, 5, 10, 15, 20 or 25 ppm d i e l d r i n ; these females were caged w i t h males during weeks 4-6 of exposure. The e f f e c t s were f u r t h e r i n v e s t i g a t e d using v i r g i n females, one or more of I l l these r a t i o n s , and the same schemes of exposure and mating. Female mor-t a l i t y occurred only at 20 and 25 ppm. D i e l d r i n had no e f f e c t on: the number of parous females which bred; f e t a l m o r t a l i t y ; the d u r a t i o n of gesta-t i o n ; p a r t u r i t i o n . I n f e r t i l i t y r e s u l t e d i n 13% of the parous females fed 10 or 15 ppm but not i n the s u r v i v o r s at other doses. The l i t t e r s i z e of diparous females was reduced, dose-dependently, by a maximum of 17%. ' These e f f e c t s r e s u l t e d from a p r e - n i d a t i o n l e s i o n : 15 ppm decreased the number of bred, n u l l i p a r o u s females w i t h decidua 5 days post-coitum, w h i l e 25 ppm decreased the number of decidua per female. D i e l d r i n caused pre-weaning l o s s of the e n t i r e l i t t e r : 31% of the diparous c o n t r o l s l o s t t h e i r l i t t e r s compared w i t h 47% of those fed 2.5 ppm, 80% of those fed 5 ppm and 100%, of those fed > 10 ppm. Pup s u r v i v a l and growth was normal i n those l i t t e r s r a i s e d by dams fed 2.5 or 5 ppm; the _in v i v o metabolism of p e n t o b a r b i t a l was increased by 350-400% i n these pups. I t was concluded that l o s s of the l i t t e r was d i e l d r i n ' s s o l e e f f e c t o f b i o l o g i c a l importance. P u p - k i l l i n g and severe pup-neglect caused the m o r t a l i t y i n 30, 50, 67 and 100% of the l i t t e r s l o s t by diparous dams fed 10, 15, 20 and 25 ppm d i e l d r i n ; the m a j o r i t y of these l i t t e r s were l o s t w i t h i n 12 hours" of b i r t h . These causes d i d not k i l l h e a l t h y pups but only hastened the deaths of i n a t e l y i n v i a b l e pups: pups i s o l a t e d at b i r t h from primiparous dams fed 5, 10 or 15 ppm died at a s i g n i f i c a n t l y f a s t e r r a t e than c o n t r o l pups; o f pups taken from such dams and fo s t e r - n u r s e d by c o n t r o l females only 20% su r v i v e d to weaning. Pups i n the l i t t e r s l o s t by diparous dams weighed 3-13% l e s s at b i r t h than those which s u r v i v e d ; these pups became dark red s h o r t l y a f t e r b i r t h and were l i s t l e s s and i n a c t i v e u n t i l death. The pups i n the l i t t e r s l o s t by diparous dams from causes other than i n f a n t i c i d e or gross neglect Lv d i d not grow. These pups contained L i t t l e or no m i l k . The amount oi" milk synthesized by the mammary glands of primiparous mice fed 5 or 10 ppm was not reduced. Primiparous dams led 5 or 10 ppm took 477„ and 1187o longer than the c o n t r o l s to begin nursing pups. I t was concluded that pup i n v i a -b i l i t y i s the most serious reproductive l e s i o n that d i e l d r i n causes i n t h i s s t r a i n ; i t may i n t e r a c t w i t h , or be superceded by, d i e l d r i n - i n d u c e d a l t e r a -t i o n s i n the dams' behaviour. Pup i n v i a b i l i t y d i d not r e s u l t from inadequate l e v e l s of maternal progesterone: the serum c o n c e n t r a t i o n of t h i s s t e r o i d on days 3 and 14 of g e s t a t i o n was not d i f f e r e n t i n females fed 0, 5, 10 or 15 ppm d i e l d r i n . I t was concluded that these data do not support the hypothesis: A l l reproduc-t i v e l e s i o n s caused by organochlorine i n s e c t i c i d e s r e s u l t from enhanced metabolism of the sex s t e r o i d s by the i n s e c t i c i d e - i n d u c e d , mixed-function oxidase system-.. V TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES v i i i LIST OF FIGURES x i i i ACKNOWLEDGMENTS xv INTRODUCTION 1 MATERIALS AND METHODS 8 A. General Materials and Methods 8 B. D i e l d r i n ' s E f f e c t s on Some Parameters and Functions of the L i v e r 8 1. General 8 2. Preparation of Microsomes 9 3. Microsomal Metabolism of l^-c-Tmipramine 10 4. Microsomal Cytochrome P-450 13 5. Determination of Microsomal P r o t e i n 13 6. Pentobarbital Sleeping Times i n Dieldrin-Treated Mice 13 C. Reproduction Experiments: E f f e c t s of D i e l d r i n on: 14 1. Copulation, F e r t i l i t y , Fecundity and Pup S u r v i v a l i n Diparous Females 14 2. Ovulation and Implantation i n Bred, Nulliparous Females 16 3. Milk Production and Maternal Behaviour i n Primiparous Females 16 4. V i a b i l i t y of Pups Born to Primiparous Females 18 5. S u r v i v a l of Pups Born to Dieldrin-Fed Primiparous Females and Foster-Nursed by Non-Exposed Primiparous Dams 18 6. Serum Progesterone Levels During Gestation i n Dieldrin-Fed Primiparous Females 18 D. S t a t i s t i c a l Analysis of the Data 25 RESULTS 27 A. L i v e r Parameters i n Females Fed D i e l d r i n 27 1. Gross Appearance of the L i v e r 27 2. L i v e r Weight 27 3. Hepatic Microsomal Pr o t e i n 29 4. Cytochrome P-450 31 5. Summary of L i v e r Changes 33 v i P a g e B. L i v e r Function i n Females Feci Dietary D i e l d r i n 35 1' i n vivo: Pentobarbital Anesthesia 35 2. i n v i t r o : ^C-Imipramine Metabolism 36 3. Summary of Dieldrin-Induced Metabolic Changes i n Females 46 C. Regression of Dieldrin-Induced Changes in the Parameters and Function of the Female L i v e r 47 14 D. L i v e r Parameters and C-Imipramine Metabolism i n Male Mice 51 1. E f f e c t of Age . 52 2. E f f e c t of D i e l d r i n 53 E. E f f e c t of D i e l d r i n on Reproduction i n Diparous Females 57 1. Adult M o r t a l i t y 57 2. Breeding Behaviour (Copulation) 59 3. F e r t i l i t y 59 4. Gestation and P a r t u r i t i o n 61 5. L i t t e r Size and Sex Ratio at B i r t h 62 6. Post-Partum Su r v i v a l and Growth of Pups 64 7. Summary of D i e l d r i n 1 s E f f e c t s on Reproduction 83 8. Summary of Dieldrin-Induced Pup M o r t a l i t y 85 F. D i e l d r i n 1 s E f f e c t s on Ovulation and Implantation i n Bred Nulliparous Females 86 G. E f f e c t of D i e l d r i n on Pup V i a b i l i t y 88 H. S u r v i v a l and Growth of Pups Born to Primiparous Females Fed D i e l d r i n and Foster-Nursed by Unexposed Primiparous Dams 91 I. E f f e c t of Dietary D i e l d r i n on Gestational Serum Progesterone 97 J. Milk Produced by Primiparous Dams Fed D i e l d r i n 99 K. D i e l d r i n ' s E f f e c t on Maternal Behaviour i n Primiparous Females 99 DISCUSSION 103 A. Organochlorine Insecticides and Mammalian Reproduction: The L i t e r a t u r e 103 B. Reproduction: D i e l d r i n ' s E f f e c t s and Their Mechanisms 110 1. Pup I n v i a b i l i t y 111 2. I n f e r t i l i t y and Decreased L i t t e r Size 114 3. Pup M o r t a l i t y 119 v i i Page C. The L i v e r and I t s Functions: D i e l d r i n ' s E f f e c t s 126 1. L i v e r Weight and Microsomal P r o t e i n 126 2. P e n t o b a r b i t a l Metabolism: in v i v o 129 3. Imipramine Metabolism: _in v i t r o 130 SUMMARY AND CONCLUSIONS 138 LITERATURE CITED 141 V1 J. L LIST OF TABLES Loss of the r i g h t i n g r e f l e x in female SWV mice injected (ip) with sodium pentobarbital (90 mg/kgm) a f t e r eating d i e t a r y d i e l d r i n for 4 weeks. The latency to the onset was measured from the time of i n j e c t i o n while the duration was measured from the onset. The amount of IMI metabolized by hepatic microsomes prepared from SWV female mice fed the c o n t r o l r a t i o n . Where assays were done on 2 d i f f e r e n t groups of animals of the same age but at 2 d i f f e r e n t times the r e s u l t s have been presented separately. n = 2-4 for each mean. Regression of d i e l d r i n - i n d u c e d changes i n the weight of the l i v e r : 12-14 week old female mice were fed 20 ppm d i e t a r y d i e l d r i n for 4 weeks and then placed on the c o n t r o l d i e t for varying times before s a c r i f i c e . n = 3 except for the con-t r o l s at 6.86 weeks, where n = 2. Regression of d i e l d r i n - i n d u c e d increases i n the concentration of hepatic microsomal p r o t e i n : 12-14 week old female mice were fed 20 ppm d i e t a r y d i e l d r i n for 4 weeks and then placed on the c o n t r o l d i e t for varying times before s a c r i f i c e . n = 3 except for the controls at 6.36 weeks, where n = 2. Regression of d i e l d r i n - i n d u c e d increases i n the concentration of cytochrome P-450: 12-14 week old female mice were fed 20 ppm d i e t a r y d i e l d r i n for 4 weeks and then placed on the con-t r o l d i e t for varying times before s a c r i f i c e . The figures i n brackets express the experimental values as a per cent of c o n t r o l values, n = 3 except for the controls at 6.86 weeks, where n = 2. Regression of die l d r i n - i n d u c e d changes in the pathways of IMI metabolism: 12-14 week old female mice were fed 20 ppm di e t a r y d i e l d r i n for 4 weeks and then placed on the c o n t r o l d i e t for varying times before s a c r i f i c e , n = 3 except for the controls where at 6.86 weeks n = 2. Results are expres-sed as a % of co n t r o l values (x + SEM). The e f f e c t of age on l i v e r parameters i n SWV male mice. Values for l i v e r weight (mg l i v e r / g body weight), microsomal protein (mg protein/g l i v e r ) and cytochrome P-450 ( m j i moles/ mg microsomal protein) are the x + SEM of two mice. Mice were i n i t i a l l y 12-14 weeks old and were k i l l e d 4 and 8 weeks l a t e r . ix The e f f e c t of age on the metabolism of imipramine (IMI) to desmethylimipramine (DMI), hydroxylated imipramine (IMI-OH) and imipramine-N-oxide (IMI-N—»0) by hepatic microsomes prepared from SWV male mice. Mice were i n i t i a l l y 12-14 weeks old and were k i l l e d a f t e r 4 and 8 weeks. Values are the x + SEM of two mice. L i v e r parameters in SWV male mice fed d i e t a r y d i e l d r i n for 4 and 8 weeks. Values for l i v e r weight (mg l i v e r / g body weight), microsomal p r o t e i n (mg protein/g l i v e r ) and cyto-chrome P-450 (mj.t moles/mg microsomal protein) are the x + SEM of 2-3 mice. The values in brackets are the changes expressed as a per cent of co n t r o l l e v e l s . Mice were i n i t i a l l y 12-14 weeks o l d . Imipramine (IMI) metabolized per mg of protein by hepatic microsomes prepared from SWV male mice fed dietary d i e l d r i n for 4 and 8 weeks. Mice were i n i t i a l l y 12-14 weeks o l d . Values are the x + SEM of 2-3 mice. The metabolism of imipramine (IMI) to desmethylimipramine (DMI), imipramine-N-oxide (IMI-N-»0) and hydroxylated imipramine (IMI-OH) by hepatic microsomes prepared from SWV male mice fed d i e t a r y d i e l d r i n for 4 and 8 weeks. Mice were i n i t i a l l y 12-14 weeks o l d . Values are the x + SEM of 2-3 mice. M o r t a l i t y i n the groups of SWV female mice used to deter-mine the e f f e c t of d i e t a r y d i e l d r i n on reproduction. D i e l d r i n d i e t s were fed throughout the experiment. Mice were 12-14 weeks o l d when f i r s t placed on the d i e l d r i n r a t i o n s . The number of SWV mice found with vaginal plugs when caged with males during the 5th-6th week of exposure to d i e t a r y d i e l d r i n . The number of SWV mice fed d i e t a r y d i e l d r i n which had vaginal plugs but did not become pregnant. The e f f e c t of d i e t a r y d i e l d r i n on the number of pregnant SWV mice d e l i v e r i n g pups at term and on the number of days of gestation. The l i t t e r s i z e , number of placental scars and estimated i n t r a u t e r i n e f e t a l mortality i n SWV mice fed dietary d i e l d r i n . ( ) sample s i z e . The sexes of the pups born to SWV mice fed dietary d i e l d r i n . The age at which the pups died i n the l i t t e r s of SWV mice fed d i e l d r i n where none of the pups survived to weaning. The variances (<5^ ) i n the age at death of pups born to dams fed 5 and 10 ppm d i e t a r y d i e l d r i n and r a i s i n g no pups to weaning (28 days of age). The s u r v i v a l r a t e of pups i n l i t t e r s r a i s e d to weaning by dams fed d i e t a r y d i e l d r i n . Latency to the onset of the r i g h t i n g r e f l e x loss i n weaning mice r a i s e d by dams fed d i e t a r y d i e l d r i n . Weanlings were i n j e c t e d ( i p ) w i t h 60 mg/kg of sodium p e n t o b a r b i t a l on day 29 a f t e r b i r t h . S t a t i s t i c a l a n a l y s i s was performed on l o g -transformed data which have been de-transformed for presenta-t i o n , n = number of weanlings. CL = confidence l i m i t s . D uration of the r i g h t i n g r e f l e x l o s s i n weanling mice r a i s e d by dams fed d i e t a r y d i e l d r i n . Weanlings were i n j e c t e d ( i p ) wi t h 60 mg/kg sodium p e n t o b a r b i t a l on day 29 a f t e r b i r t h . S t a t i s t i c a l a n a l y s i s was performed on lo g - t r a n s formed data which have been de-transformed f o r p r e s e n t a t i o n . n = number of weanlings. CL = confidence l i m i t s . The e f f e c t of d i e t a r y d i e l d r i n on the number of dams which k i l l e d t h e i r pups. E f f e c t of d i e t a r y d i e l d r i n on the average b i r t h weight ( l i t t e r w e i g h t / l i t t e r s i z e ) of the pups. ( ) no. of l i t t e r s . The q u a n t i t y of milk i n the pups of dams fed d i e t a r y d i e l -d r i n and which r a i s e d no pups to weaning ( e x c l u s i v e of dams which k i l l e d t h e i r pups). N = normal m i l k l i n e s , R = reduced m i l k l i n e s , A = m i l k l i n e s absent. Where no symbol i s given a l l the pups were dead. See t e x t f or f u l l d e f i n i t i o n s . Cumulative observed time spent i n the nest during post-partum days 1-4 ( p a r t u r i t i o n = day 1) by f i v e dams fed no d i e l d r i n and which s u c c e s s f u l l y r a i s e d pups to weaning. Each dam was observed for 3 minutes twice a day. The e s t i -mated lower l i m i t for time spent i n the nest for s u c c e s s f u l dams i s presented. Cumulative observed time spent i n the nest during the post-partum period by dams fed d i e t a r y d i e l d r i n and which r a i s e d no pups to weaning ( e x c l u s i v e of those k i l l i n g t h e i r l i t t e r ) . Dams were observed for two 3 minute periods each day u n t i l a l l t h e i r pups were dead. XXVIII. The e f f e c t s of dietary d i e l d r i n on the number of mice with decidua on day 4 of gestation (plug day = day 0). V i r g i n mice (9-11 weeks old) were bred during weeks 5-6 of exposure. Means + SEM of other parameters are presented also. XXIX. XXX. XXXI. XXXII. XXXIII. V i a b i l i t y of pups born to primiparous dams fed d i e t a r y d i e l d r i n . Pups were i s o l a t e d from t h e i r dams at b i r t h and the number l i v i n g was determined at various times. (n = number of dam c o n t r i b u t i n g pups; N = number of pups removed). Dam e f f e c t on pup v i a b i l i t y . Littermates were i s o l a t e d at b i r t h from primiparous dams fed d i e t a r y d i e l d r i n and the number of l i t t e r s i n which one or more pups remained a l i v e was determined at times a f t e r removal. n = number of l i t t e r s ( S dams). The number of unexposed dams which r a i s e d foster pups and/or t h e i r own pups to weaning. V i r g i n females (4-6 weeks old) were placed on 0, 5, 10 and 15 ppm d i e t a r y d i e l d r i n and mated during weeks 5-6 of exposure. The l i t t e r of an unex-posed female was reduced to f i v e - s i x pups at b i r t h and f i v e -s i x pups ( £ 1 2 hours old) born to a donor female were added to i t w i t h i n 24 hours. The e f f e c t of f o s t e r i n g on the s u r v i v a l of pups to weaning (day 28). V i r g i n females (9-11 weeks old) were housed with males for 2 weeks. At b i r t h , the dam's l i t t e r was reduced to 6 pups and w i t h i n 24 hours, another 5-6 pups ( £ 1 2 hours old) born to another female were added to i t . The e f f e c t of 10 ppm d i e l d r i n i n the d i e t of a female on the s u r v i v a l to weaning (day 28) of i t s pups when f o s t e r -nursed by an unexposed dam. V i r g i n females (4-6 weeks old) were placed on rations containing 0.0 or 10.0 ppm d i e l d r i n and mated during weeks 5-6 of exposure. L i t t e r s of 0.0 ppm dams were reduced to 5-6 pups at b i r t h and within 24 hours, 5-6 pups ( £ 1 2 hours old) born to a 10.0 ppm female were added to i t . L i t t e r s where a l l fo s t e r pups died (3) have been excluded. XXXIV. XXXV. The average body weight (g) of s u r v i v i n g littermates ( l i t t e r weight/number of pups) in the three l i t t e r s where a l l the fostered pups (from 10 ppm dams) died before weaning. The e f f e c t of dietary d i e l d r i n on the concentration of serum progesterone in SWV mice on days 3 and 14 of gesta-t i o n . The figure i n brackets i s the number of mice ana-lyzed. x i i Table Page XXXVI. M i l k produced during an 3 hour s e c r e t o r y period (1200-2000 hours) on day 4 of l a c t a t i o n ( b i r t h = day 1) by primiparous dams fed d i e t a r y d i e l d r i n . 100 XXXVII. E f f e c t of d i e t a r y d i e l d r i n on the maternal behaviour of primiparous dams: the late n c y to begin p u p - r e t r i e v a l , nest-b u i l d i n g and nursing behaviour when the pups were replaced i n the cage a f t e r a 4 hour absence. Observations were made on l a c t a t i o n day 3„ 101 x i i i LIST OF FIGURES Figure Page 1. L i v e r weights of female SWV mice fed dietary d i e l d r i n (n = 2-4 at each point). Animals were i n i t i a l l y 12-14 weeks old. 28 2. The concentration of microsomal p r o t e i n i n the l i v e r s of female SWV mice fed dietary d i e l d r i n (n = 2-4 at each p o i n t ) . Animals were i n i t i a l l y 12-14 weeks old. See text for an explanation of the data in the shaded area. 30 3. The concentration of cytochrome P-450 i n the hepatic micro-somes of female SWV mice fed d i e t a r y d i e l d r i n (n = 2-4 at each p o i n t ) . Animals were i n i t i a l l y 12-14 weeks of age. 32 4. Hepatic cytochrome P-450 expressed as a per cent of co n t r o l values i n female SWV mice fed 5 and 20 ppm dietary d i e l d r i n . (n = 2-4 at each p o i n t ) . 34 5. Imipramine (IMI) metabolism by hepatic microsomes prepared from c o n t r o l SWV female mice (n = 2-4 at each p o i n t ) : E f f e c t of age on the r e l a t i v e a c t i v i t i e s of IMI - demethy-lase, IMI - N-oxidase and IMI - hydroxylase(s). 39 6. The r e l a t i v e rate of imipramine metabolism per mg of p r o t e i n by hepatic microsomes prepared from SWV female mice fed di e t a r y d i e l d r i n (n = 2-4 at each p o i n t ) . A l l animals were i n i t i a l l y 12-14 weeks o l d . 40 7. Metabolism of imipramine to imipramine N-oxide (IMI-N-»0) by hepatic microsomes prepared from SWV female mice fed di e t a r y d i e l d r i n (n = 2-4 at each p o i n t ) . A l l animals were i n i t i a l l y 12-14 weeks o l d . 42 8. Metabolism of imipramine to hydroxy-imipramine (IMI-OH) by hepatic microsomes prepared from SWV mice fed d i e t a r y d i e l -d r i n (n = 2-4 at each p o i n t ) . A l l animals were i n i t i a l l y 12-14 weeks o l d . 44 9. Metabolism of imipramine to desmethylimipramine (DMI) by hepatic microsomes prepared from SWV female mice fed d i e t a r y d i e l d r i n (n = 2-4 at each p o i n t ) . A l l animals were i n i t i a l l y 12-14 weeks o l d . 45 10. E f f e c t of dietary d i e l d r i n on the proportion of puerperal diparous females with l i v i n g pups. ( ) Sample s i z e . 67 11. Average body weight (B.W.) of s u r v i v i n g littermates ( l i t t e r weight/no. of pups) in l i t t e r s r a i s e d to weaning by dams fed 0, 2.5 and 5 ppm dietary d i e l d r i n . 70 xi.v Figure Page 12. The l i v e r weight of dams fed d i e l d r i n and s u c c e s s f u l l y r a i s i n g pups to weaning compared with those of unsuccessful dams and non-pregnant females. 73 13. Average body weight (B.W.) of su r v i v i n g littermates ( l i t t e r weight/no. of pups) in the l i t t e r s of dams fed dietary d i e l -d r i n and whose en t i r e l i t t e r died before weaning. The B.W. of pups r a i s e d to weaning by dams fed 0.0 ppm d i e l d r i n i s presented as co n t r o l s . 79 14. The s u r v i v a l rate of pups which were born to females fed 0.0 or 10.0 ppm dietary d i e l d r i n but foster-nursed by other dams fed 0.0 ppm d i e l d r i n . Each foster dam simultaneously nursed some of i t s own pups as a paired c o n t r o l . The number of pups are shown i n brackets. 94 15. The growth rate of pups which were born to females fed 0.0 or 10.0 ppm d i e l d r i n but foster-nursed by other dams fed 0.0 ppm d i e l d r i n . Each foster dam simultaneously nursed some of i t s own pups as a paired c o n t r o l . 98 16. A model of the proposed mechanism of cytochrome P-450 cata-lyzed hydroxylation of drugs and s t e r o i d s . Steps are l i s t e d in Arabic numerals while the intermediates are l i s t e d i n Roman numerals. (adapted from G i l l e t t e et a l . 1972). 136 XV ACKNOWLEDGMENTS I would l i k e to express my sincere gratitude to Dr. G a i l D. Bellward for having been an ideal supervisor throughout this study. I would also l i k e to thank Drs. Abbott,Reidel, Sutter and Taylor for t h e i r many useful comments and c r i t i c i s m s . I g r a t e f u l l y acknowledge r e c e i p t of an H.R. MacMillan Pre-Doctoral Fellowship, a Research Traineeship from the MRC and a Graduate Award from the CFAP. The research was funded i n part by the MRC and in part by the Faculty of Pharmaceutical Sciences. I would also l i k e to thank the Un i v e r s i t y Health Service for drawing the blood used as a source of CBG and Mr. K. Grunenberg and Mrs. L. Epps for excellent technical assistance. I would p a r t i c u l a r l y l i k e to thank S h e i l a who has contributed so much, i n so many ways, to th i s work. 1 INTRODUCTION Organochlorine i n s e c t i c i d e s , ingested c h r o n i c a l l y at ppm l e v e l s i n the diet, impair mammalian reproduction by d i s r u p t i n g the estrous c y c l e ( B a l l et a l . 1953; Huber, 1965) and by decreasing the number of pregnancies, the l i t t e r s i z e , or the neonate s u r v i v a l (Aulerich et a l . 1971; Ambrose et a l . 1953; Bernard and Gaertner, 1964; Deichmann et a l . 1971; Fitzhugh, 1943; G i l b e r t , 1969; Good et a l . 1965; Good and Ware, 1967; Harr et a l . 1970; Keplinger et a l . 1970; Kitselman, 1953; Morris, 196S; Murphy and Korschgen, 1970; Treon and Cleveland, 1955; Virgo, 1970; Ware and Good, 1967). Conney and his co-workers, i n 1966, were the f i r s t to suggest that a l t e r a t i o n s i n sex hormone metabolism — the consequence of i n s e c t i c i d e -induced increases i n the concentration and/or a c t i v i t y of enzymes wit h i n the hepatic microsomes — may be the cause of these reproductive f a i l u r e s . This view has since been r e i t e r a t e d by these investigators i n several papers (e.g. Welch et a l . 1967; Welch et a l . 1971; L e v i n et a l . 1968; Conney et a l . 1973) where they stressed that i t s su b s t a n t i a t i o n required much a d d i t i o n a l work; the work remains to be done while the hypothesis is widely c i t e d by others as a mechanistic explanation for insecticide-induced changes i n the mammalian reproductive system (Fahim et a l . 1970) and re-productive performance (e.g. Lundberg and Kihlstrom, 1973). This thesis presents an attempt to test Conney's hypothesis d i r e c t l y by i n v e s t i g a t i n g the i n t e r r e l a t i o n s h i p s between induction of the hepatic microsomal enzymes, reproductive performance and sex s t e r o i d l e v e l s i n the female SWV mouse fed dietary d i e l d r i n . Conney, and the other proponents of his hypothesis, support i t 2 ci r c u m s t a n t i a l l y by inferences drawn from other data. These inferences, t h e i r c r i t i c i s m s , and the observations upon which they are based w i l l be discussed i n the next few pages. Steroid hormones and xenobiotics, such as drugs and p e s t i c i d e s , are metabolized, under i d e n t i c a l i n v i t r o conditions, by enzyme systems within the hepatic microsomes (see reviews by Conney, 1967 and Kupfer, 1967). The systems are very s i m i l a r and may be i d e n t i c a l : they possess common biochemical requirements (Kuntzman et a l . 1964); steroids competitively i n h i b i t drug metabolism and vice versa (Tephly and Mannering, 1964 and 1968; Estabrook et a l . 1973); and cytochrome P-450, which acts as both the termi-nal oxidase (Cooper et a l . 1965; Omura et a l . 1965) and the substrate-binding enzyme (Remmer et a l . 1966; Imai and Sato, 1966; Schenkman et a l . 1967), i s necessary for the metabolism of both types of compounds ( G i l l e t t e et a l . 1972; Conney et a l . 1968; L e v i n et a l . 1969; Lu et a l . 1973). Michaelis constants for drugs and steroids (Kuntzman et a l . 1965; Trivus, 1965) indicate the l a t t e r are the better substrates, leading these, and other workers, to believe that the steroids are the natural ( i n vivo) sub-strates for the system(s). Confirmation of t h i s view and determination of the actual number of systems w i t h i n the microsomes must await the i s o l a t i o n and p u r i f i c a t i o n of the system(s) components. In any case, the pretreat-ment of animals with c e r t a i n xenobiotics r e s u l t s i n an increase i n the amount and the a c t i v i t y of enzyme systems which metabolize steroids and xenobiotics. Organochlorine i n s e c t i c i d e s are amongst the most potent of these inducing xenobiotics. The hepatic microsomes of immature mammals that have been injected for several days with sublethal doses of organochlorine 3 i n s e c t i c i d e s metabolize sex steroids, i n v i t r o , at an enhanced rate (Welch et a l . 1967); drug metabolism is s i m i l a r l y increased (Abernathy et a l . 1971; Chadwick et a l . 1971). There are associated increases in the l i v e r ' s weight and in i t s content of both microsomal protein (Juchau and Fouts, 1966) and cytochrome P-450 (Baker et a l . 1972). These are changes which r e s u l t , at least i n part, from p r o l i f e r a t i o n of the smooth endoplasmic reticulum (Fouts and Rogers, 1965); the induced increase i n cytochrome P-450 i s , however, dis p r o p o r t i o n a t e l y greater than that of the microsomal p r o t e i n (Chhabra and Fouts, 1973). Lower doses of organochlorine i n s e c t i c i d e s , administered as contaminants i n the d i e t at ppm concentrations, produce i d e n t i c a l morphological and biochemical a l t e r a t i o n s i n the l i v e r : the smooth endoplasmic reticulum hypertrophies (Baker et a l . 1972); i t s weight and microsomal p r o t e i n content increases (Hoffman, 1970; P i a t t and C o c k r i l l , 1969); and i t s cytochrome P-450 l e v e l i s elevated (Bunyan et a l . 1972). These changes are accompanied by an enhanced microsomal metabolism of drugs i n v i t r o (Hoffman, 1970; P i a t t and C o c k r i l l , 1969; Bunyan et a l . 1972), which can, however, a l s o occur i n the absence of increases i n l i v e r weight or microsomal p r o t e i n ( G i l l e t t e and Chan, 1968). The "lowest e f f e c -t i v e dose" of DDT or toxaphene for inducing an increase i n enzymatic a c t i v i t y i s less than 5 ppm ( G i l l e t t e , 1968; Kinoshita et a l . 1966) while the cyclo-diene compounds, such as a l d r i n and d i e l d r i n , are more potent s t i l l , with t h e i r e f f e c t i v e doses being less than 1 ppm (Street et a l . 1969). In sexually mature mammals, as i n immatures, sublethal (ip) doses of organochlorine i n s e c t i c i d e s increase l i v e r weight, microsomal protein, cytochrome P-450 and in v i t r o microsomal drug metabolism (Hart and Fouts, 1965; Juchau et a l . 1966; Hansen and Fouts, 1968; Mullen et a l . 1966; 4 Lcchner and Pousada, 1 9 7 1 ) . Lower doses, fed as ppm contaminants i n the feed, also increase l i v e r weight, microsomal protein and cytochrome P-450 (Wright et a l . 1 9 7 2 ; Davis et a l . 1 9 7 3 ) which, as i n other cases, i s at least p a r t i a l l y due to a hypertrophy of the smooth endoplasmic reticulum (Ortega, 1 9 6 6 ; Wright et a l . 1 9 7 2 ) ; the hepatic microsomes, as expected, exhibit enhanced drug-metabolizing a c t i v i t y i n v i t r o (Wright et a l . 1 9 7 2 ; Datta and Nelson, 1 9 6 8 ; Hart and Fouts, 1 9 6 5 ; Lechner and Pousada, 1 9 7 1 ) . In summary, the f i r s t of these data show that p r e t r e a t i n g immature animals with organochlorine i n s e c t i c i d e s increases the capacity of the hepatic microsomes to metabolize sex steroids i n v i t r o while the l a t t e r data imply that p r e t r e a t i n g the adult would have the same r e s u l t . These observations provide part of the basis for i n f e r r i n g that the organochlorine i n s e c t i c i d e s may a l t e r the _in v i v o metabolism of endogenous sex steroid s i n animals of reproductive age. The other basis r e s t s upon data showing that pretreatment with organochlorine i n s e c t i c i d e s can reduce the i n vivo responses to exogenous steroids and can decrease the weight of target organs which are normally maintained by endogenous s t e r o i d s . I t i s t h i s l a t t e r basis which can be c r i t i c i z e d on two points. F i r s t , for reasons of experi-mental convenience, most of these data have been obtained by use of sex-u a l l y immature animals and, i n the absence of data from adults, the con-clusions drav/n from them have been extrapolated to the sexually mature; the v a l i d i t y of th i s extrapolation i s hig h l y questionable. Second, the data c o l l e c t e d from mature animals are ambiguous and can be interpreted i n several ways, including some which are independent of enhanced s t e r o i d metabolism. The experiments which provide the data for th i s second basis are given below. Immature rata pretreated with organochlorine i n s e c t i c i d e s before administration of the s t e r o i d exhibit decreases in progesterone-induced anesthesia (Conney et a l . 1966), estrogen-induced enlargement of the uterus (Levin et a l . 1963; Welch et a l . 1971) and androgen-induced growth of the seminal v e s i c l e (Levin et a l . 1969). The reductions in anesthesia and uterine growth are accompanied by decreased s t e r o i d l e v e l s i n the brain and uterus re s p e c t i v e l y , and by increased hepatic microsomal s t e r o i d metabo-l i s m i n v i t r o . Immature rat s pretreated with the inducing agent, pheno-b a r b i t a l , and inj e c t e d with e i t h e r estrone or e s t r a d i o l have lower whole-body l e v e l s of these compounds and t h e i r metabolites than do co n t r o l animals (Welch et a l . 1968). These data show that p r e t r e a t i n g immature animals with organochlorine i n s e c t i c i d e s reduces the b i o l o g i c responses to exogenous sex hormones and indicate that accelerated hepatic degradation of the s t e r o i d i s the major cause of i t s reduced e f f e c t . A s i m i l a r , attenuated response to exogenous s t e r o i d occurs i n adult animals. The pretreatment of i n t a c t or ovariectomized adult mice with DDT reduces the amount of uterine enlargement caused by inje c t e d e s t r a d i o l (Fahim et a l . 1970). These workers, by extrapolating the above observations from immature animals, conclude that enhanced hepatic metabolism of the estrogen is the cause of t h i s e f f e c t a l s o . The extrapolation i s , however, of questionable v a l i d i t y since the a c t i v i t i e s of the hepatic microsomal enzymes of immature animals d i f f e r i n a c t i v i t y , both q u a l i t a t i v e l y and quan-t i t a t i v e l y , from those of adult animals (Basu et a l . 1971). Moreover, the biochemical composition of hepatic microsomes q u a n t i t a t i v e l y changes with age (Fonts and Devereux, 1972) as does the response of the smooth endo-plasmic reticulum to inducing agents (Furner et a l . 1969). 6 Fahim et a l . (1970) also observed that the uterine weight of intact, adult mice is reduced following treatment with DDT and this can be used to support the hypothesis of enhanced s t e r o i d metabolism in adults pretreated with organochlorine i n s e c t i c i d e s . This observation i s , however, ambiguous in i n t e r p r e t a t i o n since these authors present no data to show this weight reduction a c t u a l l y r e s u l t s from enhanced metabolism of the endogenous stero i d s . The reduction can be equally well explained for example, by decreased biosynthesis of the steroids, or by t h e i r a l t e r e d d i s t r i b u t i o n , or even as a d i r e c t e f f e c t of DDT on the uterus. It is c l e a r then, that while much a v a i l a b l e data may be used to support Conney's hypothesis, that use, in many cases, can be c r i t i c i z e d . In addition, a strong a p r i o r i argument against the hypothesis resides in other observations which indicate that the in vivo rate of sex s t e r o i d metabolism i s not l i m i t e d by the concentration of the enzymes. For instance, in the adult male rat the concentration of testosterone in the peripheral plasma (Rivarda et a l . 1968; F r i c k et a l . 1969) is smaller, by 1500 to 2300 times, than the apparent Michaelis constant for the ^n v i t r o hydroxylation of testosterone by t h i s animal's hepatic microsomes (Kuntzman et a l . 1965)." Under such conditions the r e a c t i o n k i n e t i c s are f i r s t order, and the rate of metabolism would be proportional to, and l i m i t e d by, the concentration of the s t e r o i d , not the concentration of the enzyme. The induction of either more enzyme or of a more a c t i v e enzyme would be of no consequence. Since the ubiquity of organochlorine i n s e c t i c i d e s and other i n -ducing agents within the environment lends considerable importance to Conney's hypothesis and since i t s v a l i d i t y is not presently cle a r , this study was undertaken in an attempt to test i t d i r e c t l y . Such a test can 7 only be achieved by measuring the concentrations of the endogenous steroids at the precise stage of reproduction disrupted by the i n s e c t i c i d e , and then a s c e r t a i n i n g whether decreases are the r e s u l t of increased metabolism or the r e s u l t of some other mechanism. For this test I chose the female house mouse (Mus musculus) of the SWV s t r a i n as the experimental animal and technical d i e l d r i n as the inducing i n s e c t i c i d e . The s p e c i f i c goals were to determine d i e l d r i n ' s e f f e c t on: 1) some of the components and the a c t i v i t y of the hepatic, micro-somal, mixed-function oxidase system; 2) the physiology of reproduction; and 3) to quantitate the endogenous sex steroids at the stages where reproductive lesions occur; and further 4) to determine the mechanism of any reductions i n the concentra-tions of the s t e r o i d s . 8 MATERIALS AND METHODS A. General Materials and Methods Swiss-Vancouver (SWV) mice, purchased at 3-5 weeks of age from the Department of Zoology, U n i v e r s i t y of B r i t i s h Columbia, were used i n a l l experiments. They were kept on a constant photoperiod of 14 hours of l i g h t and 10 hours of darkness ( l i g h t s on at 0600 hours) in quarters maintained at 22 C. Animals due to give b i r t h and those nursing pups, were housed singly; the others were caged i n groups of 2-4. Bedding material was crushed corn cobs, untreated with b a c t e r i c i d e s or other chemicals ("Lobund"; Paxton Processing Ltd., Paxton, I l l i n o i s ) . Purina Lab Chow and tap water were provided ad 1ibitum. D i e l d r i n was administered v i a the feed. Unpelleted Lab Chow was sprayed with an ethanol s o l u t i o n of tec h n i c a l d i e l d r i n , allowed to dry, tumble-mixed, and pe l l e t e d . The rations were prepared by N u t r i t i o n a l Bio-chemicals, (Cleveland, Ohio) from technical d i e l d r i n containing 86.17, HEOD''', supplied to me by S h e l l Canada L t d . (Toronto). Treated rations were stored at -20 C u n t i l use. Untreated Lab Chow was used as the control ( i . e . 0.0 ppm d i e l d r i n ) r a t i o n . B. D i e l d r i n ' s E f f e c t s on Some Parameters and Functions of the L i v e r 1. General V i r g i n females (12-14 weeks old) were placed on rations containing 0, 5, 10, 15, and 20 ppm d i e l d r i n . At i n t e r v a l s , two control animals and three treatment animals from each of two doses were s a c r i f i c e d to allow 1. HEOD: 1,2,3,4,10,10-hexachloro-6,7-epoxy-1,4,4a,5,6,7,8,8a-octahydro-l, 4-endo,exo-5,8-dimethanonaphthalene. 9 determination of the l i v e r s i z e, the concentrations of protein and cyto-chrome P-450 in the l i v e r microsomes and the i n v i t r o metabolism of ^ C - i m i -pramine. These procedures are described below. The regression of the dieldrin-induced changes i n these parameters was determined i n females fed 20 ppm d i e l d r i n for 4 weeks and then placed on the control r a t i o n for various i n t e r v a l s before s a c r i f i c e . S i m i l a r studies were conducted with male mice a l b e i t at fewer doses and time i n t e r v a l s . 2. Preparation of Microsomes Animals were weighed and then k i l l e d by decapitation. A f t e r e x c i -sion of the g a l l bladder the l i v e r was removed and immediately placed i n ic e - c o l d , aqueous 1.157» KC1 (w/v). The l i v e r was then removed from the KC1 solu t i o n , blotted dry, weighed and placed i n a Pyrex homogenizing tube set i n i c e . To this was added 12 ml of fresh, ice-cold, aqueous 1.157o KC1 per gram of l i v e r . The ti s s u e was then homogenized for exactly 1.0 minute with a Tef l o n pestle powered by a Fisher Dyna-Mixer ( s e t t i n g 4). Homogeni-zat i o n was conducted i n a cold room at 6 C. The homogenate was then centrifuged (IEC Model B-20) at 10,000g for 20 minutes at 4 C to remove c e l l u l a r debris. Nine ml of the superna-tant were further centrifuged (Beckman U l t r a c e n t r i f u g e , Model L2-65B) at 102,000g for 60 minutes at 4 C. The r e s u l t i n g microsomal p e l l e t was homo-genized i n 9 ml of ic e - c o l d 1.0 M, pH 7.4, phosphate buffer. This suspen-sion was stored at 4 C and used without further treatment. Aliquots were frozen' at -20 C and stored for protein a n a l y s i s . 10 3. Microsomal Metabolism of ^c-iwipramine The i n v i t r o , microsomal metabolism of 14C_ imipramine (^C-T.MI) was studied by use of an incubation system adapted from that of Kuntzman et a l . , (1966). ^C-imipramine and i t s metabolites were i s o l a t e d and quantitated by methods modified from those Moody et a l . (1967), Bickel and B a g g i o l i n i (1966), Dayton and Perel (1971) and Wharton et a l . (1971). a. Microsomal Incubation  Chemicals and Reagents ^C-IMI (Amersham/Searle), r e c r y s t a l l i z e d from ethyl acetate-acetone to a constant s p e c i f i c a c t i v i t y of 4.5 x 10 pC/mg, was d i l u t e d with r e c r y s t a l l i z e d IMI (Geigy) to a s p e c i f i c a c t i v i t y of 100 dpm//ig. An aqueous s o l u t i o n (20ug/ml) was prepared. ATP, NADH, NADPH, glucose-6-phosphate (G-6-P), and glucose-6-phosphate dehydrogenase (G-6-P-DH) were purchased from Sigma Chemicals and dissolved i n 0.2 M phosphate buffer (pH 7.4). MgCl2 and KCl (Fisher AR) reagents were prepared i n water. A l l solutions were stored at 6 C u n t i l use. Incubation Procedure The incubation mix consisted of 0.345 M pH 7.4, phosphate buffer 1.1 ml 0.2 M M g C l 2 0.1 ml .2.0 M KCl 0.1 ml 0.01 M ATP 0.2 ml 0.005 M NADPH 0.1 ml 0.006 M NADH . 0.1 ml 0.03 M G-6-P 0.2 ml G-6-P-DH (50 units/ml) 0.1 ml 1 4C-IMI s o l u t i o n 1.0 ml 11 and was placed i n a ground-glass stoppered bo t t l e and pre-incubated for 1.0 minute at 37 C in a water bath. To this was added 0.5 ml of microsomal suspension and the mixture was incubated i n a i r , with a g i t a t i o n , for 15 minutes; incubations were performed i n duplicate for each animal. The metabolism was stopped by adding 1.0 ml of 1.0 N NaOH. Recovery of was determined by the use of incubation mixtures to which the microsomes were added a f t e r the 1.0 N NaOH. Blank incubation mixes were r o u t i n e l y included; these had 0.5 ml of water added in place of the microsome suspen-s ion. 14 b. Q u a n t i f i c a t i o n of C-IMI and i t s Metabolites Chemicals and Reagents Heptane:isoamyl alcohol (97:3 v/v) s o l u t i o n was prepared from heptane (Eastman AR) which had been washed with 0.1 N NaOH and Fisher C e r t i f i e d isoamyl a l c o h o l . A c e t i c anhydride (Baker & Adamson A.C.S.) was d i s t i l l e d before use. Ethylene d i c h l o r i d e (Fisher C e r t i f i e d A.C.S.) was washed with 0.1 N NaOH before use. L i q u i d s c i n t i l l a t i o n counting s o l u t i o n was prepared by the method of Bray (I960). Extraction, I s o l a t i o n and Q u a n t i f i c a t i o n Six ml of heptane:isoamyl alcohol s o l u t i o n were added to the incuba-t i o n bottles which were then shaken for 1 hour on a B u r r e l l Wrist Shaker. The mixture was transfered to a 50 ml centrifuge tube, sealed with a ground glass stopper, and centrifuged (IEC Model EXD) for 10 minutes at 1000 rpm. The heptane phase, now containing the unmetabolized ^C-IMI and the metabolite, ^C-desmethylimipramine (^C-DMI), was removed; 4 ml were placed in another centrifuge tube and the balance was discarded. Ac e t i c anhydride (4-6 drops) was added to the heptane:isoamyl 12 alcohol, which, a f t e r mixing (30 seconds at s e t t i n g 10 on a Fisher Vortex Genie), was allowed to stand for 20 minutes. Five ml of 0.1 N ^50^ were added; the mixture was again vortexed and allowed to stand. The phases were separated by c e n t r i f u g a t i o n at 500 rpm for 10 minutes. A 1.0 ml aliq u o t of the acid-extracted heptane phase, now containing only ^C-DMI, and a 0.5 ml a l i q u o t of the H2SO4 phase, containing the ^^C-IHI, were re-moved for l i q u i d s c i n t i l l a t i o n counting. An a l i q u o t (0.5 ml) of the heptane-extracted incubation mix, which contained the metabolites, 2-hydroxy-imipramine (IMI-OH) and imipramine N-oxide (IMI-N-»0), was also taken for l i q u i d s c i n t i l l a t i o n counting. The IMI-N-»0 was extracted from the balance of the incubation mix by vortexing t h i s phase with 6.0 ml of ethylene d i c h l o r i d e . This was allowed to stand overnight at 6 C, and was then centrifuged at 1500 rpm for 1 hour to separate the phases. A 0.5 ml a l i q u o t of the aqueous phase, now containing only IMI-OH, was taken for l i q u i d s c i n t i l l a t i o n counting. The amount of IMI-N-*0 was determined by subtraction. Bray's s o l u t i o n was added to the removed a l i q u o t s , each of which was then counted for 30 minutes i n a Nuclear Chicago IsoCap/300 l i q u i d 14 s c i n t i l l a t i o n counter. The maximum counting e f f i c i e n c y of C i n t h i s system was 957o. Corrections for quenching were made v i a the channels - r a t i o method by use of an external standard. A f t e r corrections for blank values and recoveries, the dpm in each phase were converted to micrograms. These data were further corrected for the amount of microsomal protein (see below) in the incubation mix and the r e s u l t s were f i n a l l y expressed as micrograms/ mg microsomal protein / 1 5 minutes of incubation. 13 4. Microsomal Cytochrome P-450 Cytochrome P-450 was determined by the method of Sladek and Mannering (1966). The cytochrome P-450 in 6.0 ml of the microsome suspen-sion was reduced by adding a few mg of Na-pS-jO^Fisher P u r i f i e d ) . The re-duced cytochrome P-450 in 3 ml was then complexed with CO by gently bubbling the gas through the suspension for 1.0 minute. The di f f e r e n c e i n the absor-bance, over 500-450 mu, between the 3 ml containing the cytochrome P-450-CO complex and the 3 ml containing the reduced cytochrome P-450 was determined with a Perkin-Elmer (Coleman 124) double-beam spectrophotometer equipped with a H i t a c h i c e l l holder (Model 124-0319) maintained at 24 C. The absorbance d i f f e r e n c e was recorded on a Perkin-Elmer (Coleman 165) recorder. The absorbance of the cytochrome P-450 was converted to nanomoles by use of the molar e x t i n c t i o n c o e f f i c i e n t for membrane-bound cytochrome P-450 (91 cm _ 1 mM"1) de termined by Omura and Sato (1964). 5. Determination of Microsomal P r o t e i n P r o t e i n concentration i n the microsomal suspension was determined by the Lowry method described by Sutherland et a l . (1949) as modified by Robson et a l . (1968). Chemicals and Reagents A l k a l i n e copper s o l u t i o n was prepared by adding 1.0 ml of 2% (w/v) aqueous CuSO^'5H20 (Baker and Adamson. Reagent. A.C.S.) and 1.0 ml of 4% (w/v) aqueous Na 2C^H^0 6'2H 20 (Matheson, Coleman and B e l l . Reagent. A.C.S.) to 98 ml of 4% (w/v) aqueous Na2C03*H20 (Baker and Adamson, Reagent. A.C.S.). Phenol reagent was prepared by d i l u t i n g 2 N F o l i n - C i o c a l t e a u s o l u t i o n (Hartman-Leddon Co., Philadelphia) 1:4 in d i s t i l l e d water. Bovine serum 14 albumin was dissolved in 0.2 M phosphate buffer at concentrations ranging from 10-200 ug/ml; these standards were frozen at -20 C when not in use. Procedure The microsomal suspension was d i l u t e d 1:5 in water to place the protein concentration within the range of the albumin standards. Five ml of a l k a l i n e copper s o l u t i o n was added to 0.5 ml of the d i l u t e d suspension; t h i s was then mixed b r i e f l y on a Vortex-Genie (Fisher) and placed i n a water bath at 45 C. A f t e r exactly 12 minutes of incubation 0.5 ml of Phenol reagent (heated to 45 C) was added. The s o l u t i o n was vortexed immediately and allowed to stand at room temperature for exactly 15 minutes. The absorbance, r e l a -t i v e to a water blank, was then determined by use of a Perkin-Elmer (Coleman 124) double-beam spectrophotometer. Standard albumin solutions were treated i n the same manner i n order to generate a standard curve from which the un-known protein concentrations were estimated. 6. Pentobarbital Sleeping Times i n Dieldrin-Treated Mice V i r g i n females (12-14 weeks old) were fed co n t r o l or d i e l d r i n con-taminated rations for 4 weeks. They were then injected (ip) with sodium pentobarbital (90 mg/kg) and the latency to the loss of the r i g h t i n g r e f l e x and the duration of the loss were timed. C. Reproduction Experiments: E f f e c t s of D i e l d r i n on: 1. Copulation, F e r t i l i t y , Fecundity and Pup Su r v i v a l in Di-parous  Females V i r g i n females (4-6 weeks old) were caged with males for 2 weeks, allowed to gestate, d e l i v e r and nurse pups for 3 weeks. The successful females were retained while the unsuccessful were discarded. The former were then placed on the d i e l d r i n contaminated diets and were retained on these for the duration of the experiment. The females were caged contin-uously with f e r t i l e males during the f i f t h and s i x t h week of exposure and were examined twice d a i l y , at 0800 and 1900 hours, for the presence of copu l a t i o n plugs. Pregnancy was affirmed by p a l p i t a t i o n on day 10 (date of plu = 0) of gestation although most pregnant females exhibited discernable swelling of the abdomen by day 8. The number of pups, and t h e i r sex, was determined at b i r t h (Day 1). The dam was allowed to r e t a i n the e n t i r e l i t t e r and the number of l i v e pups was determined, by sex, each day at 0800 hours. The l i t t e r was weighed at th i s time and the average weight of a surviving pup was c a l c u l a t e d by d i v i s i o n . The quantity of milk v i s i b l e i n the pups' stomachs was rated as normal, reduced, or absent. Pups were re-moved from the dam on day 28 and, a f t e r being kept overnight without food, they were injected (ip) with, sodium pentobarbital (60 mg/kg) and the sleep-ing times measured. Because of i n s u f f i c i e n t animal f a c i l i t i e s the experiment was con-ducted i n 2 r e p l i c a t e s to obtain a reasonable sample s i z e . % General observations were made on the apparent health and behaviour of the females and the pups i n both r e p l i c a t e s . P a r t i c u l a r a t t e n t i o n was directed towards the behaviour of pregnant females during gestation, par-t u r i t i o n and the immediate post-partum period. S i m i l a r l y , p a r t i c u l a r atten t i o n was direc t e d towards the duration and normality of p a r t u r i t i o n and towards the condit i o n of the pups i n the f i r s t few hours a f t e r d e l i v e r y . In the second group, the maternal behaviour of the females was more c l o s e l y observed on days 1-14 post-partum. The presence (or absence) 16 of the dam in the nest and aggression (towards the pups) was recorded v e r b a l l y on tape every 1.33 seconds during each of two, 3.0 minute bouts, which had been chosen at random between 1400 and 1600 hours. A l l females were autopsied a f t e r s a c r i f i c e witli ether. The l i v e r and ovaries were removed, blotted dry and weighed. The number of placental scars (or fetus' and/or resorptions) were counted following which the uterus was excised, opened, blotted dry and weighed. Non-pregnant females were k i l l e d 21 days a f t e r removal of the male from the cage. Dams which did not r a i s e pups to weaning were k i l l e d on the day the l a s t pup died while those which raised pups to weaning were k i l l e d on day 28 post-partum. 2. Ovulation and Implantation in Bred, Nulliparous Females V i r g i n females (4-6 weeks old) were placed on dieldrin-contaminated or c o n t r o l rations and caged continuously with males during the f i f t h and s i x t h weeks of exposure during which they were examined twice d a i l y , at 0800 and 1900 hours, for copulation plugs. Between 0800 and 0900 hours on day 4 of gestation, mated females were injected (iv) with 0.2 ml of 0.57. (w/v) Pontamine blue 5BX (K & K Laboratories, Hollywood, C a l i f . ) and k i l l e d with ether 15 minutes l a t e r . The numbers of fresh corpora lutea and decidua. were counted. 3. Milk Production and Maternal Behaviour i n Primiparous Females V i r g i n females (4-6 weeks old) were placed on dieldrin-contaminated or control rations and caged continuously with males during the f i f t h and s i x t h week of exposure. At b i r t h ( l a c t a t i o n day 1), the l i t t e r was reduced to s i x pups and t h i s number was maintained by r e p l a c i n g dead pups with others, of the same age, taken from females fed control r a t i o n s . L7 On day 3 the L i t t e r v/as removed at 1000 hours and replaced at 1400 hours; upon replacement tlie latency to the onset of pup-retrieval, nest-bu i l d i n g and nursing behaviour by the dam was timed. These behaviour patterns were defined as: pu p - r e t r i e v a l : The pups are picked up, one at a time, by the dam, c a r r i e d to and deposited at the nest s i t e , nest-building: The female is digging, pushing, or otherwise moving the bedding to excavate a nest. Pups were us u a l l y in the nest during t h i s process, nursing: The female crouches over the pups i n any manner permitting suckling. The latency was measured to the beginning of the f i r s t , uninterrupted, 2 minute bout of nursing. Dams us u a l l y went to sleep. Milk production was measured on day 4 by a m o d i f i c a t i o n of the method described by Morag (1970). The pups were removed at 1000 hours, re-placed for a 1 liour suclcling period at 1100 hours and removed again at 1200 hours. At 2000 hours the dam was anestlietized with sodium pentobarbital (90 mg/kg, i p ) , injected (sc) with 0.5 IU oxytocin ( P i c t o c i n ; Parke-Davis), weighed and placed on i t s r i g h t flank in a cage. Six c o n t r o l pups, 9-14 days old, and starved for 8 hours were weighed and then permitted to suckle the dam for 20 minutes. The dam was then injected with another 0.5 IU oxytocin, placed on i t s l e f t flank and the pups were allowed to suckle for another 20 minutes. The dam and pups then were weighed again. The number of glands suckled was recorded. Milk production was estimated by the change i n the weight of the dam and pups. 13 4. V i a b i l i t y of Pups Born to Primiparous Females V i r g i n females (4-6 weeks old) were placed on dieldrin-contaminated or control rations and caged continuously with males during the f i f t h and s i x t h week of exposure. At b i r t h , f i v e - s i x pups were iso l a t e d from the dam, placed i n d i v i d u a l l y in p l a s t i c trays and kept at 22 C u n t i l dead. M o r t a l i t y was measured. 5. Sur v i v a l of Pups Born to Dieldrin-Fed Primiparous Females and  Foster-nursed by Non-exposed Primiparous Dams V i r g i n females (4-6 weeks old) were placed on dieldrin-contaminated or control rations and caged continuously with males during the f i f t h and s i x t h week of exposure. The l i t t e r of a control dam was reduced to f i v e -s i x pups at b i r t h . Within 24 hours, f i v e - s i x pups ( < 12 hours old) born to an exposed female, were added to the l i t t e r ; a l l pups were toe-clipped for i d e n t i f i c a t i o n . The number of l i v e pups i n each of the foster and maternal halves of the l i t t e r was determined d a i l y at 0800 hours. The foster and maternal portions of the l i t t e r were also weighed at t h i s time and the average weight of a surviving pup in each group was c a l c u l a t e d by d i v i s ion. 6. Serum Progesterone Levels During Gestation in Dieldrin-Fed  Primiparous Females a. Serum Samples V i r g i n females (4-6 weeks old) were placed on dieldrin-contaminated or control r a t i o n s . During the f i f t h and s i x t h week of exposure a male was placed i n each of the females' cages every night at 2000 hours. Males were removed at 0800 hours the next morning and the females were examined for 19 vaginal plugs. Females were bled terminally from the posterior border of the i n f e r i o r i n t r a - o r b i t a l sinus on days -8 and 14 of gestation (between 0300 and 0900 hours). Serum was prepared and stored frozen at -20 C u n t i l a nalysis (see below). Pregnancy was confirmed by uterine examination, b. Determination of Progesterone Serum progesterone was measured by a competitive protein-binding technique adapted from those of Reeves et a l . (1970) and N e i l l et a l . (1967). Materials Glassware Centrifuge tubes: 35 ml, c o n i c a l , with glass stopper and 15 ml, c o n i c a l , without stopper. Disposable cu l t u r e tubes, 12 x 75 mm; these were washed, used and discarded. A l l glassware was washed i n FL-70 Detergent (Fisher) and rinsed with tap water and d i s t i l l e d water. Solvents A l l solvents were AR grade or better and were d i s t i l l e d , at l e a s t once, before storage for future use. The ethanol (absolute) and d i e t h y l ether were d i s t i l l e d twice, the ether r e c e i v i n g i t s second d i s t i l l a t i o n the day of use. The 40-50 C b o i l i n g f r a c t i o n of petroleum ether was d i s t i l l e d weekly from M a l l i n k r o d t 1 s 30-60 C solvent. Solvents were stored i n brown bottles at room temperature; benzene was stored over sodium. Steroids Progesterone (Sigma Chemicals) was r e c r y s t a l L i z e d twice from methanol, once from ethanol and confirmed pure by TLC, in two solvent systems, and by g a s - l i q u i d chromatography. C o r t i c c s t e r o n e - l , 2 - 3 H (3H-B; 20 3 3 s p e c i f i c a c t i v i t y = 44 C/mM) and progestcrcne-7- H ( H-P; s p e c i f i c a c t i v i t y = 16 C/niM) were purchased from New England Nuclear and used without p u r i f i -c a t i o n . Working solutions of progesterone (0, 2.5, 5, 10, 20, 40 and 80 nanograms/ml), ^H-P (200 dpm/jal) and ^HB (1.0 juC/ml) were prepared in ethanol and stored in the dark at -20 C when not in use. Corticosterone-Binding Globulin (CBG) Serum was prepared from blood drawn from a 25 year old woman taking contraceptive steroids and stored i n 150 p i a l i q u o t s at -20 C. Thin-Layer Chromatography Sheets Eastman Chromagram No. 6060. S i l i c a gel with fluorescent i n d i c a t o r . F l o r i s i l (Fisher) P a r t i c l e s i z e : 60-100 mesh. The absorbent was washed ten times with ten volumes of d i s t i l l e d water; those fines not s e t t l i n g r e a d i l y , were decanted each time. A f t e r washing, the F l o r i s i l was dried for 12 hours at 105 C and stored under d e s s i c a t i o n . L i q u i d S c i n t i l l a t i o n C o c k t a i l Bray's s o l u t i o n , prepared as described by Bray (1960), was used. Method General An i n t e r n a l standard of 2000 dpm -%-P was added to a l l samples to allow c o r r e c t i o n for losses incurred during e x t r a c t i o n and p u r i f i c a t i o n . The water bath was maintained at 37 C for a l l procedures. A l l solvents were evaporated by use of the water bath and a gentle stream of G-grade nitrogen directed onto the solvent surface by means of a Ple x i g l a s manifold s i m i l a r to that of Murphy (1965). 21 Extrac t i o n To each of the 35 ml, stoppered, centrifuge tubes was added 10 /.il of 3 the H-P s o l u t i o n . This was then evaporated to dryness and the serum samples added; samples of less than 0.2 ml were made up to this volume with d i s t i l l e d water. Four ml of petroleum ether were added and the two phases mixed on a Vortex-Genie (Fisher) for 2.0 minutes at s e t t i n g 4. The phases were separated by c e n t r i f u g a t i o n (IEC Model EXD) at 1000 rpm for 10.0 minutes. The solvent was then transfered to a 15 ml centrifuge tube by use of a Pasteur pipet and evaporated to dryness. The ext r a c t i o n procedure was repeated with a second 4.0 ml of petroleum ether. The residues of the com-bined extracts were rinsed to the bottom of the tubes by 0.75 ml of petro-leum ether which was then evaporated o f f ; this was repeated with 0.25 ml of petroleum ether. P u r i f i c a t i o n : Thin Layer Chromatography Interference problems arose i n the displacement analysis i f the chromagrams were touched with bare fingers; therefore, sheets were handled only when v i n y l examination gloves (Tru-Touch; Becton, Dickinson Co.) were worn. Chromagrams were marked into s i x lanes, each 3.0 cm wide, by re-moving 0.5 mm wide bands of s i l i c a between them. A 1.0 cm band of gel was removed 18 cm from the bottom of the sheet to prevent over-development. Each plate was then washed i n d i v i d u a l l y , two times, by ascending chroma-tography with ethanol and stored under d e s s i c a t i o n u n t i l use. Chromagrams were used without a c t i v a t i o n . Progesterone solutions were applied to the o r i g i n , 3.0 cm from the bottom of the sheet, with Hamilton m i c r o l i t e r syringes. C a r r i e r solvents were evaporated with cold 22 a i r blown from a hair dryer. Ten p i of standard progesterone s o l u t i o n (0.5 pg) were spotted i n the two outside lanes. Serum extracts were spotted, with three 50 p i volumes of d i e t h y l ether, in each of the others. The sheets were checked under u l t r a v i o l e t l i g h t before development. A Desaga chamber was then l i n e d with f i l t e r paper and 60 ml ether: benzene (2:1, v/v) were added to i t . F i f t e e n minutes l a t e r a chromagram was placed i n the tank and developed; the sheet was supported on a glass plate. Fresh solvent was used for each chromagram. The chromagrams were dried and the progesterone standards located under u l t r a v i o l e t l i g h t . Points were marked 1.1 cm above and below the centres of the standards and the r e s u l t i n g h o r i z o n t a l s t r i p was cut out with a sharp s c a l p e l . The 2.2 x 3.0 cm area for each sample was then s i m i l a r l y cut out. Cuts were made by using a glass plate, devoid of f i n g e r -p r i n t s , as a s t r a i g h t edge; t h i s r e s u l t e d i n minimal disturbance of the s i l i c a g e l . TLC areas were suspended from an e l u t i n g device s i m i l a r to that of S c h r i e f f e r s et a l . (1963) and the progesterone eluted with 2.0 ml ethanol into a 12 x 75 mm tube. Samples could then be kept for up to 7 days by s e a l i n g the tubes with P a r a f i l m and s t o r i n g them under d e s s i c a t i o n at 4 C. Estimation of Procedural Losses The samples were evaporated to dryness and the residues were rinsed to the bottoms of the tubes with 0.75 ml petroleum ether. This was evapo-rated and 1.0 ml of ethanol was added following which the tubes were vor-texed (10 seconds at s e t t i n g 2) to insure d i s s o l u t i o n of the residues. Two hundred p i a l i q u o t s were taken with a Hamilton syringe and placed in 23 counting v i a l s . F i f t e e n ml of Bray's s o l u t i o n were added and the v i a l s counted for 120 minutes in a Picker Liquimat (Model 650) s c i n t i l l a t i o n 3 3 counter (maximum counting e f f i c i e n c y of 30% for H). The cpm of H-P were corrected for quenching by the external standard method and the dpm used to estimate the recovery of progesterone. Binding Solution Preparation 3 Into a 50 ml volumetric flask was pipeted 0.5 ml of the H-B solu-t i o n . The ethanol was evaporated, 45 ml of g l a s s - d i s t i l l e d water were added, and the fla s k returned to the hot water bath for 5 minutes. A f t e r the fla s k had returned to room temperature the serum was removed from the freezer and exactly 30 minutes l a t e r , 50 u l of serum were added and the s o l u t i o n made up to volume. The s o l u t i o n was kept at room temperature and used w i t h i n 4 hours of preparation. Displacement Analysis The chromagram elutant contained an unknown material, "Method-I n t e r f e r i n g Factor" (MIF), which a l t e r e d the apparent binding capacity of the 3 CBG and consequently, the displacement of H-B caused by the progesterone; t h i s e f f e c t was compensated for by adding MIF to the tubes containing the standards. A blank chromagram was washed and developed in the same manner and at the same time as those containing samples. Twelve 2.2 x 3.0 cm areas were cut from i t and eluted as usual; the eluates were pooled, 20% (corres-ponding to the recovery aliquots) was discarded and the remainder made up to 24 ml with ethanol. Two ml were placed in each of twelve 12 x 75 mm tubes. These could be stored under the same conditions as the samples. The elutant in a l l tubes, samples and standards, was then evaporated. Progesterone solutions were then pipeted into the standard tubes and also 24 evaporated. The standards, usually 0.0, 0.5, 1.0, 2.0, 4.0 and 8.0 ng, were run in duplicate; ten samples were run simultaneously with these. One ml of binding s o l u t i o n was added to the tubes which were then vortexed for exactly 15 seconds ( s e t t i n g 2) to ensure d i s s o l u t i o n of the progesterone. The tubes were then heated for exactly 5.0 minutes in the hot water bath; this caused d i s s o c i a t i o n of a l l s t e r o i d CBG complexes and ensured unbiased competition for the binding s i t e s . The tubes were trans-fered to an icebath immediately a f t e r heating and allowed to s i t for 10 minutes for binding e q u i l i b r i u m to occur. Unbound 3H-B was removed with 61.1 mg (SD =0.43; n = 10) of F l o r i s i l , added by a dispenser s i m i l a r to that of Souza et a l . (1970), and placed i n suspension, exactly 10 seconds a f t e r addition, by shaking the tube for exactly 30 seconds in a h o r i z o n t a l shaker ( P r e c i s i o n S c i e n t i f i c , Cat. No. 5855). A f t e r shaking the tube was returned to the icebath. When a l l tubes had been treated they were centrifuged (IEC model B-20; IEC Head No. 874) together at 10 C for exactly 3.0 minutes at 3500 rpm. The tubes were replaced in the icebath, allowed to stand for 15 minutes and a 0.5 ml a l i q u o t of the supernatant taken and placed in a counting v i a l . F i f t e e n ml of Bray's s o l u t i o n were added and the v i a l s counted to > 10,000 cpm. C a l c u l a t i o n of progesterone in the serum samples was by the method of Reeves et a l . (1970). The mean cpm of the two 0.0 ng standards was de-3 fined as 1007o binding of H-B and the proportion bound in a l l other tubes was c a l c u l a t e d using t h i s number. A c a l i b r a t i o n curve was obtained by 3 p l o t t i n g "7o H-B Bound" versus "ng Standard Progesterone" from which the progesterone in the sample tubes was estimated. Progesterone in the o r i g i -nal serum sample was c a l c u l a t e d by c o r r e c t i n g t h i s for losses due to 2 5 extraction, p u r i f i c a t i o n and recovery estimation, thus: ng from standard curve x 1 0 0 x 1 0 0 7» recovery x 8 0 Reagent blanks were ro u t i n e l y assayed i n the system and the reagents were never found to have a displacement e f f e c t . Assessment of the Progesterone Assay Control experiments with human male serum, devoid of detectable quantities of progesterone, and spiked with known quantities of progesterone showed that: i . The smallest quantity of serum-extracted progesterone detec-table to the displacement system was between 0 . 2 5 and 0 . 5 0 ng. i i . The c o e f f i c i e n t of v a r i a t i o n (<J/x) on duplicate samples of the same serum averaged 0 . 0 4 , i . e . 4 7 o . i i i . The estimated progesterone concentration was, on the average, 1 0 7 o higher than the known concentration. D. S t a t i s t i c a l Analysis of the Data A l l the data have been analyzed s t a t i s t i c a l l y to estimate the proba-b i l i t y that the differences between control and treatment values occurred from chance. A p r o b a b i l i t y l e v e l of £ 0 . 0 5 was chosen as i n d i c a t i n g s i g n i -ficance. Continuous, qu a n t i t a t i v e data were analyzed by analysis of variance ( i . e . F t e s t ) , a f t e r appropriate transformations were made when these were required; when more than one dose was included i n the analysis they were compared to the controls by use of Duncan's New M u l t i p l e Range Test or 2 Dunnett's d 1 Test. Discrete, q u a l i t a t i v e data were analyzed by X using Yate's procedure to correct for c o n t i n u i t y . The procedures used here are 26 f u l l y described by Sokal and Rohlf (1969) or Steel and T o r r i e (1961). Unless otherwise stated i t is the standard error ( &/\ln) which is given with the means. RESULTS A. L i v e r Parameters i n Females Fed D i e l d r i n 1. Gross Appearance of the L i v e r The appearance of the l i v e r was completely normal in a l l but one of the females. The l i v e r of that one female, k i l l e d a f t e r 8 weeks exposure to 20 ppm, was spotted with what appeared to be l i p i d deposits. No hepa-tomas of any type were seen in any of the animals. 2. L i v e r Weight The r e l a t i v e l i v e r weight of d i e l d r i n - f e d animals, presented i n Figure 1, was s i g n i f i c a n t l y increased a f t e r 2 weeks exposure to a l l doses except 5 ppm; however, even at t h i s low dose the l i v e r was c o n s i s t e n t l y , and sometimes s i g n i f i c a n t l y , larger than that of control animals. The increases were a r e s u l t of l i v e r growth and not of decreased body weight since the l a t t e r remained constant over the exposure period, within and between dosage groups; animals fed co n t r o l rations, for example, weighed 27.5 + 0.48 g and those fed 20 ppm d i e l d r i n weighed 26.9 + 0.82 g. L i v e r weight in the c o n t r o l animals was 57.9 + 0.85 mg/g body weight, and did not change with age so that the hepatomegaly in exposed animals was a consequence of d i e l d r i n alone. The i n i t i a l growth rate of the l i v e r was dose-dependent since a f t e r 5 days of exposure to 20 ppm the l i v e r was s i g n i f i c a n t l y increased to 65.5 + 0.2 mg/g body weight, whereas 10 ppm caused only a slight^. i n s i g n i f i c a n t increase and 5 ppm had no e f f e c t . The growth at a l l doses was e s s e n t i a l l y maximal a f t e r 2 weeks of exposure. The asymtotic l i v e r weight was also dose-dependent since over weeks 2-10 of exposure, animals fed 20 ppm EXPOSURE TIME (WEEKS) F i g u r e 1. L i v e r weights of female SWV mice fed d i e t a r y d i e l d r i n (n = 2-4 at each p o i n t ) . Animals were i n i t i a l l y 12-14 weeks o l d . 29 d i e l d r i n had l i v e r s which averaged 162%, of con t r o l values whereas those fed 5 ppm had l i v e r s weighing only 111% of c o n t r o l l e v e l s . 3. Hepatic Microsomal Pr o t e i n The concentration of microsomal protein i n the l i v e r s of d i e l d r i n -exposed animals i s presented i n Figure 2. The lev e l s determined for animals fed 5 and 10 ppm for 4, 6 and 8 weeks are aberrantly high, as indicated by the control values which are more than 2-fold higher than normal, and are separated, by shading, from a l l others i n the figure; these w i l l be d i s -cussed separately. Microsomal p r o t e i n concentration d i d not d i f f e r s i g n i f i c a n t l y with increasing age i n the control animals and averaged 15.3 + 0.42 mg/g l i v e r . A f t e r 3 days of exposure the l i v e r s of animals fed 20 ppm d i e l d r i n had concentrations of microsomal p r o t e i n higher than those of co n t r o l s and the maximum increase was attained by 5 days, a f t e r which the microsomal p r o t e i n remained constant at a mean of 20.0 + 0.69 mg/g l i v e r , or 29%, above con t r o l l e v e l s . Animals fed 15 ppm were examined only during the plateau phase of the increase i n protein where the concentration averaged 20.9 + 1.05 mg/g l i v e r . At the lox^er doses of 5 and 10 ppm the onset of the increase was a f t e r day 3 of exposure but by 2 weeks i t s magnitude was the same as that caused by 20 ppm. The microsomal protein concentration i n the l i v e r s of animals k i l l e d a f t e r 4, 6 and 8 weeks exposure to 5 or 10 ppm were far higher than would be expected and the appropriate control animals also had greatly elevated l e v e l s of hepatic microsomal protein (shaded area, Figure 2). The reasons for this are unknown but these apparent concentrations seem to be correct H -(TO c n ro O CL H I - 1 H - 3* CL <T> fl> p> O H O 3 O to v; H - 3 rt Hi it ro i-1 H CL P> rt i-f rt i-h 3 O H v o 3 3 O H i II ° 3 ro N> H -X i n T I J > H , H 1 o ft) P3 CO 0 rt O PJ rt H -O 3 ro P> pj i—1 o H O 13 H i O H -!2 3 3" rt ro • CL P> ro H -3 H -3 > rt 3 3* H * ro H -3 cn H 3* ro P> CL p . ro 3 CL H -rt P) H -H P) 0> I—1 pi l - i I - 1 H -ro ro co o H i H i ro ' B I—1 H -fD ro H I ro ro ?r C L m x O (/> C m m m CO mg MICROSOMAL PROTEIN/g LIVER (x ± SEM) ro DI cn cn _1_ ro o ro cn UJ o JL cn > • O x • ro _ _ O cn O cn o •O TJ *0 T3 -o •O "O "O "O - o 3 3 3 3 3 00 . . . ^ W - W W < « W v » o w 2 o * p A A o o b j-cn O ° 5 n > m _ co 2 •n •n m m z O oc 31 since the s p e c i f i c a c t i v i t i e s of enzymes within these microsomes were normal as w i l l be discussed below. At these times the dieldrin-induced increases in protein, which averaged 297o and 30% over control l e v e l s for the 5 and 10 ppm doses respectively, were the same as those found at 2 and 10.6 weeks of exposure to these doses. The average increase caused by 5 ppm over the e n t i r e 2 to 10 weeks of exposure was 35.4 + 4.9% while 10 ppm caused 34.9 + 8. 6%. The microsomal protein concentrations i n the l i v e r s were hig h l y v a r i a b l e , as evidenced by the large standard errors, and consequently the dieldrin-induced increases were s i g n i f i c a n t (P < 0.05) at only a few doses and exposure times. It can be seen, however, that the l e v e l s i n exposed animals were c o n s i s t e n t l y elevated over co n t r o l values and many of the d i f -ferences were s i g n i f i c a n t at P ( 0.10 suggesting that the increases, while v a r i a b l e and small, were r e a l and would be more r e a d i l y demonstrated by la r g e r sample s i z e s . 4. Cytochrome P-450 The cytochrome P-450 concentrations are presented i n Figure 3 . Levels i n the l i v e r s of con t r o l animals suggest that the P-450 concentra-t i o n did not change with age, although they were found to vary between assay days even when other factors were held constant. For example, two groups of animals, both 16-18 weeks old and used as controls for treatment animals exposed for 4 weeks, were assayed several months apart and had P-450 levels of 0.91 + 0.005 nmoles/mg microsomal protein and 0.63 + 0.03 nmoles/mg microsomal protein. It seems more probable that this v a r i a b i l i t y resulted from procedural differences on the days of assay rather than b i o l o g i c a l differences between ind i v i d u a l s of the two groups. nyu MOLES Cyt. P450 /mg MICROSOMAL PROTEIN (x±SEM) P — — ro cn o cn o -» -l— 1 i i ZZ 33 D i e l d r i n caused a dose-dependent increase in P-450 with 5, 10, 15 and 20 ppm causing maximal elevations to 1.29, 1.46, 1.98 and 1.96 nmoles/mg microsomal protein r e s p e c t i v e l y . The early time-course of the induction was c l e a r e s t at 20 ppm where the l e v e l , elevated at day 3, was s i g n i f i c a n t l y increased by day 5, a f t e r which l i t t l e , i f any, further increase occurred. The early changes were not so marked at 5 and 10 ppm but elevated l e v e l s , averaging 1.15 nmoles/mg microsomal protein and 1.32 nmoles/mg microsomal protein r e s p e c t i v e l y , were attained and maintained from weeks 2-10.6 of exposure. In animals fed 20 ppm d i e l d r i n , cytochrome P-450 reached maximum le v e l s i n the f i r s t 4 weeks of exposure and declined thereafter. Not so evident, due p r i m a r i l y to the pooling of the co n t r o l data, i s the fact that P-450 responds s i m i l a r l y at the lower doses. Figure 4 expresses the P-450 concentrations i n animals fed 5 and 20 ppm d i e l d r i n as a percentage of the appropriate control l e v e l s and i t can be seen that maximum concentrations were reached between 3 and 4 weeks of exposure a f t e r which they declined to about 407« of co n t r o l values by 6 and 10 weeks r e s p e c t i v e l y at the two doses. 5. Summary of L i v e r Changes The above r e s u l t s demonstrate that dietary d i e l d r i n , fed constantly at doses of 5-20 ppm, caused: a. a dose-dependent hepatomegaly of up to 627<, which was attained a f t e r 2 weeks and then maintained for at least another eight; b. an increase, at a l l doses, of 30-35% i n the weight of microsomal protein per gram of l i v e r ; the peak of t h i s response was reached at a l l doses by 2 weeks and was maintained for another eight; F i g u r e 4, Hepatic cytochrome P-450 expressed as a percent of c o n t r o l values i n female SWV mice fed 5 and 20 ppm d i e t a r y d i e l d r i n . (n = 2-4 at each p o i n t ) . c. a dose-dependent increase in the amount of cytochrome P-450 per milligram of microsomal protein which c l o s e l y p a r a l l e l e d the increase i n microsomal protein for the f i r s t 2 weeks of exposure. Maximal increases of up to 128% over co n t r o l values were reached by 3-4 weeks a f t e r which they f e l l gradually, at a l l doses, to an apparent plateau approximately 40% above normal concentrations. d. no hepatomas. B. L i v e r Function i n Females Fed Dietary D i e l d r i n 1. _in vivo: Pentobarbital Anesthesia In females fed d i e l d r i n at 5, 15, or 25 ppm for 4 weeks the duration of pentobarbital anesthesia was s i g n i f i c a n t l y decreased (Table I ) . I t i s commonly accepted that such a decrease r e f l e c t s an increase i n the o v e r a l l metabolic capacity of the 1 i v e r 1 s , cytochrome P-450 dependent, hydroxylation system. Since hydroxylation i s the major route of metabolism of the sex steroids i t may be concluded from these data that the r e q u i s i t e conditions for enhanced rates of sex s t e r o i d metabolism were met i n the l i v e r s of the d i e l d r i n - t r e a t e d animals used i n the reproduction experiments. " The data show that the maximum rate of in vivo pentobarbital metabo-lism was not li m i t e d by the amount of cytochrome P-450. The maximum rate was attained i n animals fed 5 ppm d i e l d r i n , where sleeping time was decreased by 433%, and increasing the dose 5 fold to 25 ppm did not further increase the rate (Table I ) . And yet the amount of hepatic cytochrome P-450 in the animals fed 25.0 ppm was twice that in those fed 5 p p m — 2.5 nmoles per gram of body weight compared to 1.2 nmoles per gram of body weight. Control females of comparable age had 0.8 nmoles of cytochrome P-450 per gram of 36 Table I. Loss of the r i g h t i n g r e f l e x i n female SWV mice injected (ip) with sodium pentobarbital (90 mg/kgm) a f t e r eating dietary d i e l d r i n for 4 weeks. The latency to the onset was measured from the time of ingestion while the duration was measured from the onset. Righting Reflex Loss (minutes) x + SEM Dose n onset a duration* 3 (ppm) 0 10 2.70 0.18 108.3 + 3.16 5 5 3.55 + 0.15** 25.2 + 2.65** 15 8 3.22 + 0.15* 16.5 + 3.23** 25 3 3.42 + 0.17* 19.7 + 5.24** a. 1-way anova for Doses: o v e r a l l F = 4.55, df = 3,22; P< 0.025 b. 1-way anova for Doses: o v e r a l l F = 75.04, df = 3,22; P<0.01 ** S i g n i f i c a n t l y d i f f e r e n t from co n t r o l value by Duncan's New M u l t i p l e Range Test at P<0.01 or 0.05 (*). Doses do not d i f f e r from each other. body weight. S i m i l a r l y , the maximum rate was not r e l a t e d to the t o t a l amount of microsomal protein, which for animals fed 0, 5 or 15 ppm d i e l d r i n was 0.9, 1.2 and 1.6 mg per gram of body weight r e s p e c t i v e l y . The latency to the onset of pentobarbital anesthesia was s i g n i f i -c antly increased, by 2670, at a l l three doses of d i e l d r i n (Table I ) . 2. iri v i t r o : ^C-Imipramine Metabolism The IMI-metabolizing a c t i v i t y of hepatic microsomes prepared from con t r o l animals on IS d i f f e r e n t occasions spread over 18 months, varied 37 s i g n i f i c a n t l y between assays (Table I I ) . The v a r i a t i o n , which was c l e a r l y independent of the animals' age, appears to be randomly d i s t r i b u t e d suggest-ing that i t resulted from procedural di f f e r e n c e s between the assays. This view is also supported by the v a r i a t i o n between the r e p l i c a t e s w i t h i n assays, where the mean c o e f f i c i e n t of v a r i a t i o n ( 6/x ), was 0. 106 + 0.02 and i s w i t h i n the l i m i t s of experimental error. To f a c i l i t a t e the detection of age and induction e f f e c t s , the data, a f t e r s t a t i s t i c a l a n a l y s i s , were con-verted to percentages for presentation and to remove t h i s "procedural" v a r i a t i o n . The mean rate at which microsomes from a l l the co n t r o l animals metabolized 1M1 was 11.86 + 0.62 ug/mg microsomal protein/15 minutes. The pathways by which microsomes from c o n t r o l mice metabolized IMI changed q u a n t i t a t i v e l y with the increasing age of the animal (Figure 5). The apparent a c t i v i t y of the N-demethylase system decreased s t e a d i l y to about 54% of i t s o r i g i n a l a c t i v i t y a f t e r 10 weeks while the a c t i v i t i e s of the N-oxidase and the hydroxylase(s) increased by 15 and 25%, r e s p e c t i v e l y . The decrease i n DMT production could be compensated for by the increase i n the l a t t e r pathways because of the larger proportion of IMI handled by them, so that the o v e r a l l IMI metabolism need not be decreased. The concentration of P-450 did not change during these pathway a l t e r a t i o n s i n d i c a t i n g that in non-induced microsomes, t h i s cytochrome i s eit h e r not involved i n the systems which demethylate, hydroxylate and N-oxidate IMI, or i f i t i s , i t i s not r a t e - l i m i t i n g . The o v e r a l l rate of IMI metabolism (jig IMI metabol ized/mg microsomal protein), which is the sum of that metabolized through the three pathways, was not increased by any dose of d i e l d r i n over the 10 week exposure period; t h i s i s seen i n Figure 6 where the mean rates for treatment r e p l i c a t e s have 33 Table I I . The amount of IMI metabolized by hepatic microsomes prepared from SWV female mice fed the control r a t i o n . Where assays were done on 2 d i f f e r e n t groups of animals of the same age but at 2 d i f f e r e n t times the r e s u l t s have been presented separately, n = 2-4 for each mean. Female's Age jig IMI metabol ized/mg microsomal protein/15 minutes 3' (weeks) (x + SEM) 13.143 8.45 + 0.26 16.71 + 0.79 13.429 18.23 + 0.52 8.61 + 0.75 13.714 8.67 JL i 0.25 14.143 10.90 + 0. 80 15.0 16.23 + 0.68 10.76 + 1.14 16.0 14.36 + 1.35 17.0 8.23 + 1.48 12.91 + 0.79 19.0 4.85 + 0.18 10.96 + 0.29 21.0 11.82 + 2.86 13.92 + 1.08 23.0 12.11 + 0.53 23.6 13.77 + 0.84 a. 1-way anova between groups: F = 10.85, df = 16, 17; P < 0.005 b. grand mean for a l l animals = 11.86 + 0.62 MEDIAN AGE (WEEKS) Figure 5. Imipramine (IMI) metabolism by h e p a t i c microsomes prepared from c o n t r o l SWV female mice (n = 2-4 at each p o i n t ) : E f f e c t of age on the r e l a t i v e a c t i v i t i e s of IMI - demethylase, IMI - N-oxidase and IMI - h y d r o x y l a s e ( s ) . _J o or l -2 O o Li_ O <* 2 in _ i o CD < I-LU 2 110 H 100 9 0 H 8 0 H 7 0 H 5 ppm X 10 ppm O 15 ppm • * — — 2 0 ppm A o 6 0 + 3 4 5 6 7 8 EXPOSURE TIME (WEEKS) 10 II F i g u r e 6. The r e l a t i v e r a t e of imipramine metabolism per mg of p r o t e i n by hepatic microsomes prepared from SWV female mice fed d i e t a r y d i e l d r i n (n = 2-4 at each p o i n t ) . A l l animals were i n i t i a l l y 12-14 weeks o l d . 41 been expressed as a per cent of those for the corresponding controls. The mean rates over the 10 weeks were 11.86 + 0.62 for the controls and 9.77 + 1.03, 11.92 + 1.00, 12.57 + 1.17 and 10.52 + 0.49 for those fed 5, 10, 15 and 25 ppm r e s p e c t i v e l y . Since a l l these doses elevated the microsomal concentration of cytochrome P-450 the lack of increased s p e c i f i c a c t i v i t i e s show that P-450 could be r a t e - l i m i t i n g in not more than two of the three paths and that increased rates in those where i t i s , must be compensated for by decreases in those where i t is not. The r e s u l t s , which e x h i b i t considerable v a r i a t i o n and therefore have been interpreted as much by trends as by formal s t a t i s t i c a l d i f f e r e n c e s , show that d i e l d r i n d i d i n f a c t cause changes i n the pathways of IMI metabo-lism. The formation of IMI-N- oxide was decreased a f t e r 1 day of exposure to 20 ppm d i e l d r i n (Figure 7) and this reduction continued with further exposure and the maximum decrease,307. below con t r o l l e v e l s , was reached at 6 weeks and maintained thereafter. The reduction was s i g n i f i c a n t by 8 days of exposure and at a l l times a f t e r that except for 4 weeks where the lack of s i g n i f i c a n c e was due to large v a r i a t i o n in the c o n t r o l s . At the"dose of 15 ppm, d i e l d r i n appeared to cause a s i m i l a r reduction but 5 and 10 ppm seemed to have l i t t l e e f f e c t u n t i l 8 weeks of exposure at which time both doses caused decreases in N-oxide production; of these two doses only the e f f e c t of 5 ppm was examined at 10.6 weeks and the N-oxide was further and s i g n i f i -cantly decreased in quantity. These changes, which were in opposition to the normal, age-dependent increase in N-oxide formation, occurred while the con-ce n t r a t i o n of microsomal cytochrome P-450 was elevated by a l l doses, showing that the concentration of the cytochrome is not r a t e - l i m i t i n g in the N-oxidation of IMI by dieldrin-induced microsomes. ~ I20T * SIGNIFICANTLY DIFFERENT FROM CONTROLS P<0.05 UJ CO +1 Ix O Ql f-O O o Q UJ O Q O cr o_ O t z I MOH 100 90H 80H 70 60 I 5 ppm X 10 ppm O 15 ppm • 2 0 ppm A i\ \ ..y-/"'T"^ L I* • V x * . F i g u r e 7. 1 T • ' • ' \ T . - X T 3 4 5 6 EXPOSURE TIME (WEEKS) 7 T 8 T 9 "Tr-IO n II Metabolism, of imipramine to imipramine N-oxide (IMI-N-* 0) by hep a t i c microsomes prepared from SWV female mice fed d i e t a r y d i e l d r i n (n = 2-4 at each p o i n t ) . A l l animals were i n i t i a l l y 12-14'weeks o l d . •P-f O 43 The formation of hydroxylated IMI was immediately increased f o l l o w i n g exposure to 20 ppm d i e l d r i n (Figure 8). The elevated a c t i v i t y of the hydroxylase was 307o above c o n t r o l values by day 8 of exposure and va r i e d about t h i s l e v e l f or the d u r a t i o n of the exposure p e r i o d . Except for a t r a n s i t o r y and i n s i g n i f i c a n t increase of 18% caused by 10 ppm at 2 weeks, the production of IMI-OH by the hepatic microsomes of animals fed 5 and 10 ppm was only s l i g h t l y increased d u r i n g the f i r s t 6 weeks of exposure; however at 8 weeks 5 ppm had s i g n i f i c a n t l y increased hydroxy formation and t h i s was f u r t h e r increased to about 130% of c o n t r o l values by 10.6 weeks. Increased concentrations of microsomal cytochrome P-450 accompanied the enhanced rates of h y d r o x y l a t i o n but the two d i d not c o r r e l a t e , temporally or q u a n t i t a t i v e l y . As seen i n Figure 4, 20 ppm caused the c o n c e n t r a t i o n of P-450 to increase by 200%. w i t h i n the f i r s t week of exposure w h i l e during t h i s same pe r i o d h y d r o x y l a t i o n was increased by only 30%.; s i m i l a r l y , 5 ppm, which had increased P-450 by 68% at 4 weeks, d i d not cause s i g n i f i c a n t i n -creases i n h y d r o x y l a t i o n u n t i l 8 weeks, when the c o n c e n t r a t i o n of P-450 had a c t u a l l y d e c l i n e d and s t a b i l i z e d at 40% above c o n t r o l v a l u e s . These r e s u l t s i n d i c a t e that the c o n c e n t r a t i o n of cytochrome P-450 i s not r a t e - 1 i m i t i n g i n the h y d r o x y l a t i o n of LMt d i e l d r i n - i n d u c e d microsomes. D i e l d r i n a l s o increased the r a t e of DMI formation, but only a f t e r prolonged feeding as seen i n Figure 9. The increase s t a r t e d at 4 weeks of exposure to 15 and 20 ppm and reached a maximum, 50%, above c o n t r o l values, by 8 weeks, f o l l o w i n g which i t d e c l i n e d to 35%, above c o n t r o l s at 10 weeks. At 5 and 10 ppm DMI production a l s o appeared to increase but only a f t e r 6 weeks of exposure. The microsomal c o n c e n t r a t i o n of cytochrome P-450 (Figure 4) was d e c l i n i n g or had d e c l i n e d during these changes i n demethylase a c t i v i t y - 140 T 130 * SIGNIFICANTLY DIFFERENT FROM CONTROLS P <0.05 !20 11 o H 100-J 90 A A 4 * tx *i ../ I A ' 1 80 5 ppm 10 ppm 15 ppm 20 ppm X o A 2 3 4 5 6 7 8 EXPOSURE TIME (WEEKS) 10 Figure 8. Metabolism of imipramine to hydroxy-imipramine (IMI-OH) by h e p a t i microsomes prepared from SWV mice fed d i e t a r y d i e l d r i n (n = 2-4 a each p o i n t ) . A l l animals were i n i t i a l l y 12-14 weeks o l d . * SIGNIFICANTLY DIFFERENT FROM 5* CONTROLS. P<0.05 / [ \ 0 I 2 3 4 5 6 7 8 9 10 II EXPOSURE TIME (WEEKS) F i g u r e 9. Metabolism of imipramine to desmethylimipramine (DMI) by hep a t i c microsomes prepared from SWV female mice fed d i e t a r y d i e l d r i n (n = 2-4 at each p o i n t ) . A l l animals were i n i t i a l l y 12-14 weeks o l d . 46 i n d i c a t i n g that the amount of the cytochrome was also not r a t e - l i m i t i n g i n the demethylation of Till by dieldrin-induced microsomes. 3. Summary of Dieldrin-induced Metabolic Changes in Females a. D i e l d r i n fed at 5 ppm for 4 weeks decreased pentobarbital sleep-ing time by 4307°; increasing the dose, by up to 5-fold, did not further decrease i t despite causing large a d d i t i o n a l increases i n l i v e r weight, microsomal protein concentration and cytochrome P-450 l e v e l s . b. The r e l a t i v e proportion of IMI metabolized to DMI, IMI-OH, and IMI-N-* 0 changed as a function of age i n microsomes prepared from the l i v e r s of co n t r o l animals. c. The concentration of cytochrome P-450 did not change as a func-t i o n of age showing that the concentration of the cytochrome was not r a t e - l i m i t i n g i n the demethylation, hydroxylation and N-oxidation of IMI by non-induced microsomes. d. D i e l d r i n did not cause any change i n the o v e r a l l rate of IMI metabolism. e. D i e l d r i n did cause q u a n t i t a t i v e changes in the pathways through which IMI was metabolized. These changes were c l e a r e s t at 20 ppm where: i . hydroxylation was immediately increased and by 8 days of exposure was elevated by 307»; this l e v e l was then maintained throughout the exposure period; i i . N-demethylation increased r a p i d l y by 507o from 4-8 weeks of exposure and then decreased to 357» above normal; 47 i i i . N-oxidation was immediately decreased and by 6 weeks o i exposure was 307o below control values; this l e v e l was then maintained for the balance of the exposure period. The lower doses caused s i m i l a r changes but only a f t e r longer periods of feeding, f. The cytochrome P-450 concentration was increased during these induced pathway changes showing that i t was not the r a t e - l i m i t i n enzyme for IMI-N-oxidation i n induced microsomes; the increase did not c o r r e l a t e , i n quantity or time, with the increased hydro x y l a t i o n or demethylation i n d i c a t i n g that the concentration of P-450 was not r a t e - l i m i t i n g i n these reactions e i t h e r . C. Regression of Dieldrin-Induced Changes i n the Parameters and Function  of the Female L i v e r The r e v e r s i b i l i t y of the diel d r i n - i n d u c e d changes i n the l i v e r and i t s function was determined by feeding 20 ppm dietary d i e l d r i n to females for 4 weeks. Following this they were fed the con t r o l r a t i o n for 2.0, 4.43 and 6.86 weeks before s a c r i f i c e . The l i v e r weight of these, and co n t r o l , females i s presented i n Table III which shows that 2 weeks on the con t r o l r a t i o n was i n e f f e c t i v e i n reducing the hepatomegaly caused by d i e l d r i n ; the l i v e r i n pre-exposed animals was s t i l l as large as that i n those fed d i e l d r i n continuously. A f t e r 4.43 weeks of the d i e l d r i n - f r e e d i e t , however, the l i v e r of pre-exposed females had regressed markedly, being only s l i g h t l y , and i n s i g n i f i -cantly, larger than that of the controls; t h i s regression was complete a f t e r 6.86 weeks. 48 Table I I I . Regression of dieldrin-induced changes in the weight of the l i v e r : 12-14 week old female mice were fed 20 ppm di e t a r y d i e l d r i n for 4 weeks and then placed on the control diet lor varying times before s a c r i f i c e . n = 3 except for the controls at 6.86 weeks, where n = 2. Weeks on mg l i v e r / g body weight (x it SEM) Control Diet: 2.00 a 4.43 b 6.86° controls 60.44 + 0.23 54.60 + 3.07 5S.02 + 1.57 20 ppm 80.30 + 2.93 62.70 + 1.10 56.86 + 1.31 a. F = 45.68, df = 1, 4; P<0.005 b. F = 6.15, df = 1, 4; P > 0.050 c. F = 0.32, df = 1, 3; P>0.100 The regression of the increased concentration of microsomal p r o t e i n (Table IV) was less c l e a r , p a r t l y because the increase i t s e l f was never that great or well defined (Figure 2), but i t appeared to follow the same temporal pattern. In pre-exposed animals fed c o n t r o l rations for only 2 weeks the concentration of hepatic microsomal p r o t e i n was s t i l l i n the same range as that from animals fed d i e l d r i n continuously (Figure 2) and i t was s t i l l s i g n i f i c a n t l y d i f f e r e n t from that of the controls at a p r o b a b i l i t y l e v e l of < 0.10 — as previously stated the majority of the o r i g i n a l i n -creases were s i g n i f i c a n t l y d i f f e r e n t by only t h i s l e v e l . A f t e r 4.43 weeks on the control rations, however, the microsomal protein concentration had returned to control l e v e l s . The concentrations of microsomal cytochrome P-450 are presented i n Table V. A f t e r 2 weeks on the c o n t r o l rations the pre-exposed animals had P-450 l e v e l s which were s t i l l elevated but were not quite s i g n i f i c a n t l y 49 Table IV. Regression of diel d r i n - i n d u c e d increases i n the concentration of hepatic microsomal protein: 12-14 week old female mice were fed 20 ppm dietary d i e l d r i n for 4 weeks and then placed on the control d i e t for varying times before s a c r i f i c e . n = 3 except for the controls at 6.86 weeks, where n = 2. Weeks on mg microsomal protein/g l i v e r (x + SEM) Control Diet: 2.00 a 4.43 b 6.86c controls 17.9 + 0.86 17.3 + 1.06 13.7 + 0.98 20 ppm 21.5 + 1.15 17.0 + 0.54 17.0 + 1.00 a. F = 6.94, df = 1, 4; P<0.10 b. F = 0.09, df = 1, 4; P > 0.10 c. F = 4.98, df = 1, 3; P > 0.10 Table V. Regression of diel d r i n - i n d u c e d increases i n the concentration of cytochrome P-450: 12-14 week old female mice were fed 20 ppm dietary d i e l d r i n for 4 weeks and then placed on the control diet for varying times before s a c r i f i c e . The figures i n brackets express the experimental values as a per cent of c o n t r o l values, n = 3 except for the controls at 6.86 weeks, where n = 2. Weeks on con t r o l d i e t : controls 20 ppm nmoles cyt. P-450/mg microsomal p r o t e i n (x + SEM) 2.00 1.00 + 0.08 1.23 + 0.06 (128%) 4.43 0.76 + 0.00 1.25 + 0.02 (164%) 6.36 0.72 + 0.05 1.11 + 0.07 (154%) a. F = 7.50, df = 1, 4; P • 0.063 b. F = 1051.0, df = 1, 4; P<0.005 c. F = 16.98, df = 1, 3; P<0.050 50 greater than those of the controls; however, a f t e r 4 .43 weeks of eati.ng un-contaminated feed the concentrations of P-450 were s i g n i f i c a n t l y higher i n the pre-exposed animals, being 164% of the con t r o l values, and this increase was maintained at 6.86 weeks. These proportionate changes are i n fact the opposite to those found i n animals fed d i e l d r i n continuously for comparable time periods. There the P-450 concentration was 133% of control values at 6 weeks and had decreased to 148%, and 141% of control concentration at 8 and 10 weeks r e s p e c t i v e l y . The data in Table V suggest that the cytochrome concentration r a p i d l y regresses within the f i r s t couple of weeks a f t e r ex-posure ceases, but that following t h i s the concentration again increases to le v e l s s i g n i f i c a n t l y higher than normal. Further work is required to con-firm this suggestion since i t is based on a proportionate increase which occurred while the actual concentration of P-450 was decreasing to l e v e l s considerably lower than those found i n animals exposed continuously (Figure 3 ) . The proportionate d i f f e r e n c e r e s u l t e d from a p a r a l l e l decrease i n the P-450 l e v e l s of the co n t r o l animals to values found at the extreme lower l i m i t of the d i s t r i b u t i o n of normal concentrations (Figure 3 ) . The metabolism of IMI by the hepatic microsomes of the pre-exposed animals i s presented i n Table VI where i t can be seen that a f t e r 4 .43 weeks on the control r a t i o n , the induced increase i n the a c t i v i t y of the hydroxy-l a t i o n pathway and the induced decrease i n t h e a c t i v i t y of the N-oxidation pathway no longer e x i s t . In fact, the r e s u l t s suggest that a rebound e f f e c t may occur i n these systems since a f t e r 6.86 weeks the a c t i v i t y of the hydroxylation and N-oxidation pathways are decreased and increased respec-t i v e l y by about 207o. The a c t i v i t y of the demethylation pathway was s t i l l s i g n i f i c a n t l y higher than control values a f t e r 4 .43 weeks although i t had 51 Regression of dieldrin-induced changes i n the pathways of IMI metabolism: 12-14 week old female mice were fed 20 ppm dietary d i e l d r i n for 4 weeks and then placed on the control diet for varying times before s a c r i f i c e . n = 3 except for the controls where at 6.36 weeks n = 2. Results are expressed as a % of control values (x + SEM) Weeks on Control Diet IMI Metabolized Amount Formed DMI IMI-N-* 0 IMI-OH 4.43 123.3 + 15.2 a 127. 1 + 8.9 b 99.2 + 4.9° 97 .7 + 8. l d 6.86 98.4 + 6.6 6 99. 5 + 6.6 f 125.0 + 9 . l g 81 . 8 + 9 . 5 h a. F = 2.16, df = 1, 4; P > 0.10 b. F = 7.78, df = 1, 4; P < 0.05 c F = 1.32, df = 1, 4; P > 0.10 d. F = 1.83, df = 1, 4; P>0.10 e. F = 0.03, df = l , 3; P >0.10 f. F.= 0.01, df = 1, 3; P > 0.10 g. F = 0.84, df = 1, 3; P >0.10 h. F = 3.96, df = 1, 3; P >0.10 decreased, being only 56% of that shown by the microsomes of animals fed d i e l d r i n c o n t i n u o u s l y (Figure 9); a f t e r 6.86 weeks the a c t i v i t y of t h i s pathway had returned to normal. This slowness i n r e g r e s s i n g to normal cor-r e l a t e s w i t h the slowness of the i n d u c t i o n (Figure 9). D. L i v e r Parameters and ^C-IMI Metabolism i n Male Mice To determine whether the e f f e c t s of age and d i e l d r i n are s i m i l a r i n both sexes a p r e l i m i n a r y experiment was conducted i n which males were exposed to 5 and 10 ppm d i e t a r y d i e l d r i n f o r 4 and 8 weeks p r i o r to s a c r i f i c e . The r e s u l t s , which i n d i c a t e the ex i s t e n c e of sex d i f f e r e n c e s and f u r t h e r i l l u m i -nate the r e l a t i o n s h i p between cytochrome P-450 and IMI metabolism, are i n -cluded here. 52 1. E f f e c t of Age The l i v e r parameters for control males k i l l e d at median ages of 17 and 21 weeks are presented in Table VII. The r e l a t i v e l i v e r weight of those s a c r i f i c e d at 17 weeks was 62.8 + 1-79 mg/g body weight which was s i g n i f i -c a n tly larger than the 44.4 + 0.57 mg/g body weight found for those k i l l e d at 21 weeks; the difference, which r e f l e c t s the l i v e r weight and not the body weight (27.5 +1.5 g and 27.0 + 1.00 g at the two ages) is probably due chance since i t has been my experience that l i v e r weight in males is u s u a l l y about 55 mg/g body weight and is independent of age. There were no age differences i n the concentration of microsomal protein, which was s l i g h t l y lower than that found for females, or i n the concentration of cytochrome P-450, which was s l i g h t l y higher. Table VII. The e f f e c t of age on l i v e r parameters in SWV male mice. Values for l i v e r weight (mg l i v e r / g body weight), microsomal p r o t e i n (mg protein/g l i v e r ) and cytochrome P-450 (m u moles/mg micro-somal protein) are the x + SEM of two mice. Mice were i n i t i a l l y 12-14 weeks old and were k i l l e d 4 and 8 weeks l a t e r . Week L i v e r Weight Microsomal b P r o t e i n Cytochrome P-450 C 4 62.8 + 1.79 12.2 + 1.16 . 1.13 + 0.17 8 44.4 + 0.57 13.2 + 1.00 1.08 + 0.01 a. F = b. F = c. F = 95.729, df = 1, 2; 0.446, df = 1, 2; 0.092, df = 1, 2; P <0.05 P > 0. 10 P > 0.10 Microsomes from 17 week old males metabolized IMI at the rate of 10.38 + 0.73 Jig/mg microsomal protein. A rate which was not d i f f e r e n t from 53 that of 8.97 + 0.06 j.ig/mg microsomal protein, the rate exhibited by micro-somes from the 2.1 week old males. The o v e r a l l mean rate was 9.67 + 0.51 ug/mg microsomal protein and while this rate was s i g n i f i c a n t l y lower than that shown by microsomes from the females a l l the rates for males were within the range of those for females. This indicated that the suggested sex d i f -ference requires further i n v e s t i g a t i o n . The e f f e c t of increasing age on the proportion of IMI metabolized through the three pathways is presented i n Table VIII where i t can be seen that the formation of DMI was increased, that of IMI-OH was decreased while that of IMI-N-»0 was e s s e n t i a l l y unchanged. These pathway changes are d i f f e r e n t from the decreased production of DMI and increased formation of both IMI-OH and IMI-N-»0 found in microsomes from ageing females. Since i n males the cytochrome P-450 concentration was unchanged with age, the above data show the concentration of the cytochrome was not r a t e - l i m i t i n g i n the DMI and IMI-OH pathways contained within the hepatic microsomes of d i e l d r i n -untreated males. The same conclusion was drawn for these pathways i n micro-somes from untreated females, where the data also showed that the rate of N-oxidation was not l i m i t e d by P-450; the data for males did not support this conclusion in this sex. 2. E f f e c t of D i e l d r i n The l i v e r parameters for males exposed to d i e t a r y d i e l d r i n are pre-sented in Table IX. Because of the apparently discrepant l i v e r weights in the control males any e f f e c t of d i e l d r i n was not ascertainable and the data do not warrant discussion. D i e l d r i n caused no s i g n i f i c a n t increases in the concentration of microsomal protein although the l e v e l s were c o n s i s t e n t l y 54 Table VIII. The effect, of age on the metabolism of imipramine (IMI) to desmethylimipramine (DMI) , hydroxylated imipramine (IMI-Oil) and imipramine-N-oxide (IMI-N-»0) by hepatic microsomes pre-pared from SWV male mice. Mice were i n i t i a l l y 12-14 weeks old and were k i l l e d a f t e r 4 and 8 weeks. Values are the x + SEM of two mice. % of IMI Metabolized to Week DMI3 IMI-N~O b IMI-OHc 4 . 15.0 + 0.48 45.2 + 0.94 39.8 + 1.45 8 22.2+0.73 49.0+1.52 28.8+0.69 a. F = 67.395, df = 1, 2; P<0.025 b. F = 0.341, df = 1, 2: P>0.100 c. F = 47.178, df = 1, 2; P<0.025 Table IX. L i v e r parameters i n SWV male mice fed d i e t a r y d i e l d r i n for 4 and 8 weeks. Values for l i v e r weight (mg l i v e r / g body weight), microsomal protein (mg protein/g l i v e r ) and cytochrome P-450 (m u moles/mg microsomal protein) are the x + SEM of 2-3 mice. The values i n brackets are the changes expressed as a per cent of control l e v e l s . Mice were i n i t i a l l y 12-14 weeks old. Dose (ppm) Week L i v e r Weight Microsomal Protein Cytochrome P-450 4 55.5 + 2.38 12. 8 + 1.16 (5) 1.49 + 0.12 (32) J 8 55.3 + 4.02 14.4 + 1.49 (9) 1.42 + 0.12 (32) 4 61.6 + 1.75 15.2 + 1.64 (25) 1.52 + 0.19 (41) 10 8 74.3 + 1.63* 16.0 + 3.73 (21) 1.74 + 0.48 (61) * S i g n i f i c a n t l y d i f f e r e n t from control value by Duncan's New M u l t i p l e Range Test. P<0.05 55 elevated by an average of 7%, at 5 ppm and 237, at 10 ppm; these responses are less than the 30% increase shown by females fed these doses. S i m i l a r l y , cytochrome P-450 was not s i g n i f i c a n t l y elevated in concentration by d i e l d r i n , although the l e v e l s were c o n s i s t e n t l y increased over control values; the increases of 32%, and 41%, a f t e r 4 weeks at 5 ppm and 10 ppm r e s p e c t i v e l y are less than the s i g n i f i c a n t 68%, and 98%, increases caused by these doses in the female and perhaps are a consequence of the higher baseline concentra-tions i n males. The r e s u l t s i n d i c a t e that the concentration of induced P-450 was maintained or increased over weeks 4-8 of exposure and this d i f f e r s from the decline observed in the female over t h i s period. The amount of IMI metabolized by the hepatic microsomes was not s i g n i f i c a n t l y changed by d i e l d r i n (Table X) but the standard errors are very large which, with the small sample si z e , preclude a conclusion that d i e l d r i n had no e f f e c t on the rate of metabolism. D i e l d r i n q u a n t i t a t i v e l y changed the pathways through which IMI was metabolized (Table XI). The amount of DMI formed was increased s i g n i f i -c a n t l y by both doses at both 4 and 8 weeks. The two doses did not d i f f e r s i g n i f i c a n t l y i n the magnitude of the increase they caused. In the male the increase occurred a f t e r a shorter exposure period than i t did i n the female (see Figure 9) and the magnitude was generally greater. The forma-t i o n of IMI-N-»0 was s i g n i f i c a n t l y decreased at a l l times by both doses except for that at 8 weeks exposure to 5 ppm; again, these doses appear to cause responses i n the male which occurred e a r l i e r than and were of greater magnitude than those in the female (see Figure 7). The production of IMI-OH was decreased s l i g h t l y , a l b e i t i n s i g n i f i c a n t l y , at both doses and times. This was in contrast to the s l i g h t increase these doses caused in the female (see Figure 8). 56 Table X. Imipramine (IMI) metabolized per mg of protein by hepatic micro-somes prepared from SWV male mice fed di e t a r y d i e l d r i n for 4 and 8 weeks. Mice were i n i t i a l l y 12-14 weeks old. Values are the x + SEM of 2-3 mice. Dose IMI Metabolized (ppm) Week (% of control) 4 93.3 + 25.7 5 8 87.1 + 12.0 4 80.2 + 19.8 10 8 92.8 + 7.9 Table XI. The metabolism of imipramine (IMI) to desmethylimipramine (DMI), imipramine-N-oxide (IMI-N-»0) and hydroxylated imipramine (IMI-OH) by hepatic microsomes prepared from SWV male mice fed die t a r y d i e l d r i n for 4 and 8 weeks. Mice were i n i t i a l l y 12-14 weeks o l d . Values are the x + SEM of 2-3 mice. Metabolite Y i e l d (% of control) Dose (ppm) Weeks DMI IMI-N-»0 IMI-OH 4 160.2 + 2.34*** 85.4 + 0.91** 93.3 + 2.31 5 8 139.1 + 6.35** 92.3 + 6.69 82.6 + 7.16 4 168.3+5.13*** 78.8+3.98* 98.3+9.07 10 8 149.0 + 4.78** 79.3 + 3.16* 97.5 + 9.07 * S i g n i f i c a n t l y d i f f e r e n t from control value. P<0.050 ** S i g n i f i c a n t l y d i f f e r e n t from control value. P< 0.025 ,rf~* S i g n i f i c a n t l y d i f f e r e n t from control value. P< 0.005 57 D u r i n g e x p o s u r e t o d i e l d r i n t he c o n c e n t r a t i o n o f cytochrome P-450 was n ot s i g n i f i c a n t l y i n c r e a s e d . The p r o d u c t i o n o f DMI was, w h i c h s u g g e s t s t h a t the c o n c e n t r a t i o n o f the cytochrome was not r a t e - 1 i m i t i n g i n t h e deme-t h y l a t i o n pathway. The f o r m a t i o n o f IMI-OH was not s i g n i f i c a n t l y changed s u g g e s t i n g t h a t P-450 may be r a t e - 1 i m i t i n g h e r e a l t h o u g h the q u a n t i t a t i v e r e l a t i o n s h i p between t h e two was poor and c a s t s doubt on t h i s . However, w h i l e t h e c o n c e n t r a t i o n o f P-450 i n d i e l d r i n - t r e a t e d males d i d not d i f f e r s t a t i s t i c a l l y f rom t h a t i n the c o n t r o l s t h e i n c r e a s e was o f s u f f i c i e n t mag-n i t u d e (30-607o) t o s u g g e s t i t was r e a l and t h a t a l a r g e r sample would p r o -v i d e s t a t i s t i c a l s i g n i f i c a n c e . I f t h e i n c r e a s e i n t h e c o n c e n t r a t i o n o f P-450 was r e a l i t w o u l d i n d i c a t e t h a t t h e c y t o c h r o m e might be r a t e - l i m i t i n g f o r d e m e t h y l a t i o n but n o t f o r h y d r o x y l a t i o n . F u r t h e r i n v e s t i g a t i o n i s o b v i o u s l y r e q u i r e d . I n any ev e n t , t h e o n l y f i r m c o n c l u s i o n w h i c h c o u l d be drawn was t h a t P-450 was not t h e r a t e - l i m i t i n g enzyme i n t h e N - o x i d a t i o n pathway s i n c e t h e a c t i v i t y t h e r e d e c r e a s e d s i g n i f i c a n t l y w h i l e t h e cytochrome c o n -c e n t r a t i o n was ( a t l e a s t ) c o n s t a n t . E. E f f e c t o f D i e l d r i n on R e p r o d u c t i o n i n D i p a r o u s Females • \ 1. A d u l t M o r t a l i t y The number o f fem a l e s a l i v e i n each dosage group a f t e r v a r i o u s i n t e r v a l s on t h e d i e l d r i n - c o n t a m i n a t e d r a t i o n s a r e p r e s e n t e d i n T a b l e X I I where t h e ti m e s were c h o s e n t o show t h e m o r t a l i t y w h i c h o c c u r r e d d u r i n g p a r t i c u l a r r e p r o d u c t i v e p r o c e s s e s ; f o r example, t h e d i f f e r e n c e i n t h e numbers a l i v e a f t e r 4 and 6 weeks i s the m o r t a l i t y w h i c h o c c u r r e d d u r i n g t h e p e r i o d t h e fem a l e s were caged w i t h t h e male s . C o r r e c t i o n s f o r such mor-t a l i t y have been made i n the r e p r o d u c t i o n d a t a t o e l i m i n a t e d i e l d r i n ' s a c u t e 53 Table XII. M o r t a l i t y in the groups of SWV female mice used to determine the e f f e c t of dietary d i e l d r i n on reproduction. D i e l d r i n d i e t s were fed throughout the experiment. Mice were 12-14 weeks old when f i r s t placed on the d i e l d r i n r a t i o n s . Dose (ppm) n 4 weeks 3 Number a l i v e 6 weeks^ a f t e r : 3.9 , c weeks 13 weeks^ 0 13 18 18 13 18 2.5 19 19 19 19 19 5 19 19 19 13 18 10 13 18 18 13 18 15 19 19 19 18 18 20 18 8 7 3 3 25 18 13 9 8 8 a. Females were fed experimental d i e t s for 4 weeks before being caged with males. b. Females were caged with males during weeks 5-6. c. A l l pregnant females had d e l i v e r e d by 8.9 weeks. d. A l l dams had weaned or l o s t t h e i r l i t t e r s by 13 weeks. e f f e c t which would not represent a d i r e c t e f f e c t on reproductive processes. These corrections simply consist of choosing the appropriate number of animals as the sample s i z e , for example, 8 and not 18 is the appropriate sample s i z e for the mating index (= number with vaginal plugs/number paired with males) at 20 ppm. Doses of up to 15 ppm caused no mortality since only one animal died at that dose and one other died at 5 ppm. However, 20 and 25 ppm caused heavy mortality with only 3 and 8 females out of 18, at the two doses re-59 spectively, s u r v i v i n g the 13 weeks of exposure. 2. Breeding Behaviour (Copulation) At each dose the proportion of females with copulation plugs was not d i f f e r e n t from that of the controls (Table XIII). Even females which were i n advanced stages of acute t o x i c i t y mated, as evidenced by those which were fed 20 and 25 ppm and which died 3 days a f t e r the plug was found (Table XIV). D i e l d r i n obviously did not e f f e c t behavioural estrus. Moreover, i n both control and treatment groups most of the plugs were found i n the f i r s t 8 days of the breeding period i n d i c a t i n g that d i e l d r i n did not e f f e c t the length of the estrous c y c l e . 3. F e r t i l i t y Of the females fed 0, 2.5 and 5.0 ppm d i e l d r i n a l l of those which were bred became pregnant; s i m i l a r l y , a l l of those fed 20 and 25 ppm and which did not die i n the f i r s t few days a f t e r being bred became pregnant (Table XIV). However, at 10 and 15 ppm 13% of the bred females were i n f e r -2 t i l e . A X cannot be c a l c u l a t e d for the 2 x 2 contingency table of Pregnant Not Pregnant by Dose:Control without adding a p o s i t i v e number, i n th i s case 0.01, to the zero i n the Control:Not Pregnant c e l l . When this i s done the 2 X , with Yate's Correction, for the comparison of the 10 ppm and 15 ppm doses with the controls, are 5.06 and 4.67 r e s p e c t i v e l y ; with one degree of 2 freedom these X show the decreased f e r t i l i t y , at both doses, to be s i g n i f i -cant at P <0.05. At autopsy, the u t e r i of the i n f e r t i l e animals did not contain fresh placental scars i n d i c a t i n g that the l e s i o n occurred before day 10 of g e s t a t i when formation of the a l l a n t o i c placenta is complete. The weight and gen-60 Table XIII. The number of SWV with males during d i e l d r in. mice found with vaginal plugs the 5th-6th week of exposure when caged to dietary Dose (ppm) a n Females With Plugs No. % X 2 b P 0 18 14 77.8 2.5 19 16 84.3 0.249 >0.10 5 19 11 57.9 1.663 >0.10 10 13 11 60.9 1.176 >0.10 15 19 16 84.3 0.249 >0.10 20 7 5 71.5 0.114 >0.10 25 12 9 75.0 0.031 >0.10 a. Excludes unmated females dying before 7 weeks of exposure. b. Calculated, using Yate's Correction, for the 2 x 2 contingency tables of Dose vs Control for Females With and Females Without Plugs. Table XIV. The number of SWV mice fed dietary d i e l d r plugs but did not become pregnant. in which had vaginal Dose (ppm) Number of Females Bred Not Pregnant 7 to Not Pregnant 0 14 0 0 2.5 16 0 0 5 11 0 0 10 11 2 13.2 15 16 3 13.7 20 5 l a 20.0 a 25 9 2 a 22.0 a a. These females died on day 2 of pregnancy so that t h e i r reproductive status is a c t u a l l y unknown. They are incLuded here for convenience. 61 e r a l appearance of the uterus, l i v e r and ovaries of the i n f e r t i l e animals were not d i f f e r e n t from those of the controls. The lack of i n f e r t i l e animals at 20 and 25 ppm was possibly due to the mortality these doses caused, i n conjunction with a varying threshold dose for the d i f f e r e n t toxic e f f e c t s . The higher doses might cause some animals to die, ei t h e r before or immediately a f t e r breeding, which, i f fed lower doses, would survive but be i n f e r t i l e . 4. Gestation and P a r t u r i t i o n The e f f e c t of d i e l d r i n on the- number of pregnant females which ges-tated s u c c e s s f u l l y and delivered pups is presented i n Table XV. One female fed 2.5 ppm resorbed i t s fetus' a f t e r gestation day 15 and one at each of doses 5, 15, 20 and 25 ppm died between days 14 and 18. The u t e r i of a l l the l a t t e r females contained f u l l complements of f e t u s 1 of normal gross morphology, and no resorbtion s i t e s . The length of the gestation period, 19 days, was unaffected by d i e l d r i n since i t was the same in a l l groups. These data show that once a female had become pregnant d i e l d r i n caused no- gross pathological disturbances i n gesta t i o n a l physiology except, of course, i n the 7% which died. The i n i t i a t i o n and duration of the p a r t u r i t i o n process i n the d i e l d r i n -fed females was comparable with that of normal control animals. In the SWV s t r a i n a diparous female w i l l d e l i v e r an average l i t t e r of 13 pups in approximately 30 minutes (range: 15 to 90 minutes). The d i e l d r i n - t r e a t e d females delivered pups with the same ease as the control females; none suffered from pups becoming lodged i n the vagina or from s i m i l a r problems. As w i l l be discussed below two of the treated females, both fed 25 ppm, 62 Table XV. The e f f e c t of dietary d i e l d r i n on the number of pregnant SWV mice d e l i v e r i n g pups at term and on the number of days of gestation. Dose Number of Females (ppm) Pregnant Delivered Gestation Period (days) C 0 ' 14 14 19.0 + 0.00 2.5 16 15 a 19.1 + 0.35 5 11 i o b 19.3 + 0.48 10 9 9 19.3 + 0.50 15 13 12 b 19.1 + 0.51 20 4 3 b 19.0 + 0.00 25 7 6 b 18.8 + 0.45 a. 1 female resorbed the fetus' a f t e r day 15 of pregnancy b. 1 female died between days 14 and 18 of pregnancy. c. 1-way anova: o v e r a l l F = 0.597, df = 6, 59; P>0 .10 exhibited gross disturbances i n behaviour towards the newly deli v e r e d neonate but even i n these dams the p a r t u r i t i o n processes per se were apparently normal. 5. L i t t e r Size and Sex Ratio at B i r t h The l i t t e r s i z e at b i r t h , presented i n Table XVI, tended to decrease s l i g h t l y , by a maximum of approximately 17%, with increasing doses of d i e l -d r i n ; however only amongst the females fed 25 ppm, where the decrease was largest, were the l i t t e r s s i g n i f i c a n t l y smaller than those of the c o n t r o l s . In conjunction with the decline in l i t t e r s i z e there was a general trend towards a reduction in the proportion of males born in the l i t t e r s , and 63 Table XVI. The l i t t e r s i z e , number of .placental scars and estimated i n t r a -uterine f e t a l mortality i n SWV mice fed dietary d i e l d r i n . ( ) sample s i z e . Dose L i t t e r Size Placental Scars % F e t a l (ppm) (X + SEM) (x + SEM) M o r t a l i t y 0 13.2 + 1.06 (13) 16.2 + 3.4 (9) 13.6 2.5 12.1 + 2.77 (15) 14.7 + 3.4 (7) 17.7 5 12.7 + 1.64 (10) 13.3 + 1.6 (7) 4.5 10 12.5 + 2.00 (8) 13.3 + 2.2 (9) 6.0 15 12.3 + 1.74 (11) 13.3 + 2.7 ( I D 7.6 20 11.7 + 1.15 (3) 12.0 1.7 (3) 2.5 25 11.0 + 0.63* (6) 11.7 + 2.1 (6) 5.6 a. 1-way anova: o v e r a l l F = 1.08, df = 6, 59; P>0.05 * S i g n i f i c a n t l y d i f f e r e n t from control value by Duncan's New M u l t i p l e Range Test (P < 0.05) b. Calculated as : (placental scars - l i t t e r size) * 100% placental scars amongst those females fed 25 ppm d i e l d r i n t h i s reduction was s i g n i f i c a n t (Table XVII). This shov/ed that at least one of the proximate cause(s) for the decreased l i t t e r s i z e was more detrimental to c e l l s bearing the Y chromosome. . The number of placental scars found at autopsy were used to e s t i -mate the i n t r a - u t e r i n e f e t a l mortality. This, as shown in Table XVI, was 3-fold greater i n the control animals than i n the d i e l d r i n - t r e a t e d animals which shows that such mortality was not the cause of the reduced l i t t e r s i z e s . Moreover, i t shows that the l e s i o n must occur before the completion of the a l l a n t o i c placenta (day 10). 64 Table XVII. The sexes of the pups born to SWV mice fed dietary d i e l d r i n Dose Number of: Sex Ratio of Pups (ppm) L i t t e r s Pups (males/females) 0 13 188 1.29 2.5 15 182 1.07 5.0 10 122 1.18 10.0 8 100 1.22 15.0 8 95 1.02 20.0 3 35 0.75 25.0 3 33 0.65 a 2 a. Xy = 3.98, df = 1; P<0.05 The pups born at a l l doses were morphologically normal. 6. Post-partum S u r v i v a l and Growth of Pups The mean age at death of the pups born to females which ra i s e d none to weaning i s presented i n Table XVIII where for ease of presentation, the data for the pups of a l l females wi t h i n each dosage group have been pooled. This procedure is s t a t i s t i c a l l y i n v a l i d for reasons which w i l l soon become apparent and which also preclude s t a t i s t i c a l comparisons between the pups of d i f f e r e n t doses. These data, however, do serve to demonstrate that at a l l doses the age at death was the same for male or female pups. Therefore, the s u r v i v a l data for the two sexes may be v a l i d l y pooled within each l i t t e r . This pooled data may not be further pooled between the l i t t e r s though, since as shown in Table XIX the p r o b a b i l i t y of homogenous variances 65 Table XVIII. The age at which the pups died i n the l i t t e r s of SWV mice fed d i e l d r i n where none of the pups survived to weaning. Days old at death (x + S.D.) Dose — — (ppm) Males Females Both 0.0 2.9 + 2.0 3.1 + 2.4 3.0 + 2.2 2.5 2.5 + 1.6 2.6 + 1.6 2.5 + 1.5 5.0 3.1 + 2.7 3.4 + 2.2 3.3 + 2.5 10.0 1.6 + 0.9 1.6 + 0.9 1.6 + 0.9 15.0 1.1 + 0.7 1.0 + 0.6 1.0 + 0.7 20.0 0.8 + 0.5 1.2 + 1.2 1.0 + 1.0 25.0 0.8 + 0.4 0.7 + 0.4 0.7 + 0.4 Table XIX. The variances ( 6 ) in the age at death of pups born to dams fed 5 and 10 ppm dietary d i e l d r i n and r a i s i n g no pups to weaning (28 days of age). Dose (ppm) Female No. 1 2 3 4 5 6 7 3 5.0 l i t t e r s ize 62a 15 0.34 13 0.86 14 1.41 12 0.27 11 1.11 12 1.78 15 12 18.4 0.75 Female No. 1 2 3 4 5 6 10.0 l i t t e r s ize 14 9 12 14 10 13 62 b 0.58 0.06 0.10 0.77 0.49 0.66 a. B a r t l e t t ' s b. B a r t l e t t ' s X2 = X 2 = 60.66, 21.28, df = 7; df.= 5; P< 0.005 P< 0.005 66 (<5") in the death rate of pups between the l i t t e r s of females fed 5 or 10 ppm d i e l d r i n is less than 0.005. In this instance heterogeneity of variance may be interpreted i n two ways: 1) pups in d i f f e r e n t l i t t e r s respond to the same dose of d i e l d r i n i n a logarithmic manner, the actual magnitude of which i s determined by the female; or 2) pups in d i f f e r e n t l i t t e r s die from d i e l -d r i n v i a d i f f e r e n t proximate causes — the evidence that d i e l d r i n causes pup mortality w i l l be presented s h o r t l y . In any event, the heterogeneity of variances means that the dam (= l i t t e r ) , and not the pup, is the v a l i d sample unit for analyzing d i e l d r i n ' s e f f e c t on pup s u r v i v a l . The proportion of dams whose l i t t e r s contained at l e a s t one l i v i n g pup over the e n t i r e pre-weaning period i s presented i n Figure 10. Of the con t r o l females, 69% had l i t t e r s which were u l t i m a t e l y weaned, while among those fed 2.5 ppm d i e l d r i n t h i s was reduced to 537c The pre-weaning loss of the l i t t e r was further and s i g n i f i c a n t l y reduced at 5 ppm, where only 20% of the dams weaned pups, and was complete at doses of 10 ppm and higher where no dam weaned pups. The rate of l i t t e r loss was dose-dependent since with increasing dose both the slope of the curves and the proportion of l i t t e r s l o s t on day 1 increased. The loss of the l i t t e r on day 1 appeared to r e s u l t from pups dying wi t h i n the f i r s t 24 hours post-partum, and not before or during p a r t u r i t i o n , since none of the females which were observed during b i r t h d e l i v e r e d s t i l l - b o r n pups. A l l diel d r i n - i n d u c e d pup mortality was complete by the s i x t h day post-partum. A l l of the pups born to treated females were of normal, gross morphology but a f t e r the f i r s t few hours of l i f e many became a darker red colour than normal. Moreover, v i r t u a l l y a l l of these pups were l i s t l e s s , i n a c t i v e and generally appeared to be weaker than those born to the control females. c P f-ti ^ fO Hn i-i fD CO no o fa fl> r t 3 H a si o n> C O N ft) (D K. CL CL CD r t ft) O K. c CO CL l-h H * fl) fl> CL l-i 3 o 3 r t 3* r t 3" I- 1 fl> H -< XI H - H 3 O 00 XI X3 C xi CO o i-S r t o 3 O l-h % OF DAMS WITH LIVE PUPS o H X to TP O CO cn O ro -I cm cn H cn I CD o _ 1 _ cn _ J _ O O X 4 . ^ ^ ^ 1 .. x / i z ® m o J ro ro cn p cn o cn b b b b b T> " O T I ' O T J T3 T3 "D "D T3 3 3 3 3 3 I • > DO x i i ro p cn b TJ T3 3 3 Si O I I l i I i £°iro<£o" cn w L9 63 The proportion of the l i t t e r a c t u a l l y weaned by those dams whose l i t t e r survived is presented i n Table XX. Male and female pups did not d i f f e r i n the i r s u r v i v a l at any dose so that the data for the sexes were pooled. The o v e r a l l s u r v i v a l , 75%, 93%, and 73%, for pups born to dams fed 0, 2.5 and 5.0 ppm d i e l d r i n r e s p e c t i v e l y , was not d i f f e r e n t between any of these groups. This shows that the dieldrin-induced pup mortality is an a l l or none phenomenon: exposed dams eit h e r weaned no pups or weaned the same number as the con t r o l dams. The pre-weaning growth of the weaned pups did not d i f f e r between the dams fed 0, 2.5 or 5.0 ppm d i e l d r i n ; this is evident from Figure 11 where the weight of sur v i v i n g pups is presented for each day from b i r t h to weaning. These r e s u l t s show that the metabolic processes of pups ra i s e d by dams fed d i e l d r i n before and during gestation, as well as during l a c t a t i o n , were not grossly pathologic. Moreover, since pups begin to eat s o l i d feed at about 14 days of age, they show that the pup withstood some d i r e c t ex-posure during the p e r i n a t a l period. However, pups ra i s e d by dams fed d i e l d r i n did have a l t e r e d hepatic function as evidenced by the increased latency to the onset of pentobarbital anesthesia (Table XXI) and i t s decreased duration (Table XXII). The mean latency to onset was 2.44 minutes for the pups of four c o n t r o l females and th i s was s i g n i f i c a n t l y less than the mean of 2.72 minutes for those of s i x dams fed 2.5 ppm d i e l d r i n ; w i t h i n e i t h e r of these dosage groups the latency did not d i f f e r between the pups of d i f f e r e n t dams. The late n c i e s i n onset for the pups from the two dams fed 5 ppm had heterogeneous variances even when the data was transformed into logarithms and therefore they have not been pooled or included i n the s t a t i s t i c a l a n a l y s i s . The mean latencies i n 69 Table XX. The s u r v i v a l rate o i pups i n l i t t e r s raised to weaning by dams fed dietary d i e l d r i n . 7„ S u r v i v a l (x + SD) a Dose n (ppm) ( L i t t e r s ) Males Females Pooled c 0 9 82.1 + 9.34 76.4 + 11.7 75.3 + 4.47 2.5 7 93.9 + 19.90 96.9 + 5.1 93.1 + 8.45 5.0 2 53.5 + 1.66 100 + 0.0 73.2 + 1.00 a. A l l c a l c u l a t i o n s were performed on transformed data (arc-sin) which have been de-trans formed for presentation here. b. 1-way anova for males vs females: 0.0 ppm: F = 0.219, df = 1, 16; P>0.10 2.5 ppm: F = 0.134, df = 1, 12; P>0.10 5.0 ppm: F = 67.42, df = 1, 2; P> 0.10 c. 1-way anova for dose e f f e c t : o v e r a l l F = 2.628, df = 2, 15; P>0.10 both l i t t e r s were, however, within the range of those exhibited by the pups at 2.5 ppm. The mean duration of the anesthesia d i f f e r e d s i g n i f i c a n t l y between the pups of the four i n d i v i d u a l c o n t r o l females and also between those of the two dams fed 5 ppm d i e l d r i n so that the data for a l l the pups could not be pooled, within these doses, for an analysis of variance. However i n d i v i d u a l F-tests between the pups of each c o n t r o l female and those of each female fed d i e l d r i n showed that, i n each case, the l a t t e r had s i g n i f i c a n t l y shortened pentobarbital sleeping times. D i e l d r i n at 2.5 and 5.0 ppm decreased the sleeping time by a mean of 356 and 4057o respec-t i v e l y . 70 1 3 -1 2 -CO +1 IX e 0 . 0 p p m - 9 L I T T E R S • 2 . 5 p p m - 8 L I T T E R S X 5 . 0 p p m - 2 L I T T E R S I T X T» I • 1 CO UJ cr U l I-CD U l O < cr ui > < 8 61 U . O 5 21 T • i 1 i T 5 H i 5 5 i ti 9 1 1 1 1 ' i i — i — i — r - T — i — i — i — i — i — i — i — i — i — i — i — i — i — i — | — | — | — | — , 0 f 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6 2 8 3 0 BIRTH DAYS POST-PARTUM WEANED Figure 11. Average body weight (B.W.) of s u r v i v i n g littermates ( l i t t e r weight/no. of pups) i n l i t t e r s r aised to weaning by dams fed 0, 2.5 and 5 ppm dietary d i e l d r i n . 71 Table XXI. Latency to the onset of the r i g h t i n g r e f l e x loss i n weanling mice raised by dams fed dietary d i e l d r i n . Weanlings were i n -jected (ip) with 60 mg/kg of sodium pentobarbital on day 29 af t e r b i r t h . S t a t i s t i c a l analysis was performed on log-trans-formed data which have, been de-trans formed for presentation, n = number of weanlings. CL = confidence l i m i t s . Latency to onset (minutes) Dose (ppm) 0.0 a 2.5 b c 5.0 Dam No. n X 95% CL n X 95% CL n x 95% CL 1 4 2.39 1,73, 3.29 9 3.01 2.38, 3.80 9 2.65, 2.46, 2.84 2 12 2.31 2.11, 2.54 9 2.26 1.92, 2.66 12 2.38 1.99, 2.85 3 11 2.52 2.14, 2.96 7 2.73 2.43, 3.07 4 9 2.55 2.28, 2.84 10 2.92 2.37, 3.59 5 11 2.46 2.25, 2.69 6 13 2.97 2.52, 3.52 pooled^ 36 2.44 2.29, 2.60 59 2.72 2.54, 2.91 a. 1-way anova (Dams). F = 0.60, df = 3, 33; P>0.25 b. 1-way anova (Dams). F = 1.70, df = 5, 54; P>0.10 c. Heterogeneity of 6 on transformed data. F max. = 9.45, df = 8; P<0.01 d. 1-way anova (Doses) F = 5.42, df = 1, 95; P<0.05 72 Table XXII. Duration of the r i g h t i n g r e f l e x loss in weanling mice raised by dams fed dietary d i e l d r i n . Weanlings were injected (ip) with 60 mg/kg sodium pentobarbital on day 29 a f t e r b i r t h . S t a t i s t i c a l analysis was performed on log-trans formed data which have been de-trans formed for presentation. n = number of weanlings. CL = confidence l i m i t s . Reflex Loss (minutes) Dose (ppm) 0.0* 2.5° 5.0 C n x 95% CL n x 95% CL n x 95% CL Dam No. 1 4 219.1 175.1, 274.1 9 42.2 35.9, 49.6 9 62.5 47.7, 81.9 2 12 150.2 132.3, 170.6 9 68.2 50.3, 92.5 12 40.1 29.6, 54.4 3 11 229.9 170.4, 310.2 7 53.1 42.3, 66.6 4 9 179.2 155.8, 206.1 10 46.1 45.0, 47.3 5 11 54.2 46.7, 62.9 6 13 53.5 36.7, 77.9 pooled 36 186.4 166,5, 208.9 59 52.4 50.0, 58.4 21 46.0 36.8, 57.5 a. F = 5.11, df = 3, 32; P<0.01. The pups of each dam d i f f e r (P<0.05) from those of a l l other dams by Duncan's New M u l t i p l e Range Test. b. F = 1.35, df = 5, 53; P> 0.25 c. F = 8.42, df = 1, 19; P < 0.01 The r e l a t i v e l i v e r weights of the females which s u c c e s s f u l l y raised pups to weaning are compared, i n Figure 12, to those of the unsuccessful dams and those of the non-pregnant females. As described i n Methods the animals i n the three groups d i f f e r e d in age when they were k i l l e d but since the l i v e r weight does not change with age they can s t i l l be compared ~ 165 LU </) + 1 £ 145 h-X e> UJ * SIGNIFICANTLY DIFFERENT FROM UNSUCCESSFUL, P <0.05 m NON-PREGNANT A SUCCESSFUL • UNSUCCESSFUL T 1 .5--" 125-i >-Q O CO cn \ cr LLI > / I 105 85H -I' .A* T*/ Cn E 65-2.5 15 5 10 DIETARY DIELDRIN (ppm) — r — 20 •a 1 25 Figure 12. The l i v e r weight of dams fed d i e l d r i n and su c c e s s f u l l y r a i s i n g pups to weaning compared with those of unsuccessful dams and non-pregnant females. 74 d i r e c t l y on the basis of t h e i r p h y s i o l o g i c a l d i f f e r e n c e s . A l l of the i n -creases in r e l a t i v e l i v e r weight were due to increased l i v e r weight and not to decreased body weight. The l i v e r weighed more i n a l l of these animals than in those used i n the induction study (see Figure 1) and probably re-f l e c t s the congestion caused by k i l l i n g the former females with ether. At 0 ppm the successful and unsuccessful dams possessed l i v e r s which were not d i f f e r e n t i n weight, but in both groups they were s i g n i f i c a n t l y larger, by 247°, than those of the non-pregnant females. This shows that i n the co n t r o l females the l i v e r mass was increased during gestation and that the increase was maintained throughout l a c t a t i o n . The l i v e r s of both non-pregnant and unsuccessful females increased in weight with increasing doses of d i e l d r i n and since the l i v e r s of the l a t t e r females were proportionately larger, by the same magnitude, at a l l doses, these r e s u l t s show that the hepatomegalic e f f e c t s of d i e l d r i n and reproductive processes were a d d i t i v e rather than s y n e r g i s t i c . The l i v e r s of successful dams fed 2.5 or 5 ppm d i e l d r i n did not d i f f e r i n weight from those of the successful dams fed 0 ppm d i e l d r i n ; they d i d weigh s i g n i f i c a n t l y l e s s than those of unsuccessful dams at these two doses. These data suggest that the die l d r i n - i n d u c e d hepatomegaly may be c a u s a l l y r e l a t e d to the pup mortality and indicate that only c e r t a i n increases i n l i v e r mass are compatible with successful reproduc-t i o n . Since l i t t e r loss is an a l l or none phenomenon the data further suggest, t e l e o l o g i c a l l y , that the only females which w i l l reproduce success-f u l l y w i l l be those able to contain the hepatomegaly, from a l l causes, to a maximum of about 25%. The weight of the dam's body, uterus or ovaries did not c o r r e l a t e with the pup m o r t a l i t y . I n f a n t i c i d e , which did not occur at doses of 5 ppm and lower, was found to be an important proximate mortality factor at doses of 15 ppm and higher (Table XXIII). Females observed while committing i n f a n t i c i d e ex-h i b i t e d c h a r a c t e r i s t i c behaviour patterns immediately a f t e r b i r t h which ultim a t e l y resulted in the pups dying from damage i n f l i c t e d to the o c c i p i t a l region of the s k u l l and c e n t r a l nervous system. At the termination of par-t u r i t i o n and placentophagia these females became very hyperactive and per-formed prolonged bouts of hyperkinesia; i n very rapid and jerky movements they would run about the cage, often ploughing the bedding material with the forelimbs and/or snout and stopping frequently to d i g i n i t with the fore-paws. During these bouts, pups were frequently picked up by the nape of the neck and c a r r i e d for a few seconds before being dropped into the bedding; consequently, the pups were scattered randomly about the cage. The dam, a f t e r p i c k i n g up a pup, would u s u a l l y stand for several seconds v i o l e n t l y moving i t s head up and down through an arc of greater than 90°. This, and/or the manner by which i t was held, u l t i m a t e l y r e s u l t e d i n the pup's sk i n being punctured by the dam's i n c i s o r s . Once t h i s occurred the female would p e r i o d i c a l l y l i c k the wound which, over time, exacerbated i t and u l t i m a t e l y the dam would begin eating the o c c i p i t a l region and continue into the cen-t r a l nervous system. The v i s c e r a of pups seen to be k i l l e d i n t h i s manner were never eaten — a f t e r death, the pups were t o t a l l y ignored — and since i t i s usual for females to consume the v i s c e r a of dead pups,those dams which were not a c t u a l l y seen to k i l l t h e i r pups but which had dead pups i n which only the occiput was eaten were also considered to have k i l l e d t h e i r o f f -spring. Bouts of h y p e r a c t i v i t y were interspersed with others where the dam would l i e prostrate and motionless, panting heavily, for several minutes; 7 6 Table XXIII. The e f f e c t of dietary d i e l d r i n on the number of dams which k i l l e d t h e i r pups. Number of Dams Dose (ppm) Losing Pups K i l l i n g Pups % K i l l i n g Pups 0 4 0 0 2.5 7 0 0 5.0 8 0 0 10.0 8 1 12.5 15.0 12 5 41.5 20.0 3 1 33.3 25.0 6 4 66.7 during t h i s i n a c t i v i t y the eyes were always kept open. The average weight of the pups at b i r t h is presented i n Table XXIV. In the successful l i t t e r s born to dams which were fed ei t h e r 0, 2.5 or 5 ppm d i e l d r i n and which s u c c e s s f u l l y r a i s e d pups to weaning this weight did not d i f f e r . However, at a l l these doses the weight of pups i n unsuccessful l i t t e r s was less than that i n the successful l i t t e r s , although the dif f e r e n c e was s i g n i f i c a n t only at 2.5 ppm d i e l d r i n . The pup weight i n a l l the un-successful l i t t e r s born to females fed d i e l d r i n (note that t h i s includes a l l the l i t t e r s at doses of 10 ppm and higher) was less, by 2.6% to 13.07<>, than that of the pups i n the successful l i t t e r s of females fed no d i e l d r i n ; the d i f f e r e n c e however, was s i g n i f i c a n t only at 25.0 ppm. These r e s u l t s indicate that the smaller pups do not survive and suggest that d i e l d r i n Table XXIV. E f f e c t of di e t a r y d i e l d r i n on the average b i r t h weight ( l i t t e r , w e i g h t / l i t t e r size) of the pups. ( ) no. of l i t t e r s . Average b i r t h weight (x + SEM) (g) Dose (ppm) of_ 2 . 5 b 5 . 0 ° 10.0 15.0 20.0 25.0 Dams' Pups: were weaned 1.15 + 0.031 1.16 + 0.019 1.15 + 0.00 (9) (8) (2) d died 1.08+0.015 1.11+0.033 1.10+0.011 1.12+0.012 1.10+0.032 1.07+0.084 1.00+0.051* W (6) (9) (7) (11) (3) (4) a. Weaned vs. Died F = 2. 252, df = 1, 11; P>0.10 b. Weaned vs. Died F = 5.319, df = 1, 12; P<0.05 c. Weaned vs. Died F = 2.933, df = 1, 9; P>0.10 d. 1-way anova of Weaned (0 ppm) vs Died (treatment). Overall F = 1.553, df = 6, 42; P>0.10 * S i g n i f i c a n t l y less than Weaned (0 ppm) by Dunnett's d'. P<0.05. 78 increases the. number of females which d e l i v e r small pups. The average weight of the s u r v i v i n g pups in the l i t t e r s born to un-successful, d i e l d r i n - f e d females is presented, for each day between b i r t h and death, in Figure 13 where i t is contrasted with the pup weight in the l i t t e r s of successful dams fed the control r a t i o n . The data show that pups i n the unsuccessful l i t t e r s had t h e i r growth reduced or abolished from b i r t h to death. Table XXV presents the estimation of the amount of milk contained by the pups of unsuccessful, d i e l d r i n - f e d dams. The milk content of the l i t t e r was defined as: 1) Normal i f 507„ or more of the pups had "normal" m i l k l i n e s ( i . e . approximately 4 mm wide and 7 mm long). 2) Reduced i f 1007„ of the pups had less than "normal" m i l k l i n e s ; l i t t e r s so-defined usually had only 1 pup with milk. 3) Absent i f 1007o of the pups had no milk. These d e f i n i t i o n s are such that the p r o b a b i l i t y of committing a Type I error must be minimal even though the index is crude. The r e s u l t s suggest that the milk content of the pups was reduced. General observations i n d i -cated that the pups of treated dams were very l i s t l e s s and i n a c t i v e , a condition which could either r e s u l t from or cause a reduction i n milk content. The time spent in the nest by the successful, c o n t r o l females has been cumulated for each of the f i r s t 4 days post-partum and i s presented in Table XXVI. The subtraction of two standard deviations from the means pro-vided an estimate of the lower l i m i t of nesting time associated with the successful r a i s i n g of pups; since 957« of the successful dams would spend at I 7 n I-3J •2H i-H i - c H 0.0 ppm • 2.5 ppm B — 5.0 ppm x - - - - - — 10.0 ppm o — ~ 15.0 ppm • 20.0 ppm A 25.0 ppm A A X 1 -rr 2 • MRTH 4 DAYS POST-PARTUM Figure 13. Average body weight (B.W.) of s u r v i v i n g l i t t e r m a t e s ( l i t t e r weight/no..of pups) i n the l i t t e r s of dams fed d i e t a r y d i e l d r i n and whose e n t i r e l i t t e r died before weaning. The BW of pups r a i s e d to weaning by dams fed 0.0 ppm d i e l d r i n i s presented as c o n t r o l s . 80 Table XXV. The quantity of milk i n the pups of dams fed dietary d i e l d r i n and which raised no pups to weaning (exclusive of dams which k i l l e d t h e i r pups). N = normal m i l k l i n e s , R = reduced milk-l i n e s , A = mil k l i n e s absent. Where no symbol is given a l l the pups were dead. See text for f u l l d e f i n i t i o n s . Dose Female Pup M i l k l i n e s on Post-partum Day: (ppm) Number 1 2 3 4 1 R R R R 2 R R R R 5 3 R R R 4 N N N N 5 N R R 1 R R A 2 N A 10 3 R R 4 N N A 1. A 2 A A 15 3 A A 4 R A A 5 R R A 20 1 A 2 R R A 31 Table XXVI. Cumulative observed time spent in the nest during post-partum days 1-4 ( p a r t u r i t i o n = day 1) by Hive dams fed no d i e l d r i n and which s u c c e s s f u l l y raised pups to weaning. Each dam was observed for 3 minutes twice a day. The estimated lower l i m i t for time spent i n the nest for successful dams is presented. Day Cumulat ive Time (minutes) Observed Nesting (x + SEM) a Lower Limit 1 6 5.96 + 0.04 5.78 2 12 11.36 + 0.59 8.71 3 18 16.16 + 1.19 • 10.85 4 24 20.50 + 1.14 15.40 a. Calculated as : x - 2 standard deviations. l e a s t t h i s much time i n the nest, i t follows that a female nesting for less time than this would s u c c e s s f u l l y r a i s e pups with a p r o b a b i l i t y of only 0.05. The cumulative time spent i n the nest by those unsuccessful, d i e l d r i n -fed females which did not commit i n f a n t i c i d e , i s presented i n Table XXVII. Some of these dams were in the nest for less than the time associated with the successful r a i s i n g of pups which suggests that dieldrin-induced maternal neglect was a proximate cause of pup mortal i t y . Unfortunately, since these data were c o l l e c t e d only for the second r e p l i c a t e of the experiment the sample i s small and precludes dose-response a n a l y s i s . However, although neglect occurred at a l l doses the r e s u l t s suggest i t was p a r t i c u l a r l y prevalent among those dams fed 20 and 25 ppm. The s e v e r i t y of the neglect was also greatest at 25 ppm where the two n e g l e c t f u l dams exhibited a post-82 Table XXVII. Cumulative observed time spent in the nest during the post-partum period by dams fed dietary d i e l d r i n and which raised no pups to weaning (exclusive of those k i l l i n g t h e i r l i t t e r ) . Dams were observed for two 3 minute periods each day u n t i l a l l t h e i r pups were dead. Dose Female (ppm) Number Days Observed Time (min) Dose (ppm) Female Number Days Observed Time (min) 1 4 15.1* 1 1 3.0* 2 3 15.0 2 2 8.8 5 3 3 15.0 15 3 2 11. 8 4 a 4 21.5 4 2 9.4 5 3 18.0 5 3 13.3 1 3 9.0* 20 1 1 6.0 2 2 9.0 2 3 9.0* 10 3 2 12.0 25 1 1 0.0* 4 3 10.3* 2 1 0.0* * Nesting time i s below exhibited by co n t r o l a. This dam ra i s e d pups the estimated lower l i m i t of dams which s u c c e s s f u l l y weaned to day 13. nesting time pups (Table XXVI). partum hyperkinesia s i m i l a r to that described above and t o t a l l y ignored the pups, which were scattered about the cage. They f a i l e d even to remove the extra-embryonic membranes or the placentae; such gross neglect was observed only at this dose. By combining the data of Tables XXIII and XXVII i t can be seen that dieldrin-induced changes i n the dams' behaviour were the proximate causes 8 3 of a l l the pup mortality at 25 ppm. General observations made during the c o l l e c t i o n of these data suggested that d i e l d r i n also disrupted maternal behaviour i n the dams which occupied the nest for the amount of time associated with the r a i s i n g of pups. These females were generally awake, hyperactive and spent l i t t l e time in postures permittent of nursing by the pups; in contrast, the con-t r o l females spent most of t h e i r time sleeping i n a crouched p o s i t i o n which allowed the pups to suckle. The treated dams appeared to spend more time i n grooming themselves, handling the pups and digging the bedding than did the c o n t r o l s . Such behaviour, which would prevent adequate nursing, could, of course, r e s u l t i n the pups r e c e i v i n g less milk than that required for ade-quate growth and s u r v i v a l . 7. Summary of D i e l d r i n ' s E f f e c t s on Reproduction The following r e s u l t s were obtained using females which had gestated, rai s e d t h e i r f i r s t l i t t e r and were then fed d i e t a r y d i e l d r i n at l e v e l s up to 25 ppm before and during the second reproduction c y c l e . a. S i g n i f i c a n t adult m o r t a l i t y was caused by d i e l d r i n only at 20 and 25 ppm. b. D i e l d r i n did not e f f e c t the proportion of females which were bred. c. A small, but s i g n i f i c a n t , proportion of bred females fed 10 and 15 ppm d i e l d r i n did not become pregnant. The l e s i o n occurred before day 10 of gestation. A l l the bred females which survived at the higher doses were f e r t i l e as were a l l those at the lower doses. 34 d. D i e l d r i n did not cause re s o r p t i o n of the fetuses. Pregnant females e i t h e r gestated to p a r t u r i t i o n or, i n a few cases, died with a f u l l complement of apparently normal pups in utero. e. The length of the gestation period was unaffected by d i e l d r i n . f. D i e l d r i n reduced, s l i g h t l y and dose-dependently, the size of the l i t t e r at b i r t h . The maximum reduction, 17%, which occurred at 25 ppm was the only one s t a t i s t i c a l l y s i g n i f i c a n t . g. The proportion of male pups i n the l i t t e r s d elivered by exposed females was decreased, s l i g h t l y and dose-dependently, by d i e l -d r i n . The maximum reduction occurred at 25 ppm and was the only decrease s t a t i s t i c a l l y s i g n i f i c a n t . h. D i e l d r i n caused pre-weaning m o r t a l i t y of the pups. Pup loss, w i t h i n l i t t e r s , was " a l l or none": dams e i t h e r l o s t a l l t h e i r pups or weaned a normal number. A l l dams fed concentrations of 10 ppm or higher l o s t t h e i r pups; 5 ppm resulted i n a s i g n i f i -cant increase i n the proportion l o s i n g pups and while 2.5 ppm also increased t h i s proportion the increase at t h i s dose was not s i g n i f i c a n t . i . Dams fed 2.5 or 5.0 ppm d i e l d r i n and whose young survived to weaning had l i v e r s which weighed the same as those of the con-t r o l dams which s u c c e s s f u l l y raised pups; i n add i t i o n , the successful dams fed 2.5 or 5.0 ppm d i e l d r i n had l i v e r s which weighed s i g n i f i c a n t l y less than those of the unsuccessful dams fed these same doses. These successful dams raised the same o v e r a l l pi-oportion of t h e i r l i t t e r , and the same proportion of male and female pups, as the con t r o l females. The pre-weaning 85 growth of these pups was not d i f f e r e n t from that of the con t r o l pups. At weaning the latency to the onset of pentobarbital anesthesia was s i g n i f i c a n t l y increased in these pups and i t s duration was s i g n i f i c a n t l y reduced showing that the hepatic microsomal mixed-function oxidase system was induced. 8. Summary of Dieldrin-induced Pup M o r t a l i t y a. This mortality occurred at the same rate i n male and female pups. b. It was completed wi t h i n 5 days of b i r t h when the dams were fed 2.5 ppm d i e l d r i n and i t increased i n rate with increasing dose u n t i l at 25 ppm no pups survived longer than 2 days a f t e r b i r t h and most died w i t h i n 1 day of b i r t h . c. It was r e l a t e d to the increased l i v e r weight of the dam: i n -creases of greater than 247<> were incompatible with pup s u r v i v a l . Such increases occurred i n a l l exposed females except some fed 2.5 ppm and some fed 5 ppm which r a i s e d pups (see i . above). d. A l l d i e l d r i n - f e d females that l o s t t h e i r l i t t e r s d elivered pups which weighed 370 - 137= less at b i r t h than those of females which weaned t h e i r pups. e. The post-partum behaviour of some d i e l d r i n - f e d dams resulted i n them k i l l i n g t h e i r pups. I n f a n t i c i d e was p a r t i c u l a r l y prevalent among females fed doses of 15 ppm and higher. f. In those l i t t e r s where the pups were not k i l l e d by t h e i r d i e l d r i n -fed dam but died of other proximate causes, the young did not grow before death. Within a few hours of b i r t h these pups f r e -quently became dark red i n colour, i n a c t i v e and l i s t l e s s ; they usually contained l i t t l e or no milk. 86 g. Some d i e l d r i n - f e d dams whose pups died, l o s t them from neglect. These females occupied the nest for less than the minimum time spent there by the co n t r o l females which s u c c e s s f u l l y r a i s e d pups to weaning. This neglect was so severe i n some cases, that the extra-embryonic membranes and placentae were not removed from the pups. Results e and g show that d i e l d r i n - i n d u c e d i n f a n t i c i d e and neglect were the proximate causes of pup mortality in some l i t t e r s . This disturbed maternal behaviour was p a r t i c u l a r l y important at 20 ppm and also at 25 ppm where i t was responsible for 1007<, of the losses. Results c, d and f suggest that mechanisms i n i t i a t e d w i t h i n the pups while they were s t i l l in utero may have been important i n t h e i r post-partum deaths* Result f suggests that s t a r v a t i o n from poor l a c t a t i o n , poor nursing or maternal neglect may also have been important i n the pup m o r t a l i t y . F. D i e l d r i n ' s E f f e c t on Ovulation and Implantation in Bred, Nulliparous Females Presented i n Table XXVIII are the proportions of those bred females fed 0, 15 or 25 ppm d i e l d r i n which had, on day 4 of gestation, decidua detectable with Pontamine blue. Of the controls 937„ had decidua while of those fed 15 ppm d i e l d r i n , only 677» had them. The p r o b a b i l i t y that this de-crease occurred from chance i s 0.061, which is only s l i g h t l y higher than the standard acceptance l e v e l of 0.05 and strongly suggests the reduction was r e a l . These data show, therefore, that the decreased f e r t i l i t y which occurs at t h i s dose r e s u l t s from some l e s i o n occurring at or before the early 87 Table XXVIII. The e f f e c t s of d i e t a r y d i e l d r i n on the number of mice with decidua on day 4 of g e s t a t i o n (plug day = day 0). V i r g i n mice (9-11 weeks old) were bred during weeks 5-6 of expo-sure. Means + SEM of other parameters are presented a l s o . Dose (ppm) 0 15 25 Mice w i t h Decidua 14/15 14/21 3 12/12 No. of d e c i d u a b 12.5 + 0.35 11.5 + 0.55 11.1 + 0.69-No. of corpora 12.5 + 0.35 14.1 + 0.79 11.9 + 0.74 l u t e a c % i m p l a n t a t i o n 100 81.5 '93.3 Mice without Decidua 1/15 7/21 0/12 With corpora l u t e a 1/1 5/7 No. of corpora 16.0 12.0 + 2.01 l u t e a 2 a. 2 x 2 contingency t a b l e of 0: 15 vs With:Without. X =3.60, df = 1, P = 0.061. b. 1-way anova: o v e r a l l F = 2.646, df = 2, 36; P>0.05 * S i g n i f i c a n t l y lower than c o n t r o l s by Dunnett's d'~ (P<0.05). c. 1-way anova: o v e r a l l F = 2.331, df = 2, 36; P>0.05 stages of b l a s t o c y s t i m p l a n t a t i o n . Of the exposed females which lacked decidua, 5 out of the 7 had f r e s h corpora l u t e a w i t h i n t h e i r o v aries and the number of corpora l u t e a w i t h i n these o v a r i e s was not d i f f e r e n t from those found i n the o v a r i e s of c o n t r o l animals. These data suggest that most of the i n f e r t i l e females ovulate and t h e r e f o r e , i n them, the l e s i o n must occur a f t e r o v u l a t i o n . Table XXVIII shows that a l l of the bred females fed 25 ppm d i e l d r i n 83 had decidua; this is s i m i l a r to the r e s u l t s of the i n i t i a l experiment where diparous females were used and where doses of 20 and 25 ppm did not r e s u l t i n i n f e r t i l i t y but that doses of 10 and 15 ppm did. The cause of this apparently anomalous r e s u l t i n the present experiment, is probably s i m i l a r to that discussed for the previous one, namely, the mortality at high doses, of females which, i f fed lower doses would l i v e but be i n f e r t i l e . Each group of animals used here consisted of 30 i n d i v i d u a l s of which only 16 from those fed 25 ppm survived to s a c r i f i c e — a l l survived in the other two groups — and i t seems probable that any females la c k i n g decidua were amongst those 14 which died. There were s i g n i f i c a n t l y fewer decidua i n the u t e r i of females fed 25 ppm d i e l d r i n than i n those of the con t r o l animals. This shows that the reduction i n the l i t t e r s i z e , caused by t h i s dose, r e s u l t s from a l e s i o n occurring at or before implantation. The number of corpora lutea i n the ovaries of these treated females was not d i f f e r e n t from those of the controls which shows that the l e s i o n occurs a f t e r ovulation. G. E f f e c t of D i e l d r i n on Pup V i a b i l i t y The s u r v i v a l rate of pups i s o l a t e d from t h e i r dams at b i r t h is pre-sented i n Table XXIX. There i t can be seen that of pups taken from females fed 5, 10 or 15 ppm d i e l d r i n the proportion a l i v e at (and a f t e r ) 48 hours of i s o l a t i o n was s i g n i f i c a n t l y , and dose-dependently, smaller than i n those removed from co n t r o l dams. These r e s u l t s show that d i e l d r i n administered to the dam p r i o r to and during gestation decreases the post-partum v i a b i l i t y of the pups. The r e s u l t s i n Tsble XXX show thcit t h e r e Wcis no nicitheinciticcil ly Table XXIX. V i a b i l i t y of pups born to primiparous dams fed d i e t a r y d i e l d r i n . Pups were i s o l a t e d f r o t h e i r dams at b i r t h and the number l i v i n g was determined at various times. (n = number of dam c o n t r i b u t i n g pups; N = number of pups removed). Dose (ppm) n Hours a f t e r N Removal: 8 24 32 7o of Pups A l i v e 48 56 72 30 96 104 0 40 159 98.7 94.3 69.2 34.6 13.8 3. 8 1.9 0.6 0.6 5 9 56 98.2 98.8 66.1 b e 17.9 3.6 1. 8 0 0 0 10 13 64 96.9 85.9 a 73.4 14.0° 1.6 f 0 0 0 0 15 4 24 95.8 83.3 58.3 4.2 d 0.0 0 0 0 0 2 x 2 contingency ta b l e of C o n t r o l A l i v e : C o n t r o l Dead vs Treatment Alive:Treatment Dead a. X 2 = 4.34 P < 0.05 d. x 2 = 9.10 P < 0.01 2 b. X = 5.50 P < 0.025 e. x 2 = 4.40 P < 0.05 2 c. X = 9.40 P < 0.01 f. X 2 = 7.43 P < 0.01 Table XXX. Dam e f f e c t on pup v i a b i l i t y . Littermates were iso l a t e d at b i r t h from primiparous dams fed d i e t a r y d i e l d r i n and the number of l i t t e r s i n which one or more pups remained a l i v e was determined at times a f t e r removal. n = number of l i t t e r s (= dams). °/o of L i t t e r s with L i v e Pups Dose Hours a f t e r (ppm) n Removal: 8 24 32 48 56 7 2 80 96 104 0 40 100 100 95 68 33 8 5 3 3 5 9 100 100 100 44 22 11 0 0 0 10 13 100 92 92 47 8 0 0 0 0 15 4 100 100 100 25 0 0 0 0 0 V D o 91 demonstrable dam e f f e c t on pup v i a b i l i t y which would preclude the use of the pup as a v a l i d sample u n i t . H. Sur v i v a l and Growth of Pups Born to Primiparous Females Fed D i e l d r i n and  Foster-nursed by Unexposed Primiparous Dams The number of dams r a i s i n g t h e i r own and/or foster pups is presented in Table XXXI. There were two dams which did not r a i s e foster pups and since they also f a i l e d to r a i s e t h e i r own they have been excluded from future analyses. Table XXXI shows that a l l of the dams r a i s i n g t h e i r own pups also r a i s e d foster pups born to other females fed 0 ppm d i e l d r i n . The proportion of these foster pups which were s t i l l a l i v e , p r i o r to or at weaning, was not d i f f e r e n t from that of the dams' own pups (Table XXXII and Figure 14). These ( p o s i t i v e control) data show that pups fostered from b i r t h to weaning survive at the same rate as those raised by t h e i r natural dam. Eleven dams nursed young taken from females fed 10 ppm d i e l d r i n ; three had a l l the foster pups die w i t h i n the f i r s t 4 days while eight had some survive to weaning (Table XXXI). The actual number of pups^ that each of these eight had at weaning, 28 days a f t e r b i r t h , i s presented i n Table XXXIII where i t can be seen that a mean of 84% of t h e i r own pups survived while only 23%, of the foster pups survived; the d i f f e r e n c e i s s i g n i f i c a n t . As seen in Figure 14, which presents the proportion of pups a l i v e on each day between b i r t h and weaning, 68%, of the deaths amongst the foster pups occurred within the f i r s t 6 days of l i f e , 22%, occurred between days 6 and 15, and the balance, 10%,, occurred i n the l a s t 13 days. S i m i l a r r e s u l t s were obtained with the very l i m i t e d samples at 5 ppm and 15 ppm; one female 92 Table XXXI. The number of unexposed dams which raised foster pups and/or the i r own pups to weaning. V i r g i n females (4-6 weeks old) were placed on 0, 5, 10 and 15 ppm dietary d i e l d r i n and mated during weeks 5-6 of exposure. The l i t t e r of an unexposed female was reduced to f i v e - s i x pups at b i r t h and f i v e - s i x pups (< 12 hours old) born to a donor female were added to i t within 24 hours. D i e l d r i n i n Diet of Donor Female (ppm) With Foster Number of Unexposed Females Pups Raising Own Pups Raising Foster Pups 0 7 6 6 5 1 1 1 10 12 11 8 15 2 2 1 foster-nursing pups from a 15 ppm female l o s t a l l of them while the other rai s e d only one pup, and the lone female foster-nursing pups born to a 5 ppm dam a l s o r a i s e d only one. These r e s u l t s confirm that pups born to females fed d i e l d r i n at d i e t a r y concentrations of 5 ppm and higher, before and during gestation, have experienced conditions _in utero which^result i n t h e i r post-partum i n v i a b i l i t y . These r e s u l t s also suggest that the toxico-l o g i c a l changes caused i n the pups are, i n most cases, absolute and irr e v o c a b l e . However, within 73% of the l i t t e r s born to females fed 10 or 15 ppm d i e l d r i n some pups did survive when they were r a i s e d by control females whereas none survived when they were kept with t h e i r own dams. Furthermore, the rate of mortality among the pups of these fostered l i t t e r s was lower than that of those kept by t h e i r own dams; as seen in Figure 14, less than 93 Table XXXII. The e f f e c t of f o s t e r i n g on the s u r v i v a l of pups to weaning (day 28 ) . V i r g i n females (9-11 weeks old) were housed with males for 2 weeks. At b i r t h , the dam's l i t t e r was reduced to 6 pups and within 24 hours, another 5-6 pups ( < 1 2 hours old) born to another female were added to i t . Pups A l i v e on Day 28 Own Foster Dam No. Number % Number 1 1/6 16.7 5/6 83.3 2 3/6 50.0 3/6 50.0 3 5/6 83.3 2/6 33.3 4 6/6 100.0 6/6 100.0 5 4/6 66.7 4/6 66.7 6 2/6 33.3 2/5 40.0 x + SEM 3 62.9 + 2.7 67 .1 + 2.1 a. 2-way anova for paired comparisons: ( i ) Pups a l i v e : Own vs Foster. F = 0.071, df = 1, 5; P>0.10 ( i i ) Dam e f f e c t : F = 2.613, df = 5, 5; P>0.05 A l l c a l c u l a t i o n s performed on a r c - s i n transformed data. Data have been de-trans formed for presentation. 207c, of the fostered pups taken from females fed 10 ppm d i e l d r i n were dead by day 4, whereas 1007. of those kept with t h e i r own dam were dead by t h i s time (Figure 10) . These r e s u l t s show that post-partum factors i n t e r a c t with the i n a t e l y depressed v i a b i l i t y to enhance the rate and magnitude of pup mortality. It w i l l be r e c a l l e d from Table XXXI that three of the dams did not 100-r 9CH 60A 70H UJ > 60 CO 5 0 0 _ QL 4 0 30-20 1 b--o X >o- -a—a- -o- -o- -a- -o >a»o--a«a (44) °— 0 Foster-nursed pups from 0.0 ppm dams •—• Paired controls °—° Foster-nursed pups from 10.0 ppm dams Paired controls i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i i i i i i i ^ 2 4 6 8 10 12 14 16 18 20 22 24 26 28 BIRTH DAYS P O S T - P A R T U M WEANED F i g u r e 14. The s u r v i v a l r a t e of pups which were born to females fed 0.0 or 10.0 ppm d i e t a r y d i e l d r i n but foster-nursed by other dams fed 0.0 ppm d i e l d r i n . Each f o s t e r dam sim u l -taneously nursed some of i t s own pups as a p a i r e d c o n t r o l . The number of pups are shown i n brackets. 95 Table XXXIII. The e f f e c t of 10 ppm d i e l d r i n i n the diet of a female on the s u r v i v a l to weaning (day 28) of i t s pups when foster nursed by an unexposed dam. V i r g i n females (4-6 weeks old) were placed on rations containing 0.0 or 10.0 pom d i e l d r i n and mated during weeks 5-6 of exposure. L i t t e r s of 0.0 ppm dams were reduced to 5-6 pups at b i r t h and within 24 hours, 5-6 pups ( £ 1 2 hours old) born to a 10.0 ppm female were added to i t . L i t t e r s where a l l foster pups died (3) have been excluded. Pups A l i v e on Day 28 Own Foster  Dam No. Number % Number % 1 4/5 80.0 1/5 20.0 2 5/5 100.0 1/5 20.0 3 4/5 80.0 1/5 20.0 4 6/6 100.0 2/6 33.3 5 5/6 83.3 1/6 16.7 6 2/6 33.3 1/6 16.7 7 4/6 66.7 5/6 83.3 8 5/6 83.3 1/5 20.0 x + SEM 83.5 + 1.2 28.0 + 0.25 a. 2-way anova for paired comparisons: ( i ) Pups a l i v e : Own vs Foster. F = 16.55, df = I, 7; ; P<0.005 ( i i ) Dam e f f e c t : F = 0.84, df = 7, 7: ; P> 0.100 A l l c a l c u l a t i o n s were done on a r c - s i n transformed data. Data have been de-transformed for presentation. 96 rai s e any o£ the foster pups taken from females fed 10 ppm d i e l d r i n , which they had been given to foster-nurse. The weight of the s u r v i v i n g f o s t e r pups, on the days before death, is compared with that of the dam's own pups in Table XXXIV. The foster pups did not grow p r i o r to death and exhibited the darkening of colour, l i s t l e s s n e s s and i n a c t i v i t y described previously for the pups born to exposed dams. The growth of the pups which died in those fostered l i t t e r s where some pups survived also appeared to be reduced or abolished but no data is a v a i l a b l e to substantiate this impression. The weight of a l l the pups s t i l l a l i v e on each day from b i r t h to weaning is presented i n Figure 15. The lower panel compares the growth of the pups taken from females fed 0.0 ppm d i e l d r i n and fostered by c o n t r o l dams, with that of the co n t r o l dams' own pups. The body weight of these two groups of pups was not s i g n i f i c a n t l y d i f f e r e n t on any day although the foster pups appeared to grow s l i g h t l y more slowly from about day 23 onwards. The upper panel compares the weight of the pups taken from females fed 10 ppm d i e l d r i n and fostered by co n t r o l dams with that of the co n t r o l dams' own pups. Again the weight of the foster pups was not s i g n i f i c a n t l y d i f f e r e n t from that of the co n t r o l females own pups although the l a t t e r did tend to weigh more from day 7 onwards. These data show that d i e l d r i n fed to the dam, before and during gestation, has l i t t l e or no e f f e c t on the growth of pups which survive to weaning by being foster-nursed by unexposed females. It w i l l be noted that a l l of the pups in the foster-nursing experi-ment grew more slowly and weighed much less at weaning than those raised by the diparous females in the f i r s t experiment (see Figure 11); the pup growth i n the present experiment was quite normal for primiparous females, which c h a r a c t e r i s t i c a l l y wean small, runty pups, and does not n e c e s s a r i l y indicate 97 Table XXXIV. The average body weight (g) of s u r v i v i n g l i t t e r m a t e s ( l i t t e r weight/number of pups) i n the three l i t t e r s where a i l the fos t e r e d pups (from 10 ppm dams) died before weaning. Average Body Weight of S u r v i v i n g L i t t e r m a t e s (x + SEM) Post-par turn Day Own Pups ' Foster Pups 1 1.17 + 0.05 1.16 + 0.04 2 1.27 + 0.08 1.13 + 0.02 3 1.40 + 0.07 1.24 + 0.08 4 1.67 + 0.0.0 1.19 + 0.00 p a t h o l o g i c a l c o n d i t i o n s i n e i t h e r the pups or the dams. I. E f f e c t of D i e t a r y D i e l d r i n on G e s t a t i o n a l Serum Progesterone Table XXXV presents the serum progesterone l e v e l s i n d i e l d r i n -fed females on days 8 and 14 of g e s t a t i o n . There i t can be seen that the c o n c e n t r a t i o n of progesterone i n the serum of mice fed the c o n t r o l r a t i o n was 41.4 +4.8 ng/ml on day 8 and 61.8 + 3.3 ng/ml on day 14; the concentra-t i o n i n the serum of mice fed e i t h e r 5, 10, or 15 ppm d i e t a r y d i e l d r i n was not d i f f e r e n t from these c o n c e n t r a t i o n s . These r e s u l t s show that the d i e l -d r i n - i n d u c e d decrease i n the inate v i a b i l i t y of the pups i s n e i t h e r caused by nor r e l a t e d to a decrease i n the c o n c e n t r a t i o n of endogenous progesterone d u r i n g g e s t a t i o n . The lack of a decrease i n the serum progesterone of the d i e l d r i n - f e d females i m p l i e s that one of two p o s s i b l e c o n d i t i o n s must e x i s t i r i v i v o . E i t h e r the metabolism of progesterone i s not increased or, i f i t i s , then 98 7.CH 6 . C H ^ 5 0 U J CO 4 . 0 + 1 IX CO 3 0 U J H < 2 2 . 0 -r r u i H I— 1 . 0 -O ZO-i o» ^ 6 . 0 H CD bJ 5.0 e> < or w 4.0-1 < 3.0-1 2.0-• F o s t e r - n u r s e d p u p s f r o m 1 0 . 0 p p m d a m s • P a i r e d c o n t r o l s a Fi J- J-B g • • 0 0 D • i.o ° F o s t e r - n u r s e d p u p s f r o m 0 . 0 p p m d a m s • P a i r e d c o n t r o l s s * 1 5 l T 5 5 1 S 1 I'1 'I I T • o o 1 i l i l l 1 * o ® B 5 J 1 I I I I I I I I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—1—I—I—I—I 0 ^ 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6 2 8 3 0 BIRTH DAYS P O S T - P A R T U M WEANED Figure 15. The growth rate of pups which were born to females fed 0.0 or 10.0 ppm d i e l d r i n but foster-nursed by other dams fed 0.00 ppm d i e l d r i n . Each foster dam simultaneously nursed some of i t s own pups as a paired c o n t r o l . 99 Table XXXV. The e f f e c t of dietary d i e l d r i n on the concentration of serum progesterone i n SWV mice on days 8 and 14 of gestation. The figure i n brackets is the number of mice analyzed. Progesterone: ng/ml serum (x + SEM) Dose (ppm): 0 5 10 15 Day 8 Day 14 41.4 +4.8 (6) 61.8 + 3.3 (6) 45.4 + 2.9 (6) 65.4 +6.2 (6) 40.3 + 3. (2) 51.1 + 6.5 (5) 54.7 + 11.7 70.0 + 6.0 (3) (6) a. 1-way anova F = 1.04, df = 3, 15; P>0.10 b. 1-way anova F = 1.01, df = 3, 16; P >0.10 the increase i s compensated for by an increased biosynthesis and/or a l t e r e d d i s t r i b u t i o n of the s t e r o i d . J . Milk Produced by Primiparous Females Fed D i e l d r i n The amount of milk synthesized by the mammary glands of primiparous females during a standardized 8 hour period on l a c t a t i o n day 4 i s presented in Table XXXVI. The amount synthesized by dams fed 0, 5 or 10 ppm d i e l d r i n did not d i f f e r s i g n i f i c a n t l y showing that d i e l d r i n does not i n t e r f e r e with the net ga l a c t o p o i e t i c processes i n the mammary gland. However, to e f f e c t milk release, exogenous oxytocin was required i n a l l the females and the p o s s i b i l i t y remains that d i e l d r i n may i n t e r f e r e with l a c t a t i o n a l output by impairing the release mechanisms. K. Di e l d r i n ' s E f f e c t on Maternal Behaviour i n Primiparous Females On l a c t a t i o n day 3 the females used i n the milk production experiment 100 Table XXXVI. Milk produced during an 8 hour secretory period (1200-2000 hours) on day 4 of l a c t a t i o n ( b i r t h = day 1) by primiparous dams fed dietary d i e l d r i n . Dose mg milk/gland/g of body weight a (ppm) n (x + SEM) 0 10 2.99 + 0.24 5 5 2.92 + 0.72 10 12 2.25 + 0.32 a. 1-way anova. F = 1.44, df = 2, 24; P>0.10 had t h e i r pups removed for 4 hours and a f t e r replacement of the pups these dams exhibited latencies to pup-retrieval, nest-building and nursing which are presented i n Table XXXVII. These behaviour patterns, which are d e s c r i -bed i n the Methods, follow one another i n the given order with minor v a r i a -tions. For example, a f t e r several pups have been taken to the nest s i t e the dam may then a l t e r n a t e between nest-building and pup - r e t r i e v a l , and i n fact, the search behaviour, which precedes p u p - r e t r i e v a l , is often continued, interspersed between bouts of nest-building, a f t e r a l l the pups have been r e t r i e v e d . The t r a n s i t i o n from n e s t - b u i l d i n g to nursing is usually gradual with females int e r s p e r s i n g longer bouts of nursing with shorter ones of nest- b u i l d i n g or, i n some cases, other types of behaviour outside the nest. Because of th i s gradual t r a n s i t i o n the onset of nursing was defined as the beginning of the f i r s t uninterrupted, 2 minute bout of nursing; previous observations showed that once this time was attained the dam usually con-tinued to nurse without further i n t e r r u p t i o n . 101 Table XXXVII. E f f e c t of dietary d i e l d r i n on the maternal behaviour of primiparous dams: the latency to begin pup-retrieval, nest-bui l d i n g and nursing behaviour when the pups were replaced i n the cage a f t e r a 4 hour absence. Observations were made on l a c t a t i o n day 3. Minutes of Latency (x and 95% CL) Dose (ppm) n p u p - r e t r i e v a l a n e s t - b u i l d i n g b n u r s i n g 0 0 10 0.47 (0.43, 0.51) 2.85 (3.97, 1.73) 9.66 (9.30, 10.03) 5 5 0.21 (0.15, 0.29) 2.34 (2.75, 1.92) 14.17 (12.87, 15.61) 10 12 0.25 (0.24, 0.25) 2.60 (3.70, 1.50) 21.06*(19.62, 22.63) A l l c a l c u l a t i o n s were made on log-transformed data which have been de-transformed for presentation. a. 1-way anova. F = 2.23, df = 2,' 24; P>0.10 b. F max. for log-transformed data = 23.74, df = 4; P<0.05 c. 1-way anova. F = 2.63, df = 2, 24; P>0.05. *S i g n i f i c a n t l y d i f f e r e n t from control value by Duncan's New M u l t i p l e Range Test. P<0.05. The latency to p u p - r e t r i e v a l shown by the dams fed 5 or 10 ppm d i e l d r i n was about 50%o of that shown by co n t r o l females, although the d i f -ference was not s t a t i s t i c a l l y s i g n i f i c a n t . This aspect of maternal beha-viour was unimpaired by d i e l d r i n and, i n fact, may have been stimulated by it*, although the apparent stimulation may merely have r e f l e c t e d general hyper-a c t i v i t y and hyperkinesia s i m i l a r to that noted i n the other experiments. S i m i l a r l y , the mean latency to nest-building was not d i f f e r e n t be-tween the groups of dams fed 0, 5, or 10 ppm d i e l d r i n ; unfortunately t h i s cannot be formalized because the variances were heterogeneous even a f t e r the data were transformed into logarithms and s t a t i s t i c a l a n alysis was there-fore precluded; however, the great overlapping of the confidence l i m i t s 102 c l e a r l y shows but no real differences existed. The latency to nursing was increased, dose-dependently, i n the fe-males fed 5 and 10 ppm d i e l d r i n , although only the larger increase, which occurred at 10 ppm, was s t a t i s t i c a l l y s i g n i f i c a n t . Most of the females spent the a d d i t i o n a l time between the onsets of nest-building and nursing i n n e s t - b u i l d i n g behaviour. The amount of this a c t i v i t y would gradually decrease and then, a f t e r apparently beginning to nurse, these dams would suddenly burst into another prolonged bout of nest-building. During these bouts the movements of the exposed dams were much quicker and more v i o l e n t than those of the controls. These data show that once a d i e l d r i n - f e d femal returns to the nest i t would take more time than a c o n t r o l dam to begin nursing pups again; this could r e s u l t i n decreased milk intake, by the pups. Moreover these observations suggest that pups could be d i r e c t l y damaged during the prolonged nest-building since they are trampled on by the female 103 DISCUSSION A. Organochlorine Insecticides and Mammalian Reproduction: The L i t e r a t u r e The purpose in i n i t i a t i n g this study was to test the hypothesis that dietary organochlorine i n s e c t i c i d e s impair mammalian reproduction by inducing l i v e r enzymes which metabolize the sex steroids and thereby cause d e f i c i e n c i e s i n the ph y s i o l o g i c a l l e v e l s of these hormones. In order to achieve this i t was necessary to se l e c t an animal and an i n s e c t i c i d e which would be l i k e l y to interact and r e s u l t i n several reproductive lesions for study. The l i t e r a t u r e at the time indicated that d i e l d r i n and the house mouse were the most probable choices that would serve as successful models. This, and the more recent l i t e r a t u r e , w i l l now be discussed b r i e f l y i n order that the reader may better understand why these were chosen and why the de c i s i o n was made to pursue the factors involved i n the dieldrin-induced pup mortality. In the vast majority of papers to be discussed the purpose of the in v e s t i g a t i o n was simply to evaluate the e f f e c t of a compound on the o v e r a l l reproductive performance of exposed males and exposed females. The under-l y i n g mechanisms of the lesions usually cannot be ascertained from data c o l -lected for such a purpose. However, many of these investigators have (erroneously) used terms which imply that the lesions resulted from impair-ments to s p e c i f i c processes. For example, many i n v a l i d l y used the term " f e r t i l i t y " to describe the sum of a l l the processes which they measured by the end-point of a successful p a r t u r i t i o n . Consequently, where necessary, I have taken the l i b e r t y of replacing the terms used by the o r i g i n a l authors with others which do not imply that s p e c i f i c processes were examined. In 104 addition, unless i t is otherwise indicated, the doses given r e f e r to the maximum dose employed, and, in the case of multi-generation studies, the f i r s t generation was the only one considered. The e f f e c t s of dietary DDT on reproduction i n the rat have been ex-tensively studied. Fitzhugh (1948) showed that doses of 600 ppm did not a f f e c t the number of females d e l i v e r i n g pups or the number of pups born; however, doses of greater than 50 ppm increased the pre-weaning pup mor-t a l i t y . Treon and Cleveland (1955), who published no data, claim that in the Carworth rat doses of 25 ppm af f e c t e d neither the number of females d e l i v e r i n g pups nor the l i t t e r s i z e but that doses over 2.5 ppm increased pup mortality. Ottoboni (1969), also using the Carworth rat, found that doses of 200 ppm did not a f f e c t reproduction at a l l . Duby et a l . (1971) found that i n the Sprague-Dawley rat no e f f e c t s were caused by technical DDT at 15 ppm, by o,p'-DDT at 3 ppm, or by p,p'-DDT at 12 ppm,on the number of females d e l i v e r i n g pups or on the number of pups del i v e r e d . They did not follow pup mo r t a l i t y . Wrenn et a l . (1971a) have shown that o,p'-DDT, at a dose of 40 ppm, was i n e f f e c t i v e i n decreasing the l i t t e r s i z e of Wistar rats or in decreasing the pup s u r v i v a l . These r e s u l t s show that i n the rat p r a c t i c a l doses of DDT cause only decreases i n the s u r v i v a l of pups a f t e r b i r t h . In keeping with these r e s u l t s , Van Tienhoven and Duby (1972) i n j e c -ted pregnant rats on each of day 1-3 of gestation with 900 /ig of e i t h e r o,p'-DDT or p,p*-DDT and found t h i s treatment to have no e f f e c t on the number of embryos present on day 10; moreover, they found that 2 mg of o,p'-DDT, when injected into pregnant, ovariectomized rats maintained with progesterone, did not induce implantation. 105 The e f f e c t s of dietary DDT have also been well documented in the laboratory mouse. Bernard and Gaertner (1964) found that 300 ppm decreased the number of C57/B1 females which delivered pups; 200 ppm was i n e f f e c t i v e ; pup s u r v i v a l was not studied. Ware and Good (1967), using the Balb/c mouse, found that 7 ppm did not a f f e c t the number of females d e l i v e r i n g pups or the number of pups i n each l i t t e r ; pup s u r v i v a l was not studied. Cannon and Holcomb (1963) claim that i n the Hal/wf/lcr mouse 300 ppm increased the number of pregnant females which died but did not a f f e c t the number of preg-nancies or the number of pups delivered by surviving females; t h e i r data is so poorly presented that the v a l i d i t y of these claims cannot be ascertained. Tarja'n and Keme'ny (1969) found that 3.0 ppm, fed for 5 generations, had no e f f e c t on the number of Balb/c females which became pregnant, on the number which gave b i r t h , on the l i t t e r s i z e , or on the number of pups which were weaned. The data for the f i r s t generation of a 6-generation experiment conducted by Keplinger et a l . (1970) show that 250 ppm fed to a "Swiss" mouse did not a f f e c t the number of females d e l i v e r i n g , the l i t t e r s i z e at b i r t h or the s u r v i v a l of pups. Guthrie et a l . (1971), who used a "white" mouse, mated the survivors of an (injected) LD50 dose of DDT from each gen-er a t i o n for 14 generations and found t h i s treatment to have no e f f e c t on the l i t t e r s i z e or s u r v i v a l of the pups to weaning. The above r e s u l t s show that p r a c t i c a l doses of DDT have e s s e n t i a l l y no e f f e c t on reproduction i n several s t r a i n s of mice. Recently, Lundberg and Kihlstrom (1973) found that i n j e c t i n g mice with DDT (20 mg/kg) either on day 1 alone or on both days 1 and 3 of gesta-tion resulted i n a reduction of the r a t i o of implantation s i t e s to corpora lutea on day 12. In addition, Lundberg (1973) has found that the long-term 106 d a i l y o r a l administration of DDT (0.05 mg/kg) extended the estrous cycle by a mean of 0.5 days and, in mated animals examined on day 12, reduced the implantation:corpora lutea r a t i o . These studies were done with the NMRI s t r a i n and i t w i l l be i n t e r e s t i n g to see i f further studies with this s t r a i n show these e f f e c t s to have some s i g n i f i c a n c e on the number of females which breed, on f e r t i l i t y , or on the l i t t e r s i z e at b i r t h . The e f f e c t s of 10 ppmo,p'-DDT on the domestic sheep were studied by Wrenn et a l . (1971b) who found i t to have no e f f e c t on the estrus behaviour, on the number of ewes conceiving or on the number of lambs delivered. A diet, prepared from f i s h with " n a t u r a l " residues, and which contained 0.58 ppm DDE and traces of other DDT metabolites, did not a f f e c t breeding or l i t t e r s i z e i n the ranch mink (Mustela vison) but i t did increase k i t mor-t a l i t y ( G i l b e r t , 1969). In t h i s species, A u l e r i c h et a l . (1971) found that a s i m i l a r l y prepared r a t i o n which contained 2.5 ppm " t o t a l DDT" and less than 0.15 ppm d i e l d r i n did not a f f e c t the number of females which mated; however, none of those mated gave b i r t h . Deichmann et a l . (1971) have pub-l i s h e d copious observations on the e f f e c t s of o r a l doses of p,p'-DDT i n the beagle, but the sample s i z e (3 females, 4 males) and the lack o f % c o n t r o l data make them of l i m i t e d value. The general lack of e f f e c t s of d i e t a r y DDT on mammalian reproduction indicated i t was not a useful compound to employ. Good et a l . (1965), who used Balb/c mice, showed that dietary Kepone, in doses as low as 5 ppm, decreased both the number of pairs producing l i t t e r s and the number of pups in the l i t t e r s . Huber (1965) also used the same s t r a i n and observed that the Kepone-induced decrease in the number of pregnant females was related to a con d i t i o n of constant estrus which, in 107 turn, was related to a reduction in the LH a c t i v i t y of an extract of t h e i r p i t u i t a r i e s . The e f f e c t s of Mirex, the completely chlorinated analog of Kepone, were studied by Ware and Good (1967). Their i n i t i a l r e s u l t s showed that in the Balb/c mouse 5 ppm increased adult mortality; i t did not affect either the number of surviving pairs which produced l i t t e r s or the s i z e of the l i t t e r , but i t did decrease the number of l i t t e r s produced by producing pairs (="fecundity"); l a t e r attempts to repeat the decrease i n fecundity were unsuccessful. Mirex had no effect on reproduction i n the CFW s t r a i n . Pup s u r v i v a l was not studied i n e i t h e r s t r a i n . In the "Swiss" mouse dietary toxaphene, fed at a concentration of 25 ppm, had no e f f e c t on reproduction (Keplinger et a l . 1970). Much research has been done on the e f f e c t s of d i e t a r y a l d r i n and on those of i t s epoxide, d i e l d r i n . Both compounds are i n commercial use but for t o x i c o l o g i c a l purposes they are i n essence one compound since a l d r i n i s r a p i d l y and almost completely metabolized to d i e l d r i n by the vertebrate l i v e r . In a d d i t i o n to both of these cyclodienes two others have been studied: endrin, the stereoisomer of d i e l d r i n , and t e l o d r i n . The e f f e c t s of chlordane, the d i c h l o r i n a t e d analog of the cyclodiene, heptachlor, have also been investigated. Ambrose et a l . (1953) who used "albino r a t s " , found that 320 ppm of dietary chlordane decreased both the number of pairs which delivered l i t t e r s and the number of pups that survived to weaning. In "Swiss" mice, chlordane, at 100 ppm, was found to have no e f f e c t on the number of females d e l i v e r i n g pups or on the l i t t e r s i z e but i t increased the mortality of the pups in the f i r s t 4 days a f t e r b i r t h ; lower doses had no e f f e c t (Keplinger et a l . 1970). Welch et a l . (1971) found that i n j e c t i n g Swiss-Webster (Carworth) 108 female mice with chlordane (25 mg/kg) once a week for 3 weeks p r i o r to caging them with males decreased the number which delivered pups but did not a f f e c t the l i t t e r s i z e ; pup s u r v i v a l was not studied. In Balb/c mice dietary t e l o d r i n , at a concentration of 1 ppm, did not a f f e c t either the number of pairs which produced pups or the number of pups produced (Ware and Good, 1967); pup s u r v i v a l was not studied. Good and Ware (1969) found that 5 ppm d i e t a r y endrin increased the mortality of adult CFW mice; the proportion of s u r v i v i n g females which de-l i v e r e d pups was not affected but the l i t t e r s i z e was reduced; the pup s u r v i v a l was not studied. Morris (1968) studied the e f f e c t s of endrin on reproduction i n captive deer mice (Peromyscus maniculatus osgoodi) and found that adult m o r t a l i t y was increased by doses of 2.4 ppm and higher; i n sur-v i v i n g adults fed 7.3 ppm neither the number of l i t t e r s produced nor the l i t t e r s i z e were a f f e c t e d but doses of 4.3 ppm and higher increased pup mortality. The e f f e c t s of a l d r i n on reproduction i n the rat and the dog have been investigated by two groups of workers in both species while the e f f e c t s i n mice were studied by one group. The e f f e c t of dietary a l d r i n on the estrous cycle of the rat was estimated by B a l l et a l . (1953) who found that doses of 10 ppm and higher decreased the percentage of vaginal smears i n -d i c a t i v e of estrus amongst a l l those smears taken from groups of animals. This would suggest that the number of females receptive to the male was decreased but no attempt was made to confirm t h i s . Treon and Cleveland (1955) claim, without presenting the data, that i n Carworth rats, 25 ppm decreased neither the number of females g i v i n g b i r t h nor the number of pups delivered by each female; 2.5 ppm, however, was claimed to decrease the 109 s u r v i v a l of the pups. Deichmann et a l . (1971), who used an inadequate number of beagles and presented no c o n t r o l data, claimed that 0.3 mg/kg of a l d r i n (given o r a l l y i n o i l ) blocked estrus and decreased the s u r v i v a l of the pups; in addition, the " l i b i d o " of the males was supposedly decreased; the l i t t e r s i z e was supposedly unaffected. Keplinger et a l . (1970) who. used a "Swiss" mouse in a multi-generation experiment, found that dietary a l d r i n at a con-c e n t r a t i o n of 25 ppm decreased both the number of females d e l i v e r i n g pups and the number of pups i n the l i t t e r s as well as abo l i s h i n g the s u r v i v a l of pups; at 10 ppm and lower no e f f e c t s were observed. The e f f e c t s of d i e l d r i n on reproduction have been studied mostly by use of laboratory rodents but some data are a v a i l a b l e for other species. In captive w h i t e - t a i l e d deer (Odocoileus v i r g i n i a n u s ) doses of 5 ppm and higher increased fawn mortality but doses as high as 25 ppm did not a f f e c t the number of does d e l i v e r i n g fawns or the number of fawns deli v e r e d (Murphy and Korschgen, 1970). Kitselman (1953) found that i n the dog o r a l l y administered d i e l d r i n a f f e c t e d only pup s u r v i v a l and t h i s was decreased or abolished. The f i r s t p u b l i c a t i o n on the e f f e c t s of d i e l d r i n on reproduction i n the rat was that of Treon and Cleveland (1955). They claim, without pre-senting any data, that 2.5 ppm reduced the number of (Carworth) females g i v i n g b i r t h and increased the pup mortality; doses of up to 25 ppm, how-ever, did not a f f e c t the l i t t e r s i z e . Virgo (1970) who used the Wistar rat found that 53 ppm affe c t e d neither the number of females d e l i v e r i n g pups nor the l i t t e r s i z e ; however, doses of 10 ppm increased pup m o r t a l i t y . Harr et a l . (1970) also worked with the Wistar rat and found that 5 ppm abolished pup s u r v i v a l ; i n addition, these workers claimed that higher doses also 110 decreased both the number of females which delivered pups and the number of pups delivered; however, a s t a t i s t i c a l a n alysis of t h e i r data shows that i t does not support these conclusions. In the CFW mouse, Good and Ware (1969) found that 5 ppm dietary d i e l d r i n did not a f f e c t e i t h e r the number of females which delivered pups or the number of pups i n each l i t t e r ; pup s u r v i v a l was not studied. Keplinger et a l . (1970), who used "Swiss" mice i n a multi-generation study, found that dietary d i e l d r i n fed at a concentration of 25 ppm decreased the number of females which delivered pups, the number of pups delivered and abolished pup s u r v i v a l ; the continued feeding of 10 ppm, which had increased pup mortality i n the f i r s t l i t t e r , decreased the number of females which delivered a second l i t t e r and further increased pup mortality; 3 ppm was without apprec-iable e f f e c t over 6 generations of feeding. A consideration of the above leads one to conclude: 1. Exposing the house mouse to d i e l d r i n is the procedure most l i k e l y to r e s u l t i n reproductive lesions for further study. 2. M o r t a l i t y of the o f f s p r i n g , induced by exposing the adults to di e t a r y i n s e c t i c i d e s , a. i s the most frequent l e s i o n caused by a l l compounds and, b. occurs at lower doses than any other l e s i o n and, therefore, i t is the most s i g n i f i c a n t and important e f f e c t of organochlorine i n s e c t i c i d e s on mammalian reproduction. B. Reproduction: D i e l d r i n ' s E f f e c t s and t h e i r Mechanisms The hypothesis which proposes that organochlorine i n s e c t i c i d e s im-pair mammalian reproduction by inducing l i v e r enzymes which metabolize the I l l the sex steroids and thereby cause d e f i c i e n c i e s i n the p h y s i o l o g i c a l l e v e l of these hormones may be stated either as a univer s a l proposition, " A l l reproductive lesions caused by organochlorine i n s e c t i c i d e s r e s u l t from en-hanced s t e r o i d metabolism'.1, or as a p a r t i c u l a r proposition, "Some reproduc-t i v e lesions caused by organochlorine i n s e c t i c i d e s r e s u l t from enhanced s t e r o i d metabolism". Some of the r e s u l t s of this study may be used to test the v a l i d i t y of the univer s a l proposition. 1. Pup I n v i a b i l i t y This study found that d i e l d r i n fed to the female SWV mouse, before and during gestation, at doses which markedly induced the hepatic mixed-function oxidase system also markedly reduced the inate v i a b i l i t y of i t s pups. It was further found that i n these females the concentration of serum progesterone on days 8 and 14 of gestation was not d i f f e r e n t from that i n contr o l females. This shows that the pup i n v i a b i l i t y did not r e s u l t from inadequate amounts of progesterone. These r e s u l t s do not support the uni-v e r s a l p r o p o s i t i o n . Instead, the a l t e r n a t i v e , the p a r t i c u l a r proposition, w i l l be t e n t a t i v e l y accepted. It must be r e a l i z e d , however, that the use of these r e s u l t s i n re-j e c t i n g the un i v e r s a l p r o p o s i t i o n i s not without c r i t i c i s m . One c r i t i c i s m resides i n the fact that the actual steroids required by the gestating mouse for the normal post-partum v i a b i l i t y of i t s pups are unknown. Cle a r l y , a more stringent test of the univer s a l p r o p o s i t i o n could be made by quantitating the endogenous steroids during other processes where the underlying s t e r o i d physiology i s better understood. However, i n this study the only other e f f e c t s of d i e l d r i n were on f e r t i l i t y and l i t t e r s i z e , where !. 1.2 the magnitude of the e f f e c t was so small that i t was not p r a c t i c a l to use them. Nor, in view of the serious, overriding e f f e c t s of d i e l d r i n on pup s u r v i v a l , was i t desirable. A second, more serious c r i t i c i s m , r e l a t e d to the f i r s t , i s the absence of data on the concentration of estrogens during gestation, since i t i s conceivable that these could be important in post-partum v i a b i l i t y . However, time did not permit the q u a n t i f i c a t i o n of both estrogens and progesterone and the l a t t e r was chosen for the reasons d i s -cussed next. In the mouse progesterone is e s s e n t i a l throughout gestation. Ovari-ectomy on days 3-15 of gestation causes resorbtion of the uterine contents while on days 16-19 i t causes abortion (Harris, 1927). The administration of progesterone to ovariectomized mice not only maintains gestation and permits normal f e t a l development but i t does this with or without the con-cominant administration of e s t r a d i o l ( H a l l , 1957). Progesterone i s , i n fact, the only ovarian s t e r o i d known to be required for the maintenance of gesta-t i o n i n this species (Porter, 1970). However, the l e v e l of progesterone appears to be important since low doses do not maintain gestation i n o v a r i -ectomized mice while doses administered to i n t a c t animals extends the ges-t a t i o n period and r e s u l t s i n the d e l i v e r y of dead or i n v i a b l e pups (Kroc et a l . , 1959). It does not seem unreasonable therefore, that decreases in the concentration of progesterone which are i n s u f f i c i e n t to terminate gestation might r e s u l t i n pup i n v i a b i l i t y . A possible mechanism for such a s i t u a t i o n may be found i n recent evidence showing that maternal progesterone is l i k e l y to be the major substrate for C o r t i s o l biosynthesis i n the adrenal of the human fetus (Solomon et a l . , 1967). The f e t a l adrenocorticoids appear to be important in a number of processes e s s e n t i a l for post-partum l i f e : 113 for example, the appearance of the surfactant required for functional matura-tio n of the lung (Kotas and Avery, 1971) and carbohydrate metabolism and pre-partum glycogen deposition (Dawes and Shelley, 1968). In this regard i t has been shown by Delphia and Singh (1971) that the glycogen l e v e l is decreased i n the l i v e r s of rat fetus' when the inducer, sodium pentobarbital, i s administered to the dam during gestation. The _in vivo concentration of sex steroids i n vertebrates exposed to inducers of the hepatic mixed-function oxidase system has been determined on two previous occasions. Peakall (1970) found a 347» decrease i n the con-ce n t r a t i o n of e s t r a d i o l within the plasma of female ringdoves ( S t r e p t o p e l i a  r i s o r i a ) fed 10 ppm p,p'-DDT. This decrease was p a r a l l e l e d by a 2737o i n -crease i n the _in v i t r o metabolism of e s t r a d i o l by the microsomal f r a c t i o n of the l i v e r . A possible consequence of the reduction i n estrogen was a prolongation of the i n t e r v a l between the p a i r i n g of the sexes and the la y i n g of the f i r s t egg. Southren et a l . (1969) found a 167o decrease i n the con-c e n t r a t i o n of testosterone i n the plasma of a human male r e c e i v i n g 1200 mg per day of N-phenylbarbital. Associated with the decrease was a large i n -14 crease i n the proportion of polar metabolites formed from infused C-testosterone. This, however, was more than compensated for by a decrease i n the production of other metabolites so that the o v e r a l l metabolic clearance rate of testosterone was a c t u a l l y decreased by 1170. The decrease i n the metabolic clearance rate demonstrates that the metabolism through the i n -duced pathways was at the expense of, and not in ad d i t i o n to, that through the normal pathways. The decrease i n the plasma l e v e l s of the st e r o i d therefore, did not d i r e c t l y r e s u l t from induced metabolism. 1 1 4 2. I n f e r r . i l i c y and Decreased L i t t e r S i z e I t was found that d i e l d r i n a l s o impaired f e r t i l i t y and decreased the number of pups born. I n f e r t i l i t y r e s u l t e d i n 18% of the bred females fed d i e t a r y concentrations of 10 and 1 5 ppm d i e l d r i n but i t d i d not occur i n ( s u r v i v i n g ) females fed other doses. In c o n t r a s t , the l i t t e r s i z e was re-duced i n a dose-dependent manner w i t h the maximum r e d u c t i o n of 1 7 % being caused by the highest dose, 25 ppm. A d d i t i o n a l r e s u l t s showed that both of these e f f e c t s were the consequence of one or more l e s i o n s which occurred during the period between the end of o v u l a t i o n and the beginning of the decidual response. I t must be considered p o s s i b l e that d i e l d r i n - i n d u c e d s t e r o i d d e f i c i e n c i e s might be the u l t i m a t e cause of these l e s i o n s , although t h i s i s improbable. During t h i s p e r i o d f i v e events take place: 1) t r a n s p o r t of the egg i n the oviduct towards the uterus; 2) f e r t i l i z a t i o n ; 3) t r a n s p o r t of the zygote i n the o v i d u c t ; 4) transport of the zygote i n the uterus; 5) changes i n the uterus and i n the b l a s t o c y s t i n p r e p a r a t i o n fo r i m p l a n t a t i o n . The s t e r o i d requirements of the mouse w i l l be b r i e f l y discussed f o r each of these f i v e events i n order to b e t t e r d e l i n e a t e where, and i f , d i e l d r i n - i n -duced s t e r o i d d e f i c i e n c i e s might be o p e r a t i n g . S u r v i v a l and t r a n s p o r t of the ( f e r t i l i z e d ) egg w i t h i n the o v i d u c t and uterus are not d i r e c t l y dependent upon the ovarian s t e r o i d s . This has been shown by Smithberg and Runner ( 1 9 5 6 ; 1 9 6 0 ) who s u c c e s s f u l l y induced i m p l a n t a t i o n of the embryos up to 15 days, a f t e r a b i l a t e r a l ovariectomy was 115 performed on day 1 of gestation (they used prepuberal mice induced to ovulate and mate with exogenous gonadotropins). S i m i l a r l y , ovarian hormones are not e s s e n t i a l for the s u r v i v a l of the blastocysts within the uterus. This has been demonstrated by Weitlauf and Greenwald (1968) who removed blastocysts from the uterus of females ovariectomized on day U, transfered them into pseudopregnant r e c i p i e n t s and found that a l l of the blastocysts which were recovered during the f i r s t 11 days a f t e r surgery developed into normal fetuses. Both of these pairs of in v e s t i g a t o r s have shown, however, that the post-implantation v i a b i l i t y of the embryo and fetus is d r a s t i c a l l y reduced when the pre-implantation period is allowed to extend beyond 15 days. The steroids required by the female reproductive t r a c t to permit successful insemination and f e r t i l i z a t i o n are not known for the mouse (Van Tienhoven, 1968. Chapt. 11). Steroids are probably important however, since i t has been shown i n the rabbit that c a p a c i t a t i o n of the sperm occurs at a rate dependent upon the reproductive stage of the uterus (Noyes, 1960). In the mouse progesterone i s e s s e n t i a l for the implantation of the blastocyst; the administration of estrogen alone to an ovariectomized, mated female r e s u l t s i n expulsion of the blastocysts from the vagina (Burdick and Pincus, 1935). The e s s e n t i a l i t y of progesterone in the intact animal is probably more d i r e c t l y a t t r i b u t a b l e to i t s e f f e c t s on the uterus than to i t s e f f e c t s on the zygote since the l a t t e r can remain v i a b l e for long periods without progesterone. In t h i s regard i t has been shown by Hethering-ton (1971) that when implantation is induced in ovariectomized, pregnant mice the weight of the decidua which forms is dependent upon the dose of progesterone used to maintain gestation. The r o l e of estrogen, long deemed to be e s s e n t i a l for implantation, is now c o n t r o v e r s i a l . It is well known that in mice which have been o v a r i -ectomized early in pregnancy and maintained on progesterone, the dormant blastocysts can be induced to implant with a si n g l e i n j e c t i o n of e s t r a d i o l (Smith, 1968) and that the number of implantations so induced is dependent upon the dose of e s t r a d i o l (Smith and Biggers, 1968). S i m i l a r l y , a s i n g l e dose of e s t r a d i o l w i l l terminate the delayed implantation which occurs i n pregnant, l a c t a t i n g mice bred during the post-partum estrus (Whitten, 1955). However, the absolute e s s e n t i a l i t y of estrogen for implantation is refuted by the data of Smithberg and Runner (1956; 1960), Yoshinaga and Adams (1966) and McLaren (1971) a l l of whom induced implantation i n mice ovariectomized early i n pregnancy by the administration of progesterone alone. S i m i l a r l y , the delayed implantation i n l a c t a t i n g mice was terminated x^ith progesterone alone by McCarthy (1965). The s i t u a t i o n i s further confused by recent work showing that shedding of the zona p e l l u c i d a , e s s e n t i a l for implantation, i s estrogen dependent i n ovariectomized, pregnant mice (Bergstrom, 1972). The sol v i n g of t h i s controversy necessitates the s t i l l to be attained q u a n t i f i -c ation of the endogenous steroids during e a r l y pregnancy. The pertinent data on the hormonal induction of the uterine condi-tions necessary for implantation have been reviewed by DeFeo (1967). He has found that the data regarding the e s s e n t i a l i t y of estrogen for this are also inconclusive. Despite these controversies regarding the e s s e n t i a l i t y of estrogen i t is generally believed that implantation is achieved i n the i n t a c t animal v i a the e f f e c t s of progesterone, secreted for 4 days, and estrogen, secreted as a surge some 12 hours p r i o r to n i d a t i o n (see McLaren, 1971 for the p e r t i -nent arguments). 117 Therefore, i f d i e l d r i n decreases the l i t t e r s i z e by decreasing the t i t r e s of endogenous steroids i t seems that estrogen is the compound most probably a f f e c t e d . The implantation rate is known to be proportional to the l e v e l of estrogen and the fact that most of the blastocysts did implant suggests that the progesterone t i t r e was s u f f i c i e n t for the normal pre-nidation development of the uterus to occur. The i n f e r t i l i t y caused by d i e l d r i n could however, r e s u l t from d e f i c i e n c i e s of either s t e r o i d . It must be emphasized, however, that the actual cause of the i n f e r -t i l i t y and reduced l i t t e r s i z e may involve mechanisms other than enhanced st e r o i d metabolism. For example, d i e l d r i n may have a d i r e c t toxic e f f e c t on the gametes or on the zygote. In support of this Hathaway et a l . (1967) have shown that d i e l d r i n administered to the rabbit is r a p i d l y secreted from the uterine epithelium and is immediately concentrated i n both free and attached blastocysts. It seems that t h i s mechanism is more l i k e l y to be the cause of the decreased f e r t i l i t y than i s a reduction i n the s t e r o i d l e v e l s since i t more r e a d i l y explains the decrease i n the number of males de-l i v e r e d by exposed dams which was found to be associated with the decline i n l i t t e r s i z e . Another of the more probable mechanisms would involve the blocking of the binding of the sex steroids to t h e i r s p e c i f i c p rotein re-ceptors within the reproductive t r a c t or the ce n t r a l nervous system. Wakeling and Visek (1973) have found that both d i e l d r i n and o,p'-DDT block the _in v i t r o binding of 50c-dihydrotestosterone to the 3.5S cytosol recep-tor of the rat prostate gland; the k i n e t i c s were c h a r a c t e r i s t i c of noncom-p e t i t i v e i n h i b i t i o n although the exclusion of competitive i n h i b i t i o n was not possible. The work of Smith et a l . (1972) who obtained a decrease in 3 the amount of H-testosterone bound in the prostate gland of mice o r a l l y 113 dosed with technical DDT, shows that such a mechanism may occur _in vivo. The r e s u l t s of this study have shown that several of the reproductive parameters measured in female mice fed d i e l d r i n were unaffected by this treatment. S p e c i f i c a l l y , the sexual r e c e p t i v i t y , the frequency of pregnan-ci e s s u c c e s s f u l l y maintained to term, the (lack of) f e t a l mortality, the length of the gestation period, and the onset and duration of p a r t u r i t i o n were a l l normal i n the d i e l d r i n - t r e a t e d animals. It follows from t h i s , that the biochemical and p h y s i o l o g i c a l factors involved i n the processes underlying these parameters were not pathologic; these would include of course, the concentrations of endogenous estrogens and progesterone. The sex steroids required for the events of early, middle and l a t e pregnancy have already been discussed above and l i t t l e would be gained from a d e t a i l e d consideration of the requirements of the other processes. It s u f f i c e s to say that in i n t a c t mice the behavioural estrus seems to r e s u l t from a post-ovulatory surge of progesterone which synergizes with the estrogens secreted during diestrus and proestrus (Ring, 1944; Young, 1961; Edwards, 1970), while normal p a r t u r i t i o n requires both estrogens and progesterone ( H a l l , 1957; Kroc et a l . , 1959). In this study d i e l d r i n ' s lack of e f f e c t on the behavioural estrus of the i n f e r t i l e females is p a r t i c u l a r l y relevant. It suggests that the l e s i o n between ovulation and implantation which res u l t e d in i n f e r t i l i t y was not a consequence of a reduction i n the estrogen or progesterone l e v e l s . I t seems improbable that adequate le v e l s of these steroids could be maintained through-out a cycle which culminates i n breeding but that they could not be main-tained over the next few days to e f f e c t implantation. In concluding i t is suggested that the re s u l t s and the l i t e r a t u r e 119 indicate that the le v e l s of progesterone and estrogen i n reproducing SWV female mice fed inducing doses of d i e l d r i n are not decreased at any time. F i r s t , the reduction in pup v i a b i l i t y was not due to a decrease in gesta-t i o n a l progesterone l e v e l s . Second, there was no e f f e c t on the other gesta-t i o n a l parameters which are s o l e l y dependent upon progesterone. Third, several parameters based on processes dependent upon both progesterone and estrogen were unaffected. F i n a l l y , those effected parameters which are based on processes dependent upon both progesterone and estrogen are e x p l i -cable i n terms of enhanced metabolism of estrogen alone but such increased metabolism seems u n l i k e l y . Moreover, a l l the e f f e c t s can be more r e a d i l y explained by other mechanisms. 3. Pup M o r t a l i t y D i e l d r i n i n the d i e t of the female, before and during a l l reproduc-t i v e processes, decreased the s u r v i v a l of the pups from b i r t h to weaning. S t a t i s t i c a l a n alysis of the r e s u l t s showed that the lowest dose e f f e c t i v e i n causing t h i s phenomenon l i e s between 2 . 5 and 5 . 0 ppm; however, an increased sample s i z e would probably f i n d the lowest e f f e c t i v e dose i s a c t u a l l y below 2 . 5 ppm. The lowest dose which caused 1 0 0 7 „ pup loss was found to l i e be-tween 5 . 0 and 1 0 . 0 ppm. As was discussed previously, the l i t e r a t u r e shows that pre-weaning m o r t a l i t y of the pups is the most important l e s i o n caused by a l l the organochlorine i n s e c t i c i d e s . Pup loss was found to be an " a l l or none" phenomenon. Females fed dietary concentrations of 2 . 5 or 5 ppm, doses below that which resulted in 1 0 0 7 o pup loss, either l o s t a l l th e i r pups or else weaned a number not d i f f e r -ent from that weaned by control animals. In addition, the growth of the pups 120 in these treatment l i t t e r s where pups survived to weaning was not d i f f e r e n t from the growth of pups raised by control dams. However, these treatment pups were not e n t i r e l y i d e n t i c a l to the control pups since the a c t i v i t y of the mixed-function oxidase system within t h e i r l i v e r s was greatly increased. Darby (1971) found a s i m i l a r s i t u a t i o n when he administered phenobarbital to the dams of suckling r a t s . I would now l i k e to discuss those factors which this study i d e n t i -f i e d as important in the dieldrin-induced pup m o r t a l i t y . In general terms the factors are of two types, pre-partum and post-partum. Pre-partum factors are those which influence the embryo or fetus _in utero i n such a manner that i t s inate a b i l i t y to survive a f t e r b i r t h i s decreased. Post-partum factors are those which a f f e c t the pup a f t e r b i r t h . It w i l l become apparent that these two sets of factors may i n t e r a c t i n a very complex manner. During the following d i s c u s s i o n i t should be borne i n mind that the pups which died not only weighed less at b i r t h than those which l i v e d but they also f a i l e d to grow p r i o r to death. These phenomena could r e s u l t from any or a l l of the possible mechanisms which w i l l be discussed; they w i l l , therefore, be used to support none. S i m i l a r l y , i n dams fed 2.5 „or 5.0 ppm d i e l d r i n the c o r r e l a t i o n s between maternal hepatomegaly and s u r v i v a l of the l i t t e r s , while i n t e r e s t i n g and useful for future i n v e s t i g a t i o n s , i s not i n -d i c a t i v e of any p a r t i c u l a r mechanism and i t also w i l l be ignored. D i e l d r i n , i n the d i e t of the dam, was found to decrease the inate v i a b i l i t y of the pups and the threshold dose for this e f f e c t was less than 5 ppm. To decrease v i a b i l i t y i t is axiomatic that d i e l d r i n must be interfe-r i n g with p h y s i o l o g i c a l and biochemical prosesses wi t h i n the o f f s p r i n g . Since d i e l d r i n i s known to cross the mouse placenta (Backstrom et a l . , 1965) 121 this interference may be a d i r e c t e f f e c t . It may, however, also be i n d i r e c t , acting through some disturbance of the females gestational metabolism which i n turn impairs development of the embryo or fetus. Regardless of the directness of the path, the number of processes which might be involved is extremely large and most may be found i n Hodge et a l . (1967) who have re-v. viewed much of the t o x i c o l o g i c a l l i t e r a t u r e on d i e l d r i n . Whatever the mechanism(s) that are operating i t is c l e a r that a reduction in the l e v e l of gestational progesterone is not involved. Moreover, the l i s t l e s s n e s s and i n a c t i v i t y shown immediately a f t e r b i r t h by the pups which died, indicates that stimulation of the c e n t r a l nervous system is also not involved. This is i n contrast to the s i t u a t i o n i n adult mammals where the acute mortality is a d i r e c t consequence of neural h y p e r a c t i v i t y in the hypothalamus and vagus (Hodge et a l . , 1967; Gowdey et a l . , 1954; Gowdey and Stavraky,. 1955). Virgo (1970) working with rats and Harris et a l . (1966) working with sheep have a l s o found that d i e l d r i n administered to the dam r e s u l t e d i n a reduction of the inate v i a b i l i t y of the young. Eighty per cent of the pups born to treated females and then f o s t e r -nursed by control dams died. This shows that the induced i n v i a b i l i t y i s b a s i c a l l y i r r e v e r s i b l e and demonstrates the importance of the pre-partum e f f e c t ( s ) . However, post-partum e f f e c t s were also found and these i n t e r -acted with the i n v i a b i l i t y to hasten the mortality of the pups. At the highest doses marked changes i n maternal behaviour occurred so that i n f a n t i c i d e and gross neglect of the pups were the proximate causes of a l l the mortality. More subtle behavioural changes were found at the lower doses where the dam spent less time i n the nest with the pups and, a f t e r returning to the nest, i t required more time to begin nursing. A l l 122 these changes were associated with and appeared to r e s u l t from (uncontrol-lable) h y p e r a c t i v i t y . Such h y p e r a c t i v i t y might have a r i s e n from CNS stimu-l a t i o n by d i e l d r i n . Since these dams had not shown h y p e r a c t i v i t y p r i o r to p a r t u r i t i o n i t is suggested that progesterone, which at high doses possesses general anesthetic properties, may depress the CNS neurons i n such a way that following i t s removal the s e n s i t i v i t y of the neurons to sti m u l a t i o n i s potentiated. However, maternal behaviour i s mediated by the sex steroids and these behavioural changes may r e f l e c t an enhancement of progesterone metabo-l i s m i n the post-partum mouse. Davis and Gandelman (1972) have shown that i n mice e x h i b i t i n g normal maternal behaviour an i n j e c t i o n of testosterone w i l l induce them to k i l l t h e i r pups. S i m i l a r l y , injected e s t r a d i o l induces the neglect of and/or the k i l l i n g of the pups i n mice which previously had shown normal maternal behaviour (Gandelman, 1973). Hydroxylated metabolites of progesterone, normally produced by enzyme systems wi t h i n the ovarian microsomes and which require NADPH, molecular oxygen and cytochrome P-450 (Abraham,1971; McMurty and Hagerman, 1972), are the d i r e c t metabolic pre-cursors of a l l the major androgens and the estrogen, e s t r i o l . The androgens are, i n turn, the precursors of the other two major estrogens, estrone and e s t r a d i o l (Abraham, 1971). In the post-partum mouse an increase i n the production of hydroxylated progesterone by the induced, hepatic mixed-function oxidase system could, by increasing the amount of precursor a v a i l -able for conversion, elevate the l e v e l s of androgens and/or estrogens and thereby cause the aberrant behaviour shown by the dam. The pups which survived for several days c h a r a c t e r i s t i c a l l y contained l i t t l e or no milk. However, the mill; production of d i e l d r i n - t r e a t e d dams 123 was found to be the same as that of untreated dams thus showing that t h i s lack of milk was not due to an e f f e c t of d i e l d r i n which reduced the a b i l i t y of the mammary gland to synthesize milk. Gannon et a l . (1959) and Gannon and Decker (1960) have found that d i e l d r i n and i t s metabolic precursor, a l d r i n , do not a f f e c t milk production in the dairy cow. Deichmann et a l . (1971), on the other hand, claim that d i e l d r i n administered to the beagle i n h i b i t e d the development of the mammary gland and decreased, or eliminated, i t s production of milk. The treated females produced the same amount of milk as the control females and t h i s implies that the development of the mammary glands i n the treated females was the same as that i n the co n t r o l s . This further supports the (previously discussed) view that d i e l d r i n did not a l t e r the l e v e l s of estrogens or progesterone during the estrous cycle or gestation. Both of these steroids are necessary for the normal development of the mammary glands; estrogen for the extension of the duct system and progesterone for the pro-l i f e r a t i o n of the secretory a l v e o l a r epithelium (Cowie and Folley, 1961; Anderson et a l . , 1961; Anderson and Turner, 1963). While I was unable to exclude the p o s s i b i l i t y that d i e l d r i n may decrease the a v a i l a b i l i t y of the milk to the pups by blocking the milk ejec-t i o n r e f l e x t h i s p o s s i b i l i t y seems u n l i k e l y . Atropine administered systemi-c a l l y to l a c t a t i n g mice has been shown to block the suckling-induced release of hypophyseal oxytocin (Oba, 1971) and this implies that at least some of the c o n t r o l l i n g neural pathways are c h o l i n e r g i c . Since d i e l d r i n ' s e f f e c t s are predominantly c h o l i n e r g i c (Gowdey et a l . , 1954; Gowdey and Stavrady, 1955) i t seems more l i k e l y that i t would a c t u a l l y f a c i l i t a t e the release of oxytocin and hence the release of milk. 124 It is well established that the organochlorine i n s e c t i c i d e s and th e i r metabolites are excreted i n the milk of many mammalian species (Henderson, 1965; Saha, 1969; Ottoboni and Ferguson, 1969). The concentra-t i o n i n the milk is frequently much higher than that i n the dam's feed; Harr et a l . (1970), for example, found that the l e v e l of d i e l d r i n i n milk curds taken from the stomachs of nursing rats was concentrated by up to 17-f o l d . This has led some workers (e.g. Morris, 1968) to suggest that the insecticide-induced pup mortality may r e s u l t from some acute e f f e c t of the i n s e c t i c i d e which had been ingested with the milk. In the current study the pups which died did not show the convulsions or tremoring t y p i c a l of acute organochlorine i n s e c t i c i d e t o x i c i t y and th i s indicated that such a mechan-ism was not operating here. The general i n s e n s i t i v i t y of young mammals to CNS i n t o x i c a t i o n supports t h i s view. Lu et a l . (1965) have shown that i n the rat the acute LD50 for d i e l d r i n i s 168 mg/kg for a pup at day 1 of l i f e , and t h i s i s 4.5-fold greater than the LD50 for adults which i s 37 mg/kg; s i m i l a r r e s u l t s were obtained with DDT. The r e l a t i v e i n s e n s i t i v i t y of the ra t pup to DDT i s due p a r t l y to an inate unresponsiveness of the CNS and p a r t l y to a lack of c e r t a i n l i p i d s which are necessary for the absorption of toxic doses from the plasma (Henderson and Woolley, 1969). It has been i m p l i c i t l y assumed i n the preceding d i s c u s s i o n that d i e l d r i n , i n exerting i t s e f f e c t s on reproduction, did not act on the males. The males were, however, exposed to the same dieldrin-contaminated rations as the females when the two sexes were housed together during the 2 week mating period. In the female such an exposure period was s u f f i c i e n t to cause maximal increases in l i v e r weight, microsomal protein and LMI-hydroxy-l a t i o n . As s i m i l a r e f f e c t s might be expected i n the male the p o s s i b i l i t y of 125 dieldrin-induced male e f f e c t s on the reproduction parameters must be con-sidered. The p o s s i b i l i t y that d i e l d r i n impaired the f e r t i l i t y of the male may be discarded since a l l those males which bred females that did not become pregnant also bred at least two other females, during the same mating period, which did. The decrease i n the l i t t e r s i z e , which was caused by a los s between ovulation and implantation, could have resulted from (the t e s t i s of the d i e l d r i n - t r e a t e d males producing an increased number of) defective sperm which might not have f e r t i l i z e d the egg or which might have caused i t s pre-implantation death. It i s u n l i k e l y , however, that pre-implantation death would r e s u l t from the sperm since i n rabbits which have been mated by heat-stressed ( i . e . i n f e r t i l e ) males, pre-implantation m o r t a l i t y of the egg occurs only during the period when the sperm are so i n v i a b l e that no young at a l l are produced (Howarth et a l . , 1965; Burfening and Uirberg, 1968). Similar r e s u l t s have been found for the mouse by Burfening et a l . (1970). The decrease i n the v i a b i l i t y of the pups could also have res u l t e d from an e f f e c t of d i e l d r i n on the male. In t h i s regard i t has been shown by Wijerat ne et a l . (1970) and by Rahnefield and Sweirstra (1970) that the s i r e has a small but s i g n i f i c a n t e f f e c t on pre-weaning m o r t a l i t y i n swine. Some investigators have implicated organochlorine i n s e c t i c i d e s i n apparent disturbances of male reproductive function. Huber (1965) found that the l i t t e r s i z e of c o n t r o l female mice bred by Kepone-treated males was decreased, although a h i s t o l o g i c a l examination of the testes revealed no apparent abnormalities in spermatogenesis or i n the i n t e r s t i t i a l c e l l s . In man, c l i n i c a l impotence developed in several farmworkers using d i e l d r i n (amongst 16 other chemical pesticides) and abated a f t e r 2-12 months of 126 therapy with methyl-testosterone (Espir et a l . , 1970). The data of Ambrose et a l . (1953) provide evidence for an i n t e r e s t i n g s i t u a t i o n where an i n t e r -a c t i o n e f f e c t occurs between exposed males and exposed females. They found that male rats fed chlordane for 1 month and then caged with control females s i r e d the same number of l i t t e r s as did the co n t r o l males. Analogous re-s u l t s were obtained when the same numbers of exposed and unexposed females deli v e r e d pups a f t e r being caged with control males. However, when both sexes were pre-exposed and then caged together the number of l i t t e r s which was produced decreased. In contrast to these r e s u l t s , Virgo (1970) found that the f e r t i l i t y , l i t t e r s i z e and pup s u r v i v a l i n untreated female rats was not af f e c t e d by breeding them with d i e l d r i n - f e d males. C. The L i v e r and i t s Functions: D i e l d r i n ' s E f f e c t s : 1. L i v e r Weight and Microsomal P r o t e i n The r e s u l t s show that d i e t a r y d i e l d r i n causes an enlargement of the l i v e r i n female SWV. mice. The "no - e f f e c t " dose i s below 5 ppm, the lowest dose tested. Both the l i v e r ' s rate of enlargement and i t s f i n a l s i z e were found to be dose-dependent although maximal responses to a l l doses were attained by 2 weeks of exposure. Maximal responses, which were maintained for the duration of exposure, were 30% and 62%, above con t r o l values at 10 ppm and 20 ppm r e s p e c t i v e l y . The maximally enlarged l i v e r regressed to control s i z e i n treated animals taken o f f the dieldrin-contaminated feed and fed the control r a t i o n for 4 weeks. These responses are almost iden-t i c a l to those of Wright et a l . (1972) who worked with male CF1 mice and who have- published the only other data on the hepatic e f f e c t s of d i e l d r i n in t h i s species. They found that 10 ppm caused a maximal increase of 30%,-127 407o i n l i v e r weight, which was attained between 1 and 4 weeks of exposure and then maintained for 1 year. One ppm, the only other dose they used, was i n e f f e c t i v e . Wright et a l . (op c i t ) compared these hepatic responses with those they obtained for the rat, the beagle and the Rhesus monkey, and with those previously published for the rat, and found that the l i v e r of the mouse i s much more susceptible to dieldrin-induced enlargement than is that of the other species. The concentration of microsomal protein i n the l i v e r was found to be increased by d i e l d r i n . The increase was not marked however, nor was i t dose-dependent over the range used. The maximal response to a l l doses was a 307<>-35% increase above con t r o l values and t h i s was also achieved by 2 weeks of exposure and maintained thereafter. Mice, with maximally induced concentrations of hepatic microsomal p r o t e i n and which were taken o f f the d i e l d r i n contaminated rations and fed the control r a t i o n , had c o n t r o l l e v e l s of microsomal p r o t e i n i n t h e i r l i v e r s when k i l l e d 4 weeks l a t e r . Wright et ' a l . (bp c i t ) found that the maximum increase in the hepatic microsomal protein of CF1 males fed 10 ppm dietary d i e l d r i n was not attained u n t i l 4 weeks (or l a t e r ) of exposure but the magnitude of the increase was, however, greater, being 507<, above control l e v e l s . This may ind i c a t e a s t r a i n d i f f e r -ence in response since i n the SWV s t r a i n the male was found to be even less responsive than the female. Wright et a l . (op c i t ) also found that the dose which caused maximum responses was r e l a t i v e l y low; they increased the dose from 10 ppm to 50 ppm but did not find a further increase i n the con-centration of microsomal protein. They found, as did the present study, that regression of the induced microsomal protein could be achieved by feed-ing the animal for 4 weeks with non-contaminated food; t h i s was a faster 123 rate than could be attained i n the dog but was slower than that attained i n the rat. Wright et a l . (op c i t ) compared the i r r e s u l t s for the mouse with those for the rat, dog and monkey and found the hepatic microsomal protein of the mouse to be the least susceptible to inductive increases. This was p a r a l l e l e d by a v i s i b l y smaller hypertrophy of the SER in mouse hepatocytes as evidenced i n electron micrographs. In SWV female mice fed 15 ppm or 20 ppm d i e l d r i n the weight of the l i v e r increased d i s p r o p o r t i o n a t e l y to i t s concentration of microsomal pro-tein. One possible i n t e r p r e t a t i o n of th i s fact would be that hyperplasia of the hepatocytes occurred. Wright et a l . (cp c i t ) have also suggested that t h i s occurs i n mice fed d i e l d r i n since they found that the increase i n l i v e r weight was 507» greater than the increase i n the microsomal protein/DNA r a t i o , and also that the DNA content of the l i v e r was increased a f t e r 7 days of exposure. In contrast, the rat requires prolonged exposure to inducing xenobiotics to increase the number of mito t i c d i v i s i o n s and the amount of hepatic DNA (Conney, 1967). It is current opinion that l i v e r enlargement, per se, i s not neces-s a r i l y i n d i c a t i v e of hepatoxicity; rather, i n the absence of other t o x i c i t y symptoms, i t i s considered to be adaptive (Goldberg, 1966; Hutterer et a l . , 1969). In t h i s study gross ( i . e . acute) d i e l d r i n t o x i c i t y was found only at doses of 20 ppm and above suggesting that the l i v e r changes caused by the lower doses were not pathologic. This suggestion would have to be con-firmed by studies on such more s e n s i t i v e indicators of hepatic t o x i c i t y as l i v e r glucose-6-phosphatase or serum glutamic-oxalic transaminase. Wright et a l . (1972) found the former enzyme was not affe c t e d by feeding 10 ppn d i e l d r i n to mice. 129 Hepatomas were not observed in any of the animals which were examined and confirms previous work which shows that they occur only a f t e r about 1 year of continuous exposure to organochlorine i n s e c t i c i d e s (e.g. a l d r i n : Davis and Fitzhugh, 1962. DDT: Tarja'n and Kemeny, 1969; Stevenson and Walker, 1969). Add i t i o n a l work on the hepatic c a r c i n o g e n i c i t y of d i e l d r i n and other chemicals which induce l i v e r enzymes has been recently published by Walker et a l . (1973) and by Thorpe and Walker (1973). The l a t t e r authors review the d i v e r s i t y of factors known to cause hepatomas in the mouse and discuss the current views on the t o x i c o l o g i c a l s i g n i f i c a n c e of chemically-induced tumours in this species. 2. Pentobarbital Metabolism: rn vivo D i e l d r i n caused a 4307o increase i n the in vivo, metabolism of pento-b a r b i t a l . This large and maximal increase was caused by the lowest, d i e t a r y l e v e l tested, 5 ppm, which would have r e s u l t e d i n an average 25 g mouse re c e i v i n g an approximate d a i l y dose of 1 mg/kg. Since i n inductive respon-ses there are well known differences between species, s t r a i n s , sexes and ages (Conney, 1967; Cram et a l . , 1965; Turner et a l . , 1969; Hospador et a l . , 1971;Joriet a l . , 1971; Kato et a l . , 1970; Yaffe et a l . , 1968) which are further modified by environmental factors ( V e s e l l , 1963; V e s e l l et a l . , 1973) i t is v i r t u a l l y impossible to compare these r e s u l t s with others. They do, however, appear to be compatible with the previous data which show that the cyclodienes and t h e i r anologs are probably the most potent inducers of the mixed-function oxidase system in mice. Hart and Fouts (1963) found that a s i n g l e i n j e c t i o n of endrin, d i e l d r i n ' s stereoisomer, at a dose of 6.25 mg/kg, increased the _in vivo metabolism of hexobarbital in CF1 mice L30 by 2557» 24 hours l a t e r ; chlordane, at 25 mg/kg, only increased i t by 14970. This is p a r t i c u l a r l y s i g n i f i c a n t since chlordane, a cyclodiene anolog, is i t s e l f a very potent inducer. For example, Cram and Fouts (1967) found that 50 mg/kg injected into male CF1 mice on 3 consecutive days increased i n vivo hexobarbital metabolism by 8607o; the same treatment resulted i n only a 3807, increase in hexobarbital metabolism in Swiss-Webster males which i l l u s t r a t e s the magnitude of the s t r a i n d ifferences which e x i s t . Cram and Fouts (op c i t ) found that 3 consecutive d a i l y i n j e c t i o n s of DDT (100 mg/kg) were i n e f f e c t i v e i n stimulating hexobarbital metabolism i n eit h e r s t r a i n , although t h i s treatment increased i t by 2507„ i n adult male Sprague-Dawley rats (Hart and Fouts, 1963). Abernathy et a l . (1971), who used a d i f f e r e n t s t r a i n of mice, found that 3 consecutive d a i l y i n j e c t i o n s of DDT (100 mg/kg) increased hexobarbital metabolism by 1807» but this was a considerably smaller response than the 3407» increase caused by the same dose of pheno-b a r b i t a l . The maximal stimulation of pentobarbital metabolism by d i e l d r i n was not l i m i t e d by the t o t a l amount of eit h e r cytochrome P-450 or microsomal p r o t e i n i n the animals. This is hardly s u r p r i s i n g as i t is frequently impossible to r e l a t e _in vivo metabolism to the biochemical parameters of l i v e r preparations ( G i l l e t t e et a l . , 1972). Some of the factors i n f l u e n c i n g i n vivo drug metabolism have been discussed by G i l l e t t e (1971). 3. Imipramine Metabolism i n v i t r o The studies of Bickel and his associates (Bickel, 1972a; B i c k e l , 1972b; Bi c k e l et a l . , 1968) have elucidated the _in v i t r o metabolism of IMI by the hepatic microsomes of the rat, the pig, the Guinea pig and the horse. 131 They have found that the NADPIi- and 07-dependent enzyme system which cata-lyzes the N-demethylation and ary1-hydroxylation reactions can be d i f f e r e n -t i a t e d , in several ways, from the NADPH- and 09-dependent system which cata-lyzes the N-oxidation reaction. For example, SKF-525A i n h i b i t s both the demethylation and hydroxylation reactions but does not i n h i b i t N-oxidation, while neotetrazolium i n h i b i t s the N-oxidation r e a c t i o n but does not i n h i b i t demethylation or hydroxylation. Moreover, i n the presence of O2 and N^, CO i n h i b i t s both the demethylation and hydroxylation reactions but does not i n h i b i t the N-oxidation react i o n . It has been concluded from these, and s i m i l a r observations, that both desmethylimipramine and 2-hydroxy-imipramine are formed by an enzyme system which is dependent upon cytochrome P-450 but that imipramine N-oxide i s formed by another enzyme system, not dependent upon cytochrome P-450. An oxidase s p e c i f i c for amines has been i s o l a t e d from porcine hepatic microsomes by Zieger et a l . (1969), who have shown that this f l a v o p r o t e i n w i l l metabolize Imipramine and a large number of other amines. In a l l cases where the.metabolite of a t e r t i a r y amine was i d e n t i f i e d i t was found to be the N-oxide suggesting that the imipramine metabolite, which they did attempt to i s o l a t e , was probably also the N-oxide. Masters and Z e i g l e r (1971) have shown that t h i s , mixed-function, amine oxidase is not the same f l a v o p r o t e i n as the NADPH-cytochrome c reductase which i s involved i n the P-450 system. The a c t i v i t y of the N-oxidation pathway i n the hepatic microsomes of the SWV female mice used here was not dependent upon the concentration of P-450 cytochrome, t h i s was indicated by the comparative e f f e c t s of age and d i e l d r i n induction. The a c t i v i t y of the N-oxidation pathway was found to increase with age while age had no a f f e c t on the concentration of cytochrome 132 P-450. S i m i l a r l y , d i e l d r i n induced large increases in the concentration of cytochrome P-450 but the a c t i v i t y of the N-oxidation pathway was not i n -creased. In a d d i t i o n to the age-dependent increase in the a c t i v i t y of the N-oxidation pathway, the hepatic microsomes prepared from the females also exhibited a concomitant increase i n the a c t i v i t y of the hydroxylation path-way and a decrease i n the a c t i v i t y of the demethylation pathway. The hepatic microsomes of the older males, i n contrast, showed an increase i n the ac-t i v i t y of the demethylation system and a decrease i n the a c t i v i t y of the hydroxylation system while the a c t i v i t y of the N-oxidation system was un-changed. Such a sex d i f f e r e n c e has not been previously reported for the metabolism of imipramine. Age e f f e c t s have been described by Das and Z i e g l e r (1970) who found that the a c t i v i t y of the N-oxidation system which metabolized N,N-dimethylaniline increased as the r a t (male: Houston-Cheek) aged from 13-22 weeks while the a c t i v i t y of the N-demethylation system de-creased over t h i s period. The d i e l d r i n treatment did not increase the a c t i v i t y of the N-oxidation pathway; in fact, i t decreased with such treatment, perhaps as a consequence of the increased a c t i v i t y i n the demethylation and hydroxylation pathways or perhaps as a r e s u l t of a dieldrin-mediated i n h i b i t i o n of the N-oxidation system. Nakazawa (1970) likewise found that hepatic microsomes prepared from phenobarbital treated rats did not exhibit increases in the a c t i v i t y of the imipramine N-oxidation pathway. D i e l d r i n was found to increase the a c t i v i t y of the cytochrome P-450 dependent system which metabolizes IMI to DMI and hydroxy-IMI. However, the inductive responses were temporally d i s t i n c t with the increase in hydroxy-133 l a t i o n occurring immediately and the increase in demethylation not occurring for some weeks. Moreover, in microsomes prepared from co n t r o l animals the responses to age were q u a n t i t a t i v e l y d i s t i n c t ; demethylation decreased with age while hydroxylation increased. It would appear that the demethylation and hydroxylation pathways may have d i f f e r e n t components and/or rate-l i m i t i n g steps. Nakazawa (1970), who found the r e l a t i v e production of DMI and hydroxy-IMI to vary as a function of the Mg ~ concentration and pheno-b a r b i t a l pretreatment, also suggests these metabolites may be produced by d i f f e r e n t enzyme systems. A v a r i e t y of other hepatic microsomal oxidative enzymes are also increased i n a c t i v i t y by d i e l d r i n pre-treatment. Wright et a l . (1972) found i t increased the a c t i v i t y of the chlorfenvinphos O-de-ethylase i n mice, r a t , dogs and monkeys. The a n i l i n e hydroxylase system is stimulated by d i e l d r i n i n Guinea pigs (Wagstaff and Street, 1971) and i n rats (Hutter et a l . , 1969; Stevens et a l . , 1973). In rats various expoxidases are found to be stimulated ( G i l l e t t and Chan, 1963) as i s ethylmorphine N-de-ethylase (Stevens et a l . , 1973). The die l d r i n - i n d u c e d a c t i v i t y of the IMI hydroxylase ancS demethylase system(s) was absent 6 weeks a f t e r the cessation of d i e l d r i n treatment. This agrees with the complete regression of the dieldrin-induced a c t i v i t y of the chlorfenvinphos O-de-ethylase system in male CF1 mice not exposed for 6 weeks (Wright et a l . , 1972). Dieldrin-induced increases i n enzymatic activ-i t y do not appear to be permanent i n t h i s species. In contrast, Wright et a l . (op c i t ) have found that regression in the dog is so slow that inductive changes can almost be considered permanent in that species. The a c t i v i t i e s of the microsomal systems which demethylated or 134 hydroxylated IMI did not c o r r e l a t e with the concentration of cytochrome P-450 in the microsomes. This applied to hepatic microsomes prepared from both control and d i e l d r i n - f e d mice. Such a lack of c o r r e l a t i o n implies that the concentration of cytochrome P-450 is not rate-1imiting i n these reac-tions — an implication which is in accord with the current views on the reg u l a t i o n of the cytochrome P-450 redox system and which is probably true. However, i t must be viewed with some caution for i t is possible that the concentration of cytochrome P-450 may have been rat e - 1 i m i t i n g i n these studies but that t h i s s i t u a t i o n was not detectable for the reason discussed next. Stevens et a l . (1973) have found that d i e l d r i n binds very r a p i d l y and very t i g h t l y to the cytochrome P-450 of r a t hepatocyte microsomes and thereby i n h i b i t s the epoxidation of i t s precursor, a l d r i n ( G i l l e t t e and Chan, 1968). I t may be in f e r r e d from the magnitude of the s p e c t r a l change which occurs when d i e l d r i n i s added to suspensions of mouse hepatocyte microsomes (Mailman and Hodgson, 1972) that d i e l d r i n binds s i m i l a r l y to mouse microsomes. Baldwin, et a l . (1972) have found that chronic exposure of the mouse to HEOD ( d i e l d r i n ) does not stimulate the metabolism of a single, high dose of ^ C-HEOD i n d i c a t i n g that d i e l d r i n is not metabolized to any appreciable extent by cytochrome P-450 in th i s species. These data suggest, therefore, that d i e l d r i n may i n h i b i t oxidative reactions, including the demethylation and hydroxylation of IMI, in mouse l i v e r microsomes by binding with the cytochrome. Under these conditions the concentration of unbound cytochrome P-450 might well be rate - 1 i m i t i n g i n the metabolism of IMI. The method for quantitating cytochrome P-450 measures i t a l l , bound and unbound (Estabrook et a l . , 1973a), and i t is therefore impossible to 135 exclude this p o s s i b i l i t y . That the concentration of cytochrome P-450 is not r a t e - l i m i t i n g in the demethylation and hydroxylation of IMI is i n accord with the view of G i l l e t t e and Gram (1969). They, for reasons analogous to that used here, have rejected the older view whereby the l i m i t i n g step i n drug or s t e r o i d metabolism was believed to be the d i s s o c i a t i o n of the cytochrome P-450 — product complex, i . e . the amount of cytochrome P-450 which i s a v a i l a b l e to the substrate. This i s Step 6 i n Figure 16, which shows a cu r r e n t l y accepted model of the P-450 redox system. They (Gigon et a l . , 1969) found that the diffe r e n c e s i n the rates of reduction of cytochrome P-450 i n the presence and absence of a substrate d i f f e r e d by the same order of magnitude as the rate of substrate metabolism. They suggested therefore, that the o v e r a l l rate of metabolism i s l i m i t e d by the rate of reduction of the P-450-substrate complex (Step 2). This view i s now generally accepted although the mechan-isms which have been proposed to account for th i s step being r a t e - l i m i t i n g (e.g. d i f f e r e n t forms of cytochrome P-450 with inherently d i f f e r e n t rates of reduction; d e f i c i e n c i e s of NADPH or of NADPH cytochrome c reductase; etc.) are c o n t r o v e r s i a l and unclear. Estabrook et a l . (1973b) have recently suggested that the rate-l i m i t i n g step of cytochrome P-450 catalyzed oxidations i n the hepatic micro-somes of inducer-treated animals may a c t u a l l y be Step 4, the a d d i t i o n of the second electron. Their studies have produced s p e c t r a l evidence for the existence of the oxycytochrome P-450 "-substrate complex (Intermediate III in Figure 16) i n microsomal suspensions f o r t i f i e d with NADPH and O2. The existence of th i s complex i n detectable quantities is believed to be incom-p a t i b l e with the hypothesis that i n s e r t i o n of the f i r s t e l e c t r o n i s rate-.136 I I NADPH—Cytochrome e Reductase Cytochrome P—450° Substrate 1 Cytochrome P—450 + J Substrate Substrate I I I Cytochrome P—450** Product Cytochrome P—450 — 0 2 Substrate -OH Cytochrome P—450+ 3 V Product—OH • Cytochrome' P—450+—O IV Substrate ; Cytochrome b s ? NADPH Cyt c Reductase ? NADH—Flavoprotein ? F i g u r e 16. A model of the proposed mechanism of cytochrome P-450 c a t a l y z e d h y d r o x y l a t i o n of drugs and s t e r o i d s . Steps are l i s t e d i n A r a b i c numerals w h i l e the intermediates are l i s t e d i n Roman numerals (adapted from G i l l e t t e et a l . , 1972). • +3 Step 1. Cytochrome P-450 combines w i t h the s u b s t r a t e to form a c y t o -chrome P-450 +^-substrate complex. +3 Step 2. The cytochrome P-450 - s u b s t r a t e complex, i s reduced to the c y t o -chrome P-450 +2-substrate complex by an e l e c t r o n from NADPH cyt o -chrome c reductase and perhaps another f l a v o p r o t e i n . Step 3. The cytochrome P - 4 5 0 + 2 - s u b s t r a t e complex combines w i t h molecular oxygen to form the oxy-cytochrome P.-450 + 2-substrate complex. Step 4. The oxy-cytochrome P-450 -s u b s t r a t e complex i s f u r t h e r reduced by the a d d i t i o n of another e l e c t r o n from an unc l e a r source. Two protons enter the system. An e l e c t r o n s h i f t occurs r e s u l t i n g i n the l i b e r a t i o n of water and intermediate IV. Step 5. Intermediate IV undergoes a molecular rearrangement such that the oxygen atom i s t r a n s f e r e d to a saturate d carbon atom on the sub-s t r a t e ; the d i s p l a c e d proton i s r e t a i n e d on the oxygen atom thereby forming a hydroxyl group. The su b s t r a t e i s now the hydroxylated product and i s s t i l l complexed w i t h cytochrome P-450 +3 (which has been r e - o x i d i z e d to the f e r r i c s t a t e ) . +3 Step 6. The cytochrome P-450 -product complex d i s s o c i a t e . 137 l i m i t i n g . They, therefore, suggest that a d d i t i o n of the second e l e c t r o n is r a t e - l i m i t i n g . The r e s u l t s of the present study are also compatible with t h i s view. Confirmation of the hypothesis of Estabrook and his associates w i l l probably require further e l u c i d a t i o n of the actual mechanisms involved in the uptake of the second electron. These mechanisms are at present un-cl e a r and highly c o n t r o v e r s i a l . Current work suggests that i f the ele c t r o n comes from NADH then cytochrome br^  is the intermediate c a r r i e r whereas i f i t comes from NADPH then another, unknown, fl a v o p r o t e i n i s the c a r r i e r (Sasame et a l . , 1973; Correia and Mannering, 1973). It is not cl e a r i f one or both of these cofactors normally donate electrons at this step. 138 SUMMARY AND CONCLUSIONS Di e l d r i n , fed at dietary concentrations of 5-20 ppm to SWV female mice, causes a dose-dependent increase i n the quantity and a c t i v i t y of the hepatic mixed-function oxidase system dependent upon NADPH and cyto-chrome P-450. This is evidenced by the following e f f e c t s caused by d i e l d r i n : a. a large, dose-dependent hepatomegaly; b. a small, dose-independent increase i n the concentration of hepatic microsomal protein; c. a large, dose-dependent increase i n the concentration of microsomal cytochrome P-450; d. a large, dose-independent increase i n the i n vivo metabolism of pentobarbital; e. a moderate, dose-dependent increase in the _in v i t r o hydroxyla-t i o n and demethylation of imipramine. Preliminary data suggest that i n a d i e l d r i n - f e d SWV female both the weight of the l i v e r and i t s concentration of microsomal protein regress to c o n t r o l l e v e l s within 4 weeks of the animal being placed on the control r a t i o n . Preliminary data suggest that the l i v e r of the SWV male responds d i f f e r e n t l y to d i e l d r i n than does that of the female. D i e l d r i n , fed at dietary concentrations of 2.5-25 ppm to SWV female mice, does not: a. a f f e c t behavioural estrus; 139 b. cause i n t r a - u t e r i n e mortality of the embryo or fetus a f t e r day 4 of gestation; c. a f f e c t the length of the gestation period; d. a f f e c t the duration or ease of p a r t u r i t i o n . D i e l d r i n , fed at dietary concentrations of 10 and 15 ppm, causes i n f e r -t i l i t y in a small (187»), but s t a t i s t i c a l l y s i g n i f i c a n t proportion of the bred females. Lower and higher doses do not cause i n f e r t i l i t y in the surviving females. The i n f e r t i l i t y r e s u l t s from a l e s i o n which occurs a f t e r ovulation but before implantation. The i n f e r t i l i t y is without b i o l o g i c a l s i g n i f i c a n c e since at these doses no pups survive to weaning. D i e l d r i n , fed at dietary concentrations of 2.5-25 ppm to SWV female mice, causes a dose-dependent reduction i n the l i t t e r s i z e at b i r t h . The maximum reduction, which occurs at 25 ppm and is s t a t i s t i c a l l y s i g n i f i c a n t , is small, being only 177<>, and b i o l o g i c a l l y i n s i g n i f i c a n t . The reduction r e s u l t s from a l e s i o n which occurs a f t e r ovulation but before implantation. D i e l d r i n , fed i n the d i e t , causes the mortality of a l l the pups born to some females; this m o r t a l i t y occurs within 5 days of b i r t h . Pup s u r v i v a l is normal i n the l i t t e r s of d i e l d r i n - f e d females where not a l l the pups die. The lowest dietary concentration that causes a s i g -n i f i c a n t increase in the number of dams which lose t h e i r l i t t e r l i e s between 2.5 and 5 ppm. The concentration which causes a l l dams to lose t h e i r l i t t e r s l i e s between 5 and 10 ppm. This pup mortality is 140 the most important e f f e c t d i e l d r i n has on reproduction i n the SWV female mouse. 8. D i e l d r i n , fed at dietary concentrations of 5 ppm and higher to female SWV mice, s i g n i f i c a n t l y reduces the v i a b i l i t y of the pups they bear. The i n v i a b i l i t y i s irrevocable i n 807=. of the pups. 9 0 I n v i a b i l i t y of the pups is the major proximate cause of pup mortality in the l i t t e r s of dams fed d i e l d r i n at di e t a r y concentrations of 10 ppm and below. At these doses dams do not k i l l t h e i r pups. The pups do not grow p r i o r to death and contain l i t t l e or no milk. This suggests that s t a r v a t i o n i s the d i r e c t cause of death. Since d i e l d r i n does not reduce the milk production of the mammary glands and since poor nurs-ing behaviour by the dam is not pronounced i t appears that the starva-t i o n r e s u l t s from the i n a b i l i t y of the pups to suckle. 10. 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