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Interaction of chronic ethanol and female sex steroids : correlation of rat hepatic enzymes and histopathology Warren, Betty Lynne 1979

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cop, / INTERACTION OF CHRONIC ETHANOL AND FEMALE SEX STEROIDS: CORRELATION OF RAT HEPATIC ENZYMES AND HISTOPATHOLOGY. by BETTY LYNNE WARREN B.Sc.(Pharm.), U n i v e r s i t y of B r i t i s h Columbia, 197^ A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES D i v i s i o n of Pharmacology and T o x i c o l o g y of the F a c u l t y of Pharmaceutical S c i e n c e s We accept t h i s t h e s i s as conforming to the r e q u i r e d standard. THE UNIVERSITY OF BRITISH COLUMBIA March, 1979 © B e t t y Lynne Warren, 1979 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 E - 6 B P 7 5 - 5 1 1 E ABSTRACT Recent r e p o r t s In the l i t e r a t u r e suggest t h a t o r a l c o n t r a c e p t i v e s t e r o i d therapy may be i m p l i c a t e d i n the develop-ment of benign h e p a t i c adenomas i n women. S i n c e estrogens and p r o g e s t i n s are known to a f f e c t l i v e r f u n c t i o n , we s t u d i e d e f f e c t s of c h r o n i c a d m i n i s t r a t i o n of the o r a l c o n t r a c e p t i v e agents mestranol and norethindrone on v a r i o u s i n d i c e s of h e p a t i c i n t e g r i t y . S e v e r a l h e p a t i c mixed f u n c t i o n oxidase a c t i v i t i e s were measured: benzo(a)pyrene hydroxylase, epoxide hydrase, a n i l i n e hydroxylase (Appendix II) and aminopyrlne N-demethylase (Appendix I I ) . In a d d i t i o n , benzo(a)pyrene hydroxylase a c t i v i t y i n l u n g t i s s u e was measured. As an i n d i c a t i o n of whether me t a b o l i t e s of the c o n t r a c e p t i v e s t e r o i d s were bound to l i v e r macromolecules, i r r e v e r s i b l e b i n d i n g of -benzopyrene was measured. Hepatic h i s t o p a t h o l o g y ( l i g h t microscopy u s i n g hematoxylin and e o s i n s t a i n and o i l r e d 0 s t a i n ) was c a r r i e d out to determine i f f u n c t i o n a l a l t e r a t i o n s c o r r e l a t e d w i t h p a t h o l o g i c a l changes i n the l i v e r . Comparisons were a l s o made between ethanol t r e a t e d and non-ethanol t r e a t e d groups to determine i f c o n t r a c e p t i v e s t e r o i d - a s s o c i a t e d h e p a t o t o x i c l t y was enhanced by or would enhance, the h e p a t o t o x i c l t y of eth a n o l ad.m i n is t r a t i o n . Female and male Wistar r a t s were pair-fed. a n u t r i t i o n a l R l i q u i d d i e t , S u s t a c a l (Mead Johnson) to which was added e i t h e r sucrose or ethanol as k-0% of c a l o r i e s . O r a l c o n t r a c e p t i v e s t e r o i d s were a d m i n i s t e r e d d a i l y i n the l i q u i d , d i e t i n the f o l l o w i n g dosest mestranol, 0 . 6 mg/kg per day, alone or i n combination with norethindrone, 5 . 0 mg/kg per day. I n i t i a l s h o r t - t e r m s t u d i e s showed t h a t the ethanol p l u s S u s t a c a l d i e t g e n e r a l l y caused enzyme i n d u c t i o n compared to the R R p l a i n S u s t a c a l d i e t or the sucrose plus S u s t a c a l d i e t i n animals t r e a t e d f o r up to 6 weeks. Animals t h a t were admin-i s t e r e d the c o n t r a c e p t i v e s t e r o i d s f o r a s i m i l a r time p e r i o d a l s o demonstrated h e p a t i c microsomal enzyme i n d u c t i o n . Enzyme a c t i v i t y i n animals that r e c e i v e d ethanol plus the c o n t r a c e p t i v e s t e r o i d s was i n c r e a s e d above that seen f o r each agent a l o n e . Chronic s t u d i e s showed t h a t ethanol a d m i n i s t r a t i o n f o r 6 months produced h e p a t o t o x i c l t y i n both male and female r a t s . H e p a t o t o x i c l t y was observed f u n c t i o n a l l y as decreased h e p a t i c benzo(a)pyrene hydroxylase a c t i v i t y and h i s t o p a t h o l o g i c a l l y as i n c r e a s e d f a t accumulation i n zone 3 of the l i v e r l o b u l e s . I t was found that a d m i n i s t r a t i o n of the c o n t r a c e p t i v e s t e r o i d s to female r a t s tended to p r o t e c t a g a i n s t e t h a n o l - a s s o c i a t e d h e p a t o t o x i c l t y . The p r o t e c t i v e e f f e c t was observed f u n c t i o n a l l y as maintenance of c o n t r o l l e v e l s of h e p a t i c benzo(a)pyrene hydroxylase a c t i v i t y and m o r p h o l o g i c a l l y as l e s s e r amounts of f a t accumulation l n the l i v e r . That i s , there tended to be a c o r r e l a t i o n between the l e v e l of h e p a t i c benzo(a)pyrene hydroxy-l a s e a c t i v i t y and h i s t o l o g i c a l f a t accumulation as an i n d i c a t i o n of e t h a n o l - a s s o c i a t e d h e p a t o t o x i c l t y . A S u s t a c a l a s s o c i a t e d phenomenon was e v i d e n t i n a l l animals l n which h l s t o p a t h o l o g y was c a r r i e d out. The " S u s t a c a l e f f e c t " was observed, as m l c r o d r o p l e t f a t accumulation l n zone 1 o f the l i v e r l o b u l e . C o n t r a c e p t i v e s t e r o i d t r e a t e d females showed the l e a s t " S u s t a c a l e f f e c t " . Microsomal enzyme a c t i v i t y d i d not appear to be a f f e c t e d by the " S u s t a c a l e f f e c t " . I t was concluded that the c o n t r a c e p t i v e s t e r o i d s a d m i n i s t e r e d i v d i d not i n c r e a s e ethanol h e p a t o t o x i c i t y . Instead, i t appeared t h a t female sex s t e r o i d s tended to a t t e n u a t e e t h a n o l - a s s o c l a t e d h e p a t o t o x i c i t y . There was no evidence to suggest t h a t the o r a l c o n t r a c e p t i v e s t e r o i d s were d i r e c t l y a s s o c i a t e d with o v e r t hepatotox i c i t y . V TABLE OF CONTENTS PAGE ABSTRACT 11 LIST OF TABLES v l i l LIST OF FIGURES AND ILLUSTRATIONS x LIST OF ABBREVIATIONS x i i INTRODUCTION 1 A. Incidence of Benign Hepatic Adenomas 1 B. C l i n i c a l Considerations of Cases 1. C l i n i c a l Presentation 5 2. Patterns of Oral Contraceptive Steroid Use 6 3. Gross and Microscopic Structure of Tumors 7 k. Laboratory Studies 8 5. Terminology and C l a s s i f i c a t i o n of Liver 9 Tumors 6. Malignant Hepatocellular Carcinoma 11 C. Evidence for Possible Contraceptive Steroid . 13 Involvement In Hepatic C e l l Adenomas D. E f f e c t of Contraceptive Steroids on Mixed 16 Function Oxidase Enzymes E. Metabolism of Contraceptive Steroids 1. S p e c i f i c Pathways of Contraceptive Steroid Metabolism a. Estrogens 20 b. Progestins 21 c. P o s s i b i l i t y of Epoxide Formation i n the Metabolism of Estrogens and Progestins 22 F. Evidence for Irre v e r s i b l e Binding of Contraceptive Steroid Metabolites 1. Accumulation of Metabolites 2h 2. Irr e v e r s i b l e Binding Studies 25 3. Effects of Irr e v e r s i b l e Binding a. General Effects 27 b. Possible Effects of Contraceptive 28 Steroid I r r e v e r s i b l e Binding v i G. E f f e c t s of E t h a n o l on the L i v e r 1. S t r u c t u r a l and F u n c t i o n a l A l t e r a t i o n s 30 2. M e t a b o l i c Routes of E t h a n o l 32 3. Role i n Enhancing H e p a t o t o x i c l t y 34 MATERIALS AND METHODS 4-0 A. Chemicals and Reagents 40 B. Animals 41 C. Treatments 1. D i e t s 4l 2. C o n t r a c e p t i v e S t e r o i d s 42 3. B i n d i n g S t u d i e s 42 D. Enzyme P r e p a r a t i o n f o r Assays 43 E. T i s s u e P r e p a r a t i o n f o r H i s t o p a t h o l o g y 43 F. T i s s u e P r e p a r a t i o n f o r B i n d i n g Assay 44 G. Enzyme Assays 1. L i v e r Benzo(a)pyrene hydroxylase 44 2. Lung Benzo(a)pyrene hydroxylase 45 3. L i v e r Epoxide Hydrase 45 H. D e t e r m i n a t i o n of I r r e v e r s i b l e B i n d i n g of 46 -Benzo(a)pyrene I. In V i t r o S t u d i e s 46 J . S t a t i s t i c s 47 RESULTS ij,8 A. Animal Data 1. Feeding P a t t e r n s 48 2. M o r b i d i t y and M o r t a l i t y 49 3. D i e t a r y Consumption 50 4. Animal Weights 50 E. In V i t r o Enzyme S t u d i e s 54 C. Short-term In Vivo S t u d i e s 1. Enzyme S t u d i e s a. E f f e c t o f D i e t 57 b. E f f e c t of C o n t r a c e p t i v e S t e r o i d s 59 2. I r r e v e r s i b l e B i n d i n g S t u d i e s 64 v i i D. Chronic In Vivo Studies 1. Enzyme Studies 67 2. Histopathology a. E f f e c t of Sucrose vs Ethanol 71 b. Contraceptive Steroid Female Rats 85 DISCUSSION 100 A. General Animal Data 100 B. Short-term In Vivo Studies 102 C. Chronic In Vivo Studies - Correlation of Enzyme 107 Studies with Hepatic Histopathology SUMMARY AND CONCLUSIONS 115 REFERENCES 117 APPENDIX I 126 APPENDIX II 128 v l i i LIST OF TABLES Table I. Number of cases of benign l i v e r tumors cited i n the l i t e r a t u r e since 19^0. PAGE Table II. Summary of cases of benign l i v e r tumors associated with o r a l contraceptive therapy, 1972-1978. 138 Table I I I . Table IV. Table V. H i s t o l o g i c a l comparison of f o c a l nodular hyperplasia and benign hepatic c e l l adenoma. 10 C a l o r i c intake of animals per day. . 51 Enzyme a c t i v i t i e s i n tissue from female •p rats fed Sustacal alone, or with sucrose or ethanol. 58 Table VI. Hepatic benzo(a)pyrene hydroxylase a c t i v i t y in steroid-treated female rats r e c e i v i n g R R Sustacal alone or Sustacal plus ethanol. 60 Table VII. Lung benzo(a)pyrene a c t i v i t y i n s t e r o i d -R treated female rats receiving Sustacal alone or Sustacal plus ethanol. 61 Table VIII. Hepatic epoxide hydrase a c t i v i t y in steroid-treated female rats r e c e i v i n g Susta-R "R c a l alone or Sustacal plus ethanol. 62 Table IX. The effects of mestranol and/or noreth-lndrone treatment on i r r e v e r s i b l e binding of C^J- "benzo(a)pyrene i n l i v e r and lung. 65 Ix Table X. Comparison of enzyme a c t i v i t i e s j female vs male r a t s . Table XI. Hepatic benzo(a)pyrene hydroxylase a c t i v i t y i n c h r o n i c a l l y t r e a t e d female r a t s . Table X I I . Lung benzo(a)pyrene hydroxylase a c t i v i t y in c h r o n i c a l l y t r e a t e d female r a t s . Table X I I I . Hepatic epoxide hydrase a c t i v i t y i n c h r o n i c a l l y t r e a t e d female r a t s . 68 70 72 73 X LIST OF FIGURES AND ILLUSTRATIONS PAGE Fi g u r e 1. F i g u r e 2. F i g u r e 3. F i g u r e h. F i g u r e 5 . F i g u r e 6 . F i g u r e ?. Fi g u r e 8 . Average weight of animals d u r i n g the treatment p e r i o d ( c a l c u l a t e d on a monthly b a s i s ) . In v i t r o e f f e c t o f c o n t r a c e p t i v e s t e r o i d s or ethanol on enzyme assays. H & E s t a i n o f female r a t l i v e r , showing normal h e p a t i c h i s t o l o g y . H & E s t a i n of female r a t l i v e r , showing some degree of p e r i p o r t a l f a t accumulation. H & E s t a i n of female r a t l i v e r , showing d i s t r i b u t i o n of f a t i n a l l 3 zones. O i l r e d 0 s t a i n of female r a t l i v e r , showing more advanced d e p o s i t i o n of p e r i p o r t a l f a t . O i l red 0 s t a i n of female r a t l i v e r , showing more advanced ge n e r a l d i s t r i b -u t i o n of f a t . H & E s t a i n of o v a r i e c t o m l z e d female r a t l i v e r , showing l a r g e f a t d r o p l e t accumu-l a t i o n and m i c r o d r o p l e t f a t accumulation l n zone 1. 52 5 5 7 5 7 6 7 7 7 8 7 9 8 1 Figure 9. H & E s t a i n of sham-operated female rat l i v e r , showing fat accumulation in the p e r i p o r t a l region. Figure 10. H & E s t a i n of ovariectomized female rat l i v e r , showing fat accumulation in the central region, as well as ln the p e r i p o r t a l and mid zonal areas. Figure 11. H.& E s t a i n of sham-operated female rat l i v e r , showing fat accumulation mainly in the p e r i p o r t a l area with some degree of mlcrodroplet fat in the p e r i c e n t r a l area. Figure 12. H & E s t a i n of male r a t l i v e r , showing only s l i g h t p e r i p o r t a l micro-droplet fat accumulation. Figure 13. H & E s t a i n of male rat l i v e r , showing only s l i g h t f a t accumulation in the p e r i c e n t r a l region. Figure 14. H & E s t a i n of male r a t l i v e r , showing only s l i g h t p e r i p o r t a l f a t depos i t ion. Figure 15. H & E s t a i n of male rat l i v e r , showing s i g n i f i c a n t large droplet fa t in zone 3« x i i Figure 16. O i l red. 0 s t a i n of female r a t l i v e r , showing some degree of zonation of fat d i s t r i b u t i o n about the pe r i p o r t a l region. 91 Figure 17. O i l red 0 s t a i n of female r at l i v e r , showing even d i s t r i b u t i o n of f a t . 92 Figure 18. O i l red 0 s t a i n of female r at l i v e r , showing marked zonation. 93 Figure 19. O i l red 0 s t a i n of female r at l i v e r , showing zonation. 9^ Flgure 20. H & E st a i n of female r at l i v e r , showing some degree of p e r i p o r t a l microdroplet f a t accumulation. 95 Figure 21. H & E s t a i n of female r at l i v e r , showing l i t t l e or no per i c e n t r a l f a t and some degree of pe r i p o r t a l f a t d i s t r i b u t i o n . 96 Figure 22. O i l red. 0 s t a i n of female r at l i v e r , showing s l i g h t accumulation of micro-droplet fat in the pe r i p o r t a l region. 98 Figure 23. O i l red 0 s t a i n of female r at l i v e r , showing uniform microdroplet fat d i s -t r i b u t i o n between the 3 zones. 99 Figure 24. Diagram of the l i v e r acinus or functional unit . 1 1 0 x l i l LIST OF ABBREVIATIONS ADP adenosine diphosphate ATP adenosine t r i p h o s p h a t e BP toenzo(a)pyrene E ethanol EH epoxide hydrase EM e l e c t r o n microscope H & E hematoxylin and e o s l n LM l i g h t microscope M mestranol MEOS microsomal e t h a n o l - o x i d i z l n g system N norethlndrone NAD n i c o t i n a m i d e adenine d l n u c l e o t l d e NADH n i c o t i n a m i d e adenine d l n u c l e o t l d e (reduced) NADPH n i c o t i n a m i d e adenine d l n u c l e o t l d e phosphate (reduced) S sucrose S.E.M. standard e r r o r o f the mean SER smooth endoplasmic r e t i c u l u m UDP u r i d i n e diphosphate x i v STRUCTURES OF ESTROGENIC AND PROGESTOGENIC COMPOUNDS PROGESTERONE NORETHINDRONE (NORETHISTERONE) ETHINYLESTRENOL NORETHYNODREL (LYNESTRENOL) ACKNO WLED GEMENT3 I am deeply g r a t e f u l to Dr. G. D. Bellward f o r her v a l u a b l e guidance throughout t h i s study. I owe s p e c i a l g r a t i t u d e to Mr. Roland Burton f o r expert a s s i s t a n c e i n p r e p a r i n g the s t a t i s t i c a l a n a l y s i s , and to Miss Peggy Warren f o r expert t e c h n i c a l and g r a p h i c a l c o n t r i b u t i o n s . 1 I. INTRODUCTION A. Incidence of Benign Hepatic C e l l Adenomas Benign hepatic c e l l adenomas have been considered to be very-rare tumors. Sherlock indicated in her 1975 textbook that they are "anatomical c u r i o s i t i e s of no c l i n i c a l importance." (1) The world l i t e r a t u r e was reviewed by Warvl (2) and only 67 cases were found up to 1944. A study carried out by Henson e_t a l . (3) at the Mayo C l i n i c revealed that i n the period 1907-195^ there were only four hepatic c e l l adenomas diagnosed out of a t o t a l of 124 benign l i v e r tumors. Edmondson (4) found only two hepatic c e l l adenomas in a review of 418 hepatic tumors i n 50,000 autopsies at the Los Angeles County General Hospital from 1918-195^. However, during the l a s t decade there has been an increased incidence of benign l i v e r tumors reported i n the l i t e r a t u r e . The most prevalent diagnoses l n t h i s group of tumors have been benign hepatic c e l l adenoma and f o c a l nodular hyperplasia. Fechner (5) i n 1977 c a r r i e d out a c r i t i c a l analysis of such reports which yielded the Information contained i n Table I. The r e s u l t s suggest that since i960 there have been Increased numbers of reports of benign l i v e r tumors, at least as Is dlscernable from the l i t e r a t u r e . The reason for the d i s t i n c t i o n between "before i960" and "after i960" is that i960 marked the beginning of large scale use of or a l contraceptives. Females have seemed to be at much higher r i s k to develop benign l i v e r tumors than males, and the majority of females involved are of c h i l d -bearing age. This age and sex d i s t r i b u t i o n of cases suggests a possible hormone-related e t i o l o g i c a l factor. For example, present estimates indicate that anywhere from 20-35 m i l l i o n American women have taken or are taking o r a l contraceptives (6) TABLE I. Number of Cases of Benign Liver Tumors Cited in the Literature Since 1 9 4 0 . ^ NUMBER OF CASES Time Period Total Focal Nodular Hyperplas ia Hepatic C e l l Adenoma INCIDENCE INCIDENCE 1940-1960 37 24 11 children 10 females (7/10 were 23-39) 3 males 8 Since i960 125 37 9 children 36 4 children 23 females 31 females (13/23 were (30/31 were 21-29) 20-40) 3 males 1 male 3 (7). I n t h i s r e g a r d , i t h a s "been s u g g e s t e d i n t h e l i t e r a t u r e t h a t o r a l c o n t r a c e p t i v e s t e r o i d t r e a t m e n t may l n some way b e a s s o c i a t e d w i t h d e v e l o p m e n t o f b e n i g n h e p a t i c t u m o r s . I n 1973 Baum e t a l . (8) r e p o r t e d a c l u s t e r o f 7 c a s e s o f h e p a t i c a d e n o m a a n d a l l w e r e a s s o c i a t e d w i t h o r a l c o n t r a c e p t i v e u s e . S i n c e t h a t t i m e t h e r e h a v e b e e n i n c r e a s i n g numbe r s o f s u c h r e p o r t s I n t h e l i t e r a t u r e . T a b l e I I r e p r e s e n t s a n e x t e n s i v e s e a r c h o f t h e l i t e r a t u r e p u b l i s h e d s i n c e 1973. I t h a s r e v e a l e d a t l e a s t 107 r e p o r t e d c a s e s o f b e n i g n l i v e r t u m o r s (65 h e p a t i c c e l l a d e n o m a s , 32 f o c a l n o d u l a r h y p e r p l a s i a , 10 o t h e r s ) a s s o c i a t e d w i t h o r a l c o n t r a c e p t i v e u s e . T h e r e a r e a l s o kZ c a s e s r e p o r t e d b y E d m o n d s o n e t a l . (9)» 27 c a s e s r e p o r t e d b y N l s s e n e t a l . (10), a n d 6 c a s e s r e p o r t e d b y G o l d f a r b (11) t h a t a r e e x c l u d e d f r o m t h e a n a l y s i s e i t h e r b e c a u s e c l i n i c a l i n f o r m a t i o n was n o t g i v e n i n t h e r e p o r t s o r b e c a u s e o f p o s s i b l e d u p l i c a t i o n o f c a s e s . The a p p a r e n t i n c r e a s e i n t h e I n c i d e n c e o f r e p o r t e d c a s e s o f b e n i g n l i v e r t u m o r s i s , h o w e v e r , d i f f i c u l t t o i n t e r p r e t f o r s e v e r a l r e a s o n s t 1. The m e t h o d o f c a s e r e p o r t i n g h a s b e e n d i f f e r e n t o v e r t h e y e a r s . Whe rea s e a r l i e r r e p o r t s w e r e e p i d e m i o l o g i c a l s u r v e y s (2) (3) W $ mo re r e c e n t r e p o r t s , e s p e c i a l l y s i n c e 1973» h a v e b e e n b a s e d o n c a s e h i s t o r i e s o f p a t i e n t s who h a v e p r e s e n t e d s y m p t o m a t l c a l l y o r i n c i d e n t a l l y w i t h b e n i g n h e p a t i c t u m o r s . I n t h i s w a y , t h e I n c r e a s e d numbe r s o f c a s e s c o u l d b e a r e s u l t o f p r e s e n t a w a r e n e s s o f t h e p r o b l e m . 2. The f a c t t h a t m o s t s t u d i e s h a v e b e e n b a s e d o n l y o n t h e s y m p t o m a t l c a l l y p r e s e n t i n g p a t i e n t s p r e c l u d e s a n y a s s e s s m e n t a n d c o m p a r i s o n s w i t h t h e t r u e ( i . e . symptomatic and asymptomatic) i n c i d e n c e of benign l i v e r tumors o c c u r r i n g i n the whole p o p u l a t i o n . 3. Furthermore, case r e p o r t s s i n c e 1973 may have been s e l e c t e d In the sense t h a t they tend to enumerate the di s e a s e i n c i d e n c e o n l y i n o r a l c o n t r a c e p t i v e - t r e a t e d women. Neve r t h e l e s s , the evidence s t i l l tends to be s u g g e s t i v e that benign h e p a t i c tumors are i n c r e a s i n g In frequency, e s p e c i a l l y l n women of c h l l d b e a r i n g age. S i n c e t h i s apparent i n c r e a s e i n in c i d e n c e o f d i s e a s e p a r a l l e l s widespread use of o r a l c o n t r a -c e p t i v e s , i t i s r a t i o n a l to i n v e s t i g a t e the p o s s i b l e c o r r e l a t i o n between i n c r e a s e d i n c i d e n c e o f benign l i v e r tumors and popular use o f c o n t r a c e p t i v e s t e r o i d s . This i s e s p e c i a l l y p e r t i n e n t because the m a j o r i t y of r e c e n t l y r e p o r t e d tumors have become manifest i n c o n t r a c e p t i v e s t e r o i d - t r e a t e d women. 5 B. C l i n i c a l C o n s i d e r a t i o n s o f C a s e s 1. C l i n i c a l P r e s e n t a t i o n T a b l e I I g i v e s a n a c c o u n t o f t h e h i s t o r i e s o f 107 c a s e s o f b e n i g n l i v e r t u m o r s r e p o r t e d i n t h e l i t e r a t u r e s i n c e 1973. P a t i e n t s t e n d e d t o p r e s e n t c l i n i c a l l y i n o n e o f t h r e e w a y s * 1) A s y m p t o m a t i c l i v e r m a s s e s w e r e f o u n d a c c i d e n t a l l y b y t h e p a t i e n t o r p h y s i c i a n o n r o u t i n e p h y s i c a l e x a m i n a t i o n , o r t h e m a s s e s w e r e f o u n d i n c i d e n t a l l y a t l a p a r o t o m y f o r a n -o t h e r i n d i c a t i o n . I n t h e s e c a s e s s u r g e r y was d o n e e l e c -t i v e l y a n d t h e t h e r a p y was u n c o m p l i c a t e d . T h i s g r o u p r e p r e s e n t s a b o u t 30% o f t h e c a s e s c i t e d . 2) A b o u t 30$ o f t h e c a s e s p r e s e n t e d w i t h v a r y i n g d e g r e e s o f a b d o m i n a l d i s c o m f o r t o r p a i n . The p a t i e n t may o r may n o t h a v e had c o n c o m i t a n t a n o r e x i a , n a u s e a , v o m i t i n g , o r a n e m i a . Symptoms w e r e u s u a l l y p r e s e n t a n y w h e r e f r o m a f e w d a y s t o s e v e r a l m o n t h s , a n d t h e p a i n was u s u a l l y l n t e r -m i t t a n t . The s ymptoms o f t e n c o u l d b e c o r r e l a t e d w i t h b o u t s o f l n t r a l e s l o n a l h e m o r r h a g e , s i n c e g r o s s p a t h o l o g y r e v e a l e d o l d a r e a s o f s c a r r i n g e x i s t i n g t o g e t h e r w i t h a r e a s o f r e c e n t h e m o r r h a g e i n t h e t u m o r . The m o s t common i n i t i a l m i s d i a g n o s i s i n t h e s e c a s e s was c h o l e c y s t i t i s . 3) The t h i r d g r o u p o f p a t i e n t s p r e s e n t e d w i t h s u d d e n r u p t u r e o f t h e t u m o r , h e m o p e r i t o n e u m , a n d s h o c k . T h e s e s i t u a t i o n s w e r e l i f e - t h r e a t e n i n g a n d h a d t o be d e a l t w i t h o n a n e m e r g e n c y b a s i s . I f t h e r e was d e l a y l n s u r g i c a l I n t e r -v e n t i o n i n t h e s e p a t i e n t s d e a t h o f t e n r e s u l t e d . S u c c e s s -f u l e m e r g e n c y p r o c e d u r e s i n c l u d e d i c o n t r o l o f h e m o r r h a g e w i t h p a c k i n g , t e m p o r a r y o c c l u s i o n o f t h e p o r t a l v e i n a n d h e p a t i c a r t e r y , c o n t r o l o f b l e e d i n g w i t h l o c a l s u t u r e s , 6 e x c i s i o n o f the tumor, or lobectomy i f a p p l i c a b l e . I f these p a t i e n t s s u r v i v e d s u r g e r y and the p o s t - o p e r a t i v e p e r i o d , they tended to do w e l l . I t i s notable t h a t b e f o r e the acute hemorrhagic episode, most p a t i e n t s experienced no symptoms even though most of the r u p t u r e d tumors were r e l a t i v e l y l a r g e and p r o b a b l y c o u l d have been d e t e c t e d . P r e o p e r a t i v e misdiagnoses i n c l u d e d r u p t u r e d e c t o p i c pregnancy and hemorrhaging p e p t i c u l c e r . These cases r e p r e s e n t about k-0% of the t o t a l number of cases r e p o r t e d and about 27% (15 out of ko) of these r e s u l t e d i n death. Of the 15 cases r e s u l t i n g l n death, 12 proved to have h e p a t i c adenoma and 1 had f o c a l nodular h y p e r p l a s i a . The two others were p r o b a b l y m i s c l a s s l f l e d and r e p r e s e n t e d , h e p a t i c adenomas. Hence there i s a s u g g e s t i o n t h a t benign h e p a t i c adenomas are more prone to r u p t u r e than are f o c a l nodular h y p e r p l a s i a s , although the numbers are too s m a l l to be c o n c l u s i v e . 2. P a t t e r n s of C o n t r a c e p t i v e S t e r o i d Use I t can be seen from Table I I t h a t no one p a r t i c u l a r s t e r o i d regimen i s i m p l i c a t e d i n tumor p r o d u c t i o n more than any o t h e r . N i s s e n et a l . (10) r e c e n t l y conducted a study on 58 cases o f l i v e r tumors where c o n t r a c e p t i v e s t e r o i d h i s t o r y was known. They found t h a t the f o l l o w i n g usage p a t t e r n s e v o l v e d i G\% (37 out of 58) of cases took mestranol as the o n l y e s t r o g e n f o r 71$ of the t o t a l accumulated months of usage f o r a l l brands 78$ (^ 5 out o f 58) of cases took a product that c o n t a i n e d mestranol f o r 82$ o f the t o t a l accumulated usage 17$ (10 out of 58) of cases took a product that c o n t a i n e d e t h i n y l e s t r a d i o l as the o n l y e s t r o g e n f o r 9.6% of t o t a l usage 7 31$ (18 out of 58) of cases used e t h i n y l e s t r a d i o l f o r 14$ of the t o t a l accumulated usage 55$ of cases took norethindrone (+ mestranol) f o r 56$ of the t o t a l usage 21 $ of cases took n o r e t h y n o d r e l (+ mestranol) f o r 18$ of the t o t a l usage 16$ of cases took n o r g e s t r e l ( + e t h i n y l e s t r a d i o l ) f o r 10% o f the t o t a l accumulated usage The apparent mestranol preponderance i s l i k e l y an a r t i f a c t due to the f a c t t h at m e s t r a n o l - c o n t a i n i n g brands of o r a l c o n t r a -c e p t i v e s were produced b e f o r e those c o n t a i n i n g e t h i n y l e s t r a d i o l . The study found t h a t the mean number of months of c o n t r a c e p t i v e usage was 58.8 months and t h a t 85$ of p a t i e n t s used the b i r t h c o n t r o l p i l l f o r more than f o u r continuous years. These f i g u r e s compare w e l l with the present study where the mean number of months of c o n t r a c e p t i v e usage was 62.4 (In the cases w i t h known c o n t r a c e p t i v e h i s t o r y ) . From these d a t a i t can be seen t h a t prolonged usage of c o n t r a c e p t i v e s t e r o i d s may be an e t i o l o g i c f a c t o r i n tumor development; whereas the s p e c i f i c s t e r o i d e n t i t y probably i s not. 3« Gross and M i c r o s c o p i c S t r u c t u r e of the Tumors G e n e r a l l y speaking, most of the tumors were s o l i t a r y nod-u l e s , although some had a complex l o b u l a t i o n and some d i d a r i s e as m u l t i p l e tumors. Tumor s i z e v a r i e d from 1 to 12 cm. i n diameter. Most l e s i o n s were a t l e a s t p a r t i a l l y encapsulated by an incomplete f i b r o u s membrane so that they were r e a d i l y d i s -t i n g u i s h a b l e from the s u r r o u n d i n g normal h e p a t i c parenchyma. Most of the tumors contained, areas o f hemmorrhage, hemorrhagic n e c r o s i s , and p o s s i b l y a d i s t i n c t s c a r . Where no degeneration had yet taken 8 p l a c e , t h e t u m o r s w e r e a l i g h t e r t a n - b r o w n c o l o r t h a n n o r m a l l i v e r . The v a s c u l a r p a t t e r n o f t h e t u m o r s was v a r i e d b u t some p a t t e r n s t e n d e d t o be p r e s e n t i n m o s t t u m o r s . One p a t t e r n e x h i b i t e d d i l a t e d t h i n - w a l l e d v e i n s a l t e r n a t i n g w i t h s m a l l v a s c u l a r b u n d l e s s u r r o u n d e d b y d e n s e c o l l a g e n . O t h e r a r e a s c o n t a i n e d a n a s t o m a s i n g , d i l a t e d , s i n u s o i d a l s t r u c t u r e s . H i s t o l o g i c a l l y t h e s e b e n i g n l i v e r t u m o r s a l l c o n s i s t e d o f w e l l - d i f f e r e n t i a t e d h e p a t o c y t e s o r g a n i z e d i n t o s h e e t s o r c o r d s o f t e n i n a s i n u s o i d a l p a t t e r n . The n e o p l a s t i c c e l l s t e n d e d t o be l a r g e r a n d h a v e c l e a r e r c y t o p l a s m ( d u e t o g l y c o g e n c o n t e n t ) t h a n n o r m a l h e p a t o c y t e s . M o s t c e l l s w e r e q u i t e u n i f o r m l n s i z e a l -t h o u g h s l i g h t l y more p l e o m o r p h i c c e l l s c o u l d be s e e n . M i t o t i c f i g u r e s w e r e no more common t h a n w o u l d be p r e s e n t l n n o r m a l h e p -a t i c p a r e n c h y m a . B i l e d u c t u l e s w e r e p o o r l y f o r m e d . I n some i n -s t a n c e s t h e y c o n n e c t e d t u m o r c e l l s t o s c a r t i s s u e w h i l e o t h e r d u c t u l e s w e r e c o n n e c t e d d i r e c t l y t o t u m o r c e l l s . (12) L a b o r a t o r y S t u d i e s The c a s e h i s t o r i e s a l s o i l l u s t r a t e d t h a t o n e o f t h e m a i n d i f f i c u l t i e s i n p r e o p e r a t i v e l y d i a g n o s i n g b e n i g n l i v e r t u m o r s was t h a t r o u t i n e d i a g n o s t i c s t u d i e s w e r e o f l i t t l e a s s i s t a n c e . U s u a l l y l i v e r f u n c t i o n a n d l i v e r enzyme t e s t s w e r e n o r m a l , a s w e r e s e r u m b i l i r u b i n , b l o o d c o u n t s , a l p h a - f e t o p r o t e i n , a n d h e p a t i t i s B s u r f a c e a n t i g e n . P l a i n x - r a y f i l m s a n d b a r i u m s t u d i e s ( e . g . u p p e r g a s t r o - i n t e s t i n a l s e r i e s , o r a l c h o l e c y s t o g r a m , I V P o r b a r i u m enema) w e r e r a r e l y c o n t r i b u t o r y t o t h e d i a g n o s i s . I n a d d i t i o n , p e r c u t a n e o u s l i v e r b i o p s i e s a r e c o n t r a i n d i c a t e d b e c a u s e o f t h e t u m o r s ' v a s c u l a r i t y . C o l l o i d l i v e r s c a n s may h a v e b e e n h e l p f u l , e s p e c i a l l y i f t h e r e was a s u f f i c i e n t r e t i c u l o e n d o t h e l i a l s y s t e m l n t h e l e s i o n t o t r a p t h e c o l l o i d . H o w e v e r , s m a l l t u m o r s w o u l d 9 have "been missed. The most hel p f u l studies carried out to date have been hepatic angiography. In this instance the extensive v a s c u l a r i t y of the lesions have enabled the s i t e and si z e of the nodules to be defined. (12) Hence i t can be seen that to define the true Incidence of benign hepatic tumors ln the general popula-tion and i n st e r o i d contraceptive users, s p e c i f i c laboratory tests w i l l have to be elucidated whereby prospective studies can be carried out. 5. Terminology and C l a s s i f i c a t i o n of Liver Tumors One discrepancy which contributes to the d i f f i c u l t y of studying the association between benign l i v e r tumors and contra-ceptive use is that the c l a s s i f i c a t i o n of l i v e r tumors is not s t r i c t l y standardized. As a r e s u l t , the terminology to describe similar lesions is variable, creating the s i t u a t i o n where benign l i v e r tumors have been variously referred to as f o c a l nodular hyperplasia, hepatic hamartoma, hepatic c e l l adenoma, benign hepatoma, w e l l - d i f f e r e n t i a t e d hepatoma, mixed adenoma, and hamartomatous cholanglohepatoma. Undoubtedly some of these terms describe the same le s i o n . It is l i k e l y that "focal nodular hyperplasia" and "benign hepatic c e l l adenoma" represent d i f f -erent e n t i t l e s . The other c l a s s i f i c a t i o n s may be variations of these two. Table III compares f o c a l nodular hyperplasia (FNH) and hepatic c e l l adenoma, (11) (14) It may be s i g n i f i c a n t to d i f f e r e n t i a t e c l e a r l y between hep-a t i c c e l l adenoma and f o c a l nodular hyperplasia because, as Table I suggests, the two e n t i t l e s have d i f f e r e n t age d i s t r i b u t i o n s , d i f f e r e n t incidences, and a seemingly d i f f e r e n t response to associated o r a l contraceptive steroids. Several authors have acknowledged the following points t F i r s t l y , hepatic c e l l adenomas 1 0 TABLE I I I . H i s t o l o g i c a l Comparison of Focal Nodular .Hyperplasia and Benign Hepatic C e l l Adenoma FOCAL NODULAR HYPERPLASIA BENIGN HEPATIC CELL ADENOMA prominent fibrous septa containing b i l e ducts, lymphocytes, and abnormal blood vessels, p r o l i f e r a -t i o n of portal triads may be well demarcated but not encapsulated excessive amounts of gly-cogen} may contain f a t large dense central scar with the thinner fibrous septa eminatlng from t h i s ; a d i s t i n c t l y lobulated appearance c e l l s appear normal but are sometimes t y p i c a l l y hyper-p l a s t i c and arranged i n 2-3 c e l l thick hepatic plates often is incidental f i n d -ing at autopsy - hepatic tissue adjacent -tb the nodules showed d i l a t e d sinusoids and some ischemic necrosis. There may be smooth muscle hyperplasia in media and subintima of a r t e r i o l e s and veins - t y p i c a l l y devoid of b i l e ducts; consists of near normal hepatic parenchyma with no conspicuous f i b -rous septa - p a r t i a l l y or completely encapsulated; sharply de-marcated from surrounding parenchyma - normal or Increased amounts of glycogen - no central scar composed of sheets of glycogen-fllled hep-atocytes that have small uniform nuclei; no mit-osis; no b i l e ducts frequently undergoes hem-orrhagic necros i s ; the enlarging hematoma often ruptures through the cap-sule surrounding l i v e r par-enchyma and tumor shows distended large blood vessels and sinusoids i n many areas; les ions are hypervas cular - smaller than adenomas - can grow to larger s i z e (up to 30 cm.) 11 are a p p a r e n t l y more frequent now wit h i n c r e a s e d c o n t r a c e p t i v e s t e r o i d use; whereas there has not been such an apparent i n c r e a s e i n the number of cases of f o c a l nodular h y p e r p l a s i a . Secondly, h e p a t i c c e l l adenomas are much more l i k e l y to r u p t u r e than are f o c a l nodular h y p e r p l a s i a s . (5) (11) (15) Consequently, f u r t h e r s t u d i e s l n t h i s a r e a must d e l i n e a t e the s p e c i f i c type of benign l i v e r tumor that i s under c o n s i d e r a t i o n . 6. Malignant H e p a t o c e l l u l a r Carcinoma One other h i s t o l o p a t h o l o g i c a l l e s i o n that deserves a t t e n t i o n i s the malignant h e p a t o c e l l u l a r carcinoma. In the l i t e r a t u r e up to 1976 there have been a t l e a s t nine r e p o r t s o f cases of l i v e r c e l l carcinoma a s s o c i a t e d w i t h o r a l c o n t r a c e p t i v e use. ( l 6 ) - ( 2 1 ) S i n c e t h a t time s e v e r a l a d d i t i o n a l cases have been r e p o r t e d and these w i l l be d i s c u s s e d l n a l a t e r s e c t i o n . However, i t i s not agreed upon whether h e p a t i c c e l l adenomas can transform i n t o hepato-c e l l u l a r carcinomas or whether there i s any r e l a t i o n s h i p between the n a t u r a l h i s t o r y o f the benign and malignant l e s i o n s . AmerIks (12) concludes t h a t "malignant change l n h e p a t i c c e l l adenomas appears to be an u n l i k e l y p o s s i b i l i t y , a l t h o u g h the n a t u r a l h i s t o r y o f these tumors i s not e x a c t l y d e f i n e d . " I t i s f e l t , l n t h i s r e s p e c t , t h a t h e p a t i c c e l l adenomas are completely benign except f o r a tendency toward hemorrhagic i n f a r c t i o n and r u p t u r e . N e v e r t h e l e s s , there has been a t l e a s t one case of l i v e r c e l l adenoma r e p o r t e d by G o l d f a r b (11) that showed p o s s i b l e t r a n s i t i o n to h e p a t o c e l l u l a r carclnomat areas o f severe d y s p l a s i a ; areas of p r o g r e s s i v e h y p e r p l a s i a with hepatocyte p l a t e s measuring up to 5-8 c e l l s l n t h i c k n e s s ; mitoses not evi d e n t ; the vas-c u l a t u r e had not been invaded. Such a l e s i o n i s more a t y p i c a l than the u s u a l adenoma because of the t h i c k e n i n g o f c e l l p l a t e s and because some areas showed hepatocytes arranged i r i a g l a n d -u l a r p a t t e r n . This type o f arrangement i s s u g g e s t i v e o f mal-ignant t r a n s i t i o n . Davis et a l . (19) r e p o r t e d a case l n 1975 where h e p a t i c c e l l adenoma and h e p a t o c e l l u l a r carcinoma e x i s t e d w i t h i n the same l e s i o n . The two components of the tumor were s h a r p l y demarcated and inflammation was present between them. N e o p l a s t i c growth such as i n the cases d e s c r i b e d may be a s s o c i -ated o n l y c o i n c i d e n t a l l y w i t h c o n t r a c e p t i v e s t e r o i d s . On the other hand, i t i s p o s s i b l e t h a t c a r c i n o g e n i c i t y may be l i n k e d w i t h the a c t i v i t y of drug m e t a b o l i z i n g enzymes (1), which as w i l l be d i s c u s s e d l a t e r c o u l d i n t u r n be a f f e c t e d by c o n t r a c e p t i v e s t e r o i d s . Hence, a t present i t seems prudent to c o n s i d e r the p o s s i b i l i t y o f malignant t r a n s i t i o n i n h e p a t i c c e l l adenomas and to t r e a t such l e s i o n s as "pre-malignant" ones. This approach would Include complete e x c i s i o n of the tumor wherever p o s s i b l e and d i s c o n t i n u a n c e of o r a l c o n t r a c e p t i v e therapy l n such p a t i e n t s . 13 C. Evidence f o r P o s s i b l e C o n t r a c e p t i v e S t e r o i d Involvement In  Hepatic C e l l Adenomas The accumulating evidence to p o s i t i v e l y suggest an e t i o l o g i c a l r o l e f o r o r a l c o n t r a c e p t i v e therapy i n benign l i v e r c e l l adenoma fo r m a t i o n i n c l u d e s j 1) S y n t h e t i c estrogens and p r o g e s t i n s are known to have s u b s t a n t i a l e f f e c t s on the l i v e r . These l n c l u d e i i n t e r f e r e n c e with o r g a n i c a n i o n s e c r e t i o n i n hepatocytes which can r e s u l t i n c h o l e s t a t i c j a u n d i c e , blockade of h e p a t i c venous outflow, Increased r i s k of thromboembolic d i s o r d e r s and angiomatous t r a n s f o r m a t i o n o f the h e p a t i c v a s c u l a t u r e ( v a s c u l a r narrowing due to i n t i m a l t h i c k -ening and p r o l i f e r a t i o n o f the media). (10) (22) (23) I t i s a l s o known that o r a l c o n t r a c e p t i v e s t e r o i d s can a l t e r the l e v e l s o f drug m e t a b o l i z i n g enzymes l n the l i v e r . T his evidence i s d i s c u s s e d i n the f o l l o w i n g s e c t i o n . E l e c t r o n m i c r o s c o p i c changes occur i n the b i l e c a n a l -l c u l i , the smooth and rough endoplasmic r e t i c u l u m , and i n the mi t o c h o n d r i a ( g i a n t m i t o c h o n d r i a and p a r a c r y -s t a l l l n e m i t o c h o n d r i a l i n c l u s i o n s ) . (22) S i g n i f i c a n t changes i n s t r u c t u r e and f u n c t i o n of the l i v e r c o u l d be tumorogenie. 2) There i s evidence o f c a r c i n o g e n i c p o t e n t i a l of female sex s t e r o i d s l n c e r v i c a l and endometrial t i s s u e i n humans, and l n endometrial and b r e a s t t i s s u e i n beagle dogs. (2*0 In a d d i t i o n , a n a b o l i c androgenic s t e r o i d s , which are s i m i l a r i n s t r u c t u r e to the s y n t h e t i c p r o g e s t -i n s , are a l s o known to be c a r c i n o g e n i c l n animals and i n human males. (23) 14 A n d r o g e n i c s t e r o i d s a r e a l s o a s s o c i a t e d w i t h a r a r e v a s c u l a r d i s o r d e r , p e l l o s l s h e p a t l s , w h i c h c o n s i s t s o f b l o o d f i l l e d c y s t i c s p a c e s b e t w e e n h e p a t o c y t e s w i t h o u t e n d o t h e l i a l l i n i n g s . The c o n d i t i o n I s s e e n l n w a s t i n g d i s e a s e s s u c h a s i n t e r m i n a l t u b e r c u l o s i s a n d i n a s s o c i -a t i o n w i t h a n a b o l i c s t e r o i d t h e r a p y . T h i s c o u l d , p e r h a p s , be t h e b a s i s f o r t h e v a s c u l a r c h a n g e s s e e n l n b e n i g n l i v e r t u m o r s . A n i m a l s t u d i e s (25) r e v e a l e d t h a t t h e s y n t h e t i c p r o g e s t -a t i o n a l h o r m o n e s , n o r e t h y n o d r e l a n d n o r e t h i s t e r o n e , a l o n e o r i n c o m b i n a t i o n w i t h m e s t r a n o l , c a u s e d a n i n c r e a s e l n b e n i g n a n d i n some i n s t a n c e s m a l i g n a n t h e p a t i c t u m o r s i n m a l e r a t s . F e m a l e r a t s d e v e l o p e d v a r i a t i o n l n l i v e r c e l l s i z e , c e l l u l a r s w e l l i n g , a n d o p e n i n g u p o f s i n u s o i d s when g i v e n n o r e t h y n o d r e l . I t was a l s o f o u n d t h a t m e g -e s t r o l a n d e t h i n y l e s t r a d i o l e i t h e r a l o n e o r I n c o m -b i n a t i o n c a u s e d a n i n c r e a s e i n h e p a t o c e l l u l a r c a r c i n o m a s i n f e m a l e r a t s . A s d i s c u s s e d a b o v e , l i v e r c e l l adenomas a r e more p r e -v a l e n t w h e r e l o n g - t e r m t h e r a p y ha s b e e n c a r r i e d o u t , I . e . t u m o r s h a v e f o r m e d m o s t o f t e n i n p a t i e n t s who h a v e b e e n t a k i n g o r a l c o n t r a c e p t i v e s f o r a b o u t f i v e y e a r s o r m o r e . T h i s i s t h e u s u a l p a t t e r n e x p e c t e d i n c h e m i c a l l y - I n d u c e d t u m o r s w h e r e l o n g - t e r m e x p o s u r e p r e d i s p o s e s t o n e o p l a s t i c g r o w t h . A s i g n i f i c a n t f i n d i n g r e c e n t l y a p p e a r i n g i n t h e l i t -e r a t u r e i s t h a t l n c e r t a i n c a s e s o f b e n i g n h e p a t i c c e l l a d e n o m a s , w h e r e s u r g i c a l e x c i s i o n I s i n a d v i s a b l e , d i s -c o n t i n u a n c e o f o r a l c o n t r a c e p t i v e s h a s c a u s e d r e g r e s s i o n 15 and a decrease i n tumor s i z e . (26)-(28) 7) In order to s t u d y the p o s s i b l e pathogenesis o f h e p a t i c c e l l adenomas, i t i s a l s o n ecessary to evaluate another l i n e o f r e c e n t evidence which suggests that t o x i c m e t a b o l i t e s of o r a l c o n t r a c e p t i v e s t e r o i d s are formed i n the l i v e r and are subsequently bound to t i s s u e macromolecules. ( S e c t i o n F) However, f i r s t i t i s r e l e v a n t to c o n s i d e r how c o n t r a c e p t i v e s t e r o i d s a f f e c t drug m e t a b o l i z i n g enzymes, and, i n t u r n , how these s t e r o i d s themselves are m e t a b o l i z e d . 16 D. E f f e c t o f C o n t r a c e p t i v e S t e r o i d s on Mixed F u n c t i o n  Oxidase Enzymes As mentioned above, the a c t i v i t y of d r u g - m e t a b o l i z i n g enzymes may be a l t e r e d when o r a l c o n t r a c e p t i v e s t e r o i d hormones are a d m i n i s t e r e d . These d r u g - m e t a b o l i z i n g enzymes are l o c a t e d p r i m a r i l y i n the smooth endoplasmic r e t i c u l u m of c e l l s , par-t i c u l a r l y of the l i v e r , l u n g , kidney and I n t e s t i n e . G e n e r a l l y speaking, enzymes a t these s i t e s decrease the l i p i d s o l u b i l i t y of a wide v a r i e t y o f endogenous and exogenous compounds which f a c i l i t a t e s t h e i r e x c r e t i o n . S p e c i f i c a l l y , the "mixed f u n c t i o n oxidase system" mediates such r e a c t i o n s as aromatic h y d r o x y l a t l o n , a l i p h a t i c h y d r o x y l a t l o n , N - d e a l k y l a t i o n , O - d e a l k y l a t l o n and e p o x i d a t l o n . The mixed f u n c t i o n oxidase system i s comprised o f cytochrome P-450, NADPH-cytochrome P-4-50 reductase and phos-p h a t i d y l c h o l i n e . The enzymic a c t i v i t y of t h i s system r e q u i r e s O2 and NADPR"2« Other c h a r a c t e r i s t i c s of the mixed f u n c t i o n oxidase system a r e i 1) I t can be induced eg. by pretreatment w i t h phenobarb-l t a l o r with 3-m e'thylcholanthrene In the case o f cytochrome P-450, and 2) i t can be i n h i b i t e d eg. by carbon monoxide and SKF 52 5A. In a d d i t i o n to the mixed f u n c t i o n oxygenase a c t i v i t i e s , the h e p a t i c endoplasmic r e t i c u l u m a l s o c o n t a i n s a number o f r e d u c t -ases, s u l f o k i n a s e s , the UDP-glucuronyl t r a n s f e r a s e s , and gl u t a t h i o n e S - t r a n s f e r a s e s . Although i t i s known that exogenous s t e r o i d hormones a f f e c t the mixed f u n c t i o n oxidase enzymes, i t i s c o n t r o v e r s i a l what s p e c i f i c e f f e c t s p r o g e s t i n s and estrogens e x e r t on t h i s system. The d i f f i c u l t y i n t e r p r e t i n g previous workers' r e s u l t s a r i s e s 17 because so many factors vary between studies i n the areat various animal species, d i f f e r e n t sexes of animals, d i f f e r e n t , experimental approaches, various contraceptive ster o i d regimens and combinations and d i f f e r e n t lengths of treatments. E a r l i e r studies such as those carried out by Juchau and Pouts (29)» Tephly and Mannering (30) and Freudenthal and Amerson (3D showed progestins to be i n h i b i t o r y on various i n v i t r o and in vivo enzyme a c t i v i t i e s . These workers used male rats l n their exper-imental procedures. Freudenthal and Amerson (3D measured the eff e c t of acute s t e r o i d pretreatment on pentobarbital-induced sleep; whereas, Tephly and Mannering (30) measured the oxidation of ethylmorphine and hexobarbltal by hepatic microsomes obtained from steroid-treated r a t s . Juchau and Fouts (29) determined i n h i b i t i o n i n 2 waysi 1) adding the progestins d i r e c t l y to the incubation mixture and measuring side-chain oxidation of hexobarbltal, r i n g hyd-roxylat i o n of zoxazolamlne, and p-hydroxylatlon of a n i l i n e , and 2) pretreatlng animals with the steroids, obtaining hepatic microsomes and then determining the drug metabolizing a b i l i t y of the microsomes with respect to zoxazolamlne and hexobarbltal. It is notable though that when the st e r o i d was given 1-2 hours before s a c r i f i c e competitive i n h i b i t i o n occurred, but when norethynodrel was administered 18-48 hours before s a c r i f i c e metabolism of hexobarbltal and zoxazolamlne was stimulated. Tuttenberg et a l . (32), carried out s i m i l a r studies measuring i n h i b i t i o n by progestagens on i n v i t r o metabolism of p- n i t r o -anlsole and a n i l i n e . In this case, however, microsomes were obtained from male rats pretreated with phenobarbltal. Soyka and Deckert (33) determined a c t i v i t y of p-nltroanisole 0-demeth-18 y l a t i o n i n microsomes o b t a i n e d from male, female and pregnant r a t s . They found t h a t progesterone, i t s m e t a b o l i t e s and s t r u c -t u r a l l y s i m i l a r s t e r o i d s i n h i b i t e d drug metabolism i n a l l cases. In one of the few human s t u d i e s done l n the are a , O'Malley et a l . (3*0 found t h a t d r u g - m e t a b o l l z l n g c a p a c i t y seemed to be impaired i n women t a k i n g o r a l c o n t r a c e p t i v e s t e r o i d s . They measured mean plasma a n t i p y r i n e h a l f - l i v e s and found t h a t the s t e r o i d - t r e a t e d group had a s i g n i f i c a n t l y higher value than d i d c o n t r o l s u b j e c t s . However, when phenylbutazone plasma h a l f - l i f e was used as the index o f d r u g - m e t a b o l l z l n g a b i l i t y , the I n h i b i t o r y e f f e c t s o f c o n t r a c e p t i v e s t e r o i d s were not seen. Somewhat c o n t r a s t i n g r e s u l t s were obtained by a group of workers i n I t a l y who have p u b l i s h e d s e v e r a l papers i n t h i s a r e a . J o r l e t a l . (35) a d m i n i s t e r e d progesterone, medroxyprogesterone, n o r e t h y n o d r e l , mestranol, e t h i n y l e s t r a d i o l and estrone alone or l n combination f o r k- days or JO days to female r a t s . They compared the e f f e c t o f acute and c h r o n i c treatments w i t h syn-t h e t i c and n a t u r a l l y o c c u r r i n g p r o g e s t a t i o n a l compounds, alone and together w i t h estrogens, on the metabolism of f o r e i g n compounds i n v i v o and i n v i t r o . I t was found t h a t r a t s t r e a t e d c h r o n i c a l l y w i t h medroxyprogesterone alone or l n combination w i t h e t h i n y l -e s t r a d i o l showed an i n c r e a s e d a c t i v i t y o f the three l i v e r enzymes t e s t e d JLn v i t r o t p - n l t r o a n i s o l e O-demethylase, a n i l i n e hydroxylase and amlnopyrlne N-demethylase. In v i v o s t u d i e s showed t h a t medroxyprogesterone alone or combined with e t h i n y l e s t r a d i o l and nor e t h y n o d r e l combined wi t h mestranol, a d m i n i s t e r e d f o r 30 days, a l s o i n c r e a s e d the metabolism of p e n t o b a r b i t a l . B r i a t i c o et a l . (36) and J o r l e_t a l . (37) c a r r i e d out s i m i l a r experiments but, i n a d d i t i o n , attempted to model t h e i r treatments a f t e r p a t t e r n s of 19 human use. That I s, they a d m i n i s t e r e d the s t e r o i d s c h r o n i c a l l y to r a t s , mice and guinea pigs l n r a t i o s used i n human contrac e p -t i v e m e d i c a t i o n and i n doses shown to produce an e x p e r i m e n t a l l y c o n t r o l l e d a n t l f e r t i l i t y a c t i v i t y i n the animal s p e c i e s s t u d i e d . When they measured aminopyrine N-demethylation:, a n i l i n e hyd-r o x y l a t l o n and p - n i t r o a n i s o l e O-demethylation i n v i t r o , they found that a l l enzyme a c t i v i t i e s were Induced l n r a t s and mice. The i n d u c t i o n appeared to be mediated by the progestagenlc component, and was e l i c i t e d a t s t e r o i d doses that b a r e l y p r o-duced a n t l f e r t i l i t y e f f e c t s . Other d r u g - m e t a b o l i z i n g enzymes t h a t have been s t u d i e d are styr e n e monooxygenase and s t y r e n e epoxide hydrase. T h i s system i s of i n t e r e s t because i t i s i n v o l v e d i n the h y d r a t i o n of epox-i d e s ; which, as w i l l be d i s c u s s e d l a t e r , are p o s s i b l e m e t a b o l i t e s of s t e r o i d c o n t r a c e p t i v e s . Salmona ejb a l . (38) found that the c o n t r a c e p t i v e s t e r o i d combination l y n e s t r e n o l + mestranol s t i m u l a t e s s t y r e n e monooxygenase and i n h i b i t s s t y r e n e epoxide hydrase, both processes a l l o w i n g f o r i n c r e a s e d l e v e l s of epoxide to occur. However, In a subsequent paper, J o r l e_t a l . (39) found that l y n e s t r a n o l + mestranol s t i m u l a t e d both epoxide synthetase and epoxide hydratase, as d i d n o r e t h i s t e r o n e + mes t r a n o l . The combination o f n o r e t h y n o d r e l * mestranol caused s t i m u l a t i o n o f epoxide hydratase o n l y . With so many d i f f e r e n t experimental designs and c o n f l i c t i n g r e s u l t s between s t u d i e s , i t i s d i f f i c u l t to deduce the a c t u a l e f f e c t s o f c o n t r a c e p t i v e s t e r o i d s on mixed f u n c t i o n oxidase enzymes. Hence I t would be of i n t e r e s t to c l a r i f y some of t h i s d a t a , i f p o s s i b l e . We wish to I n v e s t i g a t e s e v e r a l drug-metabol-i z i n g enzymes i n v i t r o i n female W i s t a r r a t s over c h r o n i c time 20 p e r i o d s and to attempt to c o r r e l a t e t h i s with o t h e r measures of drug metabolism and h e p a t i c i n t e g r i t y . E. Metabolism o f C o n t r a c e p t i v e S t e r o i d s I t has been p r e v i o u s l y mentioned ( S e c t i o n C) that the m e t a b o l i t e s of o r a l c o n t r a c e p t i v e s t e r o i d s may themselves be r e s p o n s i b l e f o r c e r t a i n h e p a t o t o x i c i t i e s . In t h i s s e c t i o n , the present understanding o f the metabolic f a t e of the parent s t e r o i d s i n the body i s d i s c u s s e d . Evidence which suggests the p o s s i b l e nature o f the t o x i c i t y of the m e t a b o l i t e s i s presented l n S e c t i o n F, 1. S p e c i f i c Pathways of C o n t r a c e p t i v e S t e r o i d Metabolism a. Estrogens The metabolism of estrogens i n v o l v e s the f o l l o w i n g r e a c t i o n s (4o)« 1) o x i d a t i o n - r e d u c t i o n of hydroxy or keto groups i n r i n g D, 2) h y d r o x y l a t i o n s i n p o s i t i o n s 2, 15oCi l6oC, 6<C, 6 ,^ 7<£ mediated by the mixed f u n c t i o n oxidase system, and 3) methylatlons and conjugations to produce s u l f a t e s , g l u c u r o n l d e s , or m e r c a p t u r l c a c i d d e r i v a t i v e s . The main metabolic pathway f o r e l i m i n a t i o n o f the n a t u r a l l y o c c u r r i n g estrogen, e s t r a d i o l , i s v i a l6o0-hydroxylation. Approximately 55$ o f a g i v e n dose o f e s t r a d i o l w i l l be h y d r o x y l a t e d a t the 16<C p o s i t i o n and about 33$ w i l l be h y d r o x y l a t e d a t C2 o f Ring A. (4l) However, the metabolic f a t e o f the s y n t h e t i c estrogens, 1 7 c C - e t h l n y l e s t r a d l o l and mestranol, i s somewhat d i f f e r e n t . B o l t et a l . (4l)-(4-3) have c a r r i e d out i n v i t r o and i n v i v o s t u d i e s and have shown t h a t e t h i n y l e s t r a d i o l i s predominately metabolized to C-2 hydroxylated p r o d u c t s . They found t h a t 2 - h y d r o x y l a t i o n of e t h i n y l e s t r a d i o l i s about 50% higher than 2 - h y d r o x y l a t i o n of e s t r a d i o l . (42) Measurements i n 2 human s u b j e c t s showed 64$ C-2 h y d r o x y l a t i o n and J6% C-2 h y d r o x y l a t i o n o f e t h i n y l -e s t r a d i o l i n a 28 year o l d male and 60 year o l d female r e s p e c t i v e l y . (41) A s i m i l a r p a t t e r n o f p r e f e r e n t i a l C-2 h y d r o x y l a t i o n i s p r o b a b l y seen w i t h mestranol metabolism, s i n c e e t h i n y l e s t r a d i o l i s the major m e t a b o l i t e of mestranol. (44) The d i f f e r e n c e s l n metabolism observed between the n a t u r a l and s y n t h e t i c estrogens are p r o b a b l y a r e s u l t of the presence of the 17-oC e t h i n y l s u b s t i t u e n t In the D r i n g of the s y n t h e t i c hormones. (42) (43) Whereas n a t u r a l estrogens are h y d r o x y l a t e d mainly a t C-16, the 1 7 - o C s u b s t i t u t i o n In e t h i n y l e s t r a d i o l and mestranol hinders r i n g D hydroxy-l a t i o n and r e s u l t s l n a g r e a t e r amount of C-2 h y d r o x y l a t i o n i n the s y n t h e t i c compounds, b. P r o g e s t i n s The metabolism of p r o g e s t i n s i n v o l v e s the f o l l o w i n g pathways! 1) r e d u c t i o n of the 3-keto group to 3oC and 3/f? -hydroxy groups, 2) h y d r o x y l a t i o n l n p o s i t i o n 10 ( p o s s i b l y v i a 5il0 epoxide f o r m a t i o n ) , 3) s h i f t i n g of r i n g A double bond and subsequent aromat-i z a t i o n , and 4) c o n j u g a t i o n to more p o l a r products. In a human study l n 1972, Cook et a l . (45) found t h a t 95% of the u r i n a r y m e t a b o l i t e s of n o r e t h y n o d r e l ( c o r r e s p o n d i n g 22 -•• to 20$ of the g i v e n dose) were conjugated products, 2 of which were glucuronid.es. They a l s o found that the main hydroxylated. compounds were 3oC -hydroxy and to a l e s s e r extent 3/3 -hydroxynorethynodrel. With a s h i f t l n the Ring A double bond, no r e t h y n o d r e l was metabolized "to n o r e t h i n -drone, c. P o s s i b i l i t y of Epoxide Formation i n the Metabolism of  Estrogens and P r o g e s t i n s Thompson and Horning In 19?4 (46) i n v e s t i g a t e d the 1 0-hydroxylation r e a c t i o n of norethindrone to t r y to determine i f the 5»10-epoxlde s t r u c t u r e o f n o r e t h y n o d r e l norethynodrel 10 B-hydroxynorethindrone 5,10 epoxide could g i v e r i s e to the h y d r o x y s t e r o l d . They found t h a t the 5,10-epoxide of no r e t h y n o d r e l had a s t r a i n e d and r e a c t i v e s t r u c t u r e t h at l e d to formation of 1 0-hydroxynorethindrone with no evidence of 5.10 d i o l f o r m a t i o n . Indeed, i n t h i s case, e p o x i d a t l o n was shown to be a normal me t a b o l i c r o u t e f o r e l i m i n a t i o n o f n o r e t h y n o d r e l . However, epoxide f o r m a t i o n may not always lead, to e l i m i n a t i o n of a drug. For example, the metabolic f a t e o f 2-hydroxylated estrogens d i s c u s s e d above may be the f o l l o w i n g ! 1) c o n j u g a t i o n with s u l f h y d r y l - c o n t a i n i n g compounds, e.g. g l u t a t h i o n e , c y s t e i n e , or N - a c e t y l c y s t e l n e , 2) m e t h y l a t i o n of the c a t e c h o l group by catechol-O-methyl t r a n s f e r a s e present i n l i v e r and e r y t h r o c y t e s , or 3) p o s s i b l e epoxide fo r m a t i o n l e a d i n g to i r r e v e r s i b l e b i n d i n g to t i s s u e macromolecules. In t h i s case epoxide f o r m a t i o n c o u l d l e a d to accumulation of a drug or i t s met-a b o l i t e s . These three pathways have i n c r e a s e d s i g n i f i c a n c e i n s y n t h e t i c estrogen metabolism s i n c e C 2 - h y d r o x y l a t i o n i s the prime mech-anism of t h e i r b i o t r a n s f o r m a t i o n . Whereas i n c r e a s e d c o n j u g a t i o n and m e t h y l a t i o n r e a c t i o n s are most l i k e l y of l i t t l e consequence to the organism, i n c r e a s e d I r r e v e r s i b l e b i n d i n g of m e t a b o l i t e s to t i s s u e p r o t e i n s and n u c l e i c a c i d s c o u l d have d i s t i n c t d e l e t e r i o u s e f f e c t s . 24 F. Evidence for Irr e v e r s i b l e Binding of Contraceptive Steroid- Metabolites Two l i n e s of evidence exist to suggest that synthetic estrogen and progestin metabolites may produce t o x i c i t i e s by Irr e v e r s i b l y binding to tissue macromolecules. 1) Indirect evidence i n d i c a t i n g accumulation of the metabolites i n the body. 2) Direct measurements of tissue binding. 1. Accumulation of Metabolites There have been reports of accumulation of synthetic estrogen and progestin metabolites i n animals and l n man. Bolt and Remmer (44) administered radioactive mestranol to rats and found that only 48$ of the dose of mestranol was excreted i n 3 days. About 12$ of the remaining dose could not be extracted with organic solvents or by hot acid hydrolysis i n d i c a t i n g i r r e v e r s i b l e binding. These same workers in a s i m i l a r animal study (47) recovered i n 10 days 57$ of administered r a d i o a c t i v i t y associated with mestranol or e t h i n y l e s t r a d i o l / In this case, only 5$ of the radioactive metabolites of the synthetic estrogens could be extracted, while most of the r a d i o a c t i v i t y was present in compounds not hydrolyzed by hot acid to give ether-extract-able products. In contrast to the above data, recovery of rad-i o a c t i v i t y a f t e r administration of radioactive e s t r a d i o l amount-ed to 75$ In 3 days; and 50$ of that which was not excreted resided i n extractable s u l f a t e and glucuronide conjugates. The authors suggested that the observed accumulation of radio-a c t i v i t y represented i r r e v e r s i b l e binding of metabolites to tissue. [ 4 - l 4 c 25 Human s t u d i e s ( 4 4 ) s h owed t h a t a f t e r a d m i n i s t r a t i o n o f - m e s t r a n o l , o n l y a b o u t 4 0 $ o f t h e a d m i n i s t e r e d d o s e o f r a d i o a c t i v i t y was r e c o v e r e d i n t h e u r i n e . On t h e o t h e r h a n d , C 4 - ^ c [ ] - e s t r a d i o l was n e a r l y c o m p l e t e l y r e c o v e r e d . M i l l s e t a l . ( 4 8 ) h a d p r e v i o u s l y f o u n d t h a t p a r e n t r a d l o l a b e l l e d m e s t r a n o l a n d n o r e t h i n d r o n e w e r e r a p i d l y c l e a r e d f r o m t h e p l a s m a . H o w e v e r , t h e r a d l o l a b e l l e d m e t a b o l i t e s o f t h e s t e r o i d s h ad h a l f - l i v e s o f g r e a t e r t h a n 24 h o u r s i n t h e p l a s m a o f women r e c e i v i n g a s i n g l e d o s e o f r a d l o l a b e l l e d p a r e n t c o m p o u n d . When s u b j e c t s r e c e i v e d d a i l y d o s e s o f t h e p a r e n t s t e r o i d s f o r 6 d a y s t h e r e was a p r o g r e s s i v e s t e p - w i s e I n c r e a s e i n t h e l e v e l o f m e t a b o l i t e s i n t h e b l o o d . T h e s e d a t a s u g g e s t t h a t a c c u m u l -a t i o n o f m e t a b o l i t e s o f c o n t r a c e p t i v e s t e r o i d s d o e s o c c u r i n humans a s w e l l a s l n a n i m a l s . 2. I r r e v e r s i b l e B i n d i n g S t u d i e s One e x p l a n a t i o n o f t h e a c c u m u l a t i o n o f s t e r o i d m e t a b o l i t e s i s , a s m e n t i o n e d a b o v e , t h a t r e a c t i v e m e t a b o l i t e s o f s y n -t h e t i c s t e r o i d s c o u l d b i n d i r r e v e r s i b l y t o t i s s u e m a c r o m o l -e c u l e s . B y d e f i n i t i o n , compounds b o u n d i n t h i s way c a n n o t be r e m o v e d f r o m t i s s u e s b y s o l v e n t e x t r a c t i o n o r b y h o t a c i d h y d r o l y s i s . S e v e r a l i n v e s t i g a t o r s h a v e s t u d i e d p r o p e r t i e s o f i r r e v e r s i b l e b i n d i n g o f o r a l c o n t r a c e p t i v e s t e r o i d m e t a b o l i t e s t o t i s s u e m a c r o m o l e c u l e s . K a p p u s e_t a l . l n 1973 (^9) c a r r i e d o u t i n v i t r o a n d _in v i v o e x p e r i m e n t s w i t h e t h i n y l e s t r a d i o l t o e x a m i n e I r r e v e r s i b l e b i n d i n g . When a l b u m i n was i n c u b a t e d w i t h - e t h i n y l -e s t r a d l o l a n d r a t l i v e r m i c r o s o m e s , some o f t h e r a d i o a c t i v i t y became b o u n d t o a l b u m i n a n d some became t i g h t l y b o u n d t o m i c -r o s o m a l p r o t e i n . The b o u n d r a d i o a c t i v e m e t a b o l i t e s o f e t h i n y l -26 e s t r a d i o l could not be extracted with organic solvents or by-charcoal adsorption. Hence, these authors assumed that i n t h e i r experiments, i r r e v e r s i b l e binding of e t h i n y l e s t r a d i o l metabolites to tissue proteins did Indeed occur. The i r r e v e r s i b l e binding was shown to be dependent upon 2-hydroxylation mediated by the mixed function oxidase system. T s i b r l s and McGulre (40) found that activated metabolites of natural and synthetic estrogen could not only bind to tissue proteins, but they could also i r r e v e r s i b l y bind to polyguanylic a c i d , single stranded DNA, and nucleotides in v i t r o . These workers found that cytochrome P^-450 was involved i n the production of the reactive metabolite. They presented data suggesting that estrogen epoxides may be one type of reactive metabolite involved in i r r e v e r s i b l e binding. Nelson et a l , (50) also investigated the binding of 2-hydroxy-estrogen metabolites. They determined that 2-hydroxyestradiol, 2-hydroxyestrone, and 2-hydroxy, I70Q-ethinylestradiol were converted by r a t l i v e r microsomes to reactive metabolites that became I r r e v e r s i b l y bound to microsomal protein. They showed that cytochrome P-450 was r a t e - l i m i t i n g . These workers, however, postulated that the reactive oxygen moiety at the C-2 p o s i t i o n of estrogen metabolites that was responsible for i r r e v e r s i b l e bind-ing was the superoxide anion based on r e s u l t s using a xanthine-xanthine oxidase system i n the incubation ffilxture. This study, and others (42) (4-3), showed that addition of glutathione to the incubation mixture nearly completely abolished the binding reaction of the 2-hydroxyestrogens, This is probably due to the fact that s u l f h y d r y l groups are known to bind the reactive met-abolites produced by the mixed function oxidase system (epoxides) and the xanthine-xanthine oxidase system (superoxide anion). In v i t r o and In v i v o s t u d i e s on n o r e t h y n o d r e l (51) and n o r e t h i s t e r o n e (52) have provided s i m i l a r data on I r r e v e r s i b l e b i n d i n g with r e s p e c t to the f o l l o w l n g i the requirements f o r the mixed f u n c t i o n oxidase system to a c t i v a t e m e t a b o l i t e s , i r r e v e r -s i b l y bound m e t a b o l i t e s c o u l d not be e x t r a c t e d from t i s s u e , and the presence of g l u t a t h i o n e decreased the amount of b i n d i n g seen. Kappus and Remmer (52) p o s t u l a t e d that the r e a c t i v e m e t a b o l i t e formed, i n t h e i r s t u d i e s on n o r e t h i s t e r o n e was the 4,5-epoxid.e of t h a t compound. These workers found t h a t p h e n o b a r b l t a l a d m i n i s t r a t i o n d i d not i n c r e a s e the b i n d i n g of n o r e t h i s t e r o n e m e t a b o l i t e s . In c o n t r a s t , Chen and Lee (51) found that micro-somes of animals p r e t r e a t e d with p h e n o b a r b l t a l exhibited, twice the amount of i r r e v e r s i b l e b i n d i n g of nontreated microsomes i n d i c a t i n g , i n t h i s case, that the cytochrome P-4-50 system was i n v o l v e d . These s t u d i e s show that c o n t r a c e p t i v e s t e r o i d m e t a b o l i t e s b i n d to h e p a t i c macromolecules i n experimental s i t u a t i o n s . Hence, the p o t e n t i a l f o r h e p a t o t o x i c l t i e s to a r i s e v i a t h i s mechanism i s suggested. 3. E f f e c t s of I r r e v e r s i b l e B i n d i n g a. General E f f e c t s There are few s t u d i e s which r e l a t e i r r e v e r s i b l e b i n d i n g of c o n t r a c e p t i v e s t e r o i d m e t a b o l i t e s to p o s s i b l e t o x i c e f f e c t s of these compounds. N e v e r t h e l e s s , there i s evidence l n the l i t e r a t u r e which o u t l i n e s the p o s s i b l e t o x i c nature of i r r e v e r s i b l e b i n d i n g of m e t a b o l i t e s g e n e r a l l y . In a r e c e n t review, Nelson et a l . (53) p o i n t e d out that t i s s u e damage c o u l d be a s s o c i a t e d w i t h i r r e v e r s i b l e b i n d i n g of mixed f u n c t i o n o x l d a s e - a c t l v a t e d m e t a b o l i t e s . There i s 28 frequent l o c a l i z a t i o n of tissue damage only in species, organs and tissues that contain the mixed function oxidase system. Examples of drugs which produce reactive metab-o l i t e s that bind to tissues and produce t o x i c i t i e s arei 1) i s o n l a z l d , lproniazid-associated hepatic necrosis which Is c l i n i c a l l y indistinguishable biochemically and morph-o l o g i c a l l y from acute hepatocellular Injury caused by v i r a l h e p a t i t i s , 2) acetaminophen, phenacetin-associated hepatic and renal Injury, 3) furosemlde-associated renal injury and hepatic necrosis, 4) cephalorldlne-associated renal injury and hepatic nec-ros i s , Some of these t o x i c i t i e s are more s i g n i f i c a n t when endogenous glutathione Is depleted; for instance, acet-aminophen overdosage may be treated with exogenous sulfhydryl-contalning compounds e.g.cC-mercaptoproprlonyl-glycine (54). These observations suggest that reactive metabolites are associated with the tissue damage. t>» Possible Effects of Contraceptive Steroid I r r e v e r s i b l e  Binding Since the weight of evidence suggests that activated metabolites of acetaminophen, Isonlazid, furosemlde and cephaloridlne can Induce tissue Injury v i a i r r e v e r s i b l e binding to tissue macromolecules, i t could also be possible for contraceptive steroids to cause tissue damage v i a a sim i l a r mechanism. Furthermore, l f hepatic damage resulted from Irreversible binding, the l i v e r could respond to such Injury only l n a limited number of ways. Two such responses 29 could, be h y p e r p l a s i a and p o s s i b l e v a s c u l a r changes. I f such responses d i d occur i n the "damaged" h e p a t i c t i s s u e , i t would, be worthwhile to t r y to c o r r e l a t e extent and s e v e r i t y of I r r e v e r s i b l e b i n d i n g l n the l i v e r to h i s t o p a t h o l o g i c a l changes o c c u r r i n g as a r e s u l t of t h a t damage. A d d i t i o n a l l y , there Is evidence, which w i l l be d i s c u s s e d l n the f o l l o w i n g s e c t i o n ( S e c t i o n G), to suggest that p r e - e x i s t i n g l i v e r l e s i o n s c o u l d a l t e r the course and extent of the hepato-t o x i c i t y . A known hep a t o t o x i n , e t h a n o l , w i l l be examined In t h i s r e g a r d . 30 G. E f f e c t s o f E t h a n o l o n t h e L i v e r E t h a n o l a f f e c t s a l m o s t e v e r y o r g a n s y s t e m l n t h e b o d y . T h i s i s a c c o u n t e d f o r b y t h e f a c t t h a t e t h a n o l i s f r e e l y m i s c l b l e w i t h w a t e r a n d i s r e l a t i v e l y s o l u b l e i n l i p i d s o l v e n t s . H e n c e , e t h a n o l d i f f u s e s a c r o s s c e l l membranes w i t h r e l a t i v e e a s e a n d I s r a p i d l y a n d r e a d i l y d i f f u s i b l e t h r o u g h o u t b o d y f l u i d s and t i s s u e s . (55) E t h a n o l I n g e s t i o n c a n a f f e c t t h e l i v e r , g a s t r o i n t e s t i n a l t r a c t , n e r v o u s s y s t e m , m y o c a r d i u m , s k e l e t a l m u s c l e , a d r e n a l c o r t e x , n e u r o h y p o p h y s i s , t h y r o i d a n d h e m a t o l o g i c t i s s u e s . A d d i t i o n a l l y , e t h a n o l i n g e s t i o n c a n l e a d t o p r o f o u n d a l t e r a t i o n s i n many m e t -a b o l i c p a t h w a y s o f t h e b o d y . C a r b o h y d r a t e , p r o t e i n a n d l i p i d m e t a b o l i s m a r e a l l a f f e c t e d . Some o f t h e m e t a b o l i c a l t e r a t i o n s t h a t e t h a n o l c a n p r o d u c e a r e : d e c r e a s e d g l u c o n e o g e n e s i s , I n c r e a s e d l i p o p r o t e i n s y n t h e s i s , d e c r e a s e d a l b u m i n a n d t r a n s f e r r i n s y n t h e s i s , I n c r e a s e d l i v e r a n d s e r u m t r i g l y c e r i d e l e v e l s , i n c r e a s e d a c e t -a l d e h y d e l e v e l s l n b l o o d and. t i s s u e s , I n c r e a s e d o x y g e n c o n s u m p t i o n a n d i n c r e a s e d NADH-NAD r a t i o . (56) W i t h r e g a r d , t o t h e p r e s e n t s t u d y , h o w e v e r , i t i s n e c e s s a r y t o f o c u s p r i m a r i l y o n : 1) s t r u c t u r a l a n d f u n c t i o n a l c h a n g e s l n t h e l i v e r r e s u l t i n g f r o m e t h a n o l i n g e s t i o n , 2) a l t e r a t i o n s i n h e p a t i c d r u g - m e t a b o l l z l n g enzymes p r o d u c e d b y e x p o s u r e o f h e p a t i c t i s s u e t o e t h a n o l , a n d 3) how e t h a n o l c a n e n h a n c e h e p a t o t o x i c l t y p r o d u c e d , b y o t h e r a g e n t s . ! • S t r u c t u r a l a n d F u n c t i o n a l A l t e r a t i o n s I t i s now known t h a t a l c o h o l I t s e l f c a n p r o d u c e a d i r e c t t o x i c e f f e c t o n t h e l i v e r i n d e p e n d e n t o f m a l n u t r i t i o n . T h i s d i r e c t h e p a t o t o x i c e f f e c t ha s b e e n s hown l n r a t , d o g , b a b o o n a n d man b o t h i n c h r o n i c a l c o h o l i c s a n d i n n o n a l c o h o l i c v o l u n t e e r s . I n t h e s e s t u d i e s , d i e t s u p p l e m e n t s I n c o r p o r a t i n g l a r g e a m o u n t s o f 31 p r o t e i n , m i n e r a l s , and vitamins ( i n c l u d i n g c h o l i n e c h l o r i d e ) d i d not prevent the formation of the a l c o h o l - I n d u c e d h e p a t i c l e s i o n . ( 5 7 ) G r o s s l y , a l c o h o l i n g e s t i o n r e s u l t s i n " f a t t y l i v e r " . This l e s i o n r e p r e s e n t s an abnormal accumulation of f a t w i t h i n h e p a t i c parenchymal c e l l s . The appearance of f a t vacuoles w i t h -i n c e l l s I n d i c a t e s an a b s o l u t e i n c r e a s e i n i n t r a c e l l u l a r l i p i d s . Although i t i s not completely understood, I t i s l i k e l y t h a t a l c o h o l a c t s a t more than one p o i n t l n the metabolism of f a t to cause f a t accumulation l n l i v e r c e l l s . A l c o h o l i c f a t t y change could r e s u l t from increased amounts of f a t b e i n g m o b i l i z e d from p e r i p h e r a l f a t depots, decreased f a t t y a c i d u t i l i z a t i o n l n the l i v e r , Increased s y n t h e s i s of t r i g l y c e r i d e s i n the l i v e r , or from impaired t r a n s p o r t of l i p i d s as l i p o p r o t e i n s from the l i v e r i n t o the c i r c u l a t i o n . Whatever the mechanism, f a t t y accumulation can occur even a f t e r v e r y b r i e f exposures to a l c o h o l e.g. w i t h i n 2 days l f 18 to 24 ounces o f a l c o h o l per day i s i n g e s t e d . ( 5 8 ) S m a l l e r amounts of a l c o h o l over a l o n g e r time span have the same e f f e c t . M i l d f a t t y change may not a f f e c t the gross appearance of the l i v e r and i s r e a d i l y r e v e r s i b l e when a l c o h o l i s withdrawn. However, i f accumulation of f a t i n c r e a s e s , the organ enlarges and becomes p r o g r e s s i v e l y more yellow. In extreme Instances, the l i v e r may weigh 5 to 6 kilograms and be transformed Into a b r i g h t yellow, s o f t , greasy organ. Such cases have a s s o c i a t e d h e p a t i c d y s f u n c t i o n and are most o f t e n seen l n c h r o n i c a l c o h o l i c s . In t h i s i n s t a n c e , f a t t y change may l e a d to a l c o h o l i c h e p a t i t i s and perhaps e v e n t u a l l y , to f i b r o u s s c a r r i n g of a l c o h o l i c c i r r h o s i s . M i c r o s c o p i c a l l y , f a t t y change begins with development of liposomes p r o b a b l y d e r i v e d from smooth endoplasmic r e t i c u l u m (SER). The s m a l l f a t vacuoles are l o c a t e d i n i t i a l l y l n the c y t o -32 plasm about the nucleus. L a t e r , the vacuoles c o a l e s c e and, l n advanced st a g e s , the l i v e r c e l l may l o o k l i k e an a d u l t adipose t i s s u e c e l l . I f contiguous c e l l s r u p t u r e , the hepatocytes d i e and "become n e c r o t i c , l e a d i n g to a l c o h o l i c h e p a t i t i s . J u s t as a l c o h o l can produce gross and m i c r o s c o p i c l e s i o n s i n the l i v e r independent of m a l n u t r i t i o n , a l c o h o l can a l s o induce u l t r a s t r u e t u r a l changes i n human hepatocytes independent of mal-n u t r i t i o n . The o r g a n e l l e s affected, by a l c o h o l are m i t o c h o n d r i a and smooth endoplasmic r e t i c u l u m (SER). M i t o c h o n d r i a l changes i n the l i v e r Include s w e l l i n g and d i s f i g u r a t i o n of m i t o c h o n d r i a , d i s o r i e n t a t i o n of the c r i s t a e , and l n t r a m l t o c h o n d r l a l c r y s t a l l i n e i n c l u s i o n s . These changes are a s s o c i a t e d with Increased mitochond-r i a l f r a g i l i t y and p e r m e a b i l i t y . Various m i t o c h o n d r i a l enzymes are a l s o a f f e c t e d . (57) E t h a n o l i n g e s t i o n i s a s s o c i a t e d w i t h p r o l i f e r a t i o n of the membranes of the SER, as measured by i n c r e a s e d p h o s p h o l i p i d and p r o t e i n content of hepatocytes. This i n c r e a s e l n SER i s s i m i l a r to the p r o l i f e r a t i o n seen wi t h a d m i n i s t r a t i o n of v a r i o u s t h e r a p e u t i c agents and other x e n o b l o t l c s . (57) Func-t i o n a l l y , i t may be of s i g n i f i c a n c e t h a t a l c o h o l causes pro-l i f e r a t i o n of SER, s i n c e a c e r t a i n percentage of i n g e s t e d a l c o h o l i s m etabolized by the h e p a t i c microsomal enzyme system. 2. M e t a b o l i c Routes o f E t h a n o l In the body, the f i r s t s t e p l n the metabolic c o n v e r s i o n of ethanol i s the p r o d u c t i o n o f acetaldehyde. This i n t e r m e d i a t e i s r a p i d l y converted to a c e t a t e , and most o f t h i s a c e t a t e i s degraded to carbon d i o x i d e and. water. The o x i d a t i o n of ethanol can be c a r r i e d out by three enzyme systems t h e p a t i c a l c o h o l dehydrogenase, c a t a l a s e , and the microsomal ethanol o x i d i z i n g system (MEOS). (55) 33 The main metabolic pathway o f eth a n o l o x i d a t i o n Is c a r r i e d out by he p a t i c a l c o h o l dehydrogenase l o c a t e d l n the c y t o s o l . T h i s z l n c - c o n t a l n i n g enzyme c a t a l y z e s the i n i t i a l o x i d a t i o n o f ethanol to acetaldehyde. The a c t i v i t y o f a l c o h o l dehydrogenase Is NAD dependent and the r a t e - l i m i t i n g s t e p appears to be the a v a i l a b i l i t y o f NAD, not a l c o h o l dehydrogenase. Subsequently, acetaldehyde dehydrogenase and NAD convert acetaldehyde to a c e t y l CoA and e v e n t u a l l y a c e t a t e i s produced. I t i s g e n e r a l l y agreed that there i s no s i g n i f i c a n t enhancement or i n d u c t i o n o f a l c o h o l dehydrogenase a f t e r prolonged a l c o h o l i n g e s t i o n . (55) I n h i b i t i o n o f a l c o h o l dehydrogenase w i t h p y r a z o l e w i l l reduce e t h a n o l metabolism by about 70-75 percent. (57) In some circumstances the enzyme c a t a l a s e may be a f a c t o r In ethanol metabolism. I t r e q u i r e s the presence of hydrogen peroxide (Hg*^) • This enzyme i s prob a b l y not important because i n h i b i t i o n o f c a t a l a s e does not reduce a l c o h o l metabolism. The t h i r d enzymatic pathway t h a t i s a b l e to convert e t h a n o l to acetaldehyde i s l o c a l i z e d l n h e p a t i c SER and i s r e f e r r e d to as the microsomal e t h a n o l o x i d i z i n g system (MEOS). This enzyme r e q u i r e s the presence o f NADPH or a NADPH-generatlng system. The cytochrome i s prob a b l y somewhat d i f f e r e n t , however, from cytochromes P-^50 and P^-450 i n t h a t i t e x h i b i t s d i f f e r e n t sub-s t r a t e s p e c i f i c i t y and I n h i b i t i o n c h a r a c t e r i s t i c s than P-450 and P-X-450. (57) The metabolic a c t i v i t y o f the SER i s l i k e l y to be s i g n i f i c a n t because, as d i s c u s s e d above, ethanol i n g e s t i o n r e s u l t s i n p r o l i f e r a t i o n of SER, and t h i s p r o l i f e r a t i o n u s u a l l y means that the compound i s metabolized by SER. L l e b e r suggests t h a t when no i n d u c t i o n has oc c u r r e d , 20-25 percent o f et h a n o l metabolism Is carried, out by MEOS. (57) Notably, i t has been 34 observed that regular drinkers can tolerate larger amounts of alc o h o l i c beverages than can non-drinkers, and that a l c o h o l i c s develop increased rates of blood ethanol clearance. These effects could be explained on the basis of an increased MEOS a c t i v i t y due to exposure to ethanol. Microsomal induction could also be the explanation for the following observed r e s u l t s obtained with chronic administration of ethanolj (57) 1) increased a c t i v i t y of a vari e t y of microsomal drug-det-oxifying enzymes such that ethanol Ingestion can r e s u l t l n increased clearance of meprobamate, pentobarbital, t o l -butamide, warfarin, dlphenylhydantoln and phenobarbltal, 2) increased cytochrome P-450, 3) increased NADPH cytochrome P-450 reductase, 4) Increased hepatic phospholipid content, 5) increased cholesterol ester synthesis (cholesterol synthesis is carried out by SER), and 6) Increased l i p o p r o t e i n production ln l i v e r . Hence, there Is accumulating evidence that the s t r u c t u r a l changes In the SER that ethanol produces may be associated, with functional changes in the microsomal drug-metabolizing enzyme system. S p e c i f i c effects of ethanol on various mixed function oxidase a c t i v i t i e s are mentioned l n Part III (Results) and Part IV (Discussion). 3. Role of Ethanol ln Enhancing Hepatotoxiclty It can be seen that ethanol Ingestion or administration can lead to possible compromised l i v e r function v i a production of st r u c t u r a l and biochemical defects. Ethanol administration has also been shown to induce c e r t a i n of the mixed function oxidase enzymes. (59) -(64) With these conditions p r e v a i l i n g , an animal 35 may be more s u s c e p t i b l e to a d d i t i o n a l l y a d m i n i s t e r e d h e p a t o t o x i c compounds, f o r example, i n the f o l l o w i n g ways« 1) decreased l i v e r f u n c t i o n c o u l d l e a d to accumulation of he p a t o t o x i c parent compounds, or 2) induced l e v e l s of microsomal enzymes c o u l d l e a d to form a t i o n of h e p a t o t o x i c m e t a b o l i t e s . S e v e r a l o b s e r v a t i o n s support the hypothesis that e t h a n o l enhances h e p a t o t o x i c i t y . Hasumura e_t a l . (65) found t h a t a l c o h o l pre-treatment i n c r e a s e d the h e p a t o t o x i c i t y of C C l ^ i n r a t s . These workers p a i r - f e d female r a t s f o r 4 to 5 weeks with a n u t r i t i o n a l l y adequate l i q u i d d i e t c o n t a i n i n g e i t h e r e thanol (36$ of t o t a l c a l o r i e s ) or l s o c a l o r i c carbohydrate ( c o n t r o l s ) . A dose of C C l ^ (0.5 ml/kg) was a d m i n i s t e r e d i n t r a g a s t r i c a l l y 15 hr. a f t e r e t h a n o l withdrawal. The ethanol p r e t r e a t e d r a t s showed g r e a t e r decreases i n h e p a t i c cytochrome P-450, amlnopyrlne N-demethylase, and glucose 6-phosphatase than d i d the p a i r - f e d c o n t r o l s , I n d i c a t i n g poten-t i a t i o n of C C l ^ h e p a t o t o x i c i t y . In a d d i t i o n , i r r e v e r s i b l e b i n d i n g 14 of C C l ^ m e t a b o l i t e s to microsomal p r o t e i n In v i t r o was observed to be i n c r e a s e d l n microsomes of e t h a n o l - f e d r a t s . S i n c e i t was 14 14 a l s o found t h a t the metabolism of C C l ^ to C0 2 i n v i t r o was enhanced l n the e t h a n o l - f e d r a t s , the authors suggested that the Increased CClj^ h e p a t o t o x i c i t y seen was a r e s u l t o f enhanced microsomal a c t i v a t i o n and b i o t r a n s f o r m a t i o n of C C l ^ . These f i n d i n g s c o r r e l a t e w i t h the c l i n i c a l s i t u a t i o n where a l c o h o l i c s show enhanced s u s c e p t i b i l i t y to h e p a t o t o x i c compounds. S t r u b e l t et a l . (66) c a r r i e d out s i m i l a r i n v e s t i g a t i o n s but a d m i n i s t e r e d lower dose l e v e l s of et h a n o l than d i d Hasumura. (65) Rats g i v e n 5 or 15$ e t h a n o l s o l u t i o n as the s o l e source of f l u i d ( c o r -r e s p o n d i n g to 11-25$ of t o t a l c a l o r i e s ) showed enhanced CC1. 36 h e p a t o t o x i c l t y t h a t was e v i d e n t a f t e r o n l y 1-week exposure to e t h a n o l . H e p a t o t o x i c l t y was measured l n terms of serum enzymes (SGOT, SGPT, and s o r b l t a l dehydrogenase), microsomal a n i l i n e h y d r o x y l a t l o n , and h l s t o p a t h o l o g y . I t was seen to be g r e a t e r i n the 15$ than l n the 5$ e t h a n o l group. The most advanced h e p a t o c e l l u l a r changes were seen l n the C C l ^ - t r e a t e d group r e c e i v i n g 15$ e t h a n o l i massive c e n t r l l o b u l a r n e c r o s i s , f a t d r o p l e t s and p l u r i v a c u o l a r d e g e n e r a t i o n In the s u r r o u n d i n g parenchymal c e l l s , and minimal b a l l o o n i n g of hepatocytes. The authors suggested t h a t the moderate amounts of e t h a n o l consumed by many people c o u l d p o t e n t i a t e C C l ^ h e p a t o t o x i c l t y and p r e -sumably a l s o that due to other such agents. Another f a c e t of e t h a n o l h e p a t o t o x i c l t y was r e v e a l e d by a r e t r o s p e c t i v e study t h a t was c a r r i e d out by Morgan and S h e r l o c k d u r i n g 1975. (67) One hundred p a t i e n t s a t the Royal Free H o s p i t a l i n London who had a h i s t o r y of a l c o h o l abuse and ab-normal l i v e r f u n c t i o n t e s t s were s t u d i e d . Seventy-seven of the p a t i e n t s were men and 23 were women. These workers found a d e f i n i t e sex d i f f e r e n c e i n the course of a l c o h o l i c l i v e r d i s e a s e . They found that women presented with more e s t a b l i s h e d d i s e a s e than d i d men. I t was e v i d e n t a l s o t h a t the women dev-eloped c i r r h o s i s a t a lower l e v e l of a l c o h o l intake and a f t e r a s h o r t e r p e r i o d of time than the men d i d . Men tended to develop macronodular c i r r h o s i s and be a t r i s k to develop primary h e p a t o c e l l u l a r carcinoma. Women were a t low r i s k to develop t h i s carcinoma and t h e i r c i r r h o s i s tended to be micronodular. The authors concluded that female a l c o h o l i c s appear to be more s u s c e p t i b l e not o n l y to l i v e r damage but a l s o to other types of a l c o h o l - r e l a t e d p h y s i c a l i n j u r y ( p e r i p h e r a l neuropathy, pan-c r e a t i t i s ) . 3 7 In summary, not o n l y does a l c o h o l e x e r t a d i r e c t hepato-t o x l c e f f e c t , but i t enhances h e p a t o t o x i c l t y produced, by some c o n c u r r e n t l y a d m i n i s t e r e d agents ( 6 4 , 6 5 ) . With r e s p e c t to c o n t r a c e p t i v e s t e r o i d a d m i n i s t r a t i o n , i t has a l r e a d y been d i s -cussed that a c t i v a t e d m e t a b o l i t e s are produced v i a h e p a t i c microsomal enzyme metabolism. S i n c e ethanol i s r e p o r t e d to induce h e p a t i c microsomes, i t i s p o s t u l a t e d t h a t ethanol pretreatment i n a p p r o p r i a t e doses may enhance t o x i c e f f e c t s of c o n t r a c e p t i v e s t e r o i d s i n t h i s manner. We, t h e r e f o r e , propose to t e s t the hypotheses t h a t female sex hormones and e t h a n o l i n t e r a c t to produce h e p a t o t o x i c e f f e c t s , and t h a t t h i s i n t e r -a c t i o n may be caused by i n d u c t i o n of the drug m e t a b o l i z i n g enzymes r e s u l t i n g l n a subsequent i n c r e a s e i n the f o r m a t i o n of r e a c t i v e m e t a b o l i t e s . Furthermore, we wish to c o r r e l a t e the degree of h e p a t o t o x i c l t y , as measured by b i o c h e m i c a l means, with p a t h o l o g i c a l changes e v i d e n t with l i g h t microscopy. The experiments were c a r r i e d out to i n v e s t i g a t e , i n ethanol t r e a t e d and non-treated r a t s , e f f e c t s of c o n t r a c e p t i v e s t e r o i d admin-i s t r a t i o n on v a r i o u s enzyme l e v e l s , h e p a t i c h i s t o p a t h o l o g y and i r r e v e r s i b l e t i s s u e b i n d i n g . Mestranol and norethindrone were chosen as a p p r o p r i a t e c o n t r a c e p t i v e agents to i n v e s t i g a t e , s i n c e they are used commonly i n humans and because metabolism of these agents i n the r a t seems to be s i m i l a r to that i n humans. In order to approximate c h r o n i c exposure to the c o n t r a c e p t i v e agents, the animals were t r e a t e d f o r 3 months or f o r 6 months. The i n v i t r o h y d r o x y l a t i n g c a p a b i l i t y of h e p a t i c drug m e t a b o l i z i n g enzymes was measured i n c o n t r o l and t r e a t e d r a t s u s i n g benzo(a)-pyrene (cytochrome P - 4 4 8 s u b s t r a t e ) and a n i l i n e (Type I I s u b s t r a t e ) as s u b s t r a t e s (Appendix I I ) . N-demethylation was measured u s i n g 38 amlnopyrlne (Type I compound) as the s u b s t r a t e (Appendix I I ) . S i n c e I t has been shown that r e a c t i v e epoxide Intermediates may p l a y a r o l e In s t e r o i d h e p a t o t o x i c i t y , epoxide hydrase a c t i v i t y was measured u s i n g styrene oxide as the s u b s t r a t e . I t should be noted that the h y d r o x y l a t i n g enzyme a c t i v i t i e s are l o c a t e d In the smooth endoplasmic r e t i c u l u m of l i v e r c e l l s and are dependent on NADPH and 0^ f o r a c t i v i t y , i . e . they are mixed f u n c t i o n o x i d a s e s . Epoxide hydrase a c t i v i t y i s a l s o l o c a t e d l n the smooth endoplasmic r e t i c u l u m , but Is not a mixed f u n c t i o n oxidase. In a d d i t i o n , benzo(a)pyrene hydroxylase a c t i v i t y was measured i n lung t i s s u e to see how a s i m i l a r enzyme a c t i v i t y i n a d i f f e r e n t t i s s u e responded. To study the p o s s i b l e c o r r e l a t i o n of f u n c t i o n a l a l t e r a t i o n s i n drug m e t a b o l i z i n g enzymes and/or "biochemical h e p a t o t o x i c i t y " with p a t h o l o g i c a l changes i n the c e l l , l i g h t microscopy was c a r r i e d out (H & E s t a i n , f a t s t a i n ) . H i s t o l o g i c a l s e c t i o n s were taken from the l i v e r s o f c h r o n i c a l l y t r e a t e d animals a t time o f s a c r i f i c e b e f o r e the t i s s u e was prepared f o r the enzyme assays. As a p o s s i b l e mode of h e p a t o t o x i c i t y , i r r e v e r s i b l e b i n d i n g s t u d i e s were c a r r i e d out. S t e r o i d - t r e a t e d animals were used to determine i f the c o n t r a c e p t i v e s t e r o i d s a f f e c t e d d r u g m e t a b o l i z i n g enzymes i n such a way to produce a c t i v a t e d m e t a b o l i t e s t h a t would b i n d to L H J-benzo(a)pyrene i s metabolized by cytochrome P-448 r a t h e r than P-4-50, i t was found t h a t t h i s compound would not compete f o r drug m e t a b o l i z i n g s i t e s In the c e l l , as would compounds more c l o s e l y r e l a t e d to the c o n t r a c e p t i v e s t e r o i d s . In t h i s way, we expected to l e a r n how the microsomal enzyme a c t i v i t i e s were l i v e r macromolecules and r e s u l t i n t i s s u e damage. benzo(a)pyrene was used as the b i n d i n g compound. Although 39 affected by the contraceptive ster o i d drugs, i f hepatotoxicity was evident f u n c t i o n a l l y and/or morphologically, and i f i r r e -v e rsible binding of metabolites was a possible mechanism of t o x i c i t y . These effects were compared i n animals that had received ethanol and in animals that had not, to determine i f ethanol enhanced any patterns of t o x i c i t y seen. 40 I I . MATERIALS AND METHODS A. Chemicals and Reagents Mestranol (3 methoxy-19-nor-17oC-pregna 1,3,5. (10) t r i e n -20-yn-l?-ol) and norethlndrone (17-hydroxy-19-nor-17cC-pregna-4-en-20-yn-3-one) were supplied by Ortho Pharmaceutical (Canada) Ltd. (Don M i l l s , Ontario). Purity of mestranol and s t a b i l i t y of mestranol solu t i o n were v e r i f i e d by Nujol mull infrared spectrum. Purity of norethlndrone and s t a b i l i t y of norethlndrone solut i o n were v e r i f i e d (In ethanol) by u l t r a v i o l e t spectrum. NADP, NADPH, bovine serum albumin, Trisma Base, calcium acetate, and nonlabelled benzo(a)pyrene (BP) were obtained from Sigma Chemical Co. (St. Louis, Mo.). Benzo(a)pyrene was further p u r i f i e d by d i s s o l v i n g i t in benzene, then f i l t e r i n g and r e c r y s t a l l i z I n g from cold methanol. Trichloroacetic acid was obtained from J. T. Baker Chemical Co. (Ph l l l l p s b u r g , N.J.), MgCl2«^2° ^ r o m B r i t i s h Drug House (Toronto, Ontario), and formaldehyde solu t i o n from Mallinckrodt Chemical Works (St. Louis, Mo.). -Benzo(a)pyrene (pH]-BP) prepared by exchange pro-cedure and p u r i f i e d by column and thin-layer chromatography, was obtained from Amersham Co. (Oakvllle, Ont,). It was generally l a b e l l e d and had a s p e c i f i c a c t i v i t y of 24 Ci/mmole (95 mCl/mg). L Hj-styrene oxide had been prepared in our laboratory by the method of Oesch et a l . (68) and af t e r d i l u t i o n with styrene oxide had a s p e c i f i c a c t i v i t y of approximately 36 uCi/mmole. Biofluor s c i n t i l l a t i o n c o c k t a i l (New England Nuclear, Boston, Mass.) was used and counting e f f i c i e n c y in the Searle Mark III counter (Analytic Inc., Des Plalnes, 111.) ranged between 46.5 - 49$. A n i l i n e HC1 (Eastman Kodak Co., Rochester, N.Y.) and 41 aminopyrlne (Matheson, Coleman, and. B e l l , C i n c i n n a t i , Ohio) were used as substrates for the assays described in Appendix I I . A l l other chemicals and reagents were the best avai l a b l e grades. B. Animals Wlstar rats were obtained, from Canadian Breeding Farms (Montreal, Quebec) at the following weights» normal females 180-200 g, sham-operated and ovarlectomlzed females 40 g, and normal males 250-270 g. Surgical procedures had been carried out on the females as indicated at J-k weeks of age. A l l animals were allowed to equi l i b r a t e for at le a s t 7 days In controlled l i g h t and temperature conditions (0800-2000 hr, 22° C). No treatments were instituted, u n t i l females reached a weight of 210-220 g and males a weight of 290-300 g. Animals were housed 4 or 5 to a cage i n polycarbonate cages on corncob bedding (Lobund grade, Paxton Processing Ltd., Paxton, 111.). During the e q u i l i b r a t i o n period, animals received Purina laboratory chow (Ralston Purina of Canada Ltd., Woodstock, Ont.) and water ad libitum. Cages were changed 2-3 times weekly. Animals were weighed every 8-10 days. C. Treatments 1. Diets Animals were fed a n u t r i t i o n a l l i q u i d supplement d i e t , Sustacal (Mead Johnson Canada, Candlac, Quebec) in order to f a c i l i t a t e adequate ethanol Intake, to act as a vehicle for contraceptive ster o i d administration, and to allow for control of c a l o r i e intake. The experimental and. control diets provided 14$ of t o t a l calories from protein, 13$ from f a t , 33$ from carbohyd-k2 r a t e , and kO% of c a l o r i e s from ethanol (5.6 Kcal/ml) or fi sucrose (k K c a l / g ) . The ethanol i n S u s t a c a l corresponded to an 11$ v/v mixture. C a l o r i c i n take was monitored, d a i l y . Cages of animals were p a i r - f e d such that the c o n t r o l group r e c e i v e d an i s o c a l o r i c amount of sucrose to that of i t s p a i r e d e t h a n o l group. The d i e t s were a d m i n i s t e r e d v i a the u s u a l rubber-stoppered water b o t t l e s . Animals were fed between 1500-1800 hr d a i l y . B o t t l e s and stoppers were cleaned d a i l y . Water was a v a i l a b l e ad l i b i t u m a t a l l times. 2. C o n t r a c e p t i v e S t e r o i d s Mestranol and norethindrone were g i v e n i n doses of 0.6 mg/kg - day and 5.0 mg/kg - day r e s p e c t i v e l y . Groups r e c e i v i n g the combination regimen r e c e i v e d both of these doses together. These doses are g r e a t e r i n amount than human dosage, but a c c o r d i n g to B r l a t l c o et a l . (36) these doses e x e r t m a r g i n a l : a n t l f e r t l l l t y e f f e c t s i n r a t s , t h a t i s , they are approximately e q u l - p h a r m a c o l o g l c a l doses to those i n humans. The drugs were giv e n i n the l i q u i d d i e t as aqueous suspensions and were admin-i s t e r e d i d e n t i c a l l y to sucrose and e t h a n o l matched p a i r s . Animals were t r e a t e d e i t h e r f o r 2 ,4 ,6 ,or 8 weeks, or f o r 3 or 6 months. The 3 month and 6 month animals were used f o r h i s t -opathology s t u d i e s as w e l l as f o r enzyme s t u d i e s . Drugs, e t h a n o l , and sucrose were d i s c o n t i n u e d 2k hours p r i o r to s a c r i f i c e to e l i m i n a t e the p o s s i b i l i t y of c o m p e t i t i v e i n h i b i t i o n o f enzyme a c t i v i t i e s . 3. B i n d i n g S t u d i e s Animals t h a t were used f o r b i n d i n g s t u d i e s received, an I n t r a p e r i t o n e a l I n j e c t i o n of [%J -BP (1.25 umole, 125 MCI) l n corn o i l 2k hours p r i o r to s a c r i f i c e . I f the animals were 43 d r u g - t r e a t e d , the drug was d i s c o n t i n u e d 2k hr p r i o r to i n j e c t i o n D. Enzyme P r e p a r a t i o n f o r Assays Each of the f i v e enzyme assays was c a r r i e d out on t i s s u e from the same animal, and a l l enzyme work was done on the same day. Animals were s a c r i f i c e d between O830 and. 1000 hours. They were stunned, d e c a p i t a t e d , and b l e d . Organs were pe r f u s e d w i t h ice c o l d 1.15$ KC1 and. then placed i n i c e c o l d 1.15$ KC1. A l l f o l l o w i n g procedures were c a r r i e d out a t k° C. Ti s s u e s ( l i v e r , lung) were b l o t t e d , minced and then homogenized, i n a P o t t e r -Elvejhem t i s s u e homogenlzer. L i v e r was homogenized ltk (w/v) i n KC1 f o r 1 minute 15 seconds and l u n g 112 (w/v) f o r k minutes. Homogenates were c e n t r i f u g e d a t 10,000 g f o r 10 minutes. Lung 10,000 g supernatant was used i n the BP hydroxylase assay. L i v e r supernatant was f u r t h e r c e n t r i f u g e d a t 100,000 g f o r 60 minutes to produce the microsomal f r a c t i o n . Each animal had one micro-somal p e l l e t generated f o r use in. the BP hydroxylase ( l i v e r ) , a n i l i n e hydroxylase, and aminopyrlne N-demethylase assays and another f o r use i n the epoxide hydrase assay. The epoxide hydrase p e l l e t s were washed and resuspended i n 0.5 N T r l s HCI wi t h 0 .1$ Tween 80 pH 9 . 0 . The other p e l l e t s were washed and resuspended i n 0.1 M NaKgPO^ b u f f e r pH 7 . 2 . E. T i s s u e P r e p a r a t i o n f o r H i s t o p a t h o l o g y Pathology s e c t i o n s were taken from the l i v e r of each animal a t time o f s a c r i f i c e . One s e c t i o n was taken b e f o r e p e r f u s i o n (with 1.15$ KC1) from the caudate lobe f o r e l e c t r o n microscopy (EM), and another from the same lobe was taken f o r l i g h t micro-scopy (LM). A f t e r p e r f u s i o n , a t h i r d s e c t i o n was taken from the 44 main lobe of the l i v e r for l i g h t microscopy. Specimens for EM o were fixed i n 4 C Karnosky's Fi x a t i v e , and specimens for LM were fixed ln 10$ buffered formalin with calcium acetate 2%. See Appendix I for procedures used to prepare the histology s l i d e s . F» Tissue Preparation for Binding Assay Separate groups of animals were used for the binding experiments than were used for enzyme and hlstopathology work. Animals were s a c r i f i c e d in the usual manner. Tissues were homogenized as mentioned above and homogenates were kept at 4° C G. Enzyme Assays. Five enzyme assays were carried out per animal i 1) l i v e r BP hydroxylase 2) lung BP hydroxylase 3) l i v e r epoxide hydrase (EH) 4) a n i l i n e hydroxylase (see Appendix I I ) , and 5) amlnopyrlne N-demethylase (see Appendix I I ) . 1. Liver Benzo(a)pyrene Hydroxylase Liver BP hydroxylase a c t i v i t y was measured fl u o r o m e t r i c a l l y by the method of Nebert and Gelboin (69) except that albumin was added to the incubation mixture (70). The requirements for l i n e a r i t y of substrate concentration, enzyme concentration and incubation time were previously shown i n our laboratory. The f i n a l volume of the incubation mixture was 1 ml at pH 7.4. It contained O.36 umole NADH, O.36 umole NADPH, 3 umoles MgCl 2, 8 . 7 nmoles (0.6 mg) bovine serum albumin, 150 u l enzyme pre-paration, 80 nmoles BP i n 40 u l acetone, and 25 umole T r i s buffer o pH 7.5. The incubation was carried out for 2.5 minutes at 37 C. Between 1 - 2 mg of l i v e r protein was added to the incubation 45 mixture. A c t i v i t y is expressed as pmole 3-hydroxy-benzo(a)pyrene produced per minute per mg protein. 2. Lung Benzo(a)pyrene Hydroxylase The assay for lung BP hydroxylase a c t i v i t y was carried out s i m i l a r l y except for a few variations i n the incubation noted below. The incubation mix contained 1.08 umoles NADH, 1.08 umoles NADPH, 9 umoles MgClg. 26.2 nmoles (1.8 mg) bovine serum albumin, 450 u l enzyme preparation, 80 nmoles BP in 40 u l acetone, and 25 umoles Trls buffer. The incubation was carr i e d out for o 15 minutes at 37 C. Between 3 - 6 mg of lung protein was added to the Incubation mixture. 3. Liver Epoxide Hydrase Liver EH a c t i v i t y was measured rad l o m e t r l c a l l y by the method of Oesch et a l . (68) except that the incubation period was 10 minutes. The f i n a l volume of incubation mix was 0.4 ml. It contained 12.5 umole Trls HCI buffer pH 9 with 0.025$ Tween 80, 0.08 ml d i s t i l l e d water, 0.81 umole -styrene oxide i n 0.02 ml a c e t o n i t r i l e and 0.2 ml enzyme preparation. The incubation was carried, out at 37° C for 10 minutes. Between 1.5 - 2.5 mg of l i v e r protein was added to the incubation mixture. Assay values are expressed as nmole styrene glycol formed per minute per mg protein and were adjusted for recovery. Recovery of the reaction product ranged between 86 - 98$. Protein determinations were carried out according to the method of Sutherland et a l . (71) as modified by Robson e_t a l . (72), using c r y s t a l l i n e bovine serum albumin as the standard. 46 H. Determination of Ir r e v e r s i b l e Binding of -Benzo(a)pyrene Quantitation of Ir r e v e r s i b l e binding of Injected L J H J - B P to r a t l i v e r macromolecules was determined according to the method of Jollow et a l . (73). Liver and lung homogenates were prepared as described above. To p r e c i p i t a t e protein and nucleic acids, 1 ml aliquots of each homogenate equivalent to 0. 25 g of l i v e r and 0.50 g of lung were added to 2 ml of 0.9 M t r i c h l o r o a c e t i c a c i d . S e r i a l washing and low-speed centrifug-ation of the precipitates were carried out as follows i 3 times with each of 3 ml 0.6 M t r i c h l o r o a c e t i c acid, 3 ml 80$ methanol, 3 ml acetone, and 3 ml ether, u n t i l the supernatants showed no further extraction of r a d i o a c t i v i t y . The extracted protein was dissolved l n 5 ml NaOH. A 0.5 ml ali q u o t , equivalent to 0.025 g of l i v e r and 0.05 g of lung, was taken and added along with 0.5 ml R 1.2 N HC1 to 15 ml Blofluor for counting. The values obtained are expressed as pmole -BP bound per mg protein and are corrected for background and for quenching (external standard method). 1. In Vit r o Studies Studies were carried out to determine the effects of various drug concentrations added _in v i t r o on enzyme a c t i v i t i e s . Drug concentrations tested were 10~?M - 1 0 " % . Drug solutions were added to the incubation mix i n the amounts of d i s t i l l e d water or buffer that were usually present In the assay procedure. Mestranol, norethlndrone, and the combination were added in 0.31 ml of d i s t i l l e d E^0 to the l i v e r BP hydroxylase assay, and in 0.27 ml T r i s buffer pH 7.5 to the lung BP hydroxylase assay. The same steroids were added i n 0.18 ml of Incubation mix to the 47 EH assay. Each assay was s i m i l a r l y t e s t e d i n the presence of e t h a n o l . E t h a n o l was added i n 0.31 ml d i s t i l l e d HgO, 30 u l d i s t i l l e d HgO, or 0.18 ml i n c u b a t i o n mix r e s p e c t i v e l y to the three assays. Subsequent to the a d d i t i o n of the drugs, each assay was c a r r i e d out i n the u s u a l manner. In v i t r o b i n d i n g s t u d i e s were c a r r i e d out a l s o to determine i f the presence of the drugs i n t e r f e r e d w i t h the r e c o v e r y of i r r e v e r s i b l y bound. -BP. J . S t a t i s t i c s Student's t - t e s t s f o r unpaired sample means was c a r r i e d out where i n d i c a t e d . Multi-way a n a l y s i s of v a r i a n c e and c o v a r i a n c e u s i n g a g e n e r a l l i n e a r model (UBC BMD 10V) was used to t e s t f o r i n t e r a c t i o n s of e t h a n o l , s t e r o i d s , and time l n the 3 month and 6 month animals. S i g n i f i c a n t d i f f e r e n c e s were assumed when 0.05. 48 I I I . RESULTS A. Animal Data Animals were c l o s e l y monitored ln order to observe t h e i r response to the experimental conditions. Feeding patterns during the 24-hour period, morbidity and mortality, consumption of calories and ethanol, and. animal weight gains are given below. 1. Feeding Patterns A control study was carried out in which patterns of Intake of the l i q u i d d i e t were measured. As was expected for nocturnal R animals, i t was found that animals re c e i v i n g Sustacal plus sucrose (40$ of calories) drank primarily at night. That i s , i f the l i q u i d d i e t was available at a l l times, 25$ of intake occurred between 0900 - 1900 hr and approximately 75% occurred from 1900 hr to 0900. If the l i q u i d d i e t was available only from 1700 hr to the following 0900 hr, the animals took i n an equivalent number of cal o r i e s to that of the above mentioned group. It appeared that these animals adapted by drinking more in a r e l a t i v e l y shorter period of time. On the other hand, R animals r e c e i v i n g Sustacal plus ethanol (40$ of cal o r i e s ) drank s t e a d i l y throughout the night and day. When the l i q u i d d i e t was always a v a i l a b l e , 35 - 50$ of Intake occurred between 0900 - 1900 hr. If i t was available during the night only, the animals took in only one-half the number of c a l o r i e s . It was concluded, therefore, that a l l animals would always have the l i q u i d d i e t a v a i l a b l e . A l l measurement of food Intake, changing of bottles etc. was carried, out between 1500 - 1800 hr. Since i t was also determined that animals re c e i v i n g ethanol took l n 70$ of the t o t a l number of cal o r i e s compared to animals re c e i v i n g k9 sucrose, I t was r e a l i z e d t h a t f u t u r e s t u d i e s would have to provide f o r p a i r - f e e d i n g o f i s o c a l o r i c amounts of sucrose and e t h a n o l . Water was a v a i l a b l e ad l i b i t u m to the animals but intake never exceeded 8 - 10 ml per day, and was most o f t e n 0 - 5 ml per day. 2. M o r b i d i t y and M o r t a l i t y The f o l l o w i n g l i s t i s an account of a l l m o r b i d i t y and m o r t a l i t y of animals d u r i n g the e n t i r e study. I t should be noted that a l l c o n t r a c e p t i v e - t r e a t e d animals r e c e i v e d o n l y one-half the f i n a l dose of the drug d u r i n g the f i r s t week of a d m i n i s t r a t i o n to decrease acute t o x i c i t i e s . - 8 of the s t e r o i d - t r e a t e d animals developed t r a n s i e n t a l o p e c i a - 3 animals developed s u p e r f i c i a l s k i n i n f e c t i o n s and were s a c r i f i c e d - 2 animals developed middle ear i n f e c t i o n s due to mite i n f e s t a t i o n and were s a c r i f i c e d - 7 animals d i e d of unknown causes (4 of these were i n s t e r o i d plus e t h a n o l - t r e a t e d groups, i . e . 2 mestranol plus e t h a n o l , 2 combination plus ethanol) - 6 other animals were found dead, p o s s i b l y as a r e s u l t o f r e s p i r a t o r y I n f e c t i o n , s i n c e 2 animals from the same shipment had evidence of l u n g i n f e c t i o n a t s a c r i f i c e . The most n o t i c e a b l e e f f e c t of the c o n t r a c e p t i v e s t e r o i d s on the animals was an a n o r e x i c e f f e c t . For the f i r s t s e v e r a l days a f t e r R i n i t i a t i n g s t e r o i d a d m i n i s t r a t i o n , intake o f S u s t a c a l was markedly reduced. However, over a p e r i o d of time the animals adapted to t h i s e f f e c t . 50 3. D i e t a r y C o n s u m p t i o n D a l l y i n t a k e o f c a l o r i e s was r e c o r d e d a n d a n a c c o u n t g i v e n l n m o n t h l y a v e r a g e s i s s h own l n T a b l e I V . I t i s n o t a b l e t h a t , o n a p e r g ram b a s i s , a l l g r o u p s o f a n i m a l s t o o k l n t h e same number o f c a l o r i e s d a i l y , r e g a r d l e s s o f o t h e r t r e a t m e n t s r e c e i v e d e x c e p t f o r t h e I n i t i a l 2 weeks o f c o n t r a c e p t i v e s t e r o i d R a d m i n i s t r a t i o n . A n i m a l s r e c e i v i n g e t h a n o l i n S u s t a c a l u n i f o r m l y t o o k i n a p p r o x i m a t e l y 11 mg E t O H / g r a t p e r d a y ( e . g . a b o u t 2.64 g E t O H / d a y f o r a 250 g r a t ) f o r t h e d u r a t i o n o f t h e s t u d y . The r e s u l t s i n T a b l e I V do n o t s u g g e s t t h a t t h e e t h a n o l - t r e a t e d a n i m a l s g r a d u a l l y i n c r e a s e d t h e i r c o n s u m p t i o n o f e t h a n o l , i n s p i t e o f h a v i n g a d l i b i t u m a c c e s s t o i t . ^ . A n i m a l W e i g h t s F i g u r e 1 i l l u s t r a t e s t h e 6 m o n t h p a t t e r n o f w e i g h t g a i n l n v a r i o u s g r o u p s o f a n i m a l s . I n a l l c a s e s e t h a n o l a d m i n i s t r a t i o n r e s u l t e d i n l o w e r b o d y w e i g h t s c o m p a r e d t o t h e c o r r e s p o n d i n g s u c r o s e c o n t r o l s . The d e c r e a s e i n w e i g h t g a i n c a n n o t b e e x p l a i n e d b y l o w e r c a l o r i e I n t a k e s i n c e s u c r o s e a n d e t h a n o l -t r e a t e d a n i m a l s i n g e s t e d t h e same number o f c a l o r i e s p e r d a y . O v e r a l l , e t h a n o l - t r e a t e d a n i m a l s w e i g h e d 10$ l e s s t h a n s u c r o s e -t r e a t e d a n i m a l s a t t i m e o f s a c r i f i c e . The i n i t i a l d r o p i n w e i g h t e x h i b i t e d b y t h e s t e r o i d - t r e a t e d g r o u p s i s p r o b a b l y due t o t h e a n o r e x i c e f f e c t s o f t h e d r u g s r e s u l t i n g I n a c o r r e s p o n d i n g l y l o w l e v e l o f c a l o r i e i n t a k e . I n 6 m o n t h s , c o n t r o l f e m a l e s g a i n e d 55$ o f t h e i r s t a r t i n g w e i g h t s w h i l e c o n t r o l m a l e s g a i n e d 120$ o f t h e i r s t a r t i n g w e i g h t s , i n s u c r o s e - t r e a t e d a n i m a l s . I n t h e c o r r e s p o n d i n g e t h a n o l g r o u p s , f i g u r e s w e r e 30$ a n d 80$ r e s p e c t i v e l y . S t e r o i d - t r e a t e d g r o u p s r e c e i v i n g s u c r o s e e v e n t u a l l y e x c e e d e d t h e i r s t a r t i n g w e i g h t s b y 5 - 15$ l n a b o u t 4 m o n t h s . Table IV. Ca lo r i c intake of animals per day. Each number represents a monthly average of a l l animals i n the group, expressed as per ra t per day. E = ethanol; S = sucrose ov. = ovar iectomized; sham = sham operated; M = mestranol 0.6 mg/kg/day; N = norethindrone 5.0 mg/kg/day. TREATMENTS 1st 15 days of 2nd month of 4th month of 5th month of 6th month of treatment treatment treatment treatment treatment ml c a l c a l ml c a l c a l ml c a l c a l ml c a l c a l ml c a l c a l day day g day day g day day g day day g day day g S 30.4 45.6 .212 32.8 49.2 .197 36.4 54.6 .201 39.4 59.1 .204 44.6 66.9 .207 E 25.6 38.4 .185 28.7 43.1 .184 34.2 51.3 .201 35.0 53.0 .211 36.9 55.4 .201 S ov. 32.6 48.9 .200 32.7 49.1 .181 38.1 57.2 .189 38.7 58.1 .177 45.7 68.6 .186 E ov. 43.5 65.3 .260 24.2 36.3 .133 38.7 58.1 .200 35.8 53.7 .175 40.2 60.3 .184 S sham 38.8 58.2 .240 34.9 52.4 .221 35.2 52.8 .198 33.8 50.7 .179 41.7 62.6 .207 E sham 33.5 50.3 .200 35.7 53.6 .222 36.6 54.9 .203 33.0 49.5 .183 36.8 55.2 .183 S, M 10.5 15.8 .084 26.2 39.3 .218 27.1 40.7 .189 27.0 40.5 .179 30.8 46.2 .194 E, M 8.3 12.5 .068 22.6 33.9 .192 24.7 37.1 .198 24.1 36.2 .197 24.6 36.9 .182 S, N 17.1 25.7 .127 23.5 35.3 .181 21.5 32.3 .159 26.9 40.4 .187 31.1 46.7 .200 E,: N 13.3 20.0 .103 24.5 36.9 .192 21.7 32.6 .178 24.9 37.4 .193 28.1 42.2 .199 S, M + N 14.2 21.3 .118 27.3 41.0 .202 27.5 41.4 .190 29.5 44.3 .190 26.6 39.9 .180 S, M + N 13.6 20.4 .111 26.9 40.4 .220 27.6 41.4 .222 28.5 42.8 .216 27.3 41.0 .200 S male E male 41.3 62.0 .220 55.5 83.3 .210 62.2 93.3 JL9 59.7 89.6 .170 64.1 96.2 .170 34.9 52.4 .200 52.0 78.0 .210 55.5 83.3 .19 54.9 82.4 .180 59.5 89.4 .190 t o f a c e page 52 F i g u r e 1. Average weight o f an imals d u r i n g the treatment p e r i o d ( c a l c u l a t e d on a monthly b a s i s ) . S u c r o s e - » » ; e t h a n o l = o — — O ; M = mestranol} N = n o r e t h i n d r o n e . 53 S i m i l a r groups r e c e i v i n g e thanol d i d not exceed t h e i r s t a r t i n g weights even a t 6 months. 54 B. I n V i t r o Enzyme S t u d i e s E t h a n o l a n d t h e o r a l c o n t r a c e p t i v e s t e r o i d s m e s t r a n o l and. n o r e t h i n d r o n e w e r e a d d e d In v i t r o t o t h e v a r i o u s I n c u b a t i o n m e d i a , a n d t h e i r e f f e c t s o n enzyme a c t i v i t i e s w e r e d e t e r m i n e d . ( F i g u r e 2) . S i n c e m e s t r a n o l a n d n o r e t h i n d r o n e a r e b o t h m e t a b o l i z e d b y t h e m i x e d f u n c t i o n o x i d a s e s y s t e m , some c o m p e t i t i v e i n h i b i t i o n w o u l d be e x p e c t e d , a t h i g h e r l e v e l s . T h i s i s , i n f a c t , w h a t was s e e n ( F i g u r e 2a, 2b). H e p a t i c a n d l u n g BP h y d r o x y l a s e a c t i v i t y was 85 - 90$ o f c o n t r o l a t 10 ^ M -5 -3 n o r e t h i n d r o n e a n d 10 M n o r e t h i n d r o n e p l u s m e s t r a n o l . A t 10 M -3 n o r e t h i n d r o n e a n d 10 M n o r e t h i n d r o n e p l u s m e s t r a n o l , h e p a t i c and l u n g BP h y d r o x y l a s e e x h i b i t e d a p p r o x i m a t e l y 55$ i n h i b i t i o n . H e p a t i c EH a c t i v i t y was much l e s s s e n s i t i v e t h a n t h e BP h y d r o x y -l a s e a c t i v i t i e s t o a d d i t i o n o f t h e c o n t r a c e p t i v e s t e r o i d s ( F i g u r e 2c). A g a i n , t h i s w o u l d be p r e d i c t e d s i n c e EH i s n o t a m i x e d f u n c t i o n o x i d a s e e n z y m e . I n t h i s c a s e , t h e g r e a t e s t d e c r e a s e -3 i n a c t i v i t y was e v i d e n t w i t h 10 M m e s t r a n o l . T h i s d e g r e e o f l o w e r i n g o f EH a c t i v i t y p r o b a b l y d o e s n o t r e p r e s e n t s i g n i f i c a n t I n h i b i t i o n . S i m i l a r l y , EH a c t i v i t y was n o t a l t e r e d b y a d d i t i o n o f 7 -3 10 M - 10 M e t h a n o l ( F i g u r e 2 f ) . A l s o , l i t t l e o r no c h a n g e i n h e p a t i c BP h y d r o x y l a s e a c t i v i t y o c c u r r e d w i t h e t h a n o l . E t h a n o l d i d , h o w e v e r , c a u s e a p p r o x i m a t e l y 70$ i n h i b i t i o n o f l u n g BP h y d r o x y l a s e a c t i v i t y a t a c o n c e n t r a t i o n o f 10 ^ M ( F i g u r e 2e). L u n g BP h y d r o x y l a s e a c t i v i t y w o u l d p r e d i c t a b l y be more s e n s i t i v e t o t h e i n v i t r o e f f e c t s o f e t h a n o l t h a n w o u l d h e p a t i c BP h y d r o x y -l a s e . T h i s s i t u a t i o n i s p o s s i b l e s i n c e l u n g BP h y d r o x y l a s e l e v e l s o f a c t i v i t y a r e 0.6$ o f h e p a t i c BP h y d r o x y l a s e l e v e l s , a n d h e n c e w o u l d p r o b a b l y be mo re s u s c e p t i b l e t o a d d e d t o x i c compounds o r t o f a c e page 5 5 Figure 2. In v i t r o e f f e c t of contraceptive steroids or ethanol on enzyme assays, BP = "benzo(a)pyrene; M = mestranol; N = norethindrone; n = 2-4. a.HEPATIC BP HYDROXYLASE 175 1 as \ 125. 75. 2 5 . A|Vl-rN ii b u u u d. HEPATIC BP HYDROXYLASE 1 5 0 1 ethanol 100 • j t II a J 0 io"7 io"3 io"3 b. LUNG BP HYDROXYLASE e. LUNG BP HYDROXYLASE 4.0 - r * — A 3.0. A ethanol 1.0. I v n y i 0 io'7 io*5 io"3 E \ c E \ T3 CU E o u 09 c 09 _09 O E e C. HEPATIC EPOXIDE HYDRASE 3.5^. 9^. > g — - I . A R - . 2 . 5 J " | A / • • N ' A M + N M 1 . 5 . 0.5, i t I 1 f. HEPATIC EPOXIDE HYDRASE 3.0 -fc -A- • A — " A ethanol 2 . 0 J 0 10 r7 10 io*: DRUG CONCENTRATION ( M ) 56 d i r e c t molecular competition. In addition, Warren and Bellward have shown (74) that lung BP hydroxylase is s i g n i f i c a n t l y more sensit i v e to induction by 3 - m e t h y l c h o l a n t h r e n e than is l i v e r BP hydroxylase. Therefore, i t may be the case that lung BP hydroxylase is more sensi t i v e also to i n h i b i t o r y Influences. It should be noted that the assay procedures used i n the i n v i t r o studies necessitated adding the drugs either ln water or i n an aqueous buffer. Since the steroids have low s o l u b i l i t y i n water, i t was necessary to add them to the incubation mixtures as homogenous suspensions. At the higher concentrations tested -4 -3 (10 M, 10 M) undissolved drug p a r t i c l e s were present. This might Influence the assay procedure to some degree and may explain, In part, the r e s u l t s seen at these drug l e v e l s . However, at the probable tissue concentrations i n vivo this e f f e c t of the steroids would not be relevant. Therefore, It was concluded that the In v i t r o effects of the steroids and of ethanol would not a l t e r in vivo enzyme a c t i v i t i e s . To further protect against possible extraction of high drug lev e l s from the tissues into the assay, treatments were discontinued 24 hr p r i o r to s a c r i f i c e of the animals. Blood ethanol determinations (Appendix II) supported this view, because at 24 hr a f t e r ethanol administration there were no detectable blood ethanol l e v e l s . 57 C. Short-term In Vivo S t u d i e s 1. Enzyme S t u d i e s a. E f f e c t o f D i e t R Normal female r a t s were f e d S u s t a c a l , alone or a l o n g with sucrose or e t h a n o l , f o r up to 6 weeks to determine i f d i f f e r e n t d i e t s a l t e r e d the microsomal enzyme a c t i v i t i e s . Hepatic BP hydroxylase, l u n g BP hydroxylase, and h e p a t i c EH a c t i v i t i e s were measured (Table V) as w e l l as h e p a t i c a n i l i n e hydroxylase and amlnopyrlne N-demethylase a c t i v i t i e s (Appendix I I ) , No s i g n i f i c a n t e f f e c t s on h e p a t i c and l u n g BP hydroxylase a c t i v i t i e s were e v i d e n t with any of the d i e t s or time periods t e s t e d . T h i s data i s i n agreement wi t h Basu et a l , (75) who found t h a t e l e v a t e d c o n c e n t r a t i o n s of sucrose d i d not change aromatic hydroxylase a c t i v i t y of mature r a t s . However, I t was unexpected to f i n d t h a t ethanol d i d not Induce h e p a t i c BP hydroxylase e s p e c i a l l y a t the 2 week l e v e l . On c l o s e r examination, i t was found t h a t , a t the 2 week time p e r i o d 8 of the 16 animals t h a t R r e c e i v e d ethanol p l u s S u s t a c a l showed s i g n i f i c a n t l y (p<0.05) induced l i v e r BP hydroxylase (169.1 - 12,7) compared to animals t h a t r e c e i v e d S u s t a c a l a l o n e . Although the value o b t a i n e d from the 16 animals (124.6 - 13.2) does not I n d i c a t e enzyme i n d u c t i o n , i t i s notable' t h a t the h i g h e r value (n=8, 169-12 . 0 ) i s i n a s i m i l a r range to the S u s t a c a l plus e t h a n o l values a t 4 and 6 weeks. As mentioned above, the BP hydroxylase a c t i v i t i e s seen d i d not change between 2, 4, and 6 weeks wit h any of the three d i e t s . In a d d i t i o n , BP hydroxylase a c t i v i t i e s were not s i g n i f i c a n t l y Table V. Enzyme act iv i t ies in tissue from female rats fed Sustacal alone, or with sucrose or ethanol. 3enzo(a)pyrene (BP) hydroxylase act iv i ty is expressed as pmole 3-hydroxybenzo(a)pyrene formed per minute per mg protein ± S . E . M . ; epoxide hydrase act ivi ty is expressed as nmole styrene glycol formed per minute per mg protein ± S.E.M. Brackets ( ) indicate n value. Significance was determined at p<0.05 using the Student's t-test for unpaired sample means, and is represented by paired lower-case letters.*Sucrose and ethanol were supplied .as 40% of calories. DIET BP hydroxylase act iv i ty ( l iver) BP hydroxylase act iv i ty (lung) Epoxide hydrase act iv i ty (l iver) 2 week 4 week 6 week 2 week 4 week 6 week 2 week 4 week 6 week Plain Sustacal^ 1 3 4 . 8 ± 4.5 1 4 2 . 2 ± 9.9 1 3 8 . 8 ± 1 0 . 6 (16) (4) (4) 0.84 ± 0 . 1 7 - 0.84 ± 0 . 1 4 (15) (4) 3 . 6 0 ± 0.57 3.57 ± 0.45 2.51 ± 0 . 3 7 (4) (4) (4) [b] Sucrose* + Sustacal 1 4 7 . 3 ± 2.5 157.3 ± 8 . 1 139.9 ± 10.6 (12) (4) (3) 0 . 7 9 ± 0.07 0.92 ± 1.10 0.84 ±'6.14 (12) (4) (3) [a] 3 . 4 4 ± 0.33 4.70 ± 0.09 2.56 ± 0 . 1 9 (4) (4) (3) [c] [c] Ethanol* + Sustacal 1 2 4 . 6 ± 1 3 . 2 1 5 9 . 6 ± 7 . 2 164.8 ± 8.9 (16) (4) (3) 1 . 2 1 ± 0.06 1.45 ±0 .15 1 . 4 6 ± 0 . 4 5 (12) (4) (3) [a] 4 . 7 0 ± 0.47 5.92 ± 0.64 3.55 ± 0 . 4 0 (4) (4) (3) [b,d] [d] 59 R d i f f e r e n t i n S u s t a c a l t r e a t e d animals as compared to R Lab-Chow t r e a t e d animals. Lung BP hydroxylase a c t i v i t y was found to be s i m i l a r l n R R S u s t a c a l alone and S u s t a c a l plus sucrose t r e a t e d animals a t a l l time p e r i o d s . Enzyme a c t i v i t y tended to Increase i n e t h a n o l t r e a t e d groups however, and a t 2 weeks, the e l e v a t i o n of l u n g BP hydroxylase a c t i v i t y due to e t h a n o l (1.21 - 0.06, n=12) was s i g n i f i c a n t when compared to the a c t i v i t y i n sucrose t r e a t e d animals (0.79 - 0.07, n=12). There were some s i g n i f i c a n t d i f f e r e n c e s seen i n h e p a t i c EH a c t i v i t i e s between groups r e c e i v i n g the three d i f f e r e n t d i e t s . The g e n e r a l tendency w i t h each of the d i e t s was f o r enzyme a c t i v i t y to r e a c h a peak a t about 4 weeks and d e c l i n e by the 6 week time p e r i o d . Sucrose In the d i e t caused a s i g n i f i c a n t e l e v a t i o n of EH a c t i v i t y a t 4 weeks compared to 2 weeks, and ethanol caused i n d u c t i o n a t 4 weeks compared to 6 weeks. The a d d i t i o n of e t h a n o l i n the d i e t tended to cause an i n c r e a s e i n enzyme a c t i v i t y R over that seen w i t h the S u s t a c a l alone d i e t . A t 4 weeks, the i n c r e a s e i n EH a c t i v i t y due to ethanol was s i g n i f i c a n t a t p<0.05. "b. E f f e c t of C o n t r a c e p t i v e S t e r o i d s Mestranol 0.6 mg/kg - day and/or norethlndrone 5.0 mg/kg - day were added to both the S u s t a c a l alone and R S u s t a c a l plus ethanol d i e t s and were ad m i n i s t e r e d to female r a t s d a l l y f o r up to 8 weeks. Enzyme a c t i v i t i e s R ' R of the S u s t a c a l plus s t e r o i d , and S u s t a c a l plus e t h a n o l and s t e r o i d groups are compared l n Table VI, VII and V I I I . R Hepatic BP hydroxylase a c t i v i t y (Table VI) i n the S u s t a c a l Table VI. Hepatic benzo(a)pyrene (BP) hydroxylase act iv i ty in steroid-treated female rats receiving Sustacal R alone or Sustacal R plus ethanol. Act ivi ty is expressed as pmole 3-hydroxy-benzo(a)pyrene formed per minute per mg protein ± S.E.M. M = mestranol; N = norethindrone; n = 4, except where indicated in brackets ( ) . Significance was determined at p<0.05 using the Student's t-test for unpaired sample means and is represented by paired lower-case let ters . DIET BP hydroxylase act ivi ty (liver) STEROID 2 week 4 week 6 week 8 week Sustacal R only (controls) 134.8 + 4.5 (15) 142.2 ± 9.9 [a,b] 138. 8 ± 10.6 -Sustacal R + M 150.0 + 28.0 131.3 ± 10.4 111. 0 ± 10.5 -Sustacal R + N 142.8 [c] + 7.7 81.1 ± 2.9 [a,c,d] 118. [d] 9 ± 5.4 -Sustacal R + 173.0 ± 25.2 79.4 ± 11.3 131.6 ± 7.6 M + N [e] [b,e,f] [f] Sustacal R + Ethanol 124.6 ± [g,h] 13. 18 (16) 159.6 + 7 .2 164. 8 + 8 .9 (3) — Sustacal R + Ethanol + M 177-1 ± 31. 7 202.4 [i ,k] + 13 .5 (3) 160. 1 + 23 .9 (3) 119 [k] .2 + 8 .3 Sustacal R + Ethanol + N 312.6 ± [g,l,m] 28. 5 (3) 159.7 [m] + 16 .3 227. 7 + 24 .5 202 [1] .7 + 16 .7 Sustacal R + Ethanol + 310.0 ± [h,n,o] 26. 3 228.4 [j] + 24 .2 (3) 145. [n] 9 + 15 .9 141 [o] .2 + 13 .9 M + N 61 Table VII. Lung benzo(a)pyrene (BP) hydroxylase act iv i ty in steroid-treated female rats receiving Sustacal R alone or SustacalR- plus ethanol. Act iv i ty is expressed as pmole 3-hydroxybenzo(a)pyrene formed per minute per mg protein ± S.E.M. M = mestranol; N = norethindrone; n = 4, except where indicated in brackets ( ) . Significance was determined at p <0.05 using the Student's t-test for unpaired sample means and is represented by paired lower-case le t ters . DIET + STEROID BP hydroxylase act iv i ty (lung) 2 week 4 week 6 week 8 week Sustacal R only (controls) 0.84 ± 0.17 (15) - 0.84 ± 0.14 [a] -Sustacal R + M 0.85 ± 0.22 2.30 ± 0.42 0.68 ± 0.17 -Sustacal R + N 0.77 ± 0.14 0.74 ± 0.29 1.03 ± 0.23 -Sustacal R + M + N 0.86 ± 0.18 0.73 ± 0.07 3.15 ± 0.38 [a] -Sustacal R + Ethanol 1.21 ± 0.06 (12) 1.45 ± 0.15 1.46 ± 0.45 (3) -Sustacal R + Ethanol + M 1.55 ± 0.43 1.32 ± 0.09 (3) 2.21 ± 0.81 (3) 0.81 ± 0.18 Sustacal + Ethanol + N - 1.12 ± 0.33 1.50 ± 0.28 0.74 ± 0.16 Sustacal R + Ethanol + M + N 1.12 ± 0.23 (3) 1.17 ± 0.27 (3) 1.39 ± 0.33 1.24 ± 0.35 Table VIII. Hepatic epoxide hydrase act iv i ty in steroid-treated female rats receiving Sustacal R alone or Sustacal R plus ethanol. Activity is expressed as nmole styrene glycol formed per minute per mg protein ± S .E .M. . M = mestranol; N = norethindrone; n = 4, except where indicated. Significance was determined at p<0.05 using the Student's t-test for unpaired sample means and is represented by paired lower-case letters . DIET Epoxide hydrase act ivi ty (liver) + • STEROID 2 week 4 week 6 week 8 week •n Sustacal only 3.60 + 0.57 3.57 ± 0.45 2.51 ± 0.37 (controls) ta] [b,c] Sustacal R + M 3.70 + 0.46 5.77 ± 0.30 4.10 ± 0.20 [a,d] [b] Sustacal R + N 3.70 + 0.46 3.20 ± 0.35 3.50 ± 0.49 [d] Sustacal R + 3.54 + 0.49 4.00 ± 0.21 4.28 ± 0.23 M + N [c] Sustacal R + 4.70 ± 0.47 5.92 ± 0.64 3.55 ± 0.40 (3) Ethanol [e] [f] [f,g] Sustacal* + 4.95 ± 0.30 6.54 ± 0.23 (3) 5.09 ± 0.51 (3) 4.17 ± 0.68 Ethanol + M [h] [h,i] [i] Sustacal* + 7.60 ± 0.47 6.52 ± 0.23 6.40 ± 0.22 5.51 ± 0.52 Ethanol + N [e,j] [g] [j] Sustacal R + Ethanol + • M + N 5.71 ± 0.39 5.89 + 0.71 4.94 ± 0.42 5.54 ± 0.57 63 plus s t e r o i d t r e a t e d animals was not s t a t i s t i c a l l y d i f f e r e n t from c o n t r o l a t 2 weeks, i . e . the c o n t r a c e p t i v e s t e r o i d s (mestranol, norethindrone, mestranol + norethindrone) d i d not cause s i g n i f i c a n t enzyme i n d u c t i o n a t t h i s time p e r i o d . However, the c o n t r o l v alue f o r 2 week S u s t a c a l treatment as g i v e n i n the t a b l e (134.8 - 4.5) was determined over a p e r i o d of months as a r e s u l t of 4 separate experiments. R A 2 week S u s t a c a l d e t e r m i n a t i o n was c a r r i e d out a t the same R time as the 2 week S u s t a c a l plus s t e r o i d d e t e r m i n a t i o n s . At t h a t p o i n t l n time the h e p a t i c BP hydroxylase a c t i v i t y was 74.3 ± 5 . 8 , n=4. I f t h i s value i s compared to the 2 week s t e r o i d values (150.0 - 28.0, 142.8 - 7.7, 173.0 - 25.2) then i t i s e v i d e n t t h a t each of the s t e r o i d s treatments caused s i g n i f i c a n t i n d u c t i o n of l i v e r BP hydroxylase. I f t h i s same c o n t r o l value i s used as a b a s i s of comparison, i t can a l s o be seen that i n the case of norethindrone and mestranol plus norethindrone there was a 2 week r h y t h m i c a l f l u c t u a t i o n i n enzyme l e v e l s . A t 2 weeks there was i n d u c t i o n , f o l l owed by a r e t u r n to " c o n t r o l " l e v e l s a t 4 weeks, f o l l o w e d l n t u r n by a l e s s e r but s t i l l s i g n i f i c a n t i n d u c t i o n a t 6 weeks. When eth a n o l was added to the d i e t , s i g n i f i c a n t i n d u c t i o n of h e p a t i c BP hydroxylase was e v i d e n t a t 2 weeks of norethindrone and mestranol plus norethindrone therapy, and a t 4 weeks of mestranol therapy. These values were s i g n i f i c a n t (p<0.05) whether they were compared to t h e i r c o r r e s p o n d i n g 2 week ethanol values or to t h e i r c o r r e s p o n d i n g non-ethanol s t e r o i d t r e a t e d groups. Longer treatment with s t e r o i d s and etha n o l tended to decrease the induced enzyme l e v e l s , u n t i l a t 8 weeks, s i g n i f i c a n t decreases were e v i d e n t w i t h a l l three 64 s t e r o i d plus ethanol regimes. S i g n i f i c a n t induction of BP hydroxylase a c t i v i t y (Table VII) i n lung tissue was evident at 4 weeks of Sustacal plus mestranol therapy, and at 6 weeks of mest-ranol + norethlndrone therapy. In the ethanol plus steroi d treated groups, there were no s i g n i f i c a n t a l t e r -ations l n enzyme a c t i v i t y , although there was a general tendency for ethanol treated groups to have higher l e v e l s of lung BP hydroxylase than non-ethanol treated groups. Epoxide hydrase a c t i v i t y was s i g n i f i c a n t l y induced with mestranol treatment for 4 and 6 weeks, and with mestranol plus norethlndrone treatment for 6 weeks (Table VIII). There were marked effects on enzyme a c t i v i t y when ethanol was added to the steroi d treatments. In the ethanol treated animals there was s i g n i f i c a n t Induction at 2 and 6 weeks with norethlndrone. S i g n i f i c a n t decreases in hepatic EH a c t i v i t y with time were not seen ln the mestranol plus norethlndrone group, as they were for the mestranol, norethlndrone, and ethanol only treated groups. 2. I r r e v e r s i b l e Binding Studies Female rats were administered mestranol and/or norethlndrone for up to 6 weeks ln the Sustacal d i e t . Twenty-four hours C3H ] - , perltoneally. I r r e v e r s i b l y bound L 3HJ -BP and l l t e s were determined. The data obtained from the Irreversible binding studies were generally non-conclusive because of the large standard errors (Table IX). These large standard errors may have arisen, at least In the lung as a r e s u l t of interference with the assay by the steroids. In v i t r o studies (not shown) Indicated prior to s a c r i f i c e , a dose of L J BP was administered l n t r a -JE -BP metabo-65 Table IX. The effects of mestranol (M) and/or norethlndrone (N) treatment on irreversible binding of [^H]-benzo(a)pyrene (BP) in l iver and lung. Binding is expressed as pmole [^H]-BP bound per mg protein ± S .E .M. ; n = 4 except where indicated in brackets ( ) . STEROID Irreversible binding of [3H]-BP in l iver TREATMENT 2 week 4 week 6 week - 1.22 + 0.04 (8) - -M 1.20 + 0.14 2.25 + 0.80 2. 88 + 0.56 N 1. 24 + 0.28 0.95 + 0.15 0. 97 + 0.40 M + N 3.30 + 1.92 1.24 + 0.15 0. 99 + 0.39 Irreversible binding of [JH]-BP in lung 2 week 4 week 6 week M N M + N 0.65 ± 0.03 (8) 1.28 ± 0.18 3.16 ± 1.73 3.39 ± 1.07 0.67 ± 0.74 1.66 ± 0.21 1.79 ± 0.46 1.41 ± 0.61 0.90 ± 0.52 66 that adding v a r i o u s steroid, combinations to the p r e c i p i t a t e d l u n g homogenate decreased, l e v e l s of b i n d i n g by about o n e - h a l f . This e f f e c t was not seen i n l i v e r t i s s u e . Because of the d i f f i c u l t i e s encountered w i t h t h i s b i n d i n g assay, i t was decided not to c a r r y out s i m i l a r s t u d i e s l n c h r o n i c a l l y treated, animals. 67 D. Chronic In Vivo S t u d i e s 1. Enzyme S t u d i e s R R The S u s t a c a l plus sucrose d i e t and the S u s t a c a l p l u s ethanol d i e t were a d m i n i s t e r e d f o r up to 6 months to normal female r a t s , sham-operated and o v a r l e c t o m l z e d female r a t s , and. to normal male r a t s . Eenzpyrene hydroxylase a c t i v i t i e s l n l i v e r and l u n g were measured as was h e p a t i c EH a c t i v i t y . R e s u l t s are c o n t a i n e d i n Table X. A n i l i n e hydroxylase and aminopyrine N-demethylase were a l s o measured (Appendix I I ) . S i g n i f i c a n t decreases l n h e p a t i c BP hydroxylase a c t i v i t y were ev i d e n t l n normal female r a t s t h a t r e c e i v e d e t h a n o l therapy f o r 6 months (p < 0 . 0 5 » Student's t - t e s t ) . The l e v e l s were lower than those seen w i t h 6 month treatment w i t h the sucrose d i e t and. J month treatment with the ethanol d i e t . E thanol therapy a l s o s i g n i f i c a n t l y decreased h e p a t i c BP hydroxylase i n e t h a n o l - t r e a t e d (74 . 5 3 - 4 . 0 , n=l0) o v a r l e c t o m l z e d female r a t s compared to the sucrose fed animals ( 9 7 . 8 9 - 4.2, n=8). There were marked decreases i n BP hydroxylase a c t i v i t i e s i n male r a t s with both ethanol and sucrose treatments f o r 6 months. In a d d i t i o n , e thanol therapy l n male r a t s f o r 6 months r e s u l t e d i n s i g n i f i c a n t l y lower h e p a t i c BP hydroxylase a c t i v i t y ( 4 7 6 . 5 - 27.2, n=4) than d i d sucrose f e e d i n g f o r 6 months ( 6 8 1 . 5 * 53.7, n=4). Although l u n g BP hydroxylase a c t i v i t y i n female r a t s tended to decrease with ethanol treatment the dec-rease was never s t a t i s t i c a l l y s i g n i f i c a n t . However, the enzyme a c t i v i t y i n male r a t s was s i g n i f i c a n t l y reduced i n e t h a n o l -t r e a t e d animals a t both time p e r i o d s . In c o n t r a s t , normal female r a t s ( c o n t r o l and sham-operated) e x h i b i t e d a s i g n i f i c a n t i n c r e a s e (p<:0.05) i n EH a c t i v i t y w i t h Table X. Comparison of enzyme a c t i v i t i e s : female vs male r a t s . Benzo(a)pyrene (BP) hydroxylase a c t i v i t y i s expressed as pmole 3-hydroxybenzo(a)pyrene formed per minute per mg p ro te in ; epoxide hydrase a c t i v i t y i s expressed as nmoles styrene g l y c o l formed per minute per mg p ro te in ; a l l are given ± S.E.M. Brackets ( ) i nd i ca te n va lue. S i gn i f i cance was determined using Student's t - t e s t fo r unpaired sample means at p<0.05 and i s represented by paired lower-case l e t t e r s . GONADAL STATUS DIET ( in Sustaca l R ) BP hydroxylase a c t i v i t y ( l i v e r ) BP" hydroxylase a c t i v i t y (lung) Epoxide hydrase a c t i v i t y ( l i v e r ) 3 month therapy 6 month therapy 3 month therapy 6 month therapy 3 month therapy 6 month therapy Contro l female Sucrose 118.9 ± 6.9 100.8 ± 1 0 . 9 (4) (4) [b] 1.15 ± 0.34 0.73 ± 0.14 (4) (4) 1.77 ± 0.21 1.85 ± 0.19 (4) (4) [ i ] Ethanol 151.5 ± 14.6 69.9 ± 5.5 (4) (4) [a] [a,b] 0.75 ± 0.19 0.72 ± 0.26 (4) (4) 2.31 ± 0.17 2.78 ± 0.08 (4) (4) [.1] Sham-operated Sucrose 86.04 ± 6.4 (9) 0.91 ± 0.15 (9) 1.83 ± 0.13 (9) [k,l,m] Ethanol 85.33 ± 7.4 (7) 0.80 ± 0.11 (9) 3.06 ± 0.29 (6) [k] Ovar iec-tomized female Sucrose 97.89 ± 4.2 (8) [c] 0.61 ± 0.09 (8) 2.62 ± 0.11 (8) [1] Ethanol 74.53 ± 4.0 (10) [c] 0.41 ± 0.07 (9) 2.51 ± 0.11 (5) Contro l male Sucrose 1443.0 ± 1 3 3 . 0 681.5 ± 53.7 (4) (4) [d] [d,f] 0.74 ± 0.15 1.05 ± 0.08 (4) (4) [g] [h] 3.10 ± 0.23 4.91 ± 0.83 (4) (4) Ethanol 1343.0 ± 103.0 476.5 ± 27.2 (4) (4) te] [e , f ] 0.35 ± 0.02 0.55 ± 0.11 (4) (4) [g] [h] 3.72 ± 0.42 5.34 ± 0.59 (4) (4) 69 6 month ethanol treatment compared to sucrose treatment. There was also an Increase with 6 month ethanol treatment compared with 3 month ethanol treatment i n the normal controls. Epoxide hydrase a c t i v i t y In male rats tended to increase with duration of therapy and with ethanol treatment but did not a t t a i n s t a t i s t i c a l s i g n i f i c a n c e . Normal female rats were treated c h r o n i c a l l y for up to 6 months with the contraceptive steroids mestranol (0.6 mg/kg - day) and/or norethlndrone (5.0 mg/kg - day). The drugs were administered d a l l y in the sucrose d i e t or ethanol d i e t . Hepatic BP hydroxylase a c t i v i t y , lung BP hydroxylase a c t i v i t y and hepatic EH a c t i v i t y were measured. Hepatic BP hydroxylase a c t i v i t y (Table XI) was s i g n i f i c a n t l y Increased from control (118.9 - 6.9, n=4) In animals that received sucrose plus contraceptive s t e r o i d for 3 months duration. However, l f therapy was continued to 6 months no s i g n i f i c a n t increases i n a c t i v i t y were attained above control le v e l s (100.8 - 10.9f n=4). Animals that received mestranol plus ethanol for 3 months showed a s i g n i f i c a n t l y increased l i v e r BP hydroxylase a c t i v i t y (265.0 - 23.4, n=5) over t h e i r ethanol control (151.5 - 14.6, n=4), but a decrease from the mestranol plus sucrose induced l e v e l (661.8 * 111.8, n=5). Animals that received norethlndrone or mestranol plus norethlndrone along with ethanol did not exhibit an Increase when compared to the ethanol control group (151,5 - 14,6, n=4). In contrast, animals r e c e i v i n g ethanol for 6 months showed a decrease from sucrose control l e v e l s (69.9 ± 5.5, n=4 vs 100.8 - 10,9. n=4) which, although not s i g n i f i c a n t by analysis of variance (BMD 10V), was s i g n i f i c a n t l y d i f f e r e n t by Student's t - t e s t . When ethanol was combined with steroi d therapy for this same time period, this decrease was no 70 Table XI. Hepatic benzo(a)pyrene (BP) hydroxylase a c t i v i t y i n c h r o n i c a l l y treated female r a t s . Enzyme a c t i v i t y i s expressed as pmole 3-hydroxybenzo(a)pyrene formed per min per mg p ro te in ± S.E.M. M = mestranol; N = norethindrone. Brackets ( ) i nd i ca te n va lue. S i gn i f i cance was determined at p<-0.05 ca l cu la ted by ana lys i s of var iance (BMD 10V), and i s represented by paired lower-case l e t t e r s . STEROID DIET BP hydroxylase a c t i v i t y ( l i v e r ) (In Sus taca l R ) 3 month therapy 6 month therapy Sucrose 118.9 ± 6.9 (4) 100.8 ± 10.9 (4) [a,b,c] [ d ( t - t e s t ) , e ] Ethanol 151.5 ± 14.6 (4) 69.9 ± 5.5 (4) U,g] f d , f , h , i , j ] M Sucrose 661.8 ± 111.8 (5) 109.2 ± 16.9 (7) [ a ,k , l ] [k] M Ethanol 265.0 ± 23.4 (5) 145.4 ± 14.0 (8) [g.l.m] [h>m] N Sucrose 240.9 ± 21.0 (5) 114.4 ± 8.4 (7) [b,n,o] [o] N Ethanol 170.7 ± 16.7 (5) 112.0 ± 9.4 (8) [n,p] [ i , p ] M + N Sucrose 156.3 ± 7.1 (5) 156.4 ± 12.3 (5) [ c ( t - t e s t ) ] [ e ( t - t e s t ) ] M + N Ethanol 182.7 ± 9.0 (8) [ q ( t - t e s t ) ] 135.2 ± 14.5 (4) [j,q] 71 longer e v i d e n t . In other words, the presence of mestranol and/or norethlndrone i n animals t r e a t e d f o r 6 months wi t h e t h a n o l appeared to prevent h e p a t i c BP hydroxylase from d e c r e a s i n g . Lung BP hydroxylase a c t i v i t y was s i g n i f i c a n t l y i n c r e a s e d l n mestranol t r e a t e d and norethlndrone t r e a t e d animals a t 3 months (Table X I I ) . Mestranol produced g r e a t e r e f f e c t s than n o r e t h l n d r o n e . Therapy w i t h a s i n g l e s t e r o i d had markedly s i g n i f i c a n t e f f e c t s , while therapy w i t h the combination of s t e r o i d s d i d not cause s i g n i f i c a n t changes In enzyme a c t i v i t y . A f t e r 6 months of t r e a t -ment, no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s from c o n t r o l were noted. S i g n i f i c a n t d i f f e r e n c e s l n h e p a t i c EH a c t i v i t y with c h r o n i c s t e r o i d treatment are l i s t e d i n Table X I I I . I t can be seen that mestranol, norethlndrone, and mestranol plus norethlndrone caused a s i g n i f i c a n t i n c r e a s e l n enzyme a c t i v i t y a t 3 months and a t 6 months compared to t h e i r sucrose c o n t r o l s . The same was t r u e f o r animals r e c e i v i n g the et h a n o l d i e t . This p a t t e r n i s d i f f e r e n t from t h a t o f h e p a t i c BP hydroxylase, where there was a decreased a c t i v i t y i n the 6 month et h a n o l t r e a t e d c o n t r o l group. In the case o f h e p a t i c EH, both ethanol and s t e r o i d treatment u s u a l l y produced an i n c r e a s e l n enzyme a c t i v i t y , i n most cases s t a t i s t i c a l l y s i g n i f i c a n t . The g r e a t e s t e f f e c t , however, was due to the s t e r o i d s . 2. H i s t o p a t h o l o g y The I n t e r p r e t a t i o n of the h i s t o p a t h o l o g y s e c t i o n s was c a r r i e d out by Dr. W.L. Dunn, a. E f f e c t of Sucrose vs E t h a n o l Female and male r a t s were f e d the l i q u i d S u s t a c a l d i e t f o r up to 6 months. E i t h e r sucrose or et h a n o l comprised h0% of the t o t a l number of c a l o r i e s . Twenty-four hours p r i o r to 72 Table XII. Lung benzo(a)pyrene (BP) hydroxylase act iv i ty in chronically treated female rats . Enzyme act iv i ty is expressed as pmole 3-hydroxybenzo(a)pyrene produced per min per mg protein ± S.E.M. M = mestranol; N = norethindrone. Brackets ( ) indicate N value. Significance was determined at p <0.05 as calculated by analysis of variance (BMD 10V), and is represented by paired lower-case let ters . STEROID DIET BP hydroxylase act iv i ty (lung) (in SustacalR) 3 month therapy 6 month therapy - Sucrose 1.15 ± .34 (4) [a,b(t-test)] 0.73 + .14 (4) - Ethanol 0.75 ± .19 (4) [c(t-test)] 0.72 + .26 (4) M Sucrose 24.76 ± 3.31 (5) [a,d,e,f ,g] 0.71 [g] + .17 (7) M Ethanol 3.34 ± .97 (5) [d] 1.16 + .71 (8) N Sucrose 5.05 ± .78 (5) [b,e ,h(t-test) , i ] 1.61 [i] + .66 (7) N • Ethanol 2.39 ± .27 (5) [c ,h,j( t - test)] 0.59 [j] + .11 (8) M + N Sucrose 0.83 ± .30 (5) [f] 0.67 + .12 (5) M + N Ethanol 1.16 ± .28 (8) 0.80 + .19 (4) 73 Table XIII. Hepatic epoxide hydrase act iv i ty in chronically treated female rats . Enzyme act iv i ty is expressed as n mole styrene glycol produced per min per mg protein ± S.E.M. M = mestranol; N = norethindrone. Brackets ( ) indicate n value. Significance was determined at p<0.05 as calculated by analysis of variance (BMD 10V) and is represented by paired lower-case le t ters . STEROID DIET (in SustacalR) Epoxide hydrase act iv i ty ( l iver) 3 month therapy 6 month therapy - Sucrose 1.77 ± 0.21 (4) [a,b,c] 1.85 ± 0.19 (4) [d,e,f,g] - Ethanol 2.31 ± 0.17 (4) [h ( t - tes t ) , i , j ,k ] 2.78 ± 0.08 (4) [d,h,l,m,n] M Sucrose 5.92 ± 1.23 (5) [a,o] 3.20 ± 0.31 (7) [f,o,p] M Ethanol 4.93 ± 0.66 (5) [i] 4.72 ± 0.29 (8) [1,P] N Sucrose 4.09 ± 0.24 (5) [b] 4.16 ± 0.31 (7) [f] N Ethanol 3.85 ± 0.23 (5) [j] 4.19 ± 0.28 (8) [m] M + N Sucrose 3.76 ± 0.14 (5) 3.61 ± 0.22 (5) [c] [g] M + N Ethanol 5.26 ± 0.32 (8) [k] 4.12 ± 0.44 (4) tn] 7 4 s a c r i f i c e the sucrose or et h a n o l was d i s c o n t i n u e d and S u s t a c a l alone was given. S e c t i o n s were taken f o r hematoxylin and e o s i n (H & E) s t a i n and f o r f a t s t a i n from the main lobe and caudate lobe of the l i v e r . As a b a s i s of comparison, a s e c t i o n taken from a Lab Chow f e d female r a t i s shown In F i g u r e 3 « Normal h e p a t i c h i s t o l o g y was e v i d e n t . In c o n t r a s t , the e f f e c t of the sucrose plus S u s t a c a l d i e t i n animals t r e a t e d f o r 3 months Is shown i n F i g u r e 4 . In t h i s case there was a no t a b l e degree of m i c r o d r o p l e t f a t e v i d e n t i n the p e r i p o r t a l zone, and i n some areas there was f a t i n the mld.zonal r e g i o n . Hepatocytes s u r r o u n d i n g the c e n t r a l v e i n were c l e a r o f f a t . When ethanol was gi v e n i n S u s t a c a l f o r 3 months, m i c r o d r o p l e t f a t was more evenly d i s t r i b u t e d i n a l l areas of the l o b u l e ( F i g u r e 5 ) • That i s , there was not o n l y more f a t l n the p e r i p o r t a l r e g i o n than there was In s i m i l a r l y t r e a t e d animals r e c e i v i n g sucrose, but there was more f a t i n the c e n t r a l and midzonal areas as w e l l . I f animals were maintained on the same d i e t s f o r 6 months, s i m i l a r but more advanced changes were e v i d e n t . The sucrose t r e a t e d animals ( F i g u r e 6 ) a g a i n showed a s i g n i f i c a n t degree of p e r i p o r t a l m i c r o d r o p l e t f a t accumulation w i t h the c e n t r a l v e i n area s t i l l r e l a t i v e l y c l e a r . The ethanol t r e a t e d animals ( F i g u r e 7 ) e x h i b i t e d more c e n t r a l f a t than both 3 month ethanol f ed animals and 6 month sucrose f e d animals. In a d d i t i o n , the 6 month ethanol t r e a t e d group e x h i b i t e d more p e r i p o r t a l f a t than the co r r e s p o n d i n g sucrose group. When s e c t i o n s from c o n t r o l and sham-operated female r a t s were compared with s e c t i o n s from o v a r l e c t o m i z e d female r a t s , I t can be seen t h a t the ov a r i e c t o m l z e d animals e x h i b i t e d 75 Figure 3« H & E s t a i n of female r a t l i v e r , show-ing normal hepatic histology. The animal received Lab Chow** and water ad l i b i t u m for 3 months. xlOO. 76 Figure 4. H & E s t a i n of female r a t l i v e r , show-ing some degree of pe r i p o r t a l f a t accu: i l l a t i o n . The animal received Sustacal plus sucrose for 3 months. xlOO. H & E s t a i n of female r a t l i v e r , show-ing d i s t r i b u t i o n of f a t i n a l l 3 zones. The animal r e c e i v e d Sustacal** plus e thanol f o r 3 months. xlOO. t o f a c e page 78 F i g u r e 6. O i l red 0 s t a i n of female r a t l i v e r , showing more advanced d e p o s i t i o n of p e r i p o r t a l f a t . The animal r e c e i v e d S u s t a c a l R plus sucrose f o r 6 months, a. x 100. t>. p e r i p o r t a l area (zone 1). x 400. c. p e r i c e n t r a l a r e a (zone 3)» x 400. 78 t o f a c e page 7 9 Figure ?. O i l red 0 s t a i n of female rat l i v e r , showing more advanced general d i s t -r i b u t i o n of f a t . The animal received S u s t a c a l R plus ethanol for 6 months. a. x 100. b. p e r i p o r t a l area (zone 1). x400. c. p e r i c e n t r a l area (zone 3) x^OO. 79 80 more advanced f a t accumulation than the non-ovariectomlzed animals. The sucrose f ed ovarIectomlzed animals ( F i g u r e 8) showed ex t e n s i v e p e r i p o r t a l f a t accumulation, some of which was, n o t a b l y , l a r g e d r o p l e t f a t . T h i s degree of f a t accumulation was not e v i d e n t i n s i m i l a r l y t r e a t e d non-ovar Iectomlzed animals ( F i g u r e 6 , 9 ) . A s e c t i o n taken from a sham-operated female r a t fed sucrose plus S u s t a c a l f o r 6 months ( F i g u r e 9 ) showed s i m i l a r p a t t e r n s of f a t d i s t r i b u t i o n as d i d the c o n t r o l ( F i g u r e 6 ) . That i s , the p e r i p o r t a l areas had a n o t a b l e amount of m i c r o d r o p l e t f a t formation, but the mldzonal and p e r i c e n t r a l areas were r e l a t i v e l y f r e e of f a t . I t was observed however, t h a t 2 of the 8 l i v e r s s e c t i o n e d showed some s u g g e s t i o n of l a r g e d r o p l e t f a t accumulation, which was not seen In the c o n t r o l l i v e r s . I t was a l s o e v i d e n t t h a t the ethanol f ed ov a r i e c t o m l z e d females ( F i g u r e 10) had more p e r i c e n t r a l f a t than the s i m i l a r l y t r e a t e d sucrose fed animals ( F i g u r e 8). The amount of f a t l n the p e r i p o r t a l r e g i o n s , however, x\ras not d i f f e r e n t from t h a t i n the s u c r o s e - f e d o v a r i e c t o m i z e d animals. In a d d i t i o n , the marked p e r i p o r t a l l a r g e d r o p l e t f a t accumulation was not i n evidence i n the ethanol t r e a t e d animals as i t was i n the sucrose t r e a t e d animals. Instead, there was a more uniform d i s t r i b u t i o n of m i c r o d r o p l e t f a t i n a l l a r e a s , with some p e r i p o r t a l areas showing accumulation of l a r g e f a t d r o p l e t s . The sham-operated females t r e a t e d with S u s t a c a l R plus ethanol ( F i g u r e 11) f o r 6 months e x h i b i t e d l e s s p e r i c e n t r a l f a t than the o v a r i e c t o m l z e d animals f e d •p S u s t a c a l plus ethanol ( F i g u r e 10). The p e r i p o r t a l f a t 81 Figure 8. H & E s t a i n of ovarIectomlzed female rat l i v e r , showing large f at droplet accum-ul a t i o n and microdroplet fat accumulation In zone 1. The animal was fed Sustacal 1 1 plus sucrose for 6 months. xlOO. F i g u r e 9 . H & E s t a i n o f sham-operated female r a t l i v e r , showing f a t a c c u m u l a t i o n In the p e r i p o r t a l r e g i o n . The a n i m a l was fed S u s t a c a l ^ p lus s u c r o s e f o r 6 months. x l O O . 83 H & E s t a i n of o v a r i e c t o m l z e d female r a t l i v e r , showing f a t accumulation l n the c e n t r a l r e g i o n , as w e l l as l n the p e r i p o r t a l and mldzonal areas. The animal was fed S u s t a c a l ^ plus ethanol f o r 6 months. xlOO. Figure 11. H & E s t a i n of sham-operated female ra t l i v e r , showing f a t accumulation mainly ln the p e r i p o r t a l area with some degree of microdroplet fat in the pe r i c e n t r a l area. The animal was fed S u s t a c a l 3 plus ethanol for 6 months. xlOO. 85 was m a i n l y of the m i c r o d r o p l e t type, but a few areas d i d show l a r g e d r o p l e t f a t . With the e x c e p t i o n of the s m a l l amount of l a r g e d r o p l e t p e r i p o r t a l f a t , the sham-operated females f e d ethanol ( F i g u r e 11) showed e s s e n t i a l l y the same changes as the e t h a n o l fed c o n t r o l s ( F i g u r e 7). I t i s n o t a b l e that i n s i m i l a r l y t r e a t e d male r a t s , d i f f e r e n t h i s t o l o g i c a l changes o c c u r r e d compared to female r a t s . For i n s t a n c e , there was a s u g g e s t i o n t h a t male r a t s R t r e a t e d w i t h S u s t a c a l p l u s sucrose f o r 3 months showed s l i g h t l y l e s s p e r i p o r t a l f a t accumulation than female r a t s ( F i g u r e 12 vs F i g u r e 4) although the d i f f e r e n c e was d i f f i c u l t to q u a n t i t a t e . This was a l s o the case with males f e d the sucrose d i e t f o r 6 months ( F i g u r e 14) i n comparison w i t h s i m i l a r l y t r e a t e d females ( F i g u r e 6). However, male r a t s t h a t r e c e i v e d the ethanol d i e t f o r 6 months ( F i g u r e 15) showed more a d v a n c e d p e r i c e n t r a l f a t . accumulation than the females ( F i g u r e 7). In the males there was a s i g n i f i c a n t l a r g e d r o p l e t f a t accumulation whereas i n females there was m i c r o d r o p l e t f a t accumulation. I t was a l s o noted t h a t the male r a t s t r e a t e d with the ethanol d i e t f o r 6 months had much more f a t evident than the animals t r e a t e d f o r 3 months ( F i g u r e 13). In a d d i t i o n , the animals t r e a t e d f o r the l o n g e r time p e r i o d showed l a r g e d r o p l e t f a t , while the s h o r t e r term animals had m i c r o d r o p l e t p e r i c e n t r a l f a t ( F i g u r e 15 vs F i g u r e 13). b. C o n t r a c e p t i v e S t e r o i d Treated Female Rats Female r a t s r e c e i v e d mestranol and/or norethlndrone i n the doses d e s c r i b e d p r e v i o u s l y i n the l i q u i d d i e t f o r up to 6 months. When animals r e c e i v e d mestranol i n the 8 6 F i g u r e 12. H & E s t a i n of male r a t l i v e r , show-in g o n l y s l i g h t p e r i p o r t a l microdrop-l e t accumulation. The animal r e c e i v e d S u s t a c a l 8 plus sucrose f o r 3 months. xlOO. 8 7 H &• E s t a i n of male rat l i v e r , show ing only s l i g h t f a t accumulation in the p e r i c e n t r a l region. The animal was fed S u s t a c a l 3 plus ethanol for 3 months. xlOO. 88 Figure 14. H & E s t a i n of male rat l i v e r , show-ing only s l i g h t p e r i p o r t a l f a t de-position* The animal received S u s t a c a l H plus sucrose for 6 months. xlOO. 89 F i g u r e 15. H & E s t a i n o f male r a t l i v e r , show-in g s i g n i f i c a n t l a r g e d r o p l e t f a t In zone 3« The animal was fed S u s t a c a l plus ethanol f o r 6 months. xlOO. 90 S u s t a c a l R plus sucrose d i e t for 6 months (Figure 16) there appeared to be less p e r i p o r t a l f a t deposition than there was with comparable non-steroid treated animals (Figure 6). I f mestranol was given along with ethanol, there was a very-noticeable even d i s t r i b u t i o n of fat (Figure 17). There was a f a i r amount of f a t seen i n the p e r i p o r t a l and midzonal regions, and this d i s t r i b u t i o n seemed to be in continuity with the f a t evident in the pe r i c e n t r a l region. Norethln-drone treated animals exhibited a more marked zonation of fat d i s t r i b u t i o n than did any other treatment group. In the sucrose plus norethlndrone group, a f a i r amount of fat in the p e r i p o r t a l regions was seen, but none was evident i n the central or midzonal regions (Figure 18). On the other hand, s i m i l a r l y treated animals r e c e i v i n g norethlndrone and ethanol (Figure 19) showed some f a t i n the p e r i c e n t r a l and midzonal regions. There was more marked accumulation of microdroplet f a t in the p e r i p o r t a l regions in these animals than in the corresponding norethln-drone plus sucrose animals. The animals that received the combination s t e r o i d therapy (mestranol plus norethlndrone) showed less microdroplet p e r i p o r t a l f a t than other treatment groups. This can be seen ln the animals treated for 3 months with the steroids and sucrose (Figure 20), where the p e r i p o r t a l microdroplet f a t accumulation was less than that observed in the non-steroid group (Figure 4), Animals that received the steroids and ethanol for 3 months showed l i t t l e or no evidence of fat accumulation l n the pe r i c e n t r a l region (Figure 21), and the amount of fat seen in the p e r i p o r t a l t o f a c e page 91 F i g u r e 16. O i l r e d 0 s t a i n of female r a t l i v e r , showing some degree of z o n a t i o n o f f a t d i s t r i b u t i o n about the p e r i p o r t a l r e g i o n . Animal r e c e i v e d mestranol, 0.6 mg/kg - day, i n S u s t a c a l plus sucrose f o r 6 months. a. p e r i p o r t a l a r e a (zone 1). x250. b. p e r i c e n t r a l a r e a (zone 3). x400. 91 t o f a c e page 92 Figure 17. O i l red 0 s t a i n of female r a t l i v e r , showing even d i s t r i b u t i o n of f a t . Animal received mestranol 0.6 mg/kg -day l n S u s t a c a l R plus ethanol for 6 months. a. xlOO b. p e r i p o r t a l area (zone 1). x^ J-OO. c. p e r i c e n t r a l area (zone 3). x^OO. 9 2 (c) t o f a c e page 9 3 O i l red 0 s t a i n of female r a t l i v e r , showing marked, zonation. Animal rec-eived norethindrone, 5*0 mg/kg - day, in Sustacal" plus sucrose for 6 months, a. xlOO. b. p e r i p o r t a l area (zone 1). x400. c. p e r i c e n t r a l area (zone 3). x^OO. 94 Figure 1 9 . H & E s t a i n of female rat l i v e r , show-ing zonation. Animal received nor-ethlndrone, 5 . 0 mg/kg - day, in Sustacal D I U S ethanol for 6 months. xlOO. 95 Figure 20. H & E s t a i n of female r a t l i v e r , show-ing some degree of periportal micro-droplet fat accumulation. The animal received mestranol, 0.6 mg/kg -day, plus norethindrone, 5.0 mg/kg -day, in the sucrose plus Sustacal di e t for 3 months. xlOO. 96 Figure 21. H & E stain of female rat l i v e r , show-ing l i t t l e or no pericentral fat and some degree of periportal fat dis tr ibut ion. The animal received mestranol, 0 .6 mg/kg -day, plus norethlndrone, 5.0Rmg/kg - day, ln the ethanol plus Sustacal diet for 3 months. xlOO. 97 r e g i o n was l e s s than that seen i n the corresponding n o n - s t e r o i d ethanol t r e a t e d animals ( F i g u r e 5). When compared to animals t h a t r e c e i v e d S u s t a c a l plus sucrose f o r 6 months ( F i g u r e 6 ) , the 6 month combination s t e r o i d plus sucrose t r e a t e d animals ( F i g u r e 22) a l s o showed l e s s p e r i p o r t a l f a t . Although the 6 month combination s t e r o i d and ethanol t r e a t e d animals had more f a t l n the p e r i p o r t a l and p e r i c e n t r a l r e g i o n s than the s i m i l a r s u c r o s e - t r e a t e d animals ( F i g u r e 23 vs. F i g u r e 22), t h i s degree of f a t accumulation was l e s s than was ev i d e n t i n n o n - s t e r o i d ethanol t r e a t e d animals ( F i g u r e 7 ) . In summary, the o v e r a l l d i s t r i b u t i o n o f f a t accumulation i n the ethanol and combination s t e r o i d t r e a t e d animals was q u i t e uniform throughout the l o b u l e . This appeared to be the r e s u l t of R two separate i n f l u e n c e s } 1) decreased S u s t a c a l - a s s o c i a t e d zone 1 f a t accumulation, and 2) i n c r e a s e d e t h a n o l - a s s o c i a t e d zone 3 f a t accumulation. I t should be noted t h a t there were no s i g n i f i c a n t n u c l e a r , h y p e r p l a s t i c , or inflammatory changes seen i n the s e c t i o n s . In p a r t i c u l a r , there was no evidence i n any s e c t i o n s of adenoma for m a t i o n . t o f a c e page 98 Figure 22. O i l red 0 s t a i n of female rat l i v e r , showing s l i g h t accumulation of micro-droplet f a t i n the pe r i p o r t a l region. The animal received mestranol, 0.6 mg/kg - day, plus norethindrone, 5»0 mg/kg - day, in the sucrose plus S u s t a c a l 3 d i e t for 6 months. a. xlOO. b. p e r i p o r t a l area (zone 1). x^OO. c. pe r i c e n t r a l area (zone 3)« x400. 98 t o f a c e page 9 9 Figure 23. O i l red 0 s t a i n of female r a t l i v e r , showing uniform microdroplet fa t d i s -t r i b u t i o n between the 3 zones. The animal received mestranol, 0.6 mg/kg -day, plus norethlndrone, 5.0 mg/kg - day, i n the ethanol plus S u s t a c a l 8 d i e t for 6 months. a . xlOO. b. p e r i p o r t a l area (zone 1). x^OO. c. p e r i c e n t r a l area (zone 3)» x400. 99 IV. DISCUSSION 100 A. Animal Data It became evident In the course of our studies that the ethanol-treated animals and the steroid-treated animals exhibited decreased weight gain compared to control animals despite equl-c a l o r l c ingestion of the l i q u i d d i e t . For instance, i t was found that the ethanol-fed animals Ingested 70$ of the number of calo r i e s of the sucrose-fed animals i n the uncontrolled s i t u a t i o n . This Is ln agreement with other workers' findings (76 -78). Schwetz et a l . (78) found that female rats fed 15$ v/v ethanol In the drinking water Ingested 42$ less chow-type food than animals given tap water. In our study and i n other chronic studies c a r r i e d out by Thorpe and Shorey (79) and Lieber (76), ethanol animals l o s t weight over control animals even when c a l o r i c intake was the same. Lieber suggests that the decreased weight gain ln ethanol-treated animals compared to i s o c a l o r i c a l l y - f e d carbohydrate controls may be due to energy wastage i n the course of ethanol metabolism (80). That i s , microsomal oxidation of ethanol apparently does not r e s u l t in conservation of chemical energy and res u l t s in more heat production than the body needs. Other possible routes of wasted calories with ethanol ingestion could be Interference with digestion and absorption, physiological a l t e r a t i o n s that require more energy expenditure, and enhanced protein catabolism (80). Other workers suggest that energy wastage Is the r e s u l t of an ethanol-induced hypermetabolic state of the l i v e r (81- 85). It was found that chronic treatment with alcohol increased oxygen consumption in r a t s . This e f f e c t has been attributed to an increased hepatic mitochondrial Na + - K + ATPase a c t i v i t y which 101 r e s u l t s In a decreased ATP/ADP r a t i o . In any event, our r e s u l t s show c o n s i s t e n t decreased weight g a i n i n a l l e t h a n o l - t r e a t e d animals compared to i s o c a l o r i c s u c r o s e - t r e a t e d animals. Our r e s u l t s a l s o i n d i c a t e d t h a t treatment w i t h e s t r o g e n i c and. progestogenlc compounds l e d to l a c k of weight g a i n . I t was seen ( F i g u r e 1) that a l l three c o n t r a c e p t i v e s t e r o i d regimes i n i t i a l l y produced, a decrease i n t o t a l S u s t a c a l consumption per gram weight and a weight l o s s . This was probably as a r e s u l t o f a n o r e x i a . Subsequently, these animals r e - e s t a b l i s h e d a normal c a l o r i e per gram d i e t a r y i ntake and surpassed t h e i r s t a r t i n g weights l n 3 - 4 months. However, t h e i r weights a t s a c r i f i c e were about 70$ t h a t of n o n - s t e r o i d t r e a t e d females. These f i n d i n g s agree with those of C l a r k and T a r t t e l i n (86) who found t h a t e s t -rogen treatment reduced both the r a t e of body weight g a i n and u l t i m a t e weight. These same workers a l s o observed that o v a r i -ectomlzed females had. an i n c r e a s e d r a t e of body weight g a i n and an i n c r e a s e d u l t i m a t e body weight compared to c o n t r o l s . T h e i r hypothesis t h a t the e f f e c t of ovariectomy on body weight i s due to the removal of estrogen i s in. agreement w i t h our data. We have found, t h a t ovar lectomlzed females a t t a i n e d approximately 120$ of u l t i m a t e body weight of c o n t r o l s , estrogen treatment decreased body weight g a i n , and females grew a t a slower r a t e than males. Although the s t e r o i d treatments d i d produce marked negative e f f e c t s on animal weight gains, these e f f e c t s were not the r e s u l t of c h r o n i c s t a r v a t i o n s i n c e s t e r o i d - t r e a t e d animals took i n e q u i v a l e n t numbers of c a l o r i e s per gram body weight compared, to c o n t r o l animals a f t e r the f i r s t few weeks of t r e a t -ment. I t should be noted t h a t none of the e t h a n o l - t r e a t e d animals 102 exhibited outward signs of i n e b r i a t i o n . This was expected since the dose of ethanol used (11$ v/v in Sustacal ) was chosen on the basis that i t would produce a chronic low l e v e l exposure. Blood ethanol determinations (Appendix II) indicated that serum concentrations attained were in the range of 35 mg ethanol per 100 ml blood. Schwetz et a l . (78) found that when ethanol was provided as a 15$ v/v solution in drinking water, peak blood lev e l s of alcohol were approximately 40 mg per 100 ml blood. It is apparent from Table VI (Appendix I I ) , however, that the blood determinations obtained in our study were subject to much v a r i a t i o n between animals. For instance, rats 5 and 6 showed no blood ethanol l e v e l s . This Is probably a r e s u l t of feeding patterns such that these animals may not have Ingested any of the Sustacal** plus ethanol for several hours before the experiment. Hence, the ethanol would have been metabolized by the time the blood was drawn. A more detailed analysis of blood ethanol lev e l s would require rigorous time course studies to be carried out. However, the data that we obtained provided evidence of a chronic low l e v e l dosage. Feinman e_t a l . (87) obtained blood alcohol values of 190 mg per 100 ml i n rats by the use of intravenous loading doses, B. Short-term In Vivo Studies The short-term administration of the o r a l contraceptive steroids norethlndrone and mestranol, alone and in combination, produced s i g n i f i c a n t Induction of hepatic microsomal enzymes in most cases. As discussed previously i n reference to Table VI, the magnitude of induction of hepatic BP hydroxylase by the steroids appeared somex^hat lessened as a r e s u l t of using "pooled" data for 103 the S u s t a c a l 3 control l e v e l at 2 weeks (134.8 ± 4 . 5 , n= l 6 ) . The control 2 week value that represented a single determination carried out at the same time as the 2 week Sustacal plus steroid, determinations was found to have a value of 74.3 ± 5 .8 , n=4), Since the d i f f e r e n t enzyme assays were carried out in the same animal, a sim i l a r s i t u a t i o n exists with respect to the 2 week hepatic EH control value (Table VIII). When this control value was determined at the same time as the experimental l e v e l s , enzyme a c t i v i t y was determined, to have a value of 2 .40 - 0.16 (as opposed, to 3.60 - 0 . 5 7 ) . In addition, i t must also be mentioned that the control l e v e l s at 4 weeks and 6 weeks (142.2 * 9.9 and 138.8 ± 10.6) for BP hydroxylase a c t i v i t y and for hepatic EH a c t i v i t y ( 3 .57 - 0 .45 and 2.51 - 0.37) were determined at a d i f f e r e n t point in time than were the st e r o i d determinations. Again, the 4 and 6 week values i n the tables are judged to be somewhat high, i n l i g h t of what the 2 week value was at the appropriate time. This problem in experimental design was overcome i n l a t e r studies and simultaneous controls were run thereafter whenever possible. It is relevant to consider the lower control values because i t would then appear that the 4 week norethindrone and. combination s t e r o i d determinations for BP hydroxylase (81.1 ± 2,9 and. 79.4 ± 11.3) represent control le v e l s rather than inhibited enzyme l e v e l s ; and the 2 week st e r o i d determinations for hepatic EH a c t i v i t y represent elevated enzyme l e v e l s . It can then be seen that, in this study, the o r a l contraceptive steroIds t e n d e d t o c a u s e an increase in hepatic BP hydroxylase a c t i v i t y and EH a c t i v i t y . These re s u l t s compare favorably with the studies of J o r l et a l . (35) (37 . 3 9 ) , B r l a t l c o et a l . (36), and Salmona ejt a l . (38). These workers 104 employed s i m i l a r experimental d e s i g n to the present study i n t h a t the e f f e c t s of short-term c o n t r a c e p t i v e s t e r o i d treatment (up to 30 days) In female r a t s were noted "by det e r m i n i n g i n v i t r o drug m e t a b o l i z i n g a c t i v i t i e s . Other s t u d i e s t h a t have employed d i f f e r e n t experimental designs have produced d i f f e r e n t r e s u l t s . I f , f o r i n s t a n c e , cytochrome P-450 l e v e l s a re used as the s o l e i n d i c a t o r of microsomal enzyme a c t i v i t y , c o n t r a c e p t i v e s t e r o i d combinations have been shown not to be Inducing agents (31). When progestogens were added i n v i t r o a t f a i r l y h igh c o n c e n t r a t i o n s (29» 32) drug metabolism was i n h i b i t e d p r o b a b l y due to d i r e c t c o m p e t i t i o n . A major d i s c r e p a n c y however, e s p e c i a l l y In the e a r l i e r s t u d i e s i n the area,was the use of male r a t s to determine the e f f e c t s o f female sex s t e r o i d s on drug m e t a b o l i z i n g enzymes ( 2 9 - 3 3 ) . I t has been known f o r some time (88, 89) t h a t female sex hormones a d m i n i s t e r e d to male r a t s decrease the c a p a c i t y o f var i o u s h e p a t i c microsomal enzyme a c t i v i t i e s . A d m i n i s t r a t i o n of 1 7 ^ - e s t r a d l o l to male r a t s increased h e x o b a r b l t a l s l e e p i n g time (89) and immature male r a t s given the same hormone d i d not develop l e v e l s o f ethylmorphlne N-demethylase and h e x o b a r b l t a l hydroxylase a c t i v i t i e s u s u a l l y seen l n males as they mature (89). E t h y l -morphine N-demethylase a c t i v i t y was l i k e w i s e decreased from a value o f 223 - 13 to Jk - 8.5 i n male r a t s t r e a t e d with estrogen (88). The bulk of r e c e n t data suggests t h a t c o n t r a c e p t i v e s t e r o i d s tend to induce h e p a t i c d r u g - m e t a b o l l z l n g enzymes i n females a l b e i t not to markedly h i g h l e v e l s . The short-term s t u d i e s a l s o seem to i n d i c a t e t h a t as t r e a t -ment i s continued with c o n t r a c e p t i v e s t e r o i d s f o r up to 8 weeks, h e p a t i c BP hydroxylase and EH a c t i v i t i e s tend to f a l l from induced or Increased, l e v e l s to c o n t r o l l e v e l s (Table VI, Table V I I I ) . 105 This trend i s i n agreement with Parke (90) who suggests t h a t while c o n t r a c e p t i v e s t e r o i d s do produce v a r i a b l e amounts of enzyme i n d u c t i o n in v i v o , on prolonged treatment the enzyme a c t i v i t i e s r e t u r n to normal v a l u e s . A p o s s i b l e e x p l a n a t i o n f o r t h i s t rend could be that the s t e r o i d s produced h e p a t o t o x l c e f f e c t s as d u r a t i o n of treatment i n c r e a s e d , thereby p r o d u c i n g decreased, enzyme f u n c t i o n . This p o s s i b i l i t y was c o n s i d e r e d u n l i k e l y s i n c e t o x i c i t i e s would pr o b a b l y have been manifested by l o w e r - t h a n - c o n t r o l enzyme a c t i v i t i e s ; a l s o , the I r r e v e r s i b l e b i n d i n g s t u d i e s d i d not suggest h e p a t o t o x i c l t y , and subsequent h l s t o p a t h o l o g y d i d not show evidence of marked t o x i c i t y w i t h c o n t r a c e p t i v e s t e r o i d s . Lung BP hydroxylase a c t i v i t y (Table VII) was not i n c r e a s e d to a marked extent with c o n t r a c e p t i v e s t e r o i d a d m i n i s t r a t i o n . Although l u n g t i s s u e Is very s e n s i t i v e to i n t r a p e r i t o n e a l l y a d m i n i s t e r e d 3-methylcholanthrene i n d u c t i o n (74), i t i s not s u r p r i s i n g t h a t l i t t l e i n d u c t i o n w i t h the o r a l l y a d m i n i s t e r e d s t e r o i d agents o c c u r r e d . The l u n g t i s s u e would not be exposed to as h i g h blood l e v e l s of the s t e r o i d s as the l i v e r would, e s p e c i a l l y with the o r a l a d m i n i s t r a t i o n of the drugs. In a d d i t i o n , i t may be that the l e v e l of i n d u c t i o n seen i n the l i v e r i s enhanced over what would be expected due to the presence of estrogen r e c e p t o r s i n the l i v e r (91-96). Experimental evidence suggests t h a t these cytoplasmic estrogen r e c e p t o r s b i n d the steroid, hormone a f t e r i t enters the c e l l . F o l l o w i n g a temp-e r a t u r e dependent t r a n s f o r m a t i o n that occurs l n the cytoplasm or p o s s i b l y i n the nucleus (97)* the hormone-receptor complexes b i n d to chromatin a c c e p t o r s i t e s l n the nucleus. Under the i n f l u e n c e of estrogen a t puberty l n the female r a t , the c o n c e n t r a t i o n o f 106 estrogen r e c e p t o r s i n c r e a s e s 5- to 10- f o l d (95). I t i s p o s s i b l e t h a t exogenous estrogen could produce s i m i l a r i n c r e a s e s l n e s trogen r e c e p t o r c o n c e n t r a t i o n and r e s u l t i n an accentuated i n d u c t i v e e f f e c t on dr u g - m e t a b o l i z i n g enzymes. In the present study, the r e l a t i v e l y s h o r t - t e r m adminis-t r a t i o n of ethanol produced i n c r e a s e s i n d r u g - m e t a b o l i z i n g enzymes t h a t i n s e v e r a l cases were s t a t i s t i c a l l y s i g n i f i c a n t when compared to c o n t r o l . I t can be seen i n Table V that ethanol a d m i n i s t r a t i o n i n c r e a s e d h e p a t i c BP hydroxylase and EH a c t i v i t i e s a t 2, 4 and 6 weeks. Lung BP hydroxylase a c t i v i t y was induced a t 2 weeks o f ethanol a d m i n i s t r a t i o n , and was s t i l l e l e v a t e d a t 4 and 6 weeks of ethanol treatment. A n i l i n e hydroxylase and amlnopyrine N-demethylase a c t i v i t i e s (Table I, Appendix II) were a l s o i n c r e a s e d w i t h ethanol treatment. Our r e s u l t s tend to be i n agreement with those of Rubin et a l . (59)• Rubin and L i e b e r (60), and J o l y et a l . (61) who observed t h a t benzpyrene hydroxylase, p e n t o b a r b i t a l hydroxylase, a n i l i n e h ydroxylase, amlnopyrine N-demethylase, and ethylmorphlne N-demethylase a c t i v i t i e s were Increased i n r a t s f e d e t h a n o l f o r up to 90 days. S i m i l a r trends have been noted i n human s t u d i e s (59t 60, 98). However, the other workers mentioned obtained higher induced l e v e l s of microsomal enzyme compared to ours. This c o u l d p o s s i b l y be exp l a i n e d on the b a s i s of d i e t . J o l y and He'tu (99) have shown t h a t g r e a t e r i n d u c t i o n of drug m e t a b o l i z i n g enzyme a c t i v i t i e s r e s u l t s from h i g h e r f a t d i e t s . Our S u s t a c a l d i e t c o ntained 12$ of c a l o r i e s In f a t , whereas the L i e b e r d i e t c o ntained 35-^0$ f a t . A d d i t i o n a l s t u d i e s c a r r i e d out on the microsomal e t h a n o l - o x i d l z i n g system (61) have shown th a t NADPH-cytochrome P-450 reductase and cytochrome P-450 were 107 elevated, i n female r a t s f ed "}6% of c a l o r i e s as et h a n o l f o r 5 weeks. V l l l e n e u v e et a l . (100) subsequently showed that the cytochrome P-450 from e t h a n o l - t r e a t e d r a t s had. a c a t a l y t i c a c t i v i t y d i f f e r e n t from t h a t of cytochrome P-450 obtained from c o n t r o l , p h e n o b a r b l t a l , and 3-methylcholanthrene-treated r a t s . Kunihiko and L i e b e r (101) c a r r i e d out r e c o n s t i t u t i o n experiments and confirmed these f i n d i n g s . Hence, i t i s apparent t h a t s h o r t - t e r m (up to 3 months) ethanol a d m i n i s t r a t i o n leads to adapt i v e i n c r e a s e s i n the microsomal e t h a n o l - o x i d l z i n g system. These i n c r e a s e s may be a t l e a s t p a r t l y due to q u a n t i t a t i v e and q u a l i t a t i v e changes i n cytochrome P-450.(100, 101). I t can be seen from Tables VI, V I I , and V I I I that when the c o n t r a c e p t i v e s t e r o i d s are gi v e n l n a d d i t i o n to e t h a n o l , g r e a t e r i n d u c t i o n i s ev i d e n t than when s t e r o i d s are gi v e n a l o n e . Another f e a t u r e of note evident i n Table VI and Table V I I I i s t h a t the Induced l e v e l s of h e p a t i c BP hydroxylase and h e p a t i c EH r e s u l t i n g from e t h a n o l plus s t e r o i d treatment tend, to decrease w i t h time. This p a t t e r n , as w i l l be d i s c u s s e d l n the f o l l o w i n g s e c t i o n , i s probably a r e s u l t of ethanol h e p a t o t o x i c l t y which causes decreased, l i v e r f u n c t i o n as d u r a t i o n of treatment lengthens. C. Chronic In Vivo S t u d i e s - C o r r e l a t i o n of Enzyme S t u d i e s  and. H i s t o p a t h o l o g y I t can be seen from examination of Tables X through X I I I , Table I I (Appendix I I ) , and Table I I I (Appendix II) t h a t p a t t e r n s of enzyme a c t i v i t y were markedly d i f f e r e n t i n the c h r o n i c 3 month and. 6 month s t u d i e s than they were i n the short - t e r m s t u d i e s . I t was s t a t i s t i c a l l y v e r i f i e d , by a n a l y s i s of v a r i a n c e (BMD 10V) t h a t i n the long-term s t u d i e s there was a s i g n i f i c a n t 108 4-way I n t e r a c t i o n of ethanol treatment, mestranol treatment, norethlndrone treatment, and d u r a t i o n o f treatment. T h e r e f o r e , comparisons between the s h o r t and long-term s t u d i e s can o n l y be made In a gen e r a l way. In a d d i t i o n , the s t a t i s t i c a l a n a l y s i s i n d i c a t e d t h a t the pa t t e r n s e x h i b i t e d were a r e s u l t of an i n t e r a c t i o n e f f e c t , r a t h e r than to a s i n g l e e f f e c t or to an a d d i t i v e e f f e c t o f experimental c o n d i t i o n s . In both female and male r a t s , a s i g n i f i c a n t s t a t i s t i c a l i n t e r a c t i o n of drug treatments and time was seen with r e s p e c t to he p a t i c BP hydroxylase, a n i l i n e hydroxylase, and amlnopyrine N-demethylase a c t i v i t i e s . At 3 months, ethanol g e n e r a l l y exerted an i n d u c t i v e e f f e c t compared to sucrose c o n t r o l l e v e l s . An opposite e f f e c t was seen a t s i x months, however. The enzyme a c t i v i t y i n 6 month e t h a n o l - f e d animals was decreased s i g n i f i c a n t l y below 3 month sucrose and ethanol c o n t r o l v a l u e s . I t i s hypothesized t h a t t h i s decrease i n enzyme f u n c t i o n i s a r e s u l t of e t h a n o l - a s s o c i a t e d h e p a t o t o x i c i t y . H i s t o p a t h o l o g i c a l evidence i n t h i s r e g ard i s presented below. In a d d i t i o n , female r a t s a d m i n i s t e r e d c o n t r a c e p t i v e s t e r o i d s with and without e t h a n o l (Table X, X I ) , showed a s i g n i f i c a n t s t a t i s t i c a l i n t e r a c t i o n f o r a l l the v a r i a b l e s t e s t e d ( e t h a n o l , mestranol, n o r e t h l n d r o n e , time) w i t h r e s p e c t to h e p a t i c BP hydroxylase. At the 3 month treatment p e r i o d s u c r o s e - f e d animals t r e a t e d with s t e r o i d s were found to have induced l e v e l s of BP hydroxylase (Table XI) compared to n o n - s t e r o i d t r e a t e d sucrose c o n t r o l s (118.9 * 6.9). Except f o r the combination s t e r o i d group, the 3 month e t h a n o l - f e d s t e r o i d groups showed a decreased l e v e l o f BP hydroxylase i n comparison to the cor r e s p o n d i n g s u c r o s e - f e d animals. Hence, a t 3 months, i t appeared t h a t i n the s i n g l e s t e r o i d -109 treated, groups ethanol was somehow p r e v e n t i n g enzyme i n d u c t i o n from o c c u r r i n g to the same extent as i t x\ras l n the sucrose-fed. animals. I t i s hypothesized t h a t e t h a n o l - a s s o c i a t e d h e p a t o t o x i c l t y was beg i n n i n g to become apparent a t t h i s p o i n t . A t the 6 month treatment p e r i o d , i t was notable t h a t enzyme a c t i v i t y i n the ethanol c o n t r o l s (69.9 - 5.5) was s i g n i f i c a n t l y reduced (Student's t - t e s t ) below that o f the cor r e s p o n d i n g s u c r o s e - f e d group (100.8 - 10.9). This a g a i n appears to In d i c a t e e t h a n o l - a s s o c i a t e d h e p a t o t o x i c l t y s i n c e animal age d i d not make a d i f f e r e n c e i n the s u c r o s e - t r e a t e d 6 month group. I t was s t r i k i n g t h a t a l l 6 month s t e r o i d - t r e a t e d animals had the same l e v e l of BP hydroxylase a c t i v i t y . Spec-i f i c a l l y , there was no d i f f e r e n c e between sucrose and. e t h a n o l - f e d groups. Hence, a t 6 months i t appeared that the c o n t r a c e p t i v e s t e r o i d s were p r e v e n t i n g the e t h a n o l - a s s o c i a t e d decrease i n BP hydroxylase a c t i v i t y e v i d e n t i n n o n - s t e r o i d t r e a t e d animals. Furthermore, h e p a t i c BP hydroxylase a c t i v i t y i n o v a r l e c t o m l z e d female r a t s (Table X) t r e a t e d with ethanol was s i g n i f i c a n t l y lower than the s u c r o s e - f e d group. This s i t u a t i o n was not e v i d e n t i n the p a i r - f e d sham-operated females. H i s t o p a t h o l o g y s t u d i e s c a r r i e d out i n a s s o c i a t i o n with the enzyme s t u d i e s tended to support the hypotheses put f o r t h above. In order to a p p r e c i a t e p a t h o l o g i c a l changes seen i n the t i s s u e s s t u d i e d , i t i s a p p r o p r i a t e a t t h i s p o i n t to d e s c r i b e the nature of the f u n c t i o n a l anatomy of the l i v e r l o b u l e a c c o r d i n g to Rappaport (102). F i g u r e 2k i l l u s t r a t e s a l i v e r a cinus or f u n c t i o n a l u n i t . The centre of the a c i n u s , b e i n g i n a s s o c i a t i o n w i t h the p o r t a l t r a c t , i s the bes t s u p p l i e d w i t h n u t r i e n t s and. oxygen. This a r e a i s d e f i n e d as zone 1 of the a c i n u s . The c e n t r a l v e i n area i s not s u p p l i e d as w e l l as the p o r t a l a r e a , and i s 110 \ Figure 24. Diagram of the l i v e r acinus or functional unit, represented by the dotted l i n e . The s o l i d lines outline the c l a s s i c a l hepatic lobule. CV= central vein; PT= portal t r a c t s . (103) d e f i n e d as zone 3« The midzonal r e g i o n i s zone 2. This f u n c t i o n a l d e s c r i p t i o n of the l i v e r acinus i s r e l e v a n t to h e p a t i c h i s t o p a t h o l o g y , s i n c e l i v e r damage i s o f t e n r e l a t e d to blood supply. I t i s w e l l known that c h r o n i c exposure to a l c o h o l r e s u l t s i n a form of l i v e r c e l l d e g e neration which i s known as f a t t y change. This i s a r e v e r s i b l e phenomenon, but i t o f t e n i m p l i e s severe i n j u r y and may precede c e l l death. Under the l i g h t microscope, f a t t y change i s c h a r a c t e r i z e d by the accumulation of f a t vacuoles w i t h i n the cytoplasm of hepatocytes. C h a r a c t e r -i s t i c a l l y , f a t accumulation i s seen i n zone 3 In macrodroplet form. I t i s p o s t u l a t e d t h a t zone 3 i s i n v o l v e d to a g r e a t e r extent than zones 1 and 2 because e t h a n o l - a s s o c l a t e d f a t accum-u l a t i o n i n the zone 3 c e l l s leads to c e l l u l a r s w e l l i n g which may impinge f u r t h e r on the a l r e a d y reduced blood flow to zone 3. I t can be seen i n F i g u r e 7 t h a t both female and male animals t r e a t e d with e t h a n o l f o r 6 months had more p e r i p o r t a l (zone 1) and c e n t r a l (zone 3) f a t than e i t h e r the s u c r o s e - f e d animals a t 6 months ( F i g u r e 6) or the e t h a n o l - f e d animals a t 3 months ( F i g u r e 5). The o v a r i e c t o m l z e d females had more advanced f a t accumulation than sham-operated and c o n t r o l females ( F i g u r e 8 vs F i g u r e 10). The combination s t e r o i d - t r e a t e d animals ( F i g u r e 20 and 21) a t 3 months showed l e s s p e r i p o r t a l f a t accumulation than c o r r e s p o n d i n g c o n t r o l animals ( F i g u r e s k and 5). In a d d i t i o n , animals t h a t r e c e i v e d the s t e r o i d s a l o n g with the e t h a n o l d i e t f o r 6 months had some zone 3 f a t but i t was noted to be l e s s than that seen In the n o n - s t e r o i d t r e a t e d c o n t r o l s . The degree of zone 1 f a t accumulation was o n l y s l i g h t l y e l e v a t e d , i f a t a l l , i n the ethanol plus s t e r o i d animals compared to the sucrose plus s t e r o i d animals. 112 In summary, there appeared to "be a good c o r r e l a t i o n "between hepatic histopathology and hepatic BP hydroxylase a c t i v i t y i n that: 1) h i s t o l o g i c a l l y , ethanol-associated hepatotoxiclty was evident as zone 3 fat accumulation and accentuated zone 1 fat accumulation. Functionally, hepatotoxicity was evident as dec-reased levels of hepatic BP hydroxylase a c t i v i t y , and 2) h i s t -o l o g i c a l l y , o r a l contraceptive s t e r o i d administration appeared to "protect" against ethanol-associated f a t accumulation as well as p e r i p o r t a l f a t accumulation. Functionally, the "protective" e f f e c t x-ras evident as maintenance of control levels of hepatic BP hydroxylase a c t i v i t y . It was noted that changes i n a n i l i n e hydroxylase a c t i v i t y and amlnopyrine N-demethylase a c t i v i t y with the various treatments did not correlate with the histopatholog-i c a l changes. These enzyme assays are less s e n s i t i v e because they are quantitated c o l o r i m e t r i c a l l y , and the BP hydroxylase assay is quantitated fluorometrically. Other workers have observed "protective e f f e c t s " of contra-ceptive steroids (106-110). Schwartz (106) found that 17^3 -es t r a d i o l inhibited the c y t o t o x i c i t y of 7 i12-dimethylbenz(a)-anthracene i n l i v e r and breast e p i t h e l i a l cultures. Booth et a l . (107) suggested that the mechanism of this protection was Inhibitio n of hydrocarbon epoxide synthesis by the s t e r o i d . Mgbodile and Holscher (108) noted that aflatoxin-B^-induced hepatotoxicity i n control female rats was evident as large zones of necrosis i n the p e r i p o r t a l area and extensive f a t t y inf11-t r a t l o n . Animals that received 0vral-28 for 16 days exhibited only minimal necrosis around the p e r i p o r t a l areas. These workers also found that pretreatment of rats with the contraceptive steroid, p a r t l y countered aflatoxln-B. -associated decreases in hepatic cytochrome P-450 and benzphetamine N-demethylase a c t i v i t y . Krelcsar (109) showed that ln female r a t s , progestogens protected against decreases i n enzyme a c t i v i t y , pathological uptake of water by damaged hepatocytes, and loss of protein content i n CCl^-associated chronic l i v e r injury. Our studies showed that contraceptive s t e r o i d administration also resulted in marked stimulation of hepatic epoxide hydrase a c t i v i t y (Table X I I I ) . This would serve to lower the le v e l s of toxic epoxide intermediates formed by metabolic processes and thereby "protect" the l i v e r c e l l s from toxic i r r e v e r s i b l e binding. The h i s t o l o g i c a l sections revealed another consistent feature evident i n the experimental animals. There was in a l l Sustacal -treated animals, varying degrees of zone 1 p e r i p o r t a l microdroplet fat accumulation. This w i l l be referred to as the"Sustacal e f f e c t " . This d i s t r i b u t i o n of microdroplet fat in the l i v e r is a t y p i c a l . In other situations where microdroplet is evident ( f a t t y l i v e r of pregnancy, Reye's syndrome, t e t r a -cycline t o x i c i t y ) , the d i s t r i b u t i o n is generalized, that i s , i t is present in a l l 3 zones. Generalized microdroplet fat usually indicates a poor prognosis because of severe metabolic derange-ments In the c e l l . The Sustacal -Induced p e r i p o r t a l f a t accumu-l a t i o n did not seem to a l t e r the enzyme a c t i v i t i e s tested, since R R the control Sustacal levels were si m i l a r to those in Lab Chow -fed animals. It is usually the case that dietary derangements or d e f i c i e n c i e s are manifested by p e r i p o r t a l changes i n the l i v e r , since nutrients are flowing into this region v i a the portal vein. However, It is not usual that fat accumulation Is microdroplet. Other l i q u i d diets used in ethanol studies have not been reported to be associated with s i m i l a r e f f e c t s . However, I s e r l et a l . (104) 114 used the Lieber d i e t (76) in studying prolonged ethanol ingestion, and noted, "a small amount of fine l i p i d droplets scattered, with a panlobular d i s t r i b u t i o n " to a small and variable extent. But he did not investigate this phenomenon further. A possible explanation for the "Sustacal e f f e c t " could be that the d i f f e r e n t r a t i o s of calories supplied a l t e r d i s p o s i t i o n of dietary f a t s . The l i q u i d diet based on Lieber's (76) formulation have as per cent of t o t a l c a l o r i c Intake, approximately 16$ protein, 43$ f a t , 5% sucrose, and 36% ethanol or sucrose. This d i e t has not been associated, with p e r i p o r t a l f a t d i s t r i b u t i o n . A Metrecal based die t (Drackett Company, Cin c i n n a t i , Ohio) employed, by Ruebner et a l . (110) had the following composltlom protein 15$, carbohydrate 23$, fat 21$, sucrose, other sugars, or alcohol 41$ of t o t a l c a l -o ries. With this d i e t , there were no carbohydrate-associated, f a t t y change seen in l i v e r s of treated animals. Our d i e t had 14$ protein, 12$ f a t , 33$ carbohydrate and 40$ ethanol or sucrose and was associated with p e r i p o r t a l f a t accumulation. Therefore, the Sustacal d i e t we employed had a higher carbohydrate/fat r a t i o than the other d i e t s . In addition, the Sustacal d i e t had a lower fat content than the other l i q u i d d i e t s , which more cl o s e l y approximated, the Lab Chow di e t (4.5$ f a t ) . Perhaps in some way these differences i n dietary constituents contributed to the "Sustacal e f f e c t " . As yet we have no evidence to suggest that the "Sustacal e f f e c t " is deleterious to the animal or to the drug-metabolizing c a p a b i l i t y of the l i v e r . However, this e f f e c t has not been studied in d e t a i l . In p a r t i c u l a r , i t w i l l be of Interest to observe i f any abnormalities can be seen i n electron micrographs. As mentioned under Mfethods", EM tissue samples have already been prepared, for use in this pursuit. 115 SUMMARY AND CONCLUSIONS 1. A d m i n i s t r a t i o n o f the o r a l c o n t r a c e p t i v e s t e r o i d s mestranol and norethlndrone alone and l n combination g e n e r a l l y r e s u l t e d i n Increased microsomal enzyme a c t i v i t y In female r a t s t r e a t e d f o r up to 3 months. 2. E t h a n o l a d m i n i s t r a t i o n f o r up to 3 months r e s u l t e d i n enzyme i n d u c t i o n l n female r a t s . The 5 enzyme assays c a r r i e d out were: h e p a t i c benzo(a)pyrene hydroxylase, epoxide hydrase, a n i l i n e hydroxylase and amlnopyrine N-demethylase, and lun g benzo(a)pyrene hydroxylase. 3. E t h a n o l a d m i n i s t r a t i o n f o r 6 months produced h e p a t o t o x i c i t y i n both female and male r a t s . F u n c t i o n a l l y , the hepatotox-i c i t y was e v i d e n t as lowered h e p a t i c benzo(a)pyrene hydroxylase a c t i v i t y . H i s t o l o g i c a l l y , the h e p a t o t o x i c i t y was evident as f a t accumulation i n zone 3 of the l i v e r l o b u l e s . 4. A d m i n i s t r a t i o n of the c o n t r a c e p t i v e s t e r o i d s to female r a t s tended to p r o t e c t a g a i n s t e t h a n o l - a s s o c i a t e d h e p a t o t o x i c i t y . F u n c t i o n a l l y , t h i s was e v i d e n t as maintenance of c o n t r o l l e v e l s of h e p a t i c benzo(a)pyrene hydroxylase a c t i v i t y . H i s t o l o g i c a l l y , the p r o t e c t i v e e f f e c t was evident as l e s s e r amounts of both e t h a n o l - a s s o c i a t e d f a t accumulation l n zone 3 and S u s t a c a l - a s s o c i a t e d accumulation i n zone 1. There was no evidence to suggest t h a t the c o n t r a c e p t i v e s t e r o i d s exerted h e p a t o t o x i c e f f e c t s on t h e i r own. In p a r t i c u l a r , there was no evidence to suggest t h a t o r a l c o n t r a c e p t i v e s t e r o i d s were a s s o c i a t e d with development of h e p a t i c adenomas. 5. There appeared to be a c o r r e l a t i o n between the l e v e l o f h e p a t i c benzo(a)pyrene h y d r o x y l a s e a c t i v i t y and h i s t o l o g i c a l f a t a c c u m u l a t i o n as an i n d i c a t i o n o f e t h a n o l - a s s o c i a t e d h e p a t o t o x i c i t y . 6. A l l S u s t a c a l t r e a t e d an imals e x h i b i t e d a " S u s t a c a l e f f e c t " which was e v i d e n t h i s t o l o g i c a l l y as m i c r o d r o p l e t f a t accumu-l a t i o n r e s t r i c t e d to zone 1, sometimes merging i n t o zone 2. The " S u s t a c a l e f f e c t " d i d not appear to a f f e c t microsomal enzyme a c t i v i t y . 7. O v a r i e c t o m l z e d female r a t s were more s u s c e p t i b l e to the " S u s t a c a l e f f e c t " and to e t h a n o l - a s s o c i a t e d h e p a t o t o x i c i t y . 8. I t was conc luded t h a t the c o n t r a c e p t i v e s t e r o i d s a d m i n i s t e r e d d i d not i n c r e a s e e t h a n o l h e p a t o t o x i c i t y . R a t h e r , the presence o f female sex s t e r o i d s (endogenous or exogenous) appeared to a t t e n u a t e e t h a n o l - a s s o c i a t e d h e p a t o t o x i c i t y . 117 REFERENCES 1. S h e r l o c k , S.t "Diseases of the L i v e r and B i l i a r y System", B l a c k w e l l S c i e n t i f i c P u b l i c a t i o n s . Oxford, 5 t h E d i t i o n , 1975. 2. Warvi,. W.N.i Primary Neoplasms of the L i v e r , Arch. P a t h o l . 17* 367-376, 19W. 3. Henson, S.W., Howard, K.G., Dockerty, M.B.i. 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Sladek, N.E., C h a p l i n , M.D., and Mannering, G.J.s Sex-dependent D i f f e r e n c e s i n Drug Metabolism i n - t h e Rat IV. E f f e c t of Morphine A d m i n i s t r a t i o n , Drug Metab. Dispos. 2s 293-300, 1974. 124 90. Parke, D.V.t I n d u c t i o n of the Drug-m e t a b o l l z l n g Enzymes, i n "Enzyme I n d u c t i o n " , ed. D.V. Parke, Plenum P r e s s , New York, 1975. PP. 228-235. 91. E i s e n f e l d , A.J . t L 3 H ] - E s t r a d i o l : Macromolecular I n t e r a c t i o n i n the L i v e r , Fed. Proc. 3_2: 242, 1973. 92. E i s e n f e l d , A . J . , Aten, R., Weinberger, M., Haselbacher, G.K., Halpern, K., K r a k o f f , L.: Estrogen Receptor i n the Mammalian L i v e r , S c i e n c e , 191: 862-865, 1976. 93. E i s e n f e l d , A . J . , Aten, R.F., Haselbacher, G.K., Halpern, K.: S p e c i f i c Macromolecular B i n d i n g of E s t r a d i o l i n the Mammalian L i v e r Supernatant, Biochem. Pharmacol. 26: 919-922, 1977. 94. E i s e n f e l d , A . J . , K r a k o f f , L., Aten, R.F.: Developmental C o r r e l a t i o n of Higher L e v e l s of Estrogen B i n d i n g by Macromolecules i n Rat L i v e r Supernatant and of Increases l n Plasma Renin S u b s t r a t e L e v e l s A f t e r E strogen Admin-i s t r a t i o n , Biochem. Pharmac. 26: 923-927, 1977. 95. E i s e n f e l d , A . J . , Aten, R.F. and. Weinberger, M.J.i O r a l C o n t r a c e p t i v e s - P o s s i b l e M e d i a t i o n of S i d e E f f e c t s v i a an E s t r o g e n Receptor i n L i v e r , Biochem. Pharmac. .27» 2571-2577, 1978. 96. Weinberger, M.J., Aten, R.F., E i s e n f e l d , A.J.: E s t r o g e n Receptor i n the Mammalian L i v e r . E f f e c t of Metabolism on the Amount and I d e n t i t y o f Receptor Bound E s t r o g e n , Biochem. Pharmac. 2J7_: 2469-2474, 1978. 97. L i n k i e , D.M. and S u t e r l , P.K.1 A Re-examlnatIon of the I n t e r a c t i o n of E s t r a d i o l with Target C e l l Receptors, J . S t e r o i d Biochem. £1 1071-1078, 1978. 98. Olsen, H. and Morland, J . i Ethanol-lnduced. Increase i n Drug A c e t y l a t l o n i n Man and I s o l a t e d Rat L i v e r C e l l s , B r i t . Med. J . 2: 1260-1262, 1978. 99. J o l y , J.-G. and Hetu, C : E f f e c t s of Chronic E t h a n o l A d m i n i s t r a t i o n l n the Rat: R e l a t i v e Dependency on D i e t a r y L i p i d s - I . I n d u c t i o n o f Hepatic Drug-Metabol-i z i n g Enzymes In V i t r o , Biochem. Pharm. 24: 1475-1480, 1975. 100. V i l l e n e u v e , J.-P. , Movler, P. and. J o l y , J.-G.« E t h a n o l -lnduced Cytochrome P-450: C a t a l y t i c A c t i v i t y A f t e r P a r t i a l P u r i f i c a t i o n , Biochem. Biophys. Res. Comm. 701 723-728, I976. 101. Kunihiko, 0. and. L i e b e r , C.S . i R e c o n s t l t u t i o n of the Microsomal E t h a n o l - o x i d i z l n g System, J . B i o l . Chem. 252: 7124-7131, 1977. 125 1 0 2 . Rappaport, A.M.: Anatomic C o n s i d e r a t i o n s , In"Diseases o f the L i v e r " , ed. Leon S c h l f f , J.B. L i p p l n c o t t Company, Toronto, T h i r d E d i t i o n , 1969, pp. 1 -49 . 1 0 3 . Leeson, C.R. and Leeson,. T.S.s " H i s t o l o g y " , W.B. Saunders Co., Toronto, T h i r d E d i t i o n 1976, pp. 3 7 6 - 3 7 8 . 104. I s e r i , O.A., L i e b e r , C.S. and G o t t l i e b , L.S .s The U l t r a s t r u e t u r e of F a t t y L i v e r Induced by Prolonged E t h a n o l I n g e s t i o n , Am. J . Path. 48s 535-555, I 9 6 6 . 105 . Rubin, E. and L i e b e r , C.S.s F a t t y L i v e r , A l c o h o l i c H e p a t i t i s and C i r r h o s i s Produced by A l c o h o l i n Primates, New  E n g l . J . Med. 2 9 0 : 128-135, 1974. 106. Schwartz, A.G.s The P r o t e c t i v e E f f e c t o f E s t r a d l o l - 1 7 £ A g a i n s t P o l y c y c l l c Hydrocarbon C y t o t o x i c i t y , Cancer Res. 21« 2 4 3 1 - 2 4 3 6 , October, 1973. 107. Booth, J . , K e y s e l l , G.R. and Sims, P.s E f f e c t of O e s t r a d i o l on the In V i t r o Metabolism of 7 , 1 2-Dimethylbenz a -anthracene and It s Hydroxymethyl D e r i v a t i v e s , Biochem. Pharmac. 2Js 7 3 5 - 7 4 4 , 1974. 108. Mgbodile, M.U.K. and Holscher, M.s S t u d i e s on A f l a t o x i n B, T o x i c i t y i n Female Rats P r e t r e a t e d with an O r a l Contra-c e p t i v e Agent, Fd. Cosmet. T o x i c o l . 14s 171-174, 1976. 109. K u l c s a r , A. and K u l c s a r - G e r g e l y , J.s Progestogens i n Experimental L i v e r Damage, Seventh I n t e r n a t i o n a l Congress of Pharmacology, P a r i s , 1 9 7 8 , p. 842. ( A b s t r a c t 2603) 110. Ruebner, B.H., Brayton, M.A., Freedland, R.A., Kanayama, R.f Tsao, M.s P r o d u c t i o n of a F a t t y L i v e r by E t h a n o l i n Rhesus Monkeys, Lab. Invest . 2_7_i 7 1 - 7 5 , 1 9 7 2 . 126 APPENDIX I P r e p a r a t i o n of H l s t o p a t h o l o g y S l i d e s 1 ( a c c o r d i n g to C u l l i n g ) T i s s u e specimens were f i x e d i n 10$ "buffered f o r m a l i n f o r 4 - 7 days. A s e r i e s of a l c o h o l s o l u t i o n s were used to dehydrate the t i s s u e s . Specimens were then embedded i n p a r a f f i n , cut, s t a i n e d and mounted. Dehydrating Process T i s s u e s were placed i n a s e r i e s o f ethanol s o l u t i o n s f o r a t o t a l o f 12 h r i 8 0 $ (2 h r ) , 95$ (4 h r ) , 100$ (6 h r ) . A f t e r s o a k i ng i n chl o r o f o r m f o r 3 hr, the specimens were placed In l i q u i d p a r a f f i n 60° C f o r 4 hr. They were then placed. In a / o . vacuum oven (70 C) f o r 1 hr a t 15 pounds p r e s s u r e . T i s s u e s were embedded i n p a r a f f i n b l o c k s , cut i n 5 u s e c t i o n s w i t h a s t a i n l e s s s t e e l microtome, and mounted on s l i d e s f o r s t a i n i n g . Hematoxylin and Eos In S t a i n The s l i d e s were dipped l n each of the f o l l o w i n g ! x y l o l (3 min), x y l o l (3 min), 100$ a l c o h o l (2 min), 95$ a l c o h o l (2 min), 70$ a l c o h o l (3 min), d i s t i l l e d HgO (3 min), H a r r i s hematoxylin (5l min), water (10 d i p s ) , a c i d a l c o h o l (5 d i p s ) , E^Q (5 d i p s ) , r u n n ing water (2 min), base (1 min), and water ( l i min), Nanaimo Eos in ( l | min), d i s t i l l e d HgO (2 d i p s ) , 95$ a l c o h o l (2 d i p s ) , 100$ a l c o h o l (2 d i p s ) , 100$ a l c o h o l (2 d i p s ) , x y l o l (3 min), and x y l o l (3 min). N u c l e i s t a i n e d blue and cytoplasm s t a i n e d pink. C u l l i n g , C.F.A., "Handbook of H i s t o l o g i c a l and H i s t o c h e m i c a l Techniques", Butterworth & Co., Toronto, Third. E d i t i o n , 1974. 127, The s t a i n e d s e c t i o n s were mounted with s y n t h e t i c r e s i n i n toluene (Cover Bond ) and. covered with a c o v e r s l l p . L l l l l e and Ashburn's Isopropanol O i l Red Q Method M o d i f i e d S t a i n  (Fat S t a i n ) S e c t i o n s were pl a c e d i n o i l r e d 0 s o l u t i o n i n i s o p r o p y l a l c o h o l l n a c l o s e d c o n t a i n e r f o r 10 - 15 minutes. The background was c l e a r e d i n 60% a l c o h o l . A water wash was carried, out and then n u c l e i were l i g h t l y c o u n t e r s t a i n e d with hematoxylin. A 2% s o l u t i o n of sodium b i c a r b o n a t e i n tap-water was used to blu e the s e c t i o n s . L i p i d s s t a i n e d b r i g h t red. and n u c l e i stained, b l u e . 128 APPENDIX I I A. METHODS 1. A n i l i n e Hydroxylase Assay Hepatic a n i l i n e hydroxylase a c t i v i t y was measured c o l o r -1 i m e t r i c a l l y by the method of Imai, I t o , and Sato . The f i n a l volume of the i n c u b a t i o n mix was 2.0 ml. I t contained 2.5 umole a n i l i n e , 0.25 ml phosphate b u f f e r 0.1 M pH 7.2, 0.25 ml enzyme p r e p a r a t i o n , 2.5 umole NADP, 5 umole glucose - 6-phosphate, 2 .5 u n i t s glucose - 6-phosphate dehydrogenase, 12.5 umole MgCl^» and 0 . 94 ml phosphate b u f f e r pH 7 .5. The i n c u b a t i o n was c a r r i e d out f o r 15 minutes a t 37° C 0and was stopped with 1.0 ml 20$ t r i c h l o r o a c e t i c a c i d . A f t e r c e n t r i f u g l n g o f f the p r e c i p i t a t e , a 1 ml. a l i q u o t of the supernatant was taken and 0.5 ml of 10$ NagCO^ was added, f o l l o w e d by 1.0 ml 2$ phenol i n 0.2 N NaOH. C o l o r development was measured a t 640 nm a f t e r i n c u b a t i o n f o r o 30 minutes a t 37 C. Approximately 2 - 3 mg of p r o t e i n was added to the i n c u b a t i o n mixture. A c t i v i t y i s expressed as ng p-aminophenol produced per minute per mg p r o t e i n . 2. Amlnopyrine N-demethylase Assay Amlnopyrine N-demethylase a c t i v i t y was determined c o l o r l -2 m e t r i c a l l y by the method of E l Defrawy E l Masry with m o d i f i c a t i o n s . 1 Imal, Y., I t o , A., and Sato, R . t Evidence f o r B i o c h e m i c a l l y D i f f e r e n t Types of V e s i c l e s i n the Hepatic Microsomal F r a c t i o n , J . Biochem. 60t 417-428, I966. E l Defrawy E l Masry, S., Cohen, G.M., and Mannering, J.» Sex-dependent D i f f e r e n c e s In Drug Metabolism In the Rat. I. Temporal Changes i n the Microsomal Drug-metabol.lzing System of the L i v e r d u r i n g Sexual Maturation, Drug Metab. Dispos. 2: 267-278, 1974. 129 Formaldehyde, which was produced ln the N-demethylation reaction, 3 was measured according to the method of Nash . The f i n a l volume of the incubation mix was 1.5 ml. It contained 20 umole aminopyrine, 0.2 ml enzyme preparation, 6.26 umole MgClg, 11.15 umole semicarbazlde, 1 umole NADP, 10 umole glucose - 6 -phosphate, 5 units glucose-6-phosphate dehydrogenase, 0.581 mmole Tris buffer pH 7.5, and 0.1 ml d i s t i l l e d RgO. The incubation was carried out for 20 minutes (female rats) or 10 minutes (male rats) at 37° C, and. was stopped with 0.5 ml cold 5$ zinc s u l f a t e . Saturated barium hydroxide solu t i o n was added, and the pre c i p i t a t e was centrifuged o f f . A 1,5 ml aliquot of the supernatant was sampled, and. 0.5 ml Nash Reagent was added. After incubation for 15 minutes at 60° C, color development was measured at 412 mu. Approximately 1.5 - 2 .5 mg of l i v e r protein was added to the incubation mixture. A c t i v i t y is expressed as ng formaldehyde produced, per minute per mg protein. 3. Blood Ethanol Determinations A Hewlett-Packard. 583OA gas-liquid, chromatograph f i t t e d with a 8850A gas chromatographic terminus was used. The chromato-graphic column, 10 f t . , s t a i n l e s s s t e e l , 50$ poropak Q, 50$ poropak R 80/100 was preconditioned over a 72 hr time period at 100° C progressing to 180° C with a c a r r i e r (helium) flow rate of 40 ml/min. The column was operated at a temperature of 147° C and. a flow rate of 40 ml/min. Retention time for methanol (in t e r n a l standard.) was approximately 2.04 min, and 4.15 min for ethanol. 3 Nash, T . J The Colorimetric Estimation of Formaldehyde by Means of the Hantzsch Reaction, Biochem. J. 55» 416-421, 1953. 130 Blood samples were d e p r o t e i n i z e d a c c o r d i n g to the method of Le Blanc l n a mixture c o n t a i n i n g 0.1 ml 0.3 M Ba(OE)^, 0. 1 ml 5% ZnSO^, 0.2 ml 1.25$ NaF, and 0.3 ml d i s t i l l e d HgO. Blood was c o l l e c t e d on h e p a r l n i z e d p e t r l p l a t e s . A 200 u l sample o f blood a l o n g with 100 u l 0.25 M Methanol ( I n t e r n a l standard) was c e n t r i f u g e d w i t h the d e p r o t e l n l z i n g s o l u t i o n f o r 2 minutes. A 2 u l sample of the supernatant was used f o r the chromatographic a n a l y s i s . B. RESULTS 1. A n i l i n e Hydroxylase and Amlnopyrine N-demethylase Assays R R Female r a t s were fed S u s t a c a l a l o n e , S u s t a c a l p l u s R sucrose (k0% of c a l o r i e s ) , or S u s t a c a l plus ethanol (40$ c a l o r i e s ) to determine whether the d i f f e r e n t d i e t s a f f e c t e d microsomal enzyme a c t i v i t i e s . R e s u l t s contained i n Table I Indicate t h a t both ethanol and sucrose a l t e r e d a n i l i n e hydrox-y l a s e a c t i v i t y . I t can be seen that ethanol a d m i n i s t r a t i o n f o r 2 weeks caused s i g n i f i c a n t (p<0.05) e l e v a t i o n o f a n i l i n e R hydroxylase a c t i v i t y when compared to the S u s t a c a l alone or R the S u s t a c a l plus sucrose d i e t . Enzyme a c t i v i t y was e l e v a t e d over S u s t a c a l c o n t r o l s a t 6 weeks a l s o i n animals t r e a t e d with the ethanol d i e t . Sucrose s t i m u l a t e d a n i l i n e hydroxylase a c t i v i t y a t 2 weeks and 6 weeks when compared to the S u s t a c a l alone d i e t . At k weeks, however, enzyme a c t i v i t y was s i g n i f i -R c a n t l y e l e v a t e d i n the p l a i n S u s t a c a l - t r e a t e d animals and s i g n i f i c a n t l y reduced i n the e t h a n o l - t r e a t e d animals, when compared to the r e s p e c t i v e 2 and 6 week l e v e l s . Amlnopyrine n — LeBlanc, A.E . i M i c r o d e t e r m l n a t l o n of A l c o h o l i n Blood by G a s - l i q u l d Chromatography, Can. J . P h y s i o l Pharmacol. k6\ 665-667, 1968. Table I. Enzyme act iv i t ies in female rats fed Sustacal alone or with sucrose or ethanol. Hepatic aniline hydroxylase act iv i ty is expressed as ng p-aminophenol formed per min per mg protein ± S.E.M.. Hepatic amino-pyrine N-demethylase act ivi ty is expressed as ng formaldehyde formed per min per mg protein ± S.E.M. Significance was determined at p<0.05 using the Student t-test for unpaired sample means, and is represented by paired lower case letters, n = 4 except where indicated in brackets ( ) . *Sucrose and ethanol were supplied as 40% of calories. DIET Anil ine hydroxylase act ivity Aminopyrine N-demethylase act iv i ty 2 weeks 4 weeks 6 weeks 2 weeks 4 weeks 6 weeks Plain Sustacal 8 2 + 5 113 ± 6 89 ± 1 [a,b,c] [a,d,e] j[d,f,g] 30 ± 2 29 ± 2 29 ± 2 [l,m] Sucrose* + Sustacal 121+7 95 ± 8 105 ± 3 (3) [b,h] [f] 29 ± 3 40 ± 1 [n] [l,n] Ethanol* + Sustacal R 171 ± 4 76 ± 5 118 ± 8 (3) [ c , h , i , j ] [ l , i , k ] [g,j,k] 35+3 40 + 3 37 ± 5 (3) [m] 132 N-demethylase a c t i v i t y was s i g n i f i c a n t l y e l e v a t e d a t 4 weeks i n animals r e c e i v i n g sucrose and i n animals r e c e i v i n g ethanol R as compared to the c o r r e s p o n d i n g S u s t a c a l c o n t r o l group. Four week sucrose values were a l s o e l e v a t e d when compared to t h e i r 2 week l e v e l s . I t can be seen i n Table I I t h a t l o n g e r term treatment o f animals produced d i f f e r e n t enzyme a c t i v i t i e s than treatment f o r s h o r t e r p e r i o d s (Table I ) . In a l l female animals, ethanol therapy tended to cause a decrease i n h e p a t i c a n i l i n e hydroxylase a c t i v i t y , when compared with s i m i l a r animals treated, w i t h s u c r o s e . In the case of o v a r l e c t o m l z e d females, t h i s decrease was s i g n i -f i c a n t (p«=0.05)» as was the d i f f e r e n c e between normal and o v a r l e c t o m l z e d females r e c e i v i n g e t h a n o l . However, i n males, enzyme a c t i v i t y tended to Increase with ethanol treatment. Although the i n c r e a s e s were 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 , the change from the decrease seen i n female animals was s t r i k i n g . Aminopyrine N-demethylase a c t i v i t y was s i g n i f i c a n t l y i n c r e a s e d in e t hanol t r e a t e d r a t s of both sexes as compared to s u c r o s e -t r e a t e d c o n t r o l groups a t 3 months. This enzyme was a l s o s i g n i f i c a n t l y more a c t i v e l n males than i n females, as expected. Female r a t s fed sucrose and ethanol d i e t s , were c h r o n i c a l l y t r e a t e d with mestranol and/or norethindrone f o r 3 months or 6 months (Table I I I ) . A l l animals t r e a t e d w i t h e t h a n o l f o r 3 months had lower a n i l i n e hydroxylase a c t i v i t y than the c o r r e s -ponding animals t r e a t e d f o r 3 months with sucrose. The apparent decrease seen was s i g n i f i c a n t i n the combination s t e r o i d t r e a t e d animals (mestranol plus n o r e t h i n d r o n e ) , due to the f a c t t h a t t h e i r sucrose c o n t r o l group appeared, to be induced (I63 - 11, n=5) • The r e v e r s e s i t u a t i o n occurred, a t 6 months of combination s t e r o i d Table II. Enzyme a c t i v i t i e s i n female and male rats fed Sustacal plus ethanol or sucrose. Hepatic a n i l i n e hydroxylase a c t i v i t y i s expressed as ng p-aminophenol formed per min per mg prote in ± S.E.M. Hepatic aminopyrine N-demethylase a c t i v i t y i s expressed as ng formalde-hyde formed per min per mg p ro te in ± S.E.M. S i gn i f i cance was determined at p< 0.05 using the Student 's t - t e s t f o r sample means, and i s represented by paired lower case l e t t e r s , n = 4 except where ind ica ted i n brackets ( ). GONADAL STATUS DIET ( in Sus taca l R ) A n i l i n e hydroxylase a c t i v i t y Aminopyrine N-demethylase a c t i v i t y 3 month therapy 6 month therapy 3 month therapy 6 month therapy Contro l female Sucrose 122 ± 2 (4) 98 ± 3 (4) ta] [a,b,c] 25 ± 2 (4) 23 ± 1 (4) [ i , j ] Ethanol 114 ± 5 (4) 93 ± 4 (4) [d] [d,e,f ,g] 38 ± 4 (4) 27 ± 2 (4) [ i , k ] Sham-operated female Sucrose 127 ± 6 (9) [b] - 18 ± 1 (8) Ethanol 121 ± 2 (7) tc] 26 ± 4 (7) Ovariectomized female Sucrose 101 ± 9 (8) [h] 25 ± 2 (8) Ethanol 70 ± 3 (10) [f,h] 28 ± 3 (10) Contro l male Sucrose 118 ± 5 (4) 135 ± 6 (4) tc] 89 ± 3 (4) 91 + 2 (4) U , l ] Ethanol 121 ± 9 (4) 154 ± 6 (4) tg] 134 ± 11 (4) 96 ± 3 (4) [k,l,m] [m] 134 Table III . Hepatic anil ine hydroxylase act iv i ty in chronically treated female rats . Enzyme act iv i ty is expressed as ng p-aminophenol formed per min per mg protein ± S.E.M. Significance was determined at p<0.05 using the BMD 10V analysis of variance, and is represented by paired lower-case let ters . The number of animals is indicated in brackets ( ) . M = mestranol; N = norethindrone. STEROID DIET Anil ine hydroxylase act iv i ty (in SustacalR) 3 month therapy 6 month therapy - Sucrose 122 ± 2 (4) 98 ± 3 (4) [a,b] [c] Ethanol 114 ± 5 (4) 93 ± 4 (4) [d] M Sucrose 101 ± 10 (5) 79 ± 3 (7) [c] M Ethanol 95 ± 3 (5) [e] 86 ± 5 (8) N Sucrose 76 ± 5 (5) [a,f] 94 ± [f] 4 (7) N Ethanol 72 ± 8 (5) [d,e,g,h] 106 ± [g] 3 (8) M + N Sucrose 163 ± 11 (5) [ b . i . j ] 84 ± [j,k] 3 (5) M + N Ethanol 100 ± 8 (8) [h,i] 102 ± [k] 3 (4) 135 therapy where the sucrose group exhibited a lower enzyme a c t i v i t y than the ethanol group (84 - 3, n=5 vs 102 ± 3, n=4) . At 3 months, norethlndrone-treated animals r e c e i v i n g either d i e t showed s i g n i f i c a n t l y lower a n i l i n e hydroxylase a c t i v i t i e s than th e i r corresponding ethanol sucrose controls. Hepatic amlnopyrine N-demethylase a c t i v i t i e s are given in Table IV. Norethlndrone treatment produced a s i g n i f i c a n t decrease in this enzyme a c t i v i t y also when compared to the ethanol control (16 - 1, n=4 vs 3 8 - 4 , n=4) l e v e l s . On the other hand, mestranol treatment for 3 months increased amlnopyrine N-demethylase a c t i v i t y . By 6 months of treatment, a l l groups had v i r t u a l l y the same l e v e l of amlnopyrine N-demethylase a c t i v i t y . 2. Blood Ethanol Determinations A standard curve for blood ethanol concentrations was determined (Table V). TABLE V. STANDARD CURVE FOR BLOOD ETHANOL CONCENTRATIONS Relative Ratio Concentration of (area Ethanol/area Methanol) Ethanol/100 ml blood 0.124 50 mg/100 ml 0.211 75 mg/100 ml 0.411 150 mg/100 ml In addition, blood ethanol lev e l s were determined i n animals that had been fed a 11$ v/v ethanol l n Sustacal d i e t for 2 months. Blood was collected at decapitation. It should be noted that ethanol treatment was not discontinued in these animals pr i o r to s a c r i f i c e . Results for 6 animals are contained in Table VI. 136 Table IV. Hepatic aminopyrine N-demthylase act iv i ty in chronically treated female rats . Enzyme act iv i ty is expressed as ng formaldehyde formed per min per mg protein ± S.E.M. Significance was determined at p< 0.05 using the BMD 10V analysis of variance, and is represented by paired lower-case le t ters . The number of animals per group is indicated in brackets ( ) . M = mestranol; N = norethindrone. STEROID DIET Aminopyrine N-demethylase act iv i ty (in SustacalR) 3 month therapy 6 month therapy - Sucrose 25 ± 2 [a] (4) 23 ± 1 (4) - Ethanol 38 ± 4 [b] (4) 27 ± 2 (4) M Sucrose 44 ± 4 [a,c] (5) 31 ± [c] 2 (7) M Ethanol 43 ± 2 [d] (5) 30 ± [d] 2 (8) N Sucrose 21 ± 1 (5) 27 ± 4 (7) N Ethanol 16 ± 1 [b,e] (5) 28 ± [e] 3 (8) M + N Sucrose 19 ± 2 (5) 28 ± 3 (5) [f] M + N Ethanol 28 ± 3 (8) [f] 31 ± 1 (4) 137 TABLE VI. BLOOD ETHANOL LEVELS IN RATS ADMINISTERED SUSTACAL PLUS ETHANOL (11$ v/v)FOR 2 MONTHS. (Each value represents an average of 2 determinations). Rat Concentration of Ethanol/100 ml blood #1 28.5 mg/100 ml #2 39.5 mg/100 ml #3 35.0 mg/100 ml #4 39.5 mg/100 ml #5 #6 In another experiment, 2 animals that had been given ethanol (11$ v/v) In the i r drinking water for 2 months were found, to have no detectable blood ethanol. (It should be noted, that there were a number of problems related, to obtaining blood samples from the animals, and to i n s t a b i l i t y of the gas chrom-atographic column.) S i m i l a r l y , animals that had been fed. the ethanol i n Sustacal d i e t for 3 months dr for 6 months showed no detectable blood ethanol levels when the ethanol was discon-tinued 24 hr pr i o r to s a c r i f i c e . 133 Table II. Summary of cases o f benign l i v e r tumors a s s o c i a t e d With o r a l c o n t r a c e p t i v e therapy, 1972-1978 139. Sunnnary of cases of benign l i v e r tumor s associ n ted with ora 1 contraceptive therapy, 1972-1978. A l l pat.ients are female. See end of table for l i s t of abbreviations used. AGE (YR) OF PATIENT STERIOD HISTORY OTHER PATIENT DATA AND/OR CONCURRENT THERAPY CLINICAL PRESENTATION DIAGNOSIS 26 norethynodrel + mestranol (Enovid); 2 years severe midepigastric pain, hemoper i toneum hepatic adenoma dimethisterone + e t h i n y l e s t r a d i o l (Oracon); unknown we l l u n t i l 7 hours before admission; i . v . cholan-giography and abdominal s e r i e s showed moderate hepatomegaly severe abdominal pain hepatic r a d i a t i n g to r i g h t adenoma shoulder, hemoperitoneum o.c. type unknown; 6 years w e l l u n t i l 1 month before admission; then gained weight; p o s i t i v e l i v e r scan and abdominal angiography hepatic adenoma no r g e s t r e l + e t h i n y l e s t r a d i o l (Ovral); 6 months progressive weakness, fa t i q u e , p a l l o r beginning 2 months before admission; p o s i t i v e arteriography abrupt onset of sharp, stabbing RUQ pain, anemia hepatic adenoma 29 norethynodrel + mestranol (Enovid); 5 years arteriogram showed hypervascular mass a r i s i n g from quadrate lobe of l i v e r RUQ mass s e l f - d e t e c t e d hepatic adenoma 30 / several types of o.c.; 7 years between pregnancie phenobarbital and d i u r e -t i c s f o r l*s years f o r hypertension that developed i n 3rd pregnancy sharp e p i g a s t r i c pain; hepatic weakness, f a i n t e d , adenoma hemoperitoneum norethynodrel + mestranol (Enovid) j 7 years good health u n t i l day of admission ALL CASES none showed any evidence of c i r r h o s i s , h e p a t i t i s , c ongenital e t i o l o g y RUQ pain, nausea, vomiting, hemoperitoneum (was thought to be a p p e n d i c i t i s ) hepatic adenoma no r g e s t r e l + e t h i n y l e s t r a d i o l (Ovral) f o r many yrs acute e p i g a s t r i c pain with r a d i a t i o n to r i g h t shoulder, diagnosed as acute c h o l e c y s t i t i s ; hemoperitoneum found at necropsy l i v e r - c e l l hyper-p l a s i a with abnormal v a s c u l a r i t y and areas of p e l i o s i s norethlndrone + mestranol (Ortho-Novum); seve r a l years severe RUQ pain of sudden onset - was diagnosed as acute c h o l e c y s t i t i s h e p a t i c - c e l l adenoma with ne c r o s i s i n t o which hemorrhage had occurred ( p e l i o s i s hepatis) n o r g e s t r e l + e s t r a d i o l (Orval); 5-6 years i n good hea l t h u n t i l night of admission; many years h i s t o r y of heartburn and gnawing e p i g a s t r i c recurrent pain severe upper abdominal pain, syncope, shock hemoperitoneum benign hepatoma of R lobe of l i v e r t T a b l e IT - Summary of CSHCS o f b c n u j n l i v e r t u m o r s ftssociei^d w i t h o r a l c o n t r a c e p t i v e t h e r a p y , 1972-1978. A l l p a t i e n t ? a r e f e m a l e . See end o f t a b l e f o r l i s t of a b b r e v i a t i o n s u s e d . AGE(YR) OF PATIENT 26 STERIOD HISTORY norethynodrel + mestranol (Enovid); 2 years OTHER PATIENT DATA AND/OR CONCURRENT THERAPY C L I N I C A L PRESENTATION severe midepigastric pain, hemoperitoneum DIAGNOSIS hepatic adenoma 27 dimethisterone + ethinylestradiol (Oracon); unknown well unt i l 7 hours before admission; i . v . cholan-giography and abdominal serieB showed moderate hepatomegaly severe abdominal pain hepatic radiating to right adenoma shoulder, hemoperitoneum o.c. type unknown; 6 years-well unt i l 1 month before admission; then gained weight; positive l iver scan and abdominal angiography RUO mass hepatic adenoma norgestrel + ethinylestradiol (Ovral); 6 months progressive weakness, fatique, pallor beginning 2 months before admission; positive arteriography abrupt onset of sharp, stabbing RUQ pain, anemia hepatic adenoma 29 norethynodrel + mestranol (Enovid); 5 years arteriogram showed hypervascular mass arising from quadrate lobe of l iver RUO mass self-detected hepatic adenoma 30/ / several types of ' o.c.; 7 years between pregnancies phenobarbital and diure-t ics for l*i years for hypertension that developed in 3rd pregnancy sharp epigastric pain; hepatic weakness, fainted, adenoma hemoperi toneum norethynodrel + mestranol (Enovid); 7 years good health unti l day of admission ALL CASES none showed any evidence of c irrhosis , hepatitis, congenital etiology RUQ pain, nausea, vomiting, hemoperitoneum (was thought to be appendicit is) hepatic adenoma 37 norgestrel + ethinylestradiol (Ovral) for many yra acute epigastric pain with radiation to right shoulder, diagnosed as acute cholecystit is; hemoperitoneum found at necropsy l i v e r - c e l l hyper-plasia with abnormal vascularity and areas of pel iosis 33 norethindrone •* mestranol (Ortho-Novura); Beveral years severe RUO pain of sudden onset - was diagnosed as acute cholecystit is hepatic-cell adenoma with necrosis into which hemorrhage had occurred (peliosis hepatis) norgestrel + estradiol (Orval); 5-6 years in good health unti l night severe upper abdominal benign of admission; many years pain, syncope, shock hepatoma of hi story of heartburn and hemoperitoneum R lobe of gnawing epigastric l iver recurrent pain 141 GROSS PATHOLOGICAL FINDINGS HISTOPATHOLOGY OUTCOMr. AND FOLLOW-UP massive i n t r a p e r i t o n e a l bleeding was found on coeliotomy a r i s i n g from L lobe; circumscribed yellow-tan tumor; 4.5 cn diameter; neoplasm extended cloBe to capsule at s i t e of rupture and hemorrhage acute hemorrhage i n t o peritoneum from tumor i n R lobe of l i v e n s o l i t a r y 12 cm tumori extended close to surface of l i v e r pedunculated mobile tumor ajrising from L lobe of l i v e r ; w e ll demarcated 12x6 cm tumor; 500 g. encapsulated mass; pedicle was 9 cm ALL CASESi tumors were composed of very s l i g h t l y a t y p i c a l hepatic c e l l s arranged i n cords and sometimes forming pseudoacini around secreted and retained b i l e . A l l were r e l a t i v e l y v ascular. Usual l o b u l a r a r c h i t e c t u r e not seen (no p o r t a l t r a c t s or c e n t r a l v e i n s ) . TumorB were d i s -c r e t e l y separable from adjacent l i v e r but encapsulation was incomplete. These tumor* were s o l i t a r y and were adjacent to the hepatic capsule i n one area. l e f t hepatectomy and cholecystectomy; doing well a f t e r 4 years cardiac a r r e s t a f t e r r i g h t hepatectomy; died tumor and p e d i c l e removed from L l i v e r lobe; uneventful recovery tumor in R lobe of l i v e r r i g h t hepatectomy; doing well on 2-month follow-up 13x17 cm mass i n R lobe of l i v e r ; hypervascular; d i l a t e d blood vessels on surface of l e s i o n e x c i s i o n of tumor; uneventful recovery and doing well a f t e r 3S years large nfass i n capsule i n 1 here causing i . p . hemorrhage; white 18x17 cm yellow hemorrhagic mass R lobe of l i v e r next to area, capsule ruptured R hepatectomy; lost 18,000 ml blood at surgery, post opera-t i v e problems with i n f e c t i o n , coagula-t i o n and jaundice; doing well on 15 month follow-up mass i n R lobe ( s i t e of i . p . hemorrhage); hemorrhagic, p a r t l y n e c r o t i c yellow-tan neoplasm; f a i r l y good demarcation from surrounding t i s s u e ; 10 cm i n diameter 24,000 ml blood loss at surgery; 60% R hepatectomy; at 2 months, s t i l l hepatic i n s u f f i c i e n c y massive hcrioperitoneum due to rupture of mass i n r i g h t lobe of l i v e r , poorly circumscribed; 15 cm diameterj hematoma formation l i v e r c e l l hyperplasia without l o b u l a r structure or ductule component; abnormal v a s c u l a r i t y with areas of p e l i o s i s  h epatisi no trapped b i l e i n tumor area or i n re s t of l i v e r . died at home three hours a f t e r i n i t i a l examination at surgery a 16x17x5 hemorrhagic moss i n R lobe of l i v e r was found; well circumscribed l e s i o n ; l e s i o n composed of d i l a t e d blood sinuses; tan coloured t i s s u e ; f o c a l areas of necrosis sheets of r e l a t i v e l y uniform s l i g h t l y h y p e rplastic hepatocytes; no p o r t a l areas seen; n e c r o t i c areas were p a r t l y organised and often adjacent to large blood sinuses i r i g h t hepatectomy and cholecystectomy; post-operative hcpati bed bleeding, R sub-phrenic abscess, recurrent R p l e u r a l *^rf un i nnn , l.ronrho-pneumonia; died spontaneous rupture of a large round tumor; 3-4 un i t s of blood found i n perito n e a l c a v i t y ; tumor on under-surface of r i g h t lobe of l i v e r which lay free i n RUQ beneath the l i v e r ; appeared as though the pati e n t had bled between the tumor and the l i v e r and the hemorrhage d i s s e c t e d the tumor out of the l i v e r l o c a l suturing of l i v e r bed then r i g h t hepatectomy 48 hours l a t e r ; died on follow ing day due to post-operative bleeding and shock S T E P I O D H I S T O R Y C T M r P ?A71 F.NT DATA AND/OR CONCURRENT THERAPY C L I N I C A L P R E S E N T A T I O N D1AGNOSIS noreth i sterone , 1 W . , dee Horvath, E et al , _ benign meitronol; 7 yean «J 1traitructura 1 findings hepatoma in a well differentiated hepatoma, Diaestion, 7:74, 1912V norethynodrel • gravida 6, para 4; desi- pain in RUO focal mestranol ccated thyroid 3gr/day nodular (Enovid) lor 5 years for 5-6 years; no evidence hyperplasin then none for 4 years of cirrhosis; positive l iver scan norethynodrel + gravida 2, para 2; no hemoperitoneum, shock; focal mestranol previous l iver disease or was thought to have a nodular (Enovid) for 7 years exposure to hepatotoxic perforated peptic ulcer hyperplasia agents; had intermittently taken medication for duodenal ulcer norethynodrel + gravida 2, para 2; no sudden, severe epigastric focal mestranol history of prior hepatic pain, collapse, palpable nodular (Enovid) for 5 years disease or exposure to mass in epigastrium, hyperplasia hepatotoxic agents intrahepatic hemorrhage (Ordol) for 3 years radiological examination acute pain in upper left benign and liver scan suggested a abdomen, palpable mass hepatoma D O S S i b l e mass in the left lobe of the liver lyncstrcnol + mestranol (Lyndiol), ethynodiol + mestranol (Ovulen), norethisterone + mestranol for a total of 6 years with two 12-month gaps for her 4th and 5th pregnancies tubulai ligation performed dizziness, h«:.iU.-»chc, at laparotomy; post-opera-tive arteriography showed the tumor to have an abnormal vasculature substernal pain of 3 month duration; mass in right hypochondri um 1 I V I ' I hamar toma (or benign hepatoma) norethisterone + mestranol for 8 years with two IB-month gaps for pregnancies; oral contraceptives were discontinued at time of presentation presented with an asymp-tomatic lump in upper abdomen 3 years previously (thought to be pancreatic cyst); patient remained well.-splenectomy performed when patient was 6 years old due to pulmonary siderosis; 4 years previ-ously had a ruptured ectopic pregnancy; patient had gluten enteropathy and al lergic asthma, and was taking disodium cromogly-cate and bfaclomethaaone admitted for further assessment of abdominal lump that was found by patient norethisterone • l iver scon showed no incidental at cholecyB- l iver mestranol for change in size of lesion tectomy hamartoma 8 years 2 months after i ts discovery; post angio-graphy; had a five day course of chemotherapy administered directly into celiac artery, and included 5g fluorouracil , lg cyclo-phosphamide, 2mg vincrist ine, SOnxj methotrexate in divided doses; laparotomy carried out 2 weeks after completion of chemotherapy 143 OUTCOME AND GROSS PATHOLOGICAL TINDIUGS HISTOPATHOLOGY FOLLOW-UP well encapsulated tumor weighing 870 g. well-differentiated hepatoma cells arranged in cords or acinar-like pattern; tumor cel ls showed E.M. changes associated with o.c. use (mitochondrial polymorphism, giant mitochondria, lamellar mitochondrial crystalloid inclusions) tumor in right lobe of liver; there was a subcapsular hematoma; tumor was pink, circumscribed and lobulated and 4.5 cm in diameter; cyst f i l l e d with old blood was adjacent to the tumor at surgery, large solid tumor in right lobe of l iver was found to be the source of the bleeding; mass was 10 cm in diameter; considerable hemorrhagic necrosis; mass contained a blood clot mass consisted of individual lobules as large as 7 cm; on cut section there was a large blood clot and adjacent to this were yellow-tan colored areas; no true capsule was demonstrated these 3 lesions were histologically similar; lobulated appearance due to areas of f ibrosis, mostly seen centrally; a l l contained areas of infarction necrosis, hemorrhage, and surrounding fibrosis; nodules comprised of l iver cells arranged either in cords or sheets, usually not oriented to central veins or portal areas; hepatocytes appeared relatively normal; the cytoplasm of cel ls was finely granular but at times was granular; adenomatous appearance due to sparsity of portal triads and irregular arrangement of cel ls ; nuclei were of uniform size, may have contained nucleoli; mitoses were virtual ly absent; margins were sharply demarcated, but no capsule right heptic lobec-tomy; alive at 2 yoars hemorrhage controlled by sutures, then resection of right lobe of the l iver; l iver function pro-gressed to normal and scanning showed regeneration; alive at 1 year left lobe of l iver was resected; l iver function returned to normal and scanning showed regeneration, alive after 5 years large yellowish-gray non-encapsulated hemorrhagic mass; dilated large veins seen within the tumor; no bi le stasis and no portal areas well-differentiated hepatic cel ls with vacuolated cytoplasm; cel ls arranged in cords; cords of hepatic cellB separated by dilated sinusoids left lobectomy; outcome not stated at laparotomy pale yellow solitary tumor found in right lobe of l iver; tumor had a nodular surface and was 7.5 cm in diameter laparotomy showed a solid tumor measuring 10 cm x 10 cm in the left lobe of the l iver; tumor had a nodular surface similar in a l l 3 cases; l iver tissue was arranged abnormally; . a l l tumors were well circumscribed and case 3 was encapsulated; the mass was composed of nodules of l iver ce l l cords and the nodules were separated by fibrous septa; there was no bile retention, no significant pleomorphism and no regenerative activity; there was marked proliferation of bile duct epithelium in the fibrous septa and in 2 of the cases, there was inf i l trat ion of lymphocytes into the portal tracts; numerous thin-walled dilated blood vessels were present; there was no evidence of cirrhosis in any case tumor was resected 1 month after lapar-otomy, well at 4 years biopsy done but no more of the lesion was removed; discharged on 10th post-operative day at cholecystectomy, a mass in the t ip of the right lobe of the l iver was found; tumor was a 5 era oval fleshy mass with a coarsely nodular surface tumor was resected local ly , 2 weeks after f inish of chemo-therapy; returned home 11 days post-operatively AGE (YR) or PATIENT STERIOD HISTORY OTHKK f « w i » i DATA AND/OR CONCURRENT THERAPY CLINICAL PRESENTATION DIAGNOSIS type unknownj 5 years took o .c . 'a for 5 years after the birth of her fourth child large l iver mass hepatoma (benign) sic took o.c. 's at time large l iver mass hepatoma of illness but dura- (benign) tion of therapy could sic not be determined 32 type unknown! 8 years large l iver mas hepatoma (benign) sic 27 no history of oral contraceptive medication was in ninth month of pregnancy at time of death hepatoma (benign) sic 10 34 norethisterone 1 mg on hemodialysis + mestranol SO ug years (Norinyl-l)i 1-3 tablets daily for persistent menorrhagia for 7 severe abdominal pain, l iver shock, signs of per i - hamartoma toneal i rr i ta t ion , hemoperitoneum 11 lynestrenole 2.5 mg + mestranole 0.075 mg (Lyndiol Hite); 5 years accidental finding at cholecystectomy (1972) focal nodular hyperplasia /25 lynestrenole 2.5 mg + mestranole 0.075 mg (Lyndiol Mite); 3 years ceased o.c. medication 3 years before tumor detected accidental finding at caesaxean section(1973) focal nodular hyperplasia norethisterone + methyleatrenolone for several years; norethisterone 3 rag + ethinylestradiol .05 mg for nine years unknown drugs for psychiatric problem accidental finding at uterine myoma operation detected in 1973 focal nodular hyperplasia 28 norethisterone 1 mg mestranol 0.1 mg for seven years; norethisterone 1 mg mestranol 0.05 mg for one year accidental finding at cholecystectomy focal nodular hyperplasia 12 6 chloro-6 dehydro-17«C acetoxyprog-esterone + mestranol (C-QuenB); 6 years placed on o.c.'s after 2 normal pregnancies and deliveries; had a 3rd normal pregnancy after the 6th year on o.c. 's 5 weeks post-partum; experienced sharp RUQ pain and right shoulder pain: fainted; hemo-peritoneum hepatic adenoma 13 29 type of o.c. unknown; gravida 3, para 3 patient noted mass in hepatic 5 year therapy right upper abdomen in adenoma i July 1969 but was I asymptomatic type of o.c. unknown gravida 5, para 5, mass in midepigastrium hepatic 6 year therapy about 6 months before palpated adenoma tumor detection, patient noted anorexia and morning nausea; gained 9 kg (20 lb) during this time: swelling of hands and feet; angiography showed a discrete' vascular mass 145 CROSS PATHOLOGICAL FINDINGS the extent of hemorrhage of these four tumors seemed inversely correlated with the degree of differentiation HISTOPATHOLOGY these four tumors were a l l hepato-cel lular; they varied from orderly to moderately pleomorphic OUTCOME AND FOLLOW-UP died a month after subtotal resection of liver for 2 neo-plastic masses totalling 1000 g. died immediately from hemorrhage died immediately from hemorrhage died immediately from hemorrhage l iver contained multiple, discrete, highly vascularized tumors; massive hemoperitoneum; source of hemorrhage was a ruptured intrahepatic hematoma confined to the right lobe patient died after repeated surgical intervention and maBsive transfusion (multiple lesions prevented partial hepatectomy) 1 cm lesion alive after excision of tumor 4 cm les/fon in right lobe Excision of tumor of right lobe; patient alive 8x7x5 cm lesion in right lobe tumor excised; patient committed suicide 5 cm lesion tumor excised; patient alive tumor in left lobe of l iver which was the site of rupture, mass was 6.5 cm in diameter and had penetrated through the l iver surface causing hemorrhage; clear demarcation toward the surrounding normal l iver tissue tumor consisted of cords of large, clear, regular l iver cells with interspersed sinusoidal formations; no portal tracts or lobular formations; no encapsulation almost total left lobectomy; unevent-ful recovery 12 cm diameter tumor found at celiotomy arising from left lobe of l iver; the tumor had multiple peripheral feeding vessels and tortuous vasculature 10 cm tumor in left lobe of l iver needle biopsy showed normal hepatic, architecture the 8 tumors consisted of well-differentiated hepatocytes organized in sheets and cords without lobules; cel ls often arranged in sinusoidal pattern; neoplastic cells seemed larger and clearer than non-tumor cells; clear areas represented increased glycogen content and l ip id vacuoles; occasionally some bile aggregates found between cel ls ; most cel ls uniform in size, but foci of pleomophic cel ls could be seen; no increase in number of mitotic figures over normal; bile ductules seen ln varying numbers were poorly formed often interconnected or connected directly to tumor cel ls tumor resected removing much of quadrate lobe* uneventful post-operative course; good health 5 years later uneventful post-operative course; in good health at 2*i years AGE(YR) OF PATIENT 13 cont'd STERIOD HISTORY type of o.c. unknown; 6 year therapy 146 OTHER PATIENT DATA AND/OR CONCURRENT THERAPY gravida 2, para 2 CLINICAL PRESENTATION i n c i d e n t a l f i n d i n g at hysterectomy DIAGNOSIS hepatic adenoma type of o.c. unknown; gravida 1, para 1; w e l l sudden severe RUO and hepatic 6 month therapy u n t i l Nov. 1972, then e p i g a s t r i c pain associated adenoma noticed weakness, f a t i g u e , with nausea, vomiting, p a l l o r weakness, syncope, shock 28 (Enovid); i n t e r m i t -tent therapy for 12 years gravida 4, para 4; prev i o u s l y i n good health u n t i l noted vague r i g h t upper abdominal pain; l o s t 9 kg (20 l b ) , f a t i g u e , anorexia, nausea hepatomegaly and displacement of r i g h t kidney hepati c adenoma with c e n t r a l necrosis and o l d hemorrhage type of o.c. unknown; 7 year therapy gravida 3, para 3; previous e x c e l l e n t health sudden severe e p i g a s t r i c hepatic pain, shock, i n t r a p e r i t o n - adenoma e a l hemorrhage (Enovid); f o r 7 gravida 4, para 2 RUQ pain of sudden onset hepatic years adenoma / 26 norethindrone + gravida 3, para 3 RUQ pain of sudden onset, hepatic mestranol shock adenoma (Ortho Novum), 4 yrs; norgestrel + ethiny1-e s t r a d i o l , (Ovral) 2 years various o.c.' for 7 years gravida 1, para 1; uncomplicated pregnancy without jaundice or p r u r i t i s was only i n t e r r u p t i o n i n o.c. therapy; maas revealed 1 month a f t e r r e - i n s t a t i n g o.c. therapy a f t e r d e l i v e r y mass i n RLQ; moveable, firm, nontender mass, suggestive of ovarian cyst f o c a l nodular hyperplasia 29 norethindrone 2 mg + gravida 0, para 0; no 0.1 mg mestranol previous l i v e r disease (Ortho-Novum); 6 years nontender abdominal mass f o c a l i n LUQ; i n i t i a l diagnosis nodular was ovarian or mesenteric h y p e r p l a s i a cyst ethynodiolacetate lmg +0.1 mg mestranol (Ovulen-21) for about 6 years gravida 0, para 0; l i v e r scan showed a defect i n the r i g h t lobe of the l i v e r severe RUQ pain of sudden onset that r a d i a t e d to her back; massive i n t r a h e p a t i c hemorrhage po s s i b l e f o c a l nodular hyper-p l a s i a undetectable a f t e r i n t r e -hepatic hemorrhage 147 GROSS PATHOLOGICAL FINDINGS HISTOPATHOLOGY OUTCOME AND POLLOM-UP 10 cm tumor of right lobe; tumor was grossly vascular 17 cm mass in right lobe of liver found at celiotomy and a smaller mass found in left lobe of l i v e n centre of the large tumor was necrotic and contained clotted blood at celiotomy, large mass in right lobe of liver; 2 months later, the tumor was excised 18 cm ruptured tumor occupying most of the right lobe of the liver SUMMARY OF THESE EIGHT CASES <9 r o s* pathology) areas of hemorrhage, hemorrhagic necrosis and distinct scar were seen in most of the neoplasms; in some of the tumors a central stellate scar suggested focal nodular hyperplasia rather than hepatic c e l l adenoma; no well-formed bile ducts; varied vascular patterns; e.g. dilated veins alternated with small vascular bundles surrounded by dense collagen or anastomosing, dilated angulated sinusoidal venous structures) hemorrhages seemed to occur at the interface between tumor and capsule, tumor and normal liver, or tumor and scar; hemorrhagic areas found next to zones of dilated venous-like structures; a l l tumors partly encapsulated by an incomplete fibrous membrane; most tumors solitary; complex lobulation was present in some right liver lobectomy; uneventful post-operative course to 5 months tumor excised in right lobe only; satisfactory post-operative course; in good health 20 months post-operatively doing well 3*i years pOBt-operative from tumor excision right hepatic lob-ectomy , complicated post-operative course; discharged 10 weeks pOBt-operatively; normal liver function during 3 years since operation at celiotomy, a massive hemoperitoneum found as a result of rupture of "grapefruit-sized" tumor of right lobe of liver at celiotomy, a ruptured tumor 18 cm in diameter in right lobe of liver right hepatic lob-ectomy) massive blood loss; complicated recovery; discharged 3 months after operation right hepatic lob-ectomy; complicated post-operative recovery; discharged 9 weeks after opera-tion; fully active 10 months after operation at exploratory laparotomy a 10 cm pedunculated mass arising from the left lobe of the liver was noted; tumor mass was richly vascular giving i t a spongy appearance; numerous large vascular channels; hemorrhage into adjacent liver tissue; tumor not encapsulated altered hepatocytes with vacuolated to clear cytoplasm; no evidence of nuclear atypla; loss of normal lobular pattern; absence of normal plates of hepatocytes and sparsity of bile ducts and portal areas was a characteristic feature in this and case 2; randomly distributed fibrous tracts containing vascular channels and large vessels; Kupffer cel l s present exploratory laparotomy revealed a well-differentiated hepatocytes without left partial large lobulated mass in the left lobe evidence of malignant transformation; hepatectomy; liver of the liver; 5 areas of nodular loss of normal lobular pattern; dilated function normal 3 hyperplasia; one nodule showed central sinusoids and increased vascularity months post-operation ischemic necrosis and one shoved hemorrhagic necrosis; mass not encapsulated; increased vascularity of lesion at exploratory laparotomy, a large hematoma was found In right lobe of liver; hematoma in large cystic cavity; only necrotic tissue in cystic cavity* no nodules were present at laparotomy but is possible that the intrahepatic hemorrhage was related to necrosis of a small nodule and after hemorrhage the area of nodular hyperplasia could no longer be recognised grossly hematoma waB drained; patient recovered completely A G E (YR) or R E F P A T I E N T S T E R I O D HISTORY unknown type; 2 years, 10 months 148 OTHEK PATIENT DATA AND/OR CONCURRENT THERAPY complained of f a t i g u e , lack of energy; no discomfort, pain; 2 normal d e l i v e r i e s CLINICAL PRESENTATION DIAGNOSIS palpable mass i n RUQ of benign abdomen; p o s i t i v e l i v e r hepatoma scan norgestrel + e t h i n y l -e s t r a d i o l (Ovral) f o r 6 months; discontinued for 3 months; norethlndrone + mestranol f o r 5 months f i t t e d with I.U.D. a f t e r discontinued o.c. therapy for 7 months before i l l n e s s ; p o s s i b l e sponta-neous abortion at 18 years; DfcC revealed e a r l y products of conception at present admission; r i g h t ovarian cyst discovered, abnormal corpus luteum; p o s i t i v e l i v e r scan nausea, vague upper abdominal pain noted for 3 days p r i o r to i n i t i a l examination; RUQ, RLQ tenderness f o c a l nodular hyperplasia norethlndrone + mestranol (Ortho Novum 1/80), ethynodial diacetate and mestranol (Ovulen) for 5 years gravida 6, para 5, RUQ abdominal pain f o r abortus 1; no previous 6 months and chole-hepatic disease or exposure l i t h i a s i s to hepatotoxic agents; p o s i t i v e l i v e r scan and angiography f o c a l nodular hyperplasia norethlndrone + mestranol (Ortho-Novum 2 mg) for 3 years; Ortho-Novum 1/50 for 6 months (since b i r t h of c h i l d ) gravida 1, para 1; previous good he a l t h ; p o s i t i v e abdominal arteriography and l i v e r scan acute chest and upper primary abdominal pain that hepato-p e r s i s t e d for about 15 c e l l u l a r hours; shock; hemoperi- adenoma toneurn; admi ssion diagnosis was ruptured e c t o p i c pregnancy norethlndrone + gravida 3, para 3; had mestranol upper abdominal mass that (Ortho-Novum 2 mg) had been present f o r 2 for 6 years years; l i v e r scan showed 2 f i l l i n g d efects of the l i v e r i n d i c a t i n g space occupying l e s i o n s ; p o s i t i v e angiography upper abdominal pain l i v e r c e l l associated with nausea and adenoma vomiting; RUQ mass 10 cm i n diameter found 21 megestrol acetate 4 mg + e t h i n y l o e s -t r a d i o l 50 ug for 2 years acute h e p a t i t i s 2 years previously but returned to normal l i v e r f u nction; l i v e r scan showed f i l l i n g defect i n l e f t lobe of l i v e r ; p o s i t i v e arteriography anemia on routine p h y s i c a l examination; f i r m enlarge-ment of l e f t lobe of l i v e r f o c a l nodular hyperplasia + w e l l - d i f -f e r e n t i a t e d hepato-c e l l u l a r carcinoma 149 GROSS PATHOLOGICAL FINDINGS at laparotomy, a sol id, round, light yellow mass was found in right lobe of l iver; 8 cm diameter mass; sharp demarcation from surrounding tissue but was not encapsulated HISTOPATHOLOGY OUTCOME AND FOLLOW-UP simple resection; uneventful recovery; normal function at 6 months pelvic exploratory laparotomy revealed a large biloculated ovarian cyst 12x6x10 cm and numerous firm nodules in the left lobe of the l iver; upper abdominal exploratory surgery revealed 8x6 multinodular mass in left lobe and another similar separate multinodular mass near falciforn ligament; nodules poorly circumscribed cords, cells distorted by compression; oval, round regular nuclei; sinusoids were compressed and empty as seen from percutaneous l iver biopsy; nodules were separated by wide stellate collagenous bands which contained inflammatory ce l l s , portal structures and small ducts; some nodules had central veins; hepatocytes had uniform nuclei but indistinct cytoplasmic borders; hyper-plastic Kupffer cel ls ; decreased glycogen stores part ia l left hepa-tectomy and excisional biopsy of larger l iver mass; right oophorectomy; stormy post-operative course due to bi l iary peritonit is; alive 14 months post-operatively although multiple nodules remained in left lobe of l iver at cholecystectomy, a 12cm x 12 cm tumor in left lobe of l iver found; a lcm x 1cm nodule seen on dome of right side of l iver; 6 days later, at partial hepatectomy, a 9 x 6 . 5 x 6 . 5 c m pale tan tumor that extended to the l iver surface was visible; dilated veins on the surface could also be seen; tumor was nodular with radiating fibrosis; central portion of the tumor showed hemorrhagic necrosis with associated inflammatory response; fibroblastic and bi le duct proliferation occurred surrounding this area micronodular cirrhosis with marked fibrosis in portal areas was seen; portal to portal bridging by bands of fibrous tissue that surrounded regenerating nodules of l iver cel ls; cytoplasm stained positively for glycogen; marked proliferation of bile ducts; foci of chronic inflammatory inf i l trate in portal areas; many vessels showed thickening of the intima; small arteries showed fibro-muscular proliferation in media alive and well 6 months after partial right hepatectomy and cholecystectomy hemoper/i toneum found with abdominal tap; at laparotomy a perforated necrotic tumor was found on inferior surface of caudate lobe of the l iver with massive retroperitoneal and intra-abdominal bleeding; the lesion was primarily avascular neoplastic cel ls were hepatocellular; larger than normal hepatocytes; glycogen present in cel ls part ia l resection of caudate lobe; tumor removed and.did well post-operative; alive 8 months post-opera-tively at laparotomy 2 completely separate masses were seen; 14 cm diameter and 7.5 cm diameter; large hemorrhages and necrosis were present; tumor was demarcated but not encapsulated; the surrounding l iver parenchyma and tumor showed distended large blood vessels and sinusoids in many areas trabeculae of hepatocytes with regular nuclei; no lobular arrangement, portal tracts, bile ducts; granulomatous inflammation was seen in the surrounding l iver (negative for acid fast b a c i l l i and fungi) hepatectomy (entire right lobe and part of left lobe); rapid uneventful recovery and discharged 3 weeks post-operative; doing well two months after discharge at laparoBCOpy the left lobe of the l iver was diffusely enlarged and some-what purple; 14cm mass was multilobular and well circumscribed; at hepatectomy most of the tumor was gray colored although some parts were light brown; central part of the mass was occupied by part ia l ly calcif ied fibrous tissue that radiated between the lobules; patchy hemorrhages were scattered throughout the mass, but no major hemorrhages were seen whole tumor was well demarcated from surrounding l iver tissue; true capsule not present; tumor cel ls of the l ight brown areas were fa ir ly normal but were larger than normal; no normal hepatic architecture, portal tracts or hepatic veinBj small thick-walled arteries occurred randomly throughout the tumor as did dilated thin-walled blood vessels; there were also areas of extreme di lat ion of sinusoids with formation of blood f i l l ed cysts; the gray areas of the tumor represented well-differentiated hepatocellular carcinoma; cel ls were basophilic, dysplastict nuclei were markedly pleomorphic; a few were undergoing mitotic division; normal arrangement of l iver ce l l plates was disrupted; tumor cel ls lay in hyaline stroma singly or in small groups; two components of the tumor were sharply demarcated and inflammation was present between them and sometimes a band of fibrous tissue which had been invaded by dysplastic cel ls was also present left hepatectomy; uneventful recovery and discharged 3 weeks after surgery AGE (YR) OF PATJENT 25 STERIOD HISTORY norethynodrel + mestranol for 5 years; continued UBB for 6 years more OTHER PATIENT DATA AND/OR CONCURRENT THERAPY gravida 2, para 2 CLINICAL PRESENTATION abdominal pain, shock and collapse; 2nd event, abdominal pain and massive hepatomegaly DIAGNOSIS focal nodular hyperplasia, intrahepatic hemorrhage 26 norethynodrel + gravida 2, para 2 mestranol for 7 years abdominal pain, r ig id abdomen * shock, hemo-peritoneum focal nodular hyperplasia 26 norethynodrel • mestranol for 6S years gravida 2, para 2 abdominal pain, fainting, hemoperitoneum focal nodular hyperplasia 22 norethlndrone + gravida 1, para 1 mestranol for 7 years abdominal pain, left shoulder pain, shock, hemoperitoneum focal nodular hyperplasia ethynodiol diacetate + mestranol for 4 years gravida 6, para 2 chronic recurring RUQ pain; defect on l iver scan; cholecystography normal focal nodular hyperplasia and large hemorrhagic cyst norethlndrone + mestranol for 4 years abdominal pain, nausea hamartoma, and vomiting, laparotomy normal ga l l -for acute cholecystitis bladder ethynodiol diacetate gravida 2, para 2 tumor discovered at hamartoma + mestranol for hysterectomy; later IS years evaluation with hepatic arteriogram norethlndrone + mestranol for 6 years gravida 2, para 2 tumor palpated at hysterectomy focal nodular hyperplasia 35 norethlndrone + mestranol for 5 years gravida 4, para 4 tumor palpated at hysterectomy hamartoma 20 norethisterone acetate 1 mg + ethinylestra-diol .05 mg for 1st episode of monorrhagia for 3 mo. course; off for 2 years; norethisterone 15 mg daily for 3 months for 2nd episode of menorrhagia; norethisterone 1 mg + mestranol .05 mg; 1/day for 2 months 2/day for 1 year 6/day for 2 months; norethisterone 2 mg + mestranol .1 rag bid 3<i years! 9 years prior to death was diagnosed as having mem-brano-proliferative glom-erulonephritis; G . N . was treated with diuretics. after splenectomy the patient suddenly developed severe upper abdominal pain associated with distension and shock; prednisolone, azathioprine, coagulation studies normal norethandrolone; started hemodialysis \ \ years after diagnosis of G . N . ; was anti-coagulated for dialysis and this exacerbated her menor-rhagia; this was treated with cyc l ica l hormone therapy; 2 years later developed almost continuous uterine bleeding; was treated with o.c.'s then to control vaginal bleeding; 8 years after C . N . diagnosis had bi lateral nephrectomy due to hypertension; later developed anemia, mild leukopenia and thrombo-cytopenia) l iver scan showed hepatomegaly and splenomegaly; splenectomy performed because of greatly decreased half-l i f e of red blood cel ls multiple l iver ce l l adenomas; arterio-qraphy suggested multiple tumors GROSS PATHOLOGICAL FINDINGS 151 H I S T O P A T H O L O C Y OUTCOME AND F O L L O W - U P tumor ruptured; r e s t of l i v e r e n t i r e l y normal at l e f t lobectomy> 2nd laparotomy showed 2nd tumor i n remain-ing part of l i v e n s i m i l a r to 1st tumor SUMMARY OF C A S E S no evidence of c i r r h o s i s ; marginB of tumors were sharply demarcated from surrounding hepatic t i s s u e ; microscopic areas of i n f a r c t i o n , n e c r o s i s , hemorrhage present; i n t e r l o b u l a r branches of the a f f e r e n t veins and a r t e r i e s had i n t l m a l p r o l i f e r a t i o n and swelling, hypertrophy of tunica media, and organizing thrombosis l e f t lobectomy; a 1ive a f t e r 6 years; o.c.'s not discontinued; new tumor developed in r i g h t lobe; r e s e c t i o n of 2nd tumor not done; tubal l i g a t i o n ; doing well at 5 months tumor ruptured i n t r a p e r i t o n e a l l y j hemoperitoneum averaged 2500 ml r i g h t hepatic lob-ectomy, a l i v e a f t e r 3 years tumor ruptured i n t r a p e r i t o n e a l l y ; hemoperitoneum averaged 2500 ml sublobar r e s e c t i o n and l i g a t i o n of hepati a r t e r y , r i g h t lobe; a l i v e a f t e r 5 years tumor ruptured i n t r a p e r i t o n e a l l y ; hemoperitoneum averaged 2500 ml tumor excised from l e f t l a t e r a l segment; a l i v e a f t e r 3 years tumor ruptured r i g h t lobectomy; a l i v e a f t e r 4 years tumor resected i n r i g h t lobe; chole-cystectomy; a l i v e a f t e r 6 years r e j e c t i o n of tumor adjacent to g a l l -bladder; a l i v e a f t e r 2 years tumor i n r i g h t lobe of l i v e r was 5cm; hard, i r r e g u l a r ; didn't encroach upon surface of the l i v e r needle biopsy done; a l i v e at 8 months tumor i n r i g h t lobe needle biopsy; a l i v e at 10 months emergency laparotomy showed massive hemoperitoneum due to rupture of r i g h t lobe of l i v e r ; l i v e r weighed 4945 gm inc l u d i n g a subcapsular hematoma; cut surfaces showed multiple tumors from 2-60 mm i n diameter scattered through-out both lobes of the l i v e n hematoma in centre of l e f t lobe; tumors were demarcated from surrounding normal t i s s u e , but were not encapsulated; tumors were pale brown i n c o l o r and were not lobulated; there were f o c i of hemorrhage; 64% of l i v e r was macro-s c o p i c a l l y normal; l i v e r was d i f f u s e l y enlarged due to d i f f u s e hyperplasia coupled with f o c a l nodularity tumors were composed of normal looking hepatocytes; no p o r t a l t r a c t s , no b i l e ducts, no c e n t r a l veins, no lobular pattern; hepatocytes were arranged i n cords and p l a t e s 2 or 3 c e l l s wide and were separated by sinusoids with Kupffer c e l l s ; numerous, large thin-walled blood vessels present; they consisted of e n d o t h e l i a l c e l l s with a minimum of supporting t i s s u e ; no smooth muscle or e l a s t i c f i b r e s i n v e s s e l walls; abnormal vessels confined to the tumor; the normal l i v e r showed enlarged lobules and p l a t e s of hepatocytes 2 c e l l s wide, but the lobules were a r c h i t e c t u r a l l y normal bleeding area was packed and abdomen closed; despite i n t e n s i v e blood t r a n s f u s i n g and surgery, the p a t i e n t d i e d i n 2 days 1 5 2 AGE(YR) OF PATIENT STERIOD HISTORY ethynodiol + ethinylestradiol (Bisecurin) for 5 years OTHER PATIENT DATA AND/OR CONCURRENT THERAPY i . v . cholangiography showed that a probable mass was present l iver harmartoma CLINICAL PRESENTATION DIAGNOSIS general weakness, poly-dipsia, polyuria and episodes of hypoglycemia of € months duration; recent sensitivity of right hypochondrium to pressure; palpable mass at right lower margin of liver norethindrone + mestranol (Ortho-Novum 2mg) for 7 years . one year prior to admission on routine physical examination had slightly enlarged liver (liver function normal); original diagnosis was acute cholecystitis and was treated with i . v . fluids and systemic antibiotics but symptoms worsened; oral chole-cystography showed a normal gallbladder; I .V.P. indicated a possible enlarged l iver; intra-abdominal hemorrhage was suspected; nature of the tumor was not clear at laparotomy so further surgery was delayed; positive l iver scans and angiography RUQ abdominal pain of acute onset associated with nausea, vomiting, fever; pain was colicky and radiated to the back; poorly defined mass palpable B-10 cm below costal margin l iver ce l l adenoma 29 norethynodrel mestranol (Enovid-E) for 8 years gravida 7, para 4 mass was palpated in hepato-right abdomen after 3 cellular attacks of epigastric adenoma pain 34 norethynodrel mestranol (Enovid-E) for 6 years gravida 3, para 2; 8 years previously had a right heraithyroidectomy for mixed fo l l i cu lar and papillary carcinoma; also had 2 more operations within 3 years for meta-stases of the thyroid carcinoma mass was palpated on routine examination multiple hepato-cel lular adenomas 24 dimethisterone + ethinylestradiol (Oracon) for 1 year gravida 3, para 3 right upper abdominal hepato-mass palpated after an cel lular episode of upper adenoma abdominal pain, vomit-ing, diarrhea ethynodiol diacetate + mestranol (Ovulen) for SH years gravida 2, para 1 abdominal mass hepato-cellular adenomas 153 GROSS PATHOLOGICAL FINDINGS at laparotomy a yellowish-gray sharply delineated solid growth was found beneath the liver capsule; mass measured 6x5x5 cm; tumor was not encapsulated) on the abdominal surface the tumor was covered only by the liver capsule; cut surface of the tumor was delicately lobular HIST0PATH0LOGY l iver tissue was abnormal being divided by connective tissue septa, but the septa did not completely encircle larger groups of hepatocytes; the connective tissue septa contained veins, some arterial branches, many bile ductules and lymphocytes; hepatocytes were arranged in irregular plates; cytoplasm was pale with no nuclear abnormalities; •transitional" cel ls (between ductular epithelium and hepatocytes) were found away from the septa amongst l iver cel ls; some haphazardly arranged veins were present OUTCOME AND FOLLOW-UP tumor was removed; patient's progress not stated at emergency laparotomy, a large vascular tumor was found replacing the right lobe of the l iver; no evidence of bleeding into the peritoneal cavity; at lobectomy (one week later) 16x16x12 cm tumor was seen to be well circum-scribed, not encapsulated; tumor was highly vascular with blood vessels on the surface; tumor contained a large necrotic central area f i l l e d with blood clots tumor consisted of well-differentiated l iver cel ls with single or double l iver ce l l cords; there was occasional compacted l iver ce l l arrangement; no normal lobular architecture; no portal tracts; some atypical nuclei but no mitoses found; no invasion of surrounding normal tissue or vessels right hepatic lob-ectomy and chole-cystectomy ; uneventful post-operative course surgical exploration revealed a 9 cm nodular pedunculated mass arising from right hepatic lobe; the nodule was well delineated from surrounding tissue; was pale yellow in color, incomplete capsule was present; fibrous trabeculae traversed the neoplasm; venous sinuses 2-3 cn in diameter were present neoplasm consisted of plates and cords of hepatic cel ls supported by a net-work of reticulum; plates of hepato-cytes were separated by sinusoids lined by endothelial cel ls; architecture was near normal except there were no portal areas or bi le ducts; scattered veins ware in evidence; individual tuswr cel ls looked like hepatocytes with increased amounts of fat and glycogen; nuclei wore sl ightly larger than normal and sl ightly pleomorphic; no mitotic act ivity; a pseudocapaule was present tumor was excised; uneventful recovery; normal l iver scan 3 years post-operatively surgical exploration revealed a pedunc- similar histology as above; increased wedge resection ulated mass hanging from the antero- amount of fat and glycogen present included a l l 3 tumors inferior edge of the right hepatic lobe; the mass was 6.5 cm in diameter and was well delineated; there were 2 more masses within the pedicle; had similar pathology as above surgical exploration revealed a tumor mass 9 cm ln diameter projecting from the surface of the right lobe of the l iver; appeared encapsulated and paler in color than surrounding l iver; mass was hypervascular similar histology as above; increased amounts of fat and glycogen in tumor cel ls ; dilated veins present wedge taken of right lobe and chole-cystectomy surgical exploration revealed a 10 cm similar histology as above mass was excised; nodular maaB; was pedunculated from uneventfull recovery left lobe of the l iver 154 STERIOD HISTORY norethynodrel + mestranol (Enovid) for 4 yearB; then changed to ethynodiol diacetate + mestranol (Ovulen) for 11 years (except for a period of 6 months because of hepatomegaly) OTHER PATIENT DATA AND/OR CONCURRENT THERAPY gravida 1, para 1> had only taken o.c.'s as continuous medication; no history of any kind of l iver disease; on admission for present illness had an enlarged nontender liver extending 3 cm below the right costal margin; positive arteriography CLINICAL PRESENTATION incidental finding at laparoscopic tubal l igation; hepatic surgery referred DIAGNOSIS hepatic adenoma i n i t i a l l y ; changed to focal nodular hyperplasia norethindrone + mestranol (Ortho Novum), ethynodiol diacetate + mestranol (Ovulen), norgestrel + ethyinyl estradiol (Ovral) for 9 years continuously for menstrual irregularit ies gravida 0, para 0; 10 months prior to admission had an abrupt onset of postprandial cramping, epigastric pain, and fever to 38.3 degrees C which was found to be gastroesophageal reflux and was treated with antacids and bland diet; past therapy included Vit A 50,000 i . u . daily for several weeks for acne; levothyroxine Na 0.2 mg per day for 5 years for thyroid nodule; positive celiac arter i -ography cramping, periumbilical hepatic pain and fever to 38.3 ce l l degrees C adenoma norethynodrel + mestranol (Enovid) for 5 years; discontinued after surgery (leading to decrease in size of mass) sudden severe epigastric pain; pain radiated from RUQ to right shoulder with nausea and vomiting focal nodular hyper-plasia (on frozen section) later, hepato-cellular adenoma on permanent slides but various o.c.*s for 9 years; discontinued on admission mass in RUQ found by patient 1 year prior to admission; mass didn't change slse; no symptoms except a sensation of "heaviness" when standing for long periods of time; had taken diazepam inter-mittently; had lost 13 lb in 3 months prior to admission although had normal appetite; positive angiography mass discovered by hepatic internist in routine adenomas physical examination ethynodiol diacetate + mestranol (Ovulen) for 10 years loose stools for 2 days prior to admission; severe epigastric abdominal pain for 18 hour patient was obese; positive duration associated with G.I . series and arter i - nausea and vomiting ography and l iver scan hepatic adenoma with central hemorrhage and necrosis norethynodrel + mestranol (Enovid) for 2 years; ethynodiol diacetate + mestranol (Ovulen) for 6 years para 2; in good health unti l day of admission severe abdominal pain of benign sudden onset, shock, due hepatic to hemoperitoneum adenoma norethindrone + mestranol (Ortho-Novum) for 10 years sudden onset of acute pain in RUQ and epigastrium which was persistant with occasional radiation to right shoulder; palpable mass fe l t in relation to l iver l iver ce l l adenoma GROSS PATHOLOGICAL FINDINGS 155 H1STOPATHOLOCY OUTCOME AND FOLLOW-UP at exploratory laparotomy, an 8 cn mass in the inferior portion of right lobe and another mass in the anterior superior aspect of the dome of the right lobe of the l iver were found fibrosis of the l iver parenchyma; the fibrosis in many areas was noted to be hyperplastic with no evidence of bi le ducts; lesions were not encapsulated but were demarcated from surrounding normal tissue right hepatic lob-ectomy and chole-cystectomy; on 9th day post-operative showed signs of infection and on the 14th day post-opera-tive had drainage for a right subphrenic abscess; discharged on 24th post-opera-tive day exploratory laparatomy revealed 3 hepatic masses; further evaluation showed only 2 lesions: a 6x9x7 cm tumor in the left lobe of the l iver that was grossly encapsulated by surrounding normal-appearing l iver tissue, and a 10x9x5 cm tumor in the right lobe part ial hepatectomy and cholecystectomy without incidence; uneventful post-operative course; discharged 17 days post-operatively three days after admission underwent exploratory laparotoay due to persistant symptoms; revealed a 11 cm mass mainly in right lobe of l iver although i t did extend to the left lobe / mass was not removed; patient doing well to 3 years showing decrease.in tumor size a large 12x13 cm vascular mass found at operation extending inferiorly from the free edge of the right lobe of the l iver; the mass was attached to the l iver by a pedicle; another smaller turner of about 2 cm was on the free edge of the left lobe pedunculated mass was resected as was the smaller tumor; complicated post-operative course due to a possible cerebral thrombosis; a month after discharge patient was readmitted with pulmonary embolus at operation a 5x3 cm mass in the left part ia l left hepa-lobe of the l iver was found tectomy and bi lateral tubal l igation; un-remarkable post-operative course on emergency exploratory laparotoay, a 8 cm tumor in the left lobe of the l iver was found; tumor had ruptured and was bleeding into peritoneal cavity similar to case below resection of the left lobe of the l iver; doing well after IH yearB at exploratory laparotomy l iver was tumor showed striking dilation and drainage of hematoma enlarged and a soft rounded 15 ca tumor prominence of vascular spaces; tumor was and right hepatectomy was found with a large sub-capsular sharply circumscribed, encapsulated; well 30 months later hematoma; surface of l iver was covered w a 8 composed of sheets of normal with large vessels; tumor was confined appearing hepatic parenchymal cel ls with and faxrly well circumscribed haphazardly arranged sinusoidal spaces) no portal triads or identifiable central veins were present in the tumor ACE (YR) Of PATIENT 38 STERIOD HISTORY norethynodrel + mestranol (Enovid) f o r 5 years; discontinued a (aw months before pregnancy 15b OTH2R PATIENT DATA AND/OR CONCURRENT THERAPY para 3 and 18 weeks pregnant CLINICAL PRESENTATION Incidental at hyster-ectomy and permanent a t e r i l i x a t i o n DIAGNOSIS f o c a l nodular hyperplasia 40 unknown type of o.c. f o r 3 years a f t e r admission became comatose; decerebrate r i g i d i t y and death followed Bevere headache; cerebro-vascular accident focal nodular hyperplasia 40 unknown type of o.c.'j f o r 1 year (9 years p r e v i o u s l y ) had recurrent episodes of acute c h o l e c y s t i t i s -l i k e symptoms fo r a year p r i o r to surgery I n c i d e n t a l at cholecystectomy f o c a l nodular hyperplasia 27 26 norethindrone + mestranol (Ortho Novum) for 4 years gravida 2, para 2; i n good health p r i o r to admission; p e r s i s t a n t pyrexia f o r 1 month a f t e r a laparotomy; continuous r i g h t flank pain as w e l l ; n a r c o t i c s given f o r p a i n r a m p i c i l l i n and kanamycin and pen G given; 2 weeks post-opera-t i v e increased BSP r e t e n t i o n and l i v e r scan showed 2 defects i n r i g h t lobe; p o s i t i v e arteriogram; a f t e r drainage, fever and leuko-c y t o s i s p e r s i s t e d sharp, severe RUQ pain unaffected by food or p o s i t i o n ; pain d i d not ra d i a t e hepatic adenoma 28 norethynodrel mestranol (Enovid-E) fo r 4 years para 4; p o s i t i v e l i v e r scan p a t i e n t i d e n t i f i e d palpable mass i n RUQ; mass was p a i n l e s s hepatic adenomas associated with hemorrhage' norethindrone mestranol (Ortho Novum) for 4 years no p r i o r pain, weight l o s s , anorexia, or malaise; p o s i t i v e l i v e r Bean and angiography acute crampy abdominal hepatic pain i n p e r i u m b i l i c a l area adenoma associated with watery associated d i a r r h e a , c h i l l s , f e v e r , with p e r s i s t e n t RUQ pain hemorrhage 35 norethindrone mestranol (Norinyl) f o r 3 years para 1, abortus 1; h i s t o r y of 10 years of heavy menstrual bleeding; o.c.'s were discontinued 4 months p r i o r to admission due to mild hypertension and frequent break-through bleeding; a f t e r d e t e c t i o n of the l i v e r tumor, l i v e r scan and aortogram showed a s i n g l e large vascular tumor i n c i d e n t a l at e l e c t i v e hysterectomy large mass was f o c a l nodular hy p e r p l a s i a ; small masses were hemangiomas ethynodiol d i a c e t a t e + mestranol (Ovulen) f o r 4>i year* gravida 2, para 2; 3 months a f t e r surgery, had e l e c t i v e p a r t i a l l e f t hepatectomy and b i l a t e r a l p a r t i a l salpingectomy acute m i d e p i g a s t r i c abdominal pain, mild hypotension; thought to be perforated u l c e r f o c a l nodular hy p e r p l a s i c with hemorrhage 157 GROSS PATHOLOGICAL FINDINGS on laparotomy, a 3 cm nodule was resected from the i n f e r i o r l a t e r a l border of the l i v e r HISTOPATHOLOGIC s i m i l a r to case below OUTCOME AND FOLLOW-UP e x c i s i o n a l b i o p B y of tumor; patient well a f t e r 2 years death due to subarachnoid hemorrhage; autopsy showed a subcapsular w e l l circumscribed nodule 2 cm i n diameter on a n t e r i o r aBpect of r i g h t lobe of l i v e r ; a ruptured berry aneurysm was a l s o found tumors were sharply delineated but not t r u l y encapsulated; each l e s i o n was composed of normal parenchymal c e l l s d i v i d e d i n t o lobules by fibrous septa; there were varying degrees of lymphocytic I n f i l t r a t i o n i n the septa; b i l e duct p r o l i f e r a t i o n present i n the septa; d i l a t e d , t hin-walled vascular spaces wexe present throughout the l e s i o n s i n the parenchyma as well as i n the septa cerebrovascular accident caused death at surgery a well circumscribed s i m i l a r to case above e x c i s i o n of tumor 2-3 cm nodule was seen on the a n t e r i o r surface of the l i v e r exploratory laparotomy o r i g i n a l l y d i d not i n v e s t i g a t e l i v e r ; drainage of what was thought to be a hepatic abscess was c a r r i e d out 3 weeka a f t e r i n i t i a l e x p l o r a t i o n ; 2 c a v i t i e s contained brown n e c r o t i c m a t e r i a l ; f i v e weeks a f t e r admission underwent surgery; l i v e r was resected and 2 nodules 10 cm and 6 cm i n diameter were found as were many smaller nodules d i l a t e d thin-walled v e s s e l s and sinuses present! absence of normal l i v e r a r c h i t e c t u r e ; tumor composed of c e l l s c l o s e l y resembling normal hepatocytes although c e l l s were somewhat l a r g e r ; no p o r t a l t r a c t s , b i l e ducts or c e n t r a l v e i n s ; no mitoses; i n places there was extensive f r e s h and p a r t i a l l y organized hemorrhage and n e c r o s i s ! tumor was sharply demarcated but no true capsule present! no vascular i n v a s i o n appendectomy; drainage; r i g h t hepa-tectomy 5 weeks a f t e r admission; i n good health 7 years post-operatively at surgery 2 masses were found, one d i d w e l l post-was mobile, c y s t i c and attached to the operatively l i v e r only on p o s t e r i o r aspect and the other was located i n the hilum and extended i n t o the r i g h t and l e f t l i v e r lobes; capsule of l i v e r was i n t a c t ; there was a d i s t i n c t border between the tumor and surrounding parenchyma (which was compressed by the tumor) at surgery a 5x7 m u l t i l o b u l a r mass was found i n the r i g h t upper p o r t i o n of the l i v e r : several areas of hemorrhage occupied nearly 50% of the t o t a l mass; mass was compressing the surrounding hepatic parenchyma r i g h t lobectomy; d i d w e l l post-o p e r a t i v e l y at hysterectomy a vascular tumor was found a r i s i n g from the l e f t lobe of the l i v e r , i t projected p a r t l y under the l e f t h a l f of the diaphragm; removal of the tumor was delayed; at surgery one large and two small masses were found at operation, a hemorrhaging r i g h t -sided hepatic tumor was found a l l s i x p a t i e n t s had l e s i o n s i n r i g h t hepatic lobe and three had involvement of l e f t lobe a l s o ; tumors were large , 8-10 cm i n diameter; l i v e r c e l l s were i n sheets without usual p o r t a l t r i a d s and c e n t r a l veinB; thin-walled veins wexe present; no true capsule present; i n f a r c t i o n n e c r o s i s , hemorrhage and surrounding f i b r o s i s were evident p a r t i a l r i g h t hepatectomy; doing w e l l 3 year post-o p e r a t i v e l y S T E R I O D HISTORY OTHER P A T I E N T DATA A N D / O R CONCURRENT T H E R A P Y C L I N I C A L P R E S E N T A T I O N D I A G N O S I S various o.c.'s at Intervals for 7 years gravida 2, para 2; months prior to admission com-plained of vague nagging mldepigastric and RUQ pain; had complete g . i . work-up at that time hypovolemic shock and focal diffuse abdominal pain; nodular thought to be due to hyperplasia intra-abdominal hemorrhage with of unknown etiology hemorrhage norethindrone mestranol (Ortho Novum) for IB months gravida 1, para 1; 8 months prior to admission, was evaluated for epigastric and RUO abdominal pain; radiological studies were normal at that time; intermittent bouts of pain persisted crampy mldepigastric pain and tenderness in LLO and RUQ focal nodular hyperplasia with hemorrhage ethynodiol diacetate • mestranol (Ovulen) for 4 years gravida 2, para 2; 4 mos prior to admission, com-plained of abdominal dis-non-specific recurrent abdominal pain and later diffuse abdominal pain comfort; was evaluated with radiating to back; hypo-hematology and chemistry, upper G.I . series, g a l l -bladder series, I VP, and barium enema which a l l proved to be normal tension focal nodular hyperplasia with hemorrhage ethynodiol diaoetate + mestranol (Ovulen) for 6 years gravida 0; for 2 years prior to admission had complained of inter-mittent mldepigastric pain and discomfort: barium swallow and gallbladder series were normal; positive l iver scan persistant intermittent midepigastric discomfort and pain focal nodular hyperplasia various types of o.c.'s for 6 years gravida 3, para 3; had experienced intermittent episodes of RUQ abdominal pain for about 1 year prior to admission acute onset of intra-abdominal hemorrhage focal nodular hyperplasia with hemorrhage norethynodrel mestranol (Enovid) for 3 years palpable mass l iver ce l l adenoma norethynodrel + mestranol (Enovid) for 1 year; and ethynodiol diacetate + mestranol (Ovulen) for 4 years (the 4 year course was taken after the tumor was treated) tumor therapy consisted of 5-fluorouracil 250 mg per day for 30 days via a catheter in the common hepatic artery acute pain secondary to intrahepatic hemorrhage l iver ce l l adenoma norethynodrel + mestranol (Enovid-E) for 3 years l iver ce l l adenoma CROSS PATHOLOGICAL FINDINGS 1W HISTOPATHOLOGY OUTCOME AND FOLLOW-UP a l l tumors were tan colored, soft and spongy before rupture and were easily differentiated by color and texture fr surrounding normal hepatic parenchyma at surgery, a right sided tumor was . found actively hemorrhaging see above partial right hepatectomy; doing well 2 years post-operatively at operation, a hemorrhaging right sided tumor was found and in addition, a large left-sided tumor was present see above partial right hepa-tectomy and doing well IB months post-operatively; left-sided tumor decreased in size and the vascularity lessened with discontinuation of o .c . 'a at operation, a large actively hemorrhaging right-sided tumor was found see above died six days later after total right hepatectomy, due to thrombocytopenia, hemorrhagic coagulopathy and progression to l iver and kidney failure at operation, a right sided intact hepatic tumor was identified see above total right hepa-tectomy; doing well post-operative; discontinued o.c.*s at operation, a large actively hemor-rhaging tumor was found in the right lobe of the l iver and a smaller one was found in the left lobe of the l iver; bleeding was locally controlled (11 months later, surgery was undertaken) see above total right hepa-tectomy and continued to take o .c . 's ; 4 months later under-went part ial left hepatectomy; has not discontinued o.c. medication tumor excised; after 3 years well lesion located in the hilum lesion part ial ly removed; well at 6 years two hepatic masses were found; one near the hilum one of the lesions was totally reBected; the second mass was located near the hilum of the l iver and was not completely removed; the defect produced by dissection persisted for 3 years; well after 4 years; STERIOD HISTORY i - X D U OTHER PATIENT DATA AND/OR CONCURRENT THERAPY CLINICAL PRESENTATION DIAGNOSIS several o . c . ' E for 5 years acute pain secondary to intrahepatic hemorrhage l iver ce l l adenoma ethynodiol diacetate + mestranol (Ovulen) for. 5 years acute pain secondary to intrahepatic hemorrhage l iver ce l l adenoma ethinylestrsdiol + dimethi sterone (Oraeon) for 3 years; norgestrel + ethinylestradiol (Ovral) painless mass focal nodular hyperplasia norethindrone + mestranol (Norinyl), ethynodiol diacetate + mestranol (Ovulen), norethynodrel + mestranol (Enovid), norgestrel + ethinylestradiol (Ovral), for 5 years painless mass focal nodular hyperplasia ethynodiol diacetate + mestranol (Ovulen) for 4 years; these were discontinued 2 months before pregnancy due to hepatomegaly gravida 10, para 8, abortus 1; was admitted in active labor at 42 weeks gestation; on the way to the hospital noted chest pains, nausea and air hunger; fainted enroute; l iver size seemed to be normal during pregnancy shock during active labor (shock was thought to be due to amniotic f lu id embolism) l iver adenoma norethynodrel 5mg mestranol 75mcg gradida 1, para 1; eight year history of diarrhea worsening in past l*i years; had taken ant i -cholinergic Bedatives for most of this time; tetracycline 250mg daily for 5 years for acne; upper GI series showed a mass in the RUQ displacing the small bowel to the left; patients* mother had a carcinoma of the breast resected, rheumatoid ar thr i t i s , and multiple allergies; positive l iver scan incidental finding of l iver mass 3cm below the right costal margin which was moderately tender to palpation benign vascular tumor, type undeter-mined; later determined to be hepatic adenoma chlormadinone acetate 2mg + mestranol O.OSrag for 2 years 9 months; has not taken o.c. 's or any other medi-cation for seven years prior to admission ethynodial diacetate lmg + O.lmg mestranol for 7 years no previous abdominal complaints; on physical examination abdomen was quite tender; peritoneal lavage showed gross blood; patient received 3 units of blood during surgery 2 hours after admission the hemoglobin level had fallen from 12 to lOg %; 4 units of blood were transferred during surgery severe abdominal pain and shock associated with hemoperitoneum acute onset of severe abdominal pain in RUQ; palpable mass in right side of abdomen hepatic adenoma with focal nodular hyperplasia hemorrhage into hepatic adenoma I b l GROSS PATHOLOGICAL FINDINGS H1STOPATHOLOGY OUTCOMF. AND FOLLOW-UP tumor was excised; well at 6 months tumor was excised; well at 3 months tumor was excised; well at 4 years tumor was excised; well at 6 months at necropsy, both lungs showed atalectasis without evidence of amniotic f lu id emboli; autopsy revealed a large ruptured adenoma of the right lobe of the l iver and massive hemoperitoneum patient died 1 hour after adinTssion large encapsulated tumor of the right lobe of the l iver that extended into the bed of the gallbladder; peliosis hepatis-like formation was seen subtotal hepatic lobectomy and cholecystectomy were carried out; uneventful recovery with subsiding of symptoms including diarrhea at laparotomy a tumor in the left lobe of the l iver measuring 12cm in diameter was noted to have ruptured; a second tumor was palpated in the medial aspect of the right lobe tumors were composed of sheets of hepatocytes with variably dilated sinusoids; many cel ls were binuclear and contained abundant glycogen; mitoses were infrequent; some single c e l l necrosiB and areas of hemorrhage and coagulative necrOBiB were present left segmental resec-tion was carried out to remove a l l of the tumor; the second tumor was not resected; l iver scan done 10 months after surgery showed a persistent f i l l i n g defect in the right lobe of the l iver but the patient has remained asymptomatic at. celiotomy a 14cm hemorrhagic mass was noted in the right lobe of the l iver adjacent to the left lobe; there was some blood present in the abdominal cavity similar to above a subtotal right hepatectomy was done; uneventrul recovery and has remained asymptomatic seven months post-operatively; o.c. therapy was discontinued after admisstion AGE(YR) OF PATIENT 33 cont'd STERIOD HISTORY . 25mg norgestrel + .05mg ethinyl estradiol for ten years OTHER P A T T E N T DATA A N D / O R CONCURRENT THERAPY investigation of l iver mass included a scan and hepatic angiography which were confirmatory C L I N I C A L P R E S E N T A T I O N mass in the left lobe of the l iver was diagnosed at tubal l igation D I A G N O S I S hepatic adenoma wi i focal tendency to transition to carcinoma but no definite carcinoma 0. lmg ethinyl estradiol + 25mg dimethisterone for 3 years; lmg nore-thindrone acetate + .05mg ethinyl estradiol for 5 years for investigation of l iver tumor a liver scan and hepatic arteriogram showed a vascular tumor of the right lobe of the l iver l iver tumor noted at tubal l igation hepatic adenoma with focal nodular hyperplasia norethindrone 2mg + mestranol O.lmg (Ortho-Novum 2mg) for 7 years in qood health until just before admission; hemo-globin value was 10.7g/ 100ml on admission and f e l l to 6.9g/100ml in 2 hours; the white blood ce l l count increased from 10,500 to 22,000/cu.mm during the same time period; the patient was taken to surgery for control of suspected intra-abdominal hemorrhage sudden onset of nausea, vomiting severe epigastric pain radiating to the right subcostal area and back; tenderness in the abdomen, especially in the epigastrium; suspected intra-abdominal hemorrhage benign hepatic tumor 28/ mestranol + ethynodiol diacetate for 11 months; the same combination was used intermittently for 10 months over a 3 year period and then discontinued to enable pregnancy gravida 4, para 2, abortus fullness or pressure in 2; during f i r s t pregnancy upper abdomenr absent patient noted some RUQ menses for 6 months and discomfort; after cessation i n f e r t i l i t y of o.c. therapy patient was unable to become pregnant for 2 years and presented with this complaint; physical examination showed a sizable nodular mass along the right gutter of the abdomen that extended from the right costal margin to the anterior, superior i l i a c spine; l iver scan showed a mass in the R midabdomen; ultrasonography showed the mass closely applied to the inferior aspect of the l iver R hepatic adenoma 36 34 type of o.c. unknown; therapy for 11 years; o.c.'s were not discontinued after the f i r s t admission after f i r s t admission patient went into shock and required blood trans-fusions; she remained in the intensive care unit for a week where i t was noted that she showed signs of continual intra-peritoneal bleeding along with progressive l iver enlargement; on second admission hepatic angi-ography indicated a 15 x 12cm mass in the right lobe and medial segment of the left lobe and a circumscribed 4cm mass in the left lobe of the l iver f i r s t admission was result on f i r s t of severe pain in RUQ admission radiating to the shoulder was focal brought on by a blow on nodular the back while skiing; 3 hyperplasia; years later presented with on second sudden onset of severe RUQ admission pain, which radiated to her was hepatic right shoulder, and hypo- adenoma with tensive shock necrosis and hemorrhage 163 ' GROSS PATHOLOGICAL FINDINGS HIS TO P AT H O LOG Y OUTCOME AND FOLLOW-UP at celiotomy, a pedunculated 10 x 15cm similar to above except focal dysplasia, the tumor was totally maBS that extended from the anterior hyperplasia, and features suggesting resected with a margin margin of the left lobe of the l iver focal low grade hepatocellular of normal parenchyma; was found carcinoma were noted, for example marked uneventful te^overy; thickening of hepatocellular plates in a 1 year follow-up showe<' glandular-like pattern was seen; no reoccurrence; o.c. variation between areas was notable therapy was dis -continued after admission at laparotomy a 3 x 4cm tumor was found in the right lobe of the l iver similar to above wedge resection of the tumor was carried out as was cholecystec tomy; o.c. therapy was discontinued after admission at laparotomy a 15-20cm tumor "was found in the central portion of the left lobe of the l iver extending into the right lobe; the site of hemorrhage was a 12-15cm tear in the anterior surface of the tumor the lesion was not resected and the proximal portion of the common hepatic artery was ligated as lifesaving procedure; uneventrul recovery and was discharged from hospital weeks postoperatively; doing well 6 months after survery; tumor was decreasing in size with discontinuation of o.c.'s at exploratory laparotomy a vascular tumor mass' was found; large tortuous venous channels were evident on the liver surface; the tumor adhered to the surface of the gallbladder and was seen to arise from the under-surface of the anterior edge of the right lobe of the l iver; the tumor weighed 650g and measured 15 x 10 x 7.5cm tumor was excised along with the ga l l -bladder; a bi lateral wedge resection of the ovaries was carried out also; patient was discharged on sixth post-operative day; 31 days after surgery the patient had the f i r s t menstrual period in 2 years, became pregnant 6 months later, and delivered via cesarian section at 37 weeks the f i r s t laparotomy showed a hepatic laceration that was secured and drained and a 4.5 x 4 x 3cm mass in the right lobe of the l iver which was removed; the laparotomy on second admission showed no blood in the peritoneal cavity; the l iver was enlarged and completely enveloped in scar tissue but definitive surgery was not carried out; in a subsequent operation surgical resection was carried out on the right lobe and medial segment of left lobe, but another mass was detected deep within the lateral part of the left lobe that was not removed uneventful recovery after f i r s t admission but remained on oral contraceptives; complete right lob-ectomy and part ia l left lobectomy on second admission; the one mass left behind regressed to 25% of original size 8 months after discharge and discontinuation of oral contraceptive therapy; patient has remained asymptomic 17 months post-operative JLV * f AGE(YR) OF PATIENT 37 STERIOD HISTORY type of o.c. unknown; IB month therapy OTHER PATIENT DATA AND/OR CONCURRENT THERAPY 4 days after admission serum bi l irubin and alka-line phosphatase increased; after laparotomy l iver scan showed a large defect in R lobe of l iver , selec-tive angiography of right hepatic artery showed hepatomegaly, avasular mass in right lobe, and early evidence of c ircu-latory stasis due to compression of the intra- . hepatic arteries CLINICAL PRESENTATION sudden onset of severe epigastric pain radiating to the right upper quad-rant and right shoulder; mass in RUQ; i n i t i a l diagnosis was acute cholecystitis DIAGNOSIS hepatic adenoma with secondary hemorrhage within the tumor 23 norethynodrel 2.5mg + mestranol O.lmg (Enovid-E) for 6 years 1964-1970; norethindrone + mestranol (Ortho-Novum) for 3 years (1970-1973) a l iver tumor was success-fully resected in 1967; o.c. therapy was not discontinued after resection; l iver scan on second admission showed tumor to be in the inferior aspect of the right lobe of the l iver; patient's older sister took o.c.'s and developed a l iver mass that regressed after discontinuing o.c. steriod therapy second admission (1970) recurrent was result of recurrence hepatic of tumor; i t was discovered adenoma on l iver scan for work-up of urinary tract infec-tion; no evidence of abdominal mass or tender-ness 39 ethynodiol diacetate + mestranol for 4 years angiography revealed a 12cm vascular mass in the inferior margin of the R lobe of the l iver RUQ abdominal mass pal - hepatic pated on routine physical adenoma examination 45 type and duration laboratory findings were hepatomegaly on routine hepatic of o.c. medication found to be normal; physical examination; adenoma unknown abdominal angiography l iver margin felt firm showed a massively enlarged below the right costal l iver; l iver scan showed margin an enlarged l iver with irregular areas of decreased activity involving H of the R lobe of the l iver 40 ethynodiol diacetate + mestranol (Ovulen) for 7 years gravida 1, para 1; elevated lactic dehydro-genase and serum glutamic oxaloacetic transaminase; other laboratory studies were negative (blood.work, cultures, and radiologic evaluation consisting of upper g . i . series, barium enema, IVP); intravenous antibiotic therapy for five days failed to reduce symptoms; on the sixth day a liver scan and right hepatic arteriogram suggested presence of a tumor; metronidazole was administered for the pos-s i b i l i t y of amebic l iver abscess; 14 days later, the patient developed signs of acute blood loss with the hematocrit lowering to 26% localized, sharp right subcostal pain; nausea and vomiting; shortness of breath; fever developed hepatic adenoma 165 GROSS PATHOLOGICAL FINDINGS at laparotomy the gall bladder and common bile duct were normal; hepa-tomegaly was noted and l iver biopsy was taken; at post-mortem a 12cm tumor was found in right lobe of l iver; tumor contained a large hemorrhagic area; the fatal embolus was seen in the pulmonary trunk and arteries HISTOPATHOLOGY OUTCOME AND FOLLOW-UP before surgery the patient died of cardio-respiratory arrest; death was due to a pulmonary embolus thought to have arisen in a thrombosed hepatic vein secondary to compression by tumor and hematoma at laparotomy a large orange-sized mass was found in the right lobe of the l iver well-circumscribed mass of l iver ce l l s ; normal hepatocytes were compressed at periphery of tumor; some hepatocytes were a brownish color probably due to presence of bi le; sinusoids were generally compressed; many thin-walled veins were identified; cel ls were vacuolated or clear presumably due to glycogen deposition; on electron microscopy, some mitochondria were seen to contain electron dense bodies the tumor (1970) was biopsied but not resected; o.c. medica-tion was discontued in 1973; l iver scan in 1976 showed complete regression of the tumor at laparotomy a 20cm tumor was found in the R lobe of the l iver and a biopsy was performed; surgical resection was carried out at a second operation; tumor was well demarcated but not encapsulated and compressed the surrounding normal hepatic parenchyma tumor was composed of cords of uniform cel ls with a greater than normal content of glycogen; cells were arranged in irregular trabeculae from 1-5 ce l l s thick and were separated by sinusoids lined by endothelium and Kupffer cel ls ; there were occasional foci of canalicular bi le stasis; no portal tracts or bi le ducts were present; size and density of venous channels in the tumor mass exceeded those in the surrounding l iver; some areas of the periphery contained large thick walled blood vessels that coursed circumferentially diagnosis was made at open l iver biopsy; at surgery the 20cm tumor mass in the right lobe of the l iver was seen to be hypervascular; the lesion caused compression of the surrounding l iver parenchyma; on dissection the tumor was seen to extend into the left lobe of the l iver; at postmortem the tumor was 5 x 6 x 4cm and was green in color; tumor was not encapsulated but seemed to be sharply demarcated from surrounding tissue similar to case above except for a more lobulated contour, increased glycogen content in tumor ce l l s , and the presence of scattered necrotic foci; some necrotic foci had undergone fibrosis while others showed peliosis hepatis-type formation (tumor ce l l dropout leaving blood f i l l ed spaces in continuity with sinusoids); no thick walled circumferential venous channels were present patient died in surgery due to hemorrhage emergency exploratory laparotomy revealed an extensive hemoperitoneum; a l iver mass was found in the anterior superior segment of the right lobe disorderly hyperplastic trabecular hepatic cords; dilated vascular sinusoidB; portal-to-portal bridging blood replacement during surgery was 4500ml; uneventful post-operative recovery; discharged 1 month after i n i t i a l admission 166 A G E (YR) OF P A T I E N T 40 cont * d STERIOD HISTORY ethynodiol diacetete + mestranol (Ovulen-21) continuously f o r 10 years OTHER P A T I E N T DATA A N D / O R CONCURRENT T H E R A P Y . gravida 9, para 7, abor-t i o n 2; chest x-ray showed thickening of pleura at the base of the r i g h t lung and p o s s i b l e c y s t - l i k e appearance i n r i g h t lobe of l i v e r ; laboratory t e s t s showed elevated serum a l k a l i n e phosphatase. elevated serum glutamic pyruvic transaminase, and elevated l a c t i c dehydrogenase; venogram showed throm-b o p h l e b i t i s , cholecysto-gram showed c h o l e l i t h i a s i s , and l i v e r scan revealed s i g n i f i c a n t l i v e r enlarge-ment and reduced uptake i n r i g h t lobe; s e l e c t i v e hepatic arteriogram revealed a large tumor mass i n the r i g h t lobe CLINICAL PRESENTATION RUQ abdominal pain; cough; chest pain, l e f t leg pain; edema DIAGNOSIS not p o s s i b l i because of extensive hemorrhage and r e c r o s i s norethynodrel + mestranol (Enovid) and n o r g e s t r e l + e t h i n y l e s t r a d i o l (Ovral) f o r 13 years gravida 2, para 2; upper g . i . s e r i e s , o r a l chole-cystogram, barium enema, and excretory urogram were normal; s l i g h t l y elevated serum a l k a l i n e phosphatase; p o s i t i v e l i v e r scan; s e l e c t i v e hepatic a r t e r i o -gram revealed three masses vague upper abdominal hepatic discomfort f o r 6 weeks; a adenoma 4 x 5cm mass i n RUQ was noted at t h i s time conjugated estogens (Premarin) 1.25mg d a i l y f o r 3 years f o r postmenopausal symptoms r e f e r r e d as a cancer p a t i e n t f o r actue myelo-c y t i c leukemia; there were no symptoms as c r i b a b l e to the hepatic nodule hepatic adenoma norethindrone + mestranol (Ortho-Novum) ; duration of therapy not stated during second admission, a hepatic angiogram was performed on admission f o r e l e c t i v e p l a s t i c surgery, a palp-able abdominal mass was detected; at follow-up 10 months l a t e r , another maBB was detected by pal p a t i o n i n RUQ hepatic adenoma norethindrone + mestranol f o r four years elevated white blood c e l l count, lowered hematocrit, and serum glutamic oxalo-a c e t i c transaminase and l a c t i c dehydrogenose were s l i g h t l y elevated; three weeks e a r l i e r , blood count was normal; echogram and hepatic arteriogram suggested an avascular c y s t i c mass i n the R lobe of the l i v e r ; negative t i t r e f o r amebae severe RUQ pain, lethargy, diagnosis of s l i g h t bloody d i a r r h e a , hepato-vomiting; h i s t o r y suggested c e l l u l a r a p o s s i b l e amebic abscess carcinoma was changed to hepatic adenoma Abbreviations R - r i g h t L - l e f t 0. c. - o r a l contraceptive RUQ - r i g h t upper quandrant e.m. - e l e c t r o n microscopic RLQ - r i g h t lower quadrant LUQ - L e f t upper quadrant' 1. p. - i n t r a p e r i t o n e a l D & C - d i l a t i o n and curettage G.N. - glomerulonephritis I-V.P. -= intravenous pyelogram g.i./G.I. - g a s t r o i n t e s t i n a l 167 GROSS PATHOLOGICAL FINDINGS exploratory laparotomy revealed a large, blood-fil led necrotic l iver mass HISTOPATHOLOGY OUTCOME AND FOLLOW-UP l iver tumor was excised; cholecyst-ectomy and e lective appendectomy were also carried out; right pleural effusion developed during recovery and was treated by thoracen-tesis; patient was discharged in satisfactory condition large uniform cel l s ; dilated vascular sinusoids segmental hepa-tectomy was carried out to remove one of the masses; the other 2 masses were biopsied; repeat angiogram 5 months after surgery showed no change in the size of the remaining masses at autopsy, a 3cm hepatic adenoma was found * death due to acute myelocytic leukemia f i r s t lesion was a large pedunculated mass in the left lobe of the l iver; 10 months later, 3 dist inct tumors in the right lobe of the l iver were noted to be present f i r s t mass was excised; the second operation was part ia l hepatec-tomy ; uncomplicated post-operative course; no recurrence at three years at exploratory laparotomy, 3 large cystic areas were present in the right lobe of the l iver; these areas contained bloody fluid o.c . 's were discon-tinued after surgery; 2 months later was asymptomatic: follow-up l iver scan and l iver function studies were normal but carcino-embryonic antigen was markedly elevated; patient refused further work-up 168 References for Table II-1. Baum, J.K., H o l t i , P., Bookstein, J . J . , and K l e i n , E.W.: Possible A s s o c i a t i o n Between Benign Hepatomas and Oral Contraceptives, Lancet, 2: 926-929, 1973. 2. ContostavloB, D.L.i Benign Hepatomas and Ora l Contraceptives, Lancet, 2: 1200, 1973. 3. Knapp, W.A., Ruebner, B.H.: Hepatomas and Oral Contraceptives, Lancet, 1: 270-271, 1974. 4. Kelso, D.R.: Benign Hepatomas and Oral Contraceptives, Lancet, 1: 315-316, 1974. 5. Horvath, E., Kovacs, K., Ross, R.C.: Benign Hepatoma i n a Young Woman on Contraceptive Steriods, Lancet. 1: 357-358, 1974. 6. Mays, E.F., Christopherson, H.M., Barrows, G.H.: Foc a l Nodular Hyperplasia of the L i v e r . Possible R e l a t i o n s h i p to Oral Contraceptives, Am. J . C l i n . Path, 61: 735-746, 1974. 7. Tountas, C., Paraskevas, G., D e l i g e o r g i , H.i Benign Hepatoma and Oral Contraceptives, Lancet, 1: 1351-1352, 1974. 8. O'Sullivan, J.P., and Wilding, R.P.: B r i t . Med. J , 3: 7, 1974. 9. Berg, J.W., Ketelaar, R.J., Rose, E.F., Vernon, R.G.: Hepatomas and O r a l Contraceptives, Lancet 2: 349-350, 1974. 10. Vosnides, G., Brander, W., O'Keeffe, B., Bewick, M., Ogg, C : Live r Hamartomas i n P a t i e n t s on Or a l Contraceptives, B r i t . Med. J . , 3: 580, 1974. 11. Grabowski, M., Stenrom, U. Berggvist, A.: Foc a l Nodular Hyperplasia of the L i v e r , Benign Hepatomas, Ora l Contraceptives and Other Drugs A f f e c t i n g the L i v e r , Acta Path. M i c r o b i o l . Scand. Sect. A, 83: 615-622, 1975. 12. Antoniades, K., Brooks, C.E.: Hemoperitoneum From L i v e r C e l l Adenoma i n a Pati e n t on Oral Contraceptives, Surgery, 77: 137-139, 1975. 13. 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Fechner, R.E.: Benign Hepatic Lesions and O r a l l y Administered Contraceptives. A Report of Seven Cases and a C r i t i c a l A n a l y s i s of the L i t e r a t u r e , Hum. Pathol., 8: 255-268, 1977. 31. Kent, D.R., Nissen, E.D., Nissen, S.E., Chambers, C : Maternal Death Resulting From Rupture of L i v e r Adenoma Associated w i t h l o r a l Contraceptives, Qbstet. Gynocol., 50 (Supplement): 5s-6s, 1977. 169 32. Mitchell , R . E . , Christ ie , L . G . , Camilo, M.: Hepatic Adenoma and Oral Contraceptive Therapy, Va. Med. 104: 251-254, Apr i l 1977. 33. Chan, C . K . , Detmer, D .E . : Proper Management of Hepatic Adenoma Associated with Oral Contra-ceptives, Surg. Gynec. Obstet. 144: 703-706, May 1977. 34. Turney, W.H., Shipp, R . F . , Bellegie, N . J . : Benign Liver Tumors in patients Taking Oral Contra-ceptives, Tex. Med. 73: 75-80, May 1977. 35. Keifer, U.S . , and Scott, J . C : Liver Neoplasms and the Oral Contraceptives, Am. J . Obstet.  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