Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Nuclear localization and induction of rat hepatic drug metabolizing enzymes Gontovnick, Larry Stuart 1981

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1981_A1 G66.pdf [ 8.55MB ]
Metadata
JSON: 831-1.0095513.json
JSON-LD: 831-1.0095513-ld.json
RDF/XML (Pretty): 831-1.0095513-rdf.xml
RDF/JSON: 831-1.0095513-rdf.json
Turtle: 831-1.0095513-turtle.txt
N-Triples: 831-1.0095513-rdf-ntriples.txt
Original Record: 831-1.0095513-source.json
Full Text
831-1.0095513-fulltext.txt
Citation
831-1.0095513.ris

Full Text

NUCLEAR LOCALIZATION AND INDUCTION OF RAT HEPATIC DRUG METABOLIZING ENZYMES by LARRY STUART GONTOVNICK B . S c , M c G i l l U n i v e r s i t y , 1975 M . S c , U n i v e r s i t y o f B r i t i s h Columbia, 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES ( D i v i s i o n o f Pharmacology and T o x i c o l o g y o f the F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s ) We a c c e p t t h i s t h e s i s as conforming t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1981 © L a r r y S t u a r t G o n t o v n i c k , 1981 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a gree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Qv\f\^ccN^e^v^\C^V. ^ C A ^ V ^ C ^ The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 D a t e ^vxv>-^ v ^ 9 \ - i i -ABSTRACT The n u c l e u s may be the c r i t i c a l s i t e f o r the a c t i v a t i o n o f c h e m i c a l c a r c i n o g e n s , and s u b s e q u e n t l y the i n i t i a t i o n o f n e o p l a s i a . However, i s o l a t e d n u c l e i may be c o n t a m i n a t e d w i t h e n d o p l a s m i c r e t i c u l u m , the major s i t e o f the drug m e t a b o l i z i n g enzymes. One o f the o b j e c t i v e s o f t h e p r e s e n t study was t o determine whether the enzymes i n i s o l a t e d r a t h e p a t i c n u c l e i were o f n u c l e a r o r i g i n and, i f so, to compare t h e s e enzymes w i t h t h o s e i n the microsomal f r a c t i o n . The s e l e c t i v e m a n i p u l a t i o n o f n u c l e a r enzymes would be a u s e f u l t o o l i n d e t e r m i n i n g t h e i r r o l e i n c e l l u l a r t o x i c i t y . R e c e n t r i f u g a t i o n e x p e r i m e n t s , w i t h a r y l h ydrocarbon h y d r o x y l a s e (AHH) a c t i v i t y as a marker, showed t h a t i s o l a t e d n u c l e i were n o t c o n t a m i n a t e d w i t h e n d o p l a s m i c r e t i c u l u m i n the form o f microsomes formed upon ho m o g e n i z a t i o n . However, small " t a g s " o f e n d o p l a s m i c r e t i c u l u m , c o n t i n u o u s w i t h the n u c l e a r membrane, and i n d i s c e r n a b l e i n e l e c t r o n m i c r o g r a p h s , c o u l d remain f o l l o w i n g c e n t r i f u g a t i o n and a c c o u n t f o r a l l o f the measurable enzyme a c t i v i t y i n the i s o l a t e d n u c l e i . I t was reasoned t h a t i f e n d o p l a s m i c r e t i c u l u m a c c o u n t e d f o r a l l o f the a c t i v i t y , then the r a t i o o f n u c l e a r to microsomal a c t i v i t y f o r a l l enzymes de t e r m i n e d s h o u l d be the same. The r a t i o s o f ep o x i d e h y d r o l a s e and AHH were found t o d i f f e r i n the two f r a c t i o n s . The s i m p l e s t i n t e r p r e t a t i o n o f t h e s e d a t a was t h a t drug m e t a b o l i z i n g enzymes e x i s t e d i n the n u c l e i . However., the d i s t r i b u t i o n o f drug m e t a b o l i z i n g enzymes t h r o u g h o u t the e n d o p l a s m i c r e t i c u l u m i s known t o be heterogeneous and t h e s e " t a g s " c o u l d d i f f e r from the t o t a l e n d o p l a s m i c r e t i c u l u m (microsomes) i n t h e i r enzyme make-up. Whether t h e s e enzymes a r e i n the n u c l e a r membrane, n u c l e o p l a s m , o r as " t a g s " o f e n d o p l a s m i c r e t i c u l u m , they r e p r e s e n t a c t i v i t i e s i n c l o s e p r o x i m i t y t o p o t e n t i a l t a r g e t s i t e s i n the n u c l e u s . The i n h i b i t i o n , i n d u c t i o n , and a c t i v a t i o n c h a r a c t e r i s t i c s o f n u c l e a r and microsomal enzymes were s t u d i e d w i t h - i i i -t h e goal o f s e l e c t i v e m a n i p u l a t i o n o f n u c l e a r enzymes. The enzymes i n the n u c l e a r and microsomal f r a c t i o n s were found t o d i f f e r ' o n l y i n q u a n t i t a t i v e i n d u c i b i l i t y , and were i d e n t i c a l i n a l l o t h e r r e s p e c t s . T h e r e f o r e , t h e s e l e c t i v e m a n i p u l a t i o n o f n u c l e a r enzymes was not a c h i e v e d . The i n d u c t i o n o f h e p a t i c drug m e t a b o l i z i n g enzymes i s a measure o f a l t e r e d g e n e t i c e x p r e s s i o n i n the l i v e r . Inducers o f drug m e t a b o l i z i n g enzymes have a l s o been shown t o promote n e o p l a s i a i n t h e l i v e r . T h e r e f o r e , s t u d y i n g t he i n d u c t i o n o f such enzymes may l e a d t o a f u r t h e r u n d e r s t a n d i n g o f the mechanism o f tumour promotion. P h e n o b a r b i t a l , 3 - m e t h y l c h o l a n t h r e n e and p r e g n e n o l o n e - 1 6 * - c a r b o n i t r i l e produce t h r e e d i s t i n c t i n d u c t i o n r e s p o n s e s . In the p r e s e n t s t u d y , s p i r o n o l a c t o n e and t r a n s - s t i I b e n e o x i d e were shown t o produce d i s t i n c t i n d u c t i o n r e s p o n s e s , a l s o . S p i r o n o l a c t o n e was shown t o be a d i f f e r e n t i n d u c e r based on the p r o t e i n band p a t t e r n s o b s e r v e d f o l l o w i n g SDS-p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f l i v e r microsomes. t r a n s - S t i l b e n e o x i d e was found t o produce a s i g n i f i c a n t l y d i f f e r e n t maximal l e v e l o f AHH a c t i v i t y . The o b s e r v a t i o n o f f i v e d i s t i n c t i n d u c t i o n r e s p o n s e s s u g g e s t s a t l e a s t f i v e r e c o g n i t i o n s i t e s ( r e c e p t o r s ) m e d i a t i n g t he p l e i o t r o p i c a c t i o n s o f exogenous compounds i n the l i v e r . - i v -TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i i i LIST OF FIGURES x i ACKNOWLEDGEMENTS ... XV INTRODUCTION 1 I. Chemical C a r c i n o g e n e s i s 1 1) H i s t o r i c a l A s p e c t s and Ep i d e m i o l o g y 1 2) St a g e s of I n i t i a t i o n and Promotion 3 I I . Drug M e t a b o l i z i n g Enzymes and I n i t i a t i o n o f N e o p l a s i a 5 I I I . N u c l e a r Drug M e t a b o l i z i n g Enzymes 7 IV. Promotion o f H e p a t o c a r c i n o g e n e s i s 10 V. H e p a t i c Enzyme I n d u c t i o n 12 1) R e l a t i o n s h i p t o Promotion o f H e p a t o c a r c i n o g e n e s i s 12 2) C l a s s e s o f Enzyme Inducers 13 3) t r a n s - S t i l b e n e Oxide and S p i r o n o l a c t o n e 14 4) Mechanisms o f Enzyme I n d u c t i o n 16 VI. S p e c i f i c Aims I 8 MATERIALS AND METHODS 1 9 I. Chemicals 1 9 I I . Animals 19 I I I . Treatments 20 IV. P r e p a r a t i o n o f S u b c e l l u l a r F r a c t i o n s 20 1) L i v e r P r e p a r a t i o n 20 2) Microsomal F r a c t i o n 20 3) N u c l e a r F r a c t i o n 21 -v-V. Enzyme Assays 21 1) D e t e r m i n a t i o n of AHH A c t i v i t y 21 2) D e t e r m i n a t i o n o f Epoxide H y d r o l a s e A c t i v i t y 23 3) D e t e r m i n a t i o n of N-Demethylase A c t i v i t i e s 25 4) D e t e r m i n a t i o n o f A n i l i n e H y d r o x y l a s e A c t i v i t y 26 5) D e t e r m i n a t i o n o f T e s t o s t e r o n e z ^ - H y d r o g e n a s e A c t i v i t y 27 6) D e t e r m i n a t i o n o f E t h o x y r e s o r u f i n O-Deethylase A c t i v i t y 28 7) Assay C o n d i t i o n s 29 VI. Sodium Dodecyl S u l f a t e - P o l y a c r y l a m i d e Gel E l e c t r o p h o r e s i s 29 V I I . D e t e r m i n a t i o n o f P r o t e i n s 30 V I I I . S t a t i s t i c a l A n a l y s e s 30 RESULTS 32 I. N u c l e a r L o c a l i z a t i o n and Enzymatic P r o f i l e of Rat H e p a t i c Drug M e t a b o l i z i n g Enzymes 32 1) E l e c t r o n M i c r o g r a p h s and R e c e n t r i f u g a t i o n o f H e p a t i c N u c l e i 32 2) Basal Enzyme L e v e l s : A comparison o f N u c l e i and Microsomes 36 3) AHH A c t i v i t y , Apparent Km and Vmax 36 4) Sex and Age Dependent Enzyme A c t i v i t i e s 41 5) I n h i b i t i o n and A c t i v a t i o n of AHH a c t i v i t y 41 6) A l t e r a t i o n s i n H e p a t i c AHH A c t i v i t y F o l l o w i n g Chemical A d m i n i s t r a t i o n 50 7) I n h i b i t i o n and I n d u c t i o n o f Epoxide H y d r o l a s e A c t i v i t y 54 I I . I n d u c t i o n o f Rat H e p a t i c Drug M e t a b o l i z i n g Enzymes 54 1) The P h e n o b a r b i t a l Response 54 2) The t r a n s - S t i l b e n e Oxide Response 63 3) S t u d i e s with A c e t y l m e t h a d o l 72 - v i -4) The S p i r o n o l a c t o n e Response 7 8 5) The 3 - M e t h y l c h o l a n t h r e n e Response 7 8 6) The P r e g n e n o l o n e - 1 6 o i - C a r b o n i t r i l e Response 86 7) In V i t r o I n h i b i t i o n of H e p a t i c Microsomal AHH A c t i v i t y by SKF 525-A 8 6 8) N u c l e a r Versus Microsomal I n d u c t i o n 94 9) Dose- and Time- E f f e c t S t u d i e s 9 4 10) A d d i t i o n Experiments ^ 10a. P h e n o b a r b i t a l and S p i r o n o l a c t o n e 1 0 5 10b. P h e n o b a r b i t a l and t r a n s - S t i l b e n e Oxide 1 0 8 10c. P h e n o b a r b i t a l and P r e g n e n o l o n e - 1 6 o ( - C a r b o n i t r i l e 113 l O d . Pregnenolone-16 - C a r b o n i t r i l e and 3 - M e t h y l c h o l a n t h r e n e H 8 11) . Sodium Dodecyl S u l f a t e - P o l y a c r y l a m i d e Gel E l e c t r o p h o r e s i s H 8 DISCUSSION 1 3 1 I. N u c l e a r L o c a l i z a t i o n o f Rat H e p a t i c Drug M e t a b o l i z i n g Enzymes 131 1) The Q u e s t i o n o f Microsomal C o n t a m i n a t i o n 131 2) A Comparison o f N u c l e a r and Microsomal Drug M e t a b o l i z i n g Enzymes 134 I I . The I n d u c t i o n of Rat H e p a t i c Drug M e t a b o l i z i n g Enzymes 1 3 8 1) Sex and Age Dependence o f the I n d u c t i o n of Rat H e p a t i c Microsomal Drug M e t a b o l i z i n g Enzymes 1 3 8 2) Q u a l i t a t i v e Comparison o f the Biochemical Responses Produced by P h e n o b a r b i t a l , t r a n s -S t i l b e n e O x i d e , S p i r o n o l a c t o n e , 3-Methyl-c h o l a n t h r e n e and Pregnenolone-16<*-Carbonitrile 145 - v i i -3) A More D e t a i l e d A n a l y s i s o f the P l e i o t r o p i c Responses E l i c i t e d by t h e s e Agents 146 4) R e l a t i v e C o n t r i b u t i o n o f D i f f e r e n t Forms of Cytochrome P-450 t o t h e O x i d a t i o n of S u b s t r a t e s 154 5) I n d u c t i o n Mechanism 156 SUMMARY 158 REFERENCES 161 - v i i i -LIST OF TABLES T a b l e Page I. R e c e n t r i f l i g a t i o n experiments 35 I I . Basal enzyme a c t i v i t i e s ( p e r mg p r o t e i n ) i n h e p a t i c n u c l e i and microsomes 37 I I I . Basal enzyme a c t i v i t i e s (per gram l i v e r ) i n h e p a t i c n u c l e i and microsomes 38 IV. Apparent Km and Vmax v a l u e s f o r h e p a t i c microsomal and n u c l e a r a r y l hydrocarbon h y d r o x y l a s e a c t i v i t y 44 V. The sex and age dependency of h e p a t i c a r y l hydrocarbon h y d r o x y l a s e a c t i v i t y 45 V I . H e p a t i c t e s t o s t e r o n e h y d r o g e n a s e a c t i v i t y i n a d u l t male and female r a t s 46 V I I . The e f f e c t s o f < x - n a p h t h o f l a v o n e i n v i t r o on h e p a t i c a r y l hydrocarbon h y d r o x y l a s e a c t i v i t y 49 V I I I . The e f f e c t s o f p h e n o b a r b i t a l t r e a t m e n t on h e p a t i c a r y l hydrocarbon h y d r o x y l a s e a c t i v i t y 53 IX. The e f f e c t s o f 3 - m e t h y l c h o l a n t h r e n e treatment on h e p a t i c a r y l hydrocarbon h y d r o x y l a s e a c t i v i t y 57 X. The e f f e c t s o f a c e t y l m e t h a d o l , p h e n o b a r b i t a l and t r a n s -s t i l b e n e o x i d e on h e p a t i c epoxide h y d r o l a s e a c t i v i t y i n a d u l t male r a t s 60 XI. The e f f e c t s of p h e n o b a r b i t a l t r e a t m e n t (80 mg/kg/day f o r 4 d a y s , i p ) on h e p a t i c microsomal a r y l hydrocarbon h y d r o x y l a s e (AHH) a c t i v i t y 64 X I I . The e f f e c t s of p h e n o b a r b i t a l t r e a t m e n t (80 mg/kg/day f o r 4 days, i p ) on l i v e r weight and h e p a t i c microsomal enzyme a c t i v i t i e s i n a d u l t female r a t s 69 - i x -X I I I . The e f f e c t s of t r a n s - s t i l b e n e o x i d e ( t - s o ) t r e a t m e n t (3 c o n s e c u t i v e days, i p ) on h e p a t i c microsomal a r y l h ydrocarbon h y d r o x y l a s e a c t i v i t y 73 XIV. The e f f e c t s o f t r a n s - s t i 1 bene o x i d e ( t - s o ) treatment (3 c o n s e c u t i v e days, i p ) on h e p a t i c microsomal a m i n o p y r i n e N-demethylase a c t i v i t y i n a d u l t male and a d u l t female r a t s 74 XV. The e f f e c t s of t r a n s - s t i l b e n e o xide treatment (200 mg/kg/day f o r 4 da y s , i p ) on l i v e r weight and h e p a t i c microsomal enzyme a c t i v i t i e s i n a d u l t female r a t s 75 XVI. The e f f e c t s of s p i r o n o l a c t o n e t r e a t m e n t (100 mg/kg/day f o r 4 c o n s e c u t i v e days, i p ) on the a c t i v i t i e s o f some h e p a t i c microsomal enzymes i n the a d u l t male r a t 79 XVII. The e f f e c t s o f s p i r o n o l a c t o n e t r e a t m e n t (100 mg/kg/day f o r 4 days, i p ) on l i v e r weight and h e p a t i c microsomal enzyme a c t i v i t i e s i n a d u l t female r a t s 80. X V I I I . The e f f e c t s o f 3 - m e t h y l c h o l a n t h r e n e t r e a t m e n t (20 mg/ kg/day f o r 4 da y s , i p ) on l i v e r weight and h e p a t i c microsomal enzyme a c t i v i t i e s i n a d u l t female r a t s 85 XIX. The e f f e c t s of p r e g n e n o l o n e - 1 6 o ( - c a r b o n i t r i l e a d m i n i s t r a t i o n (40 mg/kg/day f o r 4 days, i p ) on h e p a t i c microsomal a r y l hydrocarbon h y d r o x y l a s e and am i n o p y r i n e N-demethylase a c t i v i t i e s i n a d u l t male r a t s 87 XX. The e f f e c t s o f pregnenolone-16<X-carbonitri1e t r e a t m e n t (40 mg/kg/day f o r 4 days, i p ) on c e r t a i n h e p a t i c microsomal enzyme a c t i v i t i e s and l i v e r weight i n a d u l t female r a t s 88 -X-XXI. The age and sex dependency of h e p a t i c microsomal a r y l hydrocarbon h y d r o x y l a s e a c t i v i t y and c a l c u l a t e d I C 5 0 v a l u e s 92 XXII. The b a s a l a r y l hydrocarbon h y d r o x y l a s e a c t i v i t y and the c a l c u l a t e d IC50 v a l u e s f o r SKF 525-A i n h e p a t i c microsomes prepared from a d u l t p s e u d o h e r m a p h r o d i t i c (pseudo) r a t s and t h e i r male and female l i t t e r m a t e s (King-Holtzman) 93 X X I I I . Microsomal v e r s u s n u c l e a r i n d u c t i o n o f a r y l hydrocarbon h y d r o x y l a s e (AHH) a c t i v i t y 99 - x i -L I S T OF FIGURES F i g u r e Page 1. E l e c t r o n micrographs of i s o l a t e d r a t l i v e r n u c l e i 34 2. The Michael i s c u r v e s o f enzyme v e l o c i t y (Vo) versus s u b s t r a t e c o n c e n t r a t i o n ( [ A o ] ) f o r h e p a t i c n u c l e a r and microsomal a r y l hydrocarbon h y d r o x y l a s e a c t i v i t y 40 3. The H o f s t e e p l o t of enzyme v e l o c i t y (Vo) v e r s u s enzyme v e l o c i t y over s u b s t r a t e c o n c e n t r a t i o n (Vo/[Ao]) f o r h e p a t i c n u c l e a r and microsomal a r y l h ydrocarbon h y d r o x y l a s e a c t i v i t y 43 4. I n h i b i t i o n o f r a t h e p a t i c a r y l hydrocarbon h y d r o x y l a s e (AHH) a c t i v i t y by SKF 525-A i n v i t r o 48 5. The e f f e c t s of t r a n s - s t i l b e n e o x i d e on h e p a t i c a r y l h ydrocarbon h y d r o x y l a s e (AHH) a c t i v i t y 52 6. The t i m e - c o u r s e o f a r y l hydrocarbon h y d r o x y l a s e (AHH) i n d u c t i o n a f t e r p h e n o b a r b i t a l (Pb) t r e a t m e n t 56 7. I n h i b i t i o n of r a t h e p a t i c e p o x i d e h y d r o l a s e by l , 2 - e p o x y - 3 , 3 , 3 - t r i c h l o r o p r o p a n e jjn v j j t r o 59 8. The e f f e c t s of p h e n o b a r b i t a l (Pb) t r e a t m e n t (80 mg/ kg/day f o r 4 d a y s , i p ) on h e p a t i c microsomal a r y l h ydrocarbon h y d r o x y l a s e (AHH) a c t i v i t y i n immature male, a d u l t male and a d u l t female r a t s 62 9. The e f f e c t s of p h e n o b a r b i t a l (Pb) t r e a t m e n t (80 mg/ kg/day f o r 4 d a y s , i p ) on h e p a t i c microsomal a m i n o p y r i n e N-demethylase a c t i v i t y i n immature male, a d u l t male and a d u l t female r a t s 66 10. The e f f e c t s of p h e n o b a r b i t a l (Pb) t r e a t m e n t (80 mg/kg/day f o r 4 d a y s , i p ) on h e p a t i c microsomal e p o x i d e h y d r o l a s e a c t i v i t y i n immature male, - x i i -a d u l t male and a d u l t female r a t s 6 8 11. The e f f e c t s of t r a n s - s t i l b e n e o x i d e ( t - s o ) t r e a t m e n t (3 c o n s e c u t i v e days, i p ) on h e p a t i c microsomal a r y l h y drocarbon h y d r o x y l a s e (AHH) and epoxide h y d r o l a s e (EH) a c t i v i t i e s i n the r a t 71 12. The e f f e c t s of a c e t y l m e t h a d o l (LAAM) tre a t m e n t ( i n the d r i n k i n g water f o r 2 weeks) on h e p a t i c microsomal a r y l hydrocarbon h y d r o x y l a s e (AHH) and epoxide h y d r o l a s e (EH) a c t i v i t i e s i n a d u l t male and a d u l t female r a t s 77 13. The e f f e c t s o f 3 - m e t h y l c h o l a n t h r e n e (3-MC) treatment (20 mg/kg/day f o r 2 c o n s e c u t i v e days, i p ) on h e p a t i c microsomal a r y l hydrocarbon h y d r o x y l a s e (AHH) a c t i v i t y 82 14. The e f f e c t s o f 3 - m e t h y l c h o l a n t h r e n e (3-MC) tre a t m e n t (20 mg/kg/day f o r 2 days, i p ) on h e p a t i c microsomal e p o x i d e h y d r o l a s e (EH) and aminopyrine N-demethylase (APY) a c t i v i t i e s 8 4 15. I n h i b i t i o n o f a d u l t male and female r a t h e p a t i c microsomal a r y l hydrocarbon h y d r o x y l a s e (AHH) a c t i v i t y by SKF 525-A 9 0. 16. I n h i b i t i o n , by SKF 525-A, of a r y l hydrocarbon h y d r o x y l a s e (AHH) a c t i v i t y , i n h e p a t i c microsomes from p h e n o b a r b i t a l -and 3 - m e t h y l c h o l a n t h r e n e - t r e a t e d a d u l t female r a t s 96 17. The s e n s i t i v i t y of r a t h e p a t i c microsomal a r y l hydrocarbon h y d r o x y l a s e a c t i v i t y to the i n h i b i t o r y e f f e c t s of SKF 525-A i n v i t r o ^8 18. The t i m e - c o u r s e of the p h e n o b a r b i t a l response 102 19. A d o s e - r e s p o n s e study of the i n c r e a s e s i n a r y l h y drocarbon h y d r o x y l a s e a c t i v i t y , e p o x i d e h y d r o l a s e a c t i v i t y and l i v e r weight by p h e n o b a r b i t a l ( P b ) , -Xi11 -t r a n s - s t i l b e n e o x i d e ( t - s o ) and pregnenolone-1 6 < X - c a r b o n i t r i l e (PCN) 104 20. The e f f e c t s o f p h e n o b a r b i t a l (Pb) and s p i r o n o l a c t o n e ( s ) a l o n e or i n comb i n a t i o n on h e p a t i c microsomal cytochrome P-450 dependent monooxygenase a c t i v i t i e s 107 21. The e f f e c t s of p h e n o b a r b i t a l (Pb) and s p i r o n o l a c t o n e (s) a l o n e or i n comb i n a t i o n on h e p a t i c microsomal e p o x i d e h y d r o l a s e a c t i v i t y 110 22. The e f f e c t s o f p h e n o b a r b i t a l (Pb) and t r a n s - s t i l b e n e o x i d e ( t - s o ) a l o n e or i n comb i n a t i o n on h e p a t i c microsomal enzyme a c t i v i t i e s 112 23. The e f f e c t s of p h e n o b a r b i t a l (Pb) and pregnenolone-16«c-carbonitrile (PCN) alone or i n combination on h e p a t i c microsomal cytochrome P-450 dependent monooxygenase a c t i v i t i e s 115 24. The e f f e c t s of p h e n o b a r b i t a l (Pb) and pregnenolone-16<<-carbonitrile (PCN) al o n e or i n combination on h e p a t i c microsomal epoxide h y d r o l a s e a c t i v i t y 117 25. The e f f e c t s o f pregnenolone-16 - c a r b o n i t r i l e (PCN) and 3 - m e t h y l c h o l a n t h r e n e (3-MC) al o n e or i n combination on h e p a t i c microsomal cytochrome P-450 dependent monooxygenase a c t i v i t i e s 120 26. S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of l i v e r microsomes from a d u l t female r a t s t r e a t e d f o r 4 days with e i t h e r p h e n o b a r b i t a l (Pb, 80 mg/kg/day), t r a n s -s t i l b e n e o x i d e ( t - s o , 200 mg/kg/day), s p i r o n o l a c t o n e (SPIR, 200 mg/kg/day) or 3 - m e t h y l c h o l a n t h r e n e (3-MC, 20 mg/kg/day) 122 27. S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f l i v e r - X I v-microsomes from a d u l t female r a t s t r e a t e d f o r 4 days w i t h e i t h e r p h e n o b a r b i t a l (Pb, 80 mg/kg/day), t r a n s - s t i l b e n e o x i d e ( t - s o , 200 mg/kg/day), or 3 - m e t h y l c h o l a n t h r e n e (3-MC, 20 mg/kg/day) 125 28. S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of l i v e r microsomes from a d u l t female r a t s t r e a t e d f o r 7 days with e i t h e r p h e n o b a r b i t a l (Pb, 80 mg/kg/day), t r a n s - s t i l b e n e o x i d e ( t - s o , 200 mg/kg/day), s p i r o n o l a c t o n e (SPIR, 200 mg/kg/day) or 3-m e t h y l c h o l a n t h r e n e (3-MC, 20 mg/kg/day) 127 29. S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of l i v e r microsomes from a d u l t female r a t s t r e a t e d f o r 7 days with e i t h e r p h e n o b a r b i t a l (Pb, 80 mg/kg/day), p r e g n e n o l o n e - 1 6 c ( - c a r b o n i t r i l e (PCN, 40 mg/kg/day) or 3 - m e t h y l c h o l a n t h r e n e (3-MC, 20 mg/kg/day) 130 -XV-ACKNOWLEDGEMENTS I wish t o e x p r e s s my g r a t i t u d e t o Dr. G a i l B e l l w a r d f o r the e x c e l l e n t t r a i n i n g she has p r o v i d e d me i n my y e a r s as a graduate s t u d e n t . Her encouragement and a d v i c e as a t e a c h e r and a f r i e n d was a c o n s t a n t s o u r c e o f i n s p i r a t i o n . I wish t o thank Drs. Diamond, M c N e i l l , R o u f o g a l i s , and Wright f o r t h e i r c o n s t r u c t i v e c r i t i c i s m o f my r e s e a r c h and academic endeavors. I am d e e p l y a p p r e c i a t i v e o f the c o n s t a n t l o v e and s u p p o r t g i v e n t o me by my mother, f a t h e r , b r o t h e r s , and w i f e . T h e i r f a i t h i n me w i l l h e l p me t o s t r i v e f o r and a c h i e v e g r e a t e r accomplishments i n my l i f e . -1-INTRODUCTION I. Chemical C a r c i n o g e n e s i s 1) H i s t o r i c a l A s p e c t s and E p i d e m i o l o g y Chemical c a r c i n o g e n e s i s r e f e r s t o the i n d u c t i o n o f n e o p l a s i a by c h e m i c a l s p r e s e n t i n the environment. The f i r s t a s s o c i a t i o n between an e n v i r o n m e n t a l f a c t o r and c a n c e r was c r e d i t e d to the B r i t i s h p h y s i c i a n John H i l l , who r e p o r t e d i n 1761 t h a t the development o f n a s a l c a n c e r was a consequence o f e x c e s s i v e use o f t o b a c c o s n u f f (Redmond J r . 1970). P e r c i v a l P o t t , i n 1775, r e p o r t e d an u n u s u a l l y h i g h i n c i d e n c e o f c a n c e r of the s k i n o f the scrotum among men who were chimney sweeps i n t h e i r y o u t h . He suggested t h a t t h e s e "soot w a r t s " were due t o the o c c u p a t i o n a l exposure to c o a l t a r and s o o t ( P o t t , 1963). The Danish chimney sweeper's g u i l d , based on P o t t ' s o b s e r v a t i o n s , urged i t s members t o f o l l o w c e r t a i n p r o t e c t i v e measures (Clemmesen, 1951). About a c e n t u r y l a t e r , B u t l i n (1892) r e p o r t e d t h a t the low i n c i d e n c e of s c r o t a l c a n c e r among the chimney sweeps i n n o r t h e r n Europe was r e l a t e d to t h e b e t t e r p e r s o n a l hygiene and p r o t e c t i v e c l o t h i n g of t h i s group as compared to E n g l i s h chimney sweeps with a h i g h i n c i d e n c e o f t h i s c a n c e r . Thus began t h e s t u d y of o c c u p a t i o n a l and environmental c a r c i n o g e n e s i s . The development of s k i n c a n c e r i n c e r t a i n workers was shown by von Volkmann i n Germany i n 1875 and by B e l l i n S c o t l a n d i n 1876 t o be a s s o c i a t e d with c o n t a c t of the s k i n with t a r and p a r a f f i n o i l s t h a t , as known now, c o n t a i n e d p o l y c y c l i c a r o m a t i c h y d r o c a r b o n s (Haddow and Kon, 1947). A h i g h i n c i d e n c e o f b l a d d e r c a n c e r i n workers i n the s y n t h e t i c dye i n d u s t r y was r e p o r t e d by Rehn (1895). T h i s was f o l l o w e d by s e v e r a l o b s e r v a t i o n s i n many c o u n t r i e s on the a s s o c i a t i o n between human b l a d d e r c a n c e r and o c c u p a t i o n s t h a t r e s u l t e d i n r e l a t i v e l y l a r g e e xposure to a r o m a t i c amines ( C l a y s o n , 1962). These r e p o r t s on t h e a s s o c i a t i o n between human c a n c e r and chemical expo-s u r e s t i m u l a t e d attempts t o induce tumours i n l a b o r a t o r y animals with the -2-i m p l i c a t e d s u b s t a n c e s . Yamagiwa and Ichikawa (1915) s u c c e s s f u l l y induced carcinomas on the e a r s of r a b b i t s by r e p e a t e d t o p i c a l a p p l i c a t i o n s of coal t a r f o r l o n g p e r i o d s . T s u t s u i (1918) showed t h a t t a r s were a l s o c a r c i n o g e n i c f o r mouse s k i n . Subsequent i n v e s t i g a t i o n s l e d to the d i s c o v e r y of the a c t i v e agents i n t a r s , s o o t s and o i l s . It was suggested t h a t p o l y c y c l i c a r o m a t i c hydrocarbons were the a c t i v e m a t e r i a l s (Haddow and Kon, 1947). H i e g e r (1930) showed t h a t the f l u o r e s c e n c e s p e c t r a o f p r o d u c t s from the t a r s and o f s y n t h e t i c b e n z ( a ) a n t h r a c e n e d e r i v a t i v e s were a l i k e . The a c t i v i t y of t h i s f i r s t s y n t h e t i c c a r c i n o g e n was demonstrated by Kennaway and H i e g e r (1930). A c a r c i n o g e n i c hydrocarbon i s o l a t e d from c o a l t a r was i d e n t i f i e d by Cook, Hewett and H i e g e r (1933) as 3,4-benzpyrene, now c a l l e d b e n z o ( a ) p y r e n e . Yoshida (1933) showed t h a t a d m i n i s t r a t i o n of a d e r i v a t i v e of s c a r l e t r e d , 2',3-dimethyl-4-aminoazobenzene, induced l i v e r tumours i n r a t s and mice. Hueper, Wiley and Wolfe (1938) demonstrated t h e i n d u c t i o n of u r i n a r y b l a d d e r c a n c e r i n dogs fed 2-naphthylamine. The c u r r e n t l i s t o f chemical c a r c i n o g e n s spans many c l a s s e s of o r g a n i c and some i n o r g a n i c compounds. These i n c l u d e s y n t h e t i c c h e m i c a l s , n a t u r a l p r o d u c t s d e r i v e d from p l a n t s and m i c r o r g a n i s m s , and both endogenous and s y n t h e t i c hormones ( M i l l e r , 1978). In Canada, a p p r o x i m a t e l y one death i n f o u r i s caused by c a n c e r ( S t a t i s -t i c s Canada, 1978). The i n c r i m i n a t i o n of c h e m i c a l s i n the environment as a major cause o f human c a n c e r i s a r e s u l t o f s t u d i e s i n the a r e a s of e p i d e m i -o l o g y and g e o g r a p h i c p a t h o l o g y . The o v e r a l l c o n c l u s i o n from t h e s e s t u d i e s i s t h a t 70 t o 90% o f human c a n c e r s are e n v i r o n m e n t a l l y caused ( H i g g i n s o n , 1972; D o l l and V o d o p i j a , 1973; H i g g i n s o n and M u i r , 1973). A study o f Japanese immigrants to the U.S.A. was i n d i c a t i v e o f the importance of environmental f a c t o r s as the cause of c a n c e r (Haenszel and K u r i h a r a , 1968). In Japan, t h e r e i s a very high i n c i d e n c e o f stomach c a n c e r and a low i n c i d e n c e o f i n t e s t i n a l c a n c e r . The r e v e r s e i s t r u e i n t h e U.S.A. It was found t h a t i n the o f f s p r i n g - 3 -o f Japanese immigrants the i n c i d e n c e s o f t h e s e two c a n c e r s were i n t e r m e d i a t e between the i n c i d e n c e s i n the two c o u n t r i e s . In the second g e n e r a t i o n immi-g r a n t s the i n c i d e n c e s were e s s e n t i a l l y the same as i n the U.S.A. T h i s demon-s t r a t e d t h a t f o r stomach and i n t e s t i n a l c a n c e r s the major c a u s a t i v e f a c t o r was env i r o n m e n t a l r a t h e r than g e n e t i c . Over two dozen c h e m i c a l s have been i d e n t i -f i e d as causes of some human c a n c e r (Tomatis e t aj_, 1978). Emphasis has been p l a c e d on e n v i r o n m e n t a l c h e m i c a l s , d e r i v e d from i n d u s t r i a l , m edical and s o c i e t a l e x p o s u r e s , as major f a c t o r s i n the c a u s a t i o n of human c a n c e r ( M i l l e r , 1978). 2) S t a g e s o f I n i t i a t i o n and Promotion The p i o n e e r i n g s t u d i e s o f Berenblum and Shubik (1947, 1949) demonstrated t h a t c h e m i c a l c a r c i n o g e n e s i s c o u l d be d i v i d e d i n t o two d i s t i n c t s t a g e s . T h e i r f i n d i n g s i n d i c a t e d t h a t t he f i r s t s t a g e o f mouse or r a b b i t epidermal c a r c i n o g e n e s i s , termed " i n i t i a t i o n " , was e f f e c t e d by the s i n g l e d i r e c t a p p l i c a t i o n o f a c a r c i n o g e n to the s k i n . F o l l o w i n g t he a d m i n i s t r a t i o n o f the i n i t i a t i n g agent, i f a second compound, termed a "promoter", were r e p e a t e d l y a p p l i e d t o the i n i t i a t e d a r ea o f s k i n , neoplasms a r o s e . E i t h e r t r e a t m e n t a l o n e r e s u l t e d i n no neoplasms. I f the promoting phase preceded t h a t o f i n i t i a t i o n , no neoplasms r e s u l t e d . The p r o c e s s o f i n i t i a t i o n was e s s e n t i a l l y an i r r e v e r s i b l e one ( B o u t w e l l , 1976). Tumours c o u l d s t i l l be produced by r e p e t i t i v e a d m i n i s t r a t i o n o f promoter, even when the p e r i o d of time between the a d m i n i s t r a t i o n of the i n i t i a t o r and the f i r s t a p p l i c a t i o n of the promoter was s e v e r a l months to a y e a r . T h e r e f o r e , i n i t i a t e d c e l l s appeared to have a c e r t a i n "memory". The promoter was by i t s e l f c o n s i d e r e d t o be n o n c a r c i n o g e n i c , but p o s s e s s e d the p r o p e r t y of enhancing or s t i m u l a t i n g n e o p l a s t i c e x p r e s s i o n i n the i n i t i a t e d c e l l . Boutwell (1976) has proposed t h a t tumour promoters a c t th r o u g h gene a c t i v a t i o n or the r e g u l a t i o n o f g e n e t i c e x p r e s s i o n . An i n i t i a t i n g dose o f 7, 1 2 - d i m e t h y l b e n z ( a ) a n t h r a c e n e may be t h r e e - 4 -o r d e r s of magnitude l e s s than t h a t r e q u i r e d to e l i c i t a comparable number of s k i n tumours when a p p l i e d r e p e t i t i v e l y i n the absence of a promoter ( B o u t w e l l , 1976). D i b e n z ( a ) a n t h r a c e n e i s termed a complete c a r c i n o g e n s i n c e i t has the c a p a b i l i t y both of i n i t i a t i o n and promotion. A s i n g l e a p p l i c a t i o n of a low dose o f d i b e n z ( a ) a n t h r a c e n e , i n s u f f i c i e n t t o induce tumours on i t s own, i n d u c e s tumours when f o l l o w e d by r e p e t i t i v e a d m i n i s t r a t i o n of promoter. R e p e t i t i v e a p p l i c a t i o n of h i g h e r doses o f t h i s i n i t i a t o r w i l l i n d u c e tumours. Thus the i n i t i a t i o n and promotion stages of d i b e n z ( a ) a n t h r a c e n e c a r c i n o g e n e s i s can be s e p a r a t e d . Urethane has been found to induce neoplasms i n the l i v e r and l u n g , but not i n the s k i n (Berenblum and Haran-Ghera, 1957). However, a f t e r urethane a d m i n i s t r a t i o n , subsequent t r e a t m e n t o f the s k i n of mice with c r o t o n o i l , a tumor promoter, r e s u l t e d i n the appearance of tumours of the s k i n . These data suggested t h a t urethane was c a p a b l e o n l y of i n i t i a t i n g c e l l s i n the s k i n and not i n d u c i n g the remaining stages of c a r c i n o g e n e s i s . Urethane was termed an " i n c o m p l e t e " c a r c i n o g e n or "pure" i n i t i a t i n g agent f o r the s k i n w h i l e i t was a "complete" c a r c i n o g e n f o r the lung and l i v e r (Berenblum and Haran-Ghera, 1957). E v i d e n c e has a l s o been p r e s e n t e d f o r m u l t i s t a g e i n d u c t i o n o f tumours o f the mammary gland (Armuth and Berenblum, 1974), b l a d d e r ( H i c k s e t aj_, 1975) and l i v e r ( F a r b e r , 1973; Kitagawa and Sugano, 1977). S a c c h a r i n has been shown to promote n e o p l a s i a i n the b l a d d e r a f t e r i n s t i l l a t i o n of a s i n g l e dose of the i n i t i a t o r , N - m e t h y l - N - n i t r o s o u r e a i n t o the b l a d d e r . T h i s dose of i n i t i a t o r d i d not induce tumours u n l e s s animals were f e d s a c c h a r i n i n the d i e t f o r about one y e a r ( H i c k s e t aj[» 1973). A p o p u l a r working h y p o t h e s i s f o r two stage c a r c i n o g e n e s i s has been de v e l o p e d by Boutwell (1974). B r i e f l y , the i n i t i a t o r causes the f o r m a t i o n of permanent and h e r i t a b l e , but u n e x p r e s s e d , changes i n the c e l l genome ( m u t a t i o n ) , and the promoter causes the p h e n o t y p i c e x p r e s s i o n of these changes - 5 -i n genotype as a l t e r e d metabolism, s u b s e q u e n t l y as a l t e r e d morphology of the c e l l , and u l t i m a t e l y as a tumour (gene a c t i v a t i o n , "hormone-like" a c t i v i t y ) . I I . Drug M e t a b o l i z i n g Enzymes and I n i t i a t i o n o f N e o p l a s i a By the l a t e 1940's, the l i s t of c h e m i c a l s t h a t c o u l d induce c a n c e r i n e x p e r i m e n t a l a n i m a l s was a f o r m i d a b l e one. There was a g r e a t d i v e r s i t y of s t r u c t u r e among the numerous c l a s s e s of chemical c a r c i n o g e n s . S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s were not r e a d i l y d i s c e r n a b l e . How c o u l d c h e m i c a l s o f such g r e a t s t r u c t u r a l d i v e r s i t y produce the same b i o l o g i c a l e f f e c t ? M i l l e r and M i l l e r (1947) found t h a t N,N-dimethyl-4-aminoazobenzene, a l i v e r c a r c i n o g e n , was bound c o v a l e n t l y t o the h e p a t i c p r o t e i n s of r a t s f e d t h i s dye. The c o v a l e n t b i n d i n g o f the p o l c y c l i c a r o m a t i c hydrocarbons and 2 - a c e t y l a m i n o f l u o r e n e to p r o t e i n s i n t a r g e t t i s s u e s was a l s o r e p o r t e d ( M i l l e r and M i l l e r , 1952). Subsequent s t u d i e s (Wheeler and S k i p p e r , 1957; F a r b e r and Magee, 1960; S t e k o l et_ a]_, 1960; Magee and F a r b e r , 1962; Marroquin and F a r b e r , 1962; Brookes and Lawley, 1964; H e i d e l b e r g e r , 1964) demonstrated the c o v a l e n t i n t e r a c t i o n s of chemical c a r c i n o g e n s with the n u c l e i c a c i d s , RNA and DNA, i n t a r g e t t i s s u e s . The a d m i n i s t r a t i o n o f a l l c a r c i n o g e n s t h a t have been s u f f i c i e n t l y s t u d i e d has r e s u l t e d i n t h e i r c o v a l e n t i n t e r a c t i o n with the p r o t e i n , RNA and DNA o f t a r g e t t i s s u e s . Thus, the common f e a t u r e of t h e s e s t r u c t u r a l l y d i v e r s e chemical c a r c i n o g e n s was t h e i r a b i l i t y t o c o v a l e n t l y i n t e r a c t w i t h n u c l e o p h i l i c macromolecules i n t a r g e t t i s s u e s . I t was suggested ( M i l l e r , 1970) t h a t e l e c t r o p h i l i c forms, or u l t i m a t e c a r c i n o g e n s , i n i t i a t e d the c a r c i n o g e n i c p r o c e s s t h r o u g h chemical r e a c t i o n w i t h c r i t i c a l t i s s u e n u c l e o p h i l e s such as p r o t e i n , RNA and DNA. However, the m a j o r i t y of chemical c a r c i n o g e n s , with the e x c e p t i o n o f the a l k y l a t i n g a g e n t s , were c h e m i c a l l y u n r e a c t i v e m o l e c u l e s , as such. -6-Most chemical c a r c i n o g e n s were found to r e q u i r e m e t a b o l i c a c t i v a t i o n i n o r d e r to r e a c t c o v a l e n t l y with p r o t e i n , RNA and DNA ( M i l l e r , 1970; H e i d e l b e r g e r , 1975). Mammalian systems are c a p a b l e o f c o n v e r t i n g c h e m i c a l l y i n e r t compounds i n t o h i g h l y r e a c t i v e i n t e r m e d i a t e s . For example, G e l b o i n (1969) and Grover and Sims (1969) showed t h a t the l i v e r microsomal mixed f u n c -t i o n o x i d a s e system, the same enzyme system t h a t m e t a b o l i z e s d r u g s , f a t t y a c i d s , s t e r o i d s and p e s t i c i d e s (Conney, 1967) was r e q u i r e d f o r b e n z o ( a ) p y r e n e t o b i n d c o v a l e n t l y t o DNA j_n v i t r o . In the l a s t two decades t h e r e has been a major t h r u s t towards i s o l a t i n g and i d e n t i f y i n g the r e a c t i v e forms of chemical c a r c i n o g e n s . In a d d i t i o n , much a t t e n t i o n has been f o c u s e d on the enzyme pathways r e s p o n s i b l e f o r the produc-t i o n and subsequent metabolism of t h e s e r e a c t i v e m e t a b o l i t e s . For example, t h e h i g h l y r e a c t i v e d i o l - e p o x i d e (the 9,10-epoxy-7,8-dihydroxy m e t a b o l i t e o f b e n z o ( a ) p y r e n e ) has been i m p l i c a t e d as a predominant u l t i m a t e c a r c i n o g e n i c form o f b e n z o ( a ) p y r e n e ( S i m s ^ t j i l _ , 1974; W e i n s t e i n e t aj_, 1976). Microsomal cytochrome P-450 and epoxide h y d r o l a s e c a t a l y z e the o x i d a t i o n and h y d r a t i o n r e a c t i o n s t h a t are n e c e s s a r y t o t r a n s f o r m the c h e m i c a l l y n o n - r e a c t i v e b e n z o ( a ) p y r e n e i n t o a h i g h l y l a b i l e d i o l - e p o x i d e m e t a b o l i t e (Wood e t a l , 1976). The c o n v e r s i o n o f b e n z o ( a ) p y r e n e t o t h i s d i o l - e p o x i d e and i t s subse-quent c o v a l e n t i n t e r a c t i o n with n u c l e a r DNA i s thought by many to be the sequence of s t e p s i n v o l v e d i n the i n i t i a t i o n of c a n c e r by t h i s compound ( M i l l e r , 1978). A h i g h l y p o p u l a r h y p o t h e s i s t h a t might account f o r the i n i t i a t i o n of n e o p l a s i a by c h e m i c a l s i s the somatic mutation t h e o r y of c a n c e r . The concept t h a t neoplasms a r i s e from mutations i n somatic c e l l s was p o s t u l a t e d long b e f o r e t h e r e was knowledge of the m o l e c u l a r s t r u c t u r e of g e n e t i c m a t e r i a l o r the i n t e r a c t i o n between c e l l u l a r DNA and c a r c i n o g e n i c agents ( B o v e r i , 1914). In s u p p o r t of t h i s t h e o r y , c a r c i n o g e n i c agents have been shown to i n t e r a c t -7-c o v a l e n t l y w i t h DNA, enhance DNA r e p a i r , and induce gene and chromosomal mut a t i o n s i n a v a r i e t y of organisms from v i r u s e s to humans (Chu e t a]_, 1977). The development by Ames and coworkers (1973) of a r a p i d and s e n s i t i v e m u t a g e n i c i t y t e s t system, combining l i v e r microsomes f o r m e t a b o l i c a c t i v a t i o n and S a l m o n e l l a typhimurium f o r mutation d e t e c t i o n , has made i t p o s s i b l e t o examine l a r g e numbers of known c a r c i n o g e n s and s t r u c t u r a l l y r e l a t e d non-c a r c i n o g e n s . R e s u l t s o b t a i n e d i n d i c a t e d t h a t a p p r o x i m a t e l y 90% o f known c a r c i n o g e n s were mutagens i n t h e Ames system (McCann e t a t , 1975). I t appears t h a t m e t a b o l i c a c t i v a t i o n i s a n e c e s s a r y s t e p i n the i n d u c t i o n o f n e o p l a s i a by most chemical c a r c i n o g e n s . The h e p a t i c microsomal monooxygen-ases a re i n v o l v e d i n the a c t i v a t i o n o f a f l a t o x i n B^, the p o l y c y c l i c a r o m a t i c h y d r o c a r b o n s , a r o m a t i c amines, 2 - a c e t y l a m i n o f l u o r e n e , and o t h e r s ( M i l l e r , 1978). A l a r g e body of e v i d e n c e suggests t h a t t he m e t a b o l i c f o r m a t i o n o f h i g h l y r e a c t i v e i n t e r m e d i a t e s and t h e i r subsequent c o v a l e n t i n t e r a c t i o n s with n u c l e a r DNA are e s s e n t i a l s t e p s i n the i n i t i a t i o n o f c a n c e r by c h e m i c a l s . I n i t i a t i o n i s regarded by many t o be the r e s u l t of a m u t a t i o n a l event, which r e s u l t s from the c o v a l e n t i n t e r a c t i o n s o f t h e s e e l e c t r o p h i 1 i c i n t e r m e d i a t e s w i t h n u c l e a r DNA. I I I . N u c l e a r Drug M e t a b o l i z i n g Enzymes I t i s well-known t h a t t he cytochrome P-450 mixed f u n c t i o n o x i d a s e s are l o c a t e d p r e d o m i n a n t l y i n the endoplasmic r e t i c u l u m of the c e l l . However, how do h i g h l y r e a c t i v e e l e c t r o p h i 1 i c i n t e r m e d i a t e s formed i n the endoplasmic r e t i c u l u m , the apparent s i t e o f a c t i v a t i o n , s u r v i v e a sea of c y t o p l a s m i c n u c l e o p h i l e s and i n t e r a c t w i t h n u c l e a r DNA? T h i s problem would be overcome i f the a c t i v a t i o n o c c u r r e d i n the n u c l e u s . Kasper (1971) r e p o r t e d the presence o f cytochrome P-450 and a r y l hydro-carbon h y d r o x y l a s e (AHH) a c t i v i t y , an i n d i c a t o r of cytochrome P-450 mediated - 8 -b e n z o ( a ) p y r e n e o x i d a t i o n , i n the r a t h e p a t o c y t e n u c l e a r membrane. L a t e r , Fahl and coworkers (1978) demonstrated epoxide h y d r o l a s e a c t i v i t y i n t h e s e membranes. A s t r i k i n g f e a t u r e o f n u c l e a r cytochrome P-450, AHH and epoxide h y d r o l a s e was t h e i r l a c k of i n d u c i b i l i t y by p h e n o b a r b i t a l . Adequate enzyme markers t o q u a n t i t a t e endoplasmic r e t i c u l u m , the major s i t e of t h e s e drug m e t a b o l i z i n g enzymes, i n n u c l e a r p r e p a r a t i o n s do not e x i s t . However, the l a c k o f i n d u c i b i l i t y by p h e n o b a r b i t a l was taken as b i o c h e m i c a l e v i d e n c e t o s u p p o r t the s u g g e s t i o n t h a t drug m e t a b o l i z i n g enzymes e x i s t e d i n the n u c l e a r e n v e l o p e , and t h a t t h e s e a c t i v i t i e s were not s i m p l y due to endoplasmic r e t i c u l u m c o n t a m i n a t i o n of the n u c l e a r p r e p a r a t i o n s . In c o n t r a s t , s e v e r a l r e c e n t s t u d i e s have shown t h a t p h e n o b a r b i t a l i n c r e a s e d n u c l e a r cytochrome P-450 (Mukhtar et_ al_, 1979; Vizethum and Goerz, 1979), AHH ( B r e s n i c k e t j i l _ , 1977; V i v i a n i e t aj_, 1978; D a n s e t t l e e t aj_, 1979) and e p o x i d e h y d r o l a s e (Dansette e t al_, 1979; Mukhtar e t ^1_, 1979; Mukhtar e t a l , 1979a) a c t i v i t i e s . The i n c r e a s e s were about the same magnitude as t h o s e o b s e r v e d i n the microsomal f r a c t i o n . Thus, the e v i d e n c e t h a t suggested the e x i s t e n c e o f s e p a r a t e n u c l e a r enzymes was not u n i v e r s a l l y met. These l a t t e r e xperiments suggested t h a t e i t h e r the enzyme a c t i v i t i e s measured i n the n u c l e a r p r e p a r a t i o n s were s i m p l y due t o endoplasmic r e t i c u l u m c o n t a m i n a t i o n or t h a t the n u c l e a r and microsomal enzymes were s i m i l a r i n t h e i r b i o c h e m i c a l p r o p e r t i e s . Thomas and coworkers (1979) p r o v i d e d immunological e v i d e n c e of t h e s i m i l a r i t y between microsomal and n u c l e a r drug m e t a b o l i z i n g enzymes. They showed t h a t n u c l e a r and microsomal epoxide h y d r o l a s e were i m m u n o l o g i c a l l y i d e n t i c a l . They a l s o showed immunological i d e n t i t y f o r the major form of cytochrome P-450 induced by 3 - m e t h y l c h o l a n t h r e n e , namely cytochrome P-448, i n the two f r a c t i o n s . H i g h - p r e s s u r e l i q u i d c hromatographic a n a l y s i s of the m e t a b o l i t e s g e n e r a t e d by i n c u b a t i o n o f benzo(a)pyrene with n u c l e a r and microsomal enzyme p r e p a r a t i o n s d i d not i n d i c a t e any q u a l i t a t i v e d i f f e r e n c e s - 9 -( B r e s n i c k e t 1977; B r e s n i c k et a]_, 1977a; Fahl e t al_, 1978; Pezzuto e t aj_, 1978). In a d d i t i o n , both n u c l e a r and microsomal enzymes e x h i b i t e d the same s t e r e o s p e c i f i c metabolism of b e n z o ( a ) p y r e n e ( J e n n e t t e e t aj_, 1979). The i n h i b i t i o n o f v a r i o u s n u c l e a r and microsomal enzymes appeared to be q u a l i t a t i v e l y s i m i l a r as w e l l ( B r e s n i c k et aj_, 1977; Mukhtar e t al_, 1979a). Both the n u c l e i and the microsomes were shown to produce d i o l - e p o x i d e m e t a b o l i t e s o f b e n z o ( a ) p y r e n e (Pezzuto e t aj_, 1978). S i n c e t h e s e i n t e r m e d i -a t e s have been i m p l i c a t e d as i n i t i a t o r s of c a r c i n o g e n e s i s ( M i l l e r , 1978), one o f the c o n c l u s i o n s t h a t can be drawn from t h e s e s t u d i e s i s t h a t both the n u c l e i and the microsomes are p o t e n t i a l l y important s i t e s of c a r c i n o g e n a c t i -v a t i o n . In s u p p o r t o f t h i s s t a t e m e n t , a number of s t u d i e s have shown t h a t n u c l e i , l i k e microsomes, were c a p a b l e of a c t i v a t i n g b e n z o ( a ) p y r e n e (Rogan and C a v a l i e r i , 1974; Al exandrov e t aj_, 1976; J e r n s t r o m e t aj_, 1976; Pezzuto e t a l , 1976; B r e s n i c k e t a]_, 1977; B r e s n i c k e t aj_, 1977a; Pezzuto e t al_, 1977) and 7 , 8 - d i h y d r o - 7 , 8 - d i h y d r o x y b e n z o ( a ) p y r e n e ( B r e s n i c k j^t aj^, 1977a; J e r n s t r o m e t a l , 1978) to i n t e r m e d i a t e s t h a t were bound c o v a l e n t l y t o DNA. Aune and a s s o c i a t e s (1980) showed t h a t i s o l a t e d r a t l i v e r n u c l e i c o u l d a c t i v a t e 2 , 4 - d i a m i n o a n i s o l e and 2 - a m i n o f l u o r e n e to mutagenic s p e c i e s , s i m i l a r to t h a t seen with r a t l i v e r microsomes. The t o x i c o l o g i c a l s i g n i f i c a n c e of c a r c i n o g e n a c t i v a t i n g enzymes i n c l o s e p r o x i m i t y t o p o s s i b l e t a r g e t s i t e s i n the n u c l e u s has generated an i n c r e a s e d i n t e r e s t i n t h i s a r e a . However, as a r e s u l t of c o n f l i c t i n g e v i d e n c e , t h e r e remains a doubt as to the o r i g i n of t h e s e enzymes measured i n i s o l a t e d n u c l e i . Are t h e s e enzymes p r e s e n t i n the n u c l e a r membrane and/or n u c l e o p l a s m , or are they simply a r e s u l t o f microsomal c o n t a m i n a t i o n of the i s o l a t e d n u c l e a r f r a c t i o n ? One o f the o b j e c t i v e s o f t h i s study was t o determine i f the enzyme a c t i v i t i e s i n i s o l a t e d r a t h e p a t i c n u c l e i were of n u c l e a r o r i g i n . A second o b j e c t i v e o f the p r e s e n t study was t o determine i f t h e r e were any q u a n t i t a t i v e - 10-d i f f e r e n c e s between the enzymes o f n u c l e a r and microsomal o r i g i n . The s e l e c t i v e m a n i p u l a t i o n of n u c l e a r enzymes would be a u s e f u l t o o l i n c o r r o b o r a t i n g the involvement o f t h e s e enzymes i n the i n i t i a t i o n of n e o p l a s i a . IV. Promotion o f H e p a t o c a r c i n o g e n e s i s P r i o r to the experiments which i n d i c a t e d the i n i t i a t i o n and promotion s t a g e s of h e p a t o c a r c i n o g e n e s i s , a number of s t u d i e s suggested t h a t the i n c i d e n c e of n e o p l a s i a c o u l d be a l t e r e d by both exogenous ( d i e t a r y ) and endogenous (hormonal) f a c t o r s . For example, hypophysectomy was shown t o p r e v e n t 2 - a c e t y l a m i n o f l u o r e n e - i n d u c e d h e p a t i c n e o p l a s i a i n r a t s (Weisburger and W e i s b u r g e r , 1963). Thyroidectomy and adrenalectomy c o m p l e t e l y i n h i b i t e d t h e p r o d u c t i o n of hepatoma by 2 - a c e t y l a m i n o f l u o r e n e i n male r a t s ( B i e l s c h o w s k y , 1944). It was w e l l known t h a t male r a t s were more s u s c e p t i b l e t o the h e p a t o c a r c i n o g e n i c e f f e c t s o f 2 - a c e t y l a m i n o f l u o r e n e than were female r a t s ( F i r m i n g e r and Reuber, 1961). T e s t o s t e r o n e a d m i n i s t e r e d t o young c a s t r a t e d male r a t s produced an i n c r e a s e i n 2 - a c e t y l a m i n o f l u o r e n e - i n d u c e d h e p a t i c neoplasms, as compared to t h e s e r a t s a d m i n i s t e r e d c a r c i n o g e n a l o n e (Reuber, 1976). However, when t h e s e m a n i p u l a t i o n s are c a r r i e d out s i m u l t a n e o u s l y with the a d m i n i s t r a t i o n of c a r c i n o g e n , o t h e r f a c t o r s such as c a r c i n o g e n metabolism ( L o t l i k a r e t aj_, 1964; M i l l e r and M i l l e r , 1971) and DNA r e p a i r mechanisms ( P i t o t and S i r i c a , 1980) must be taken i n t o account. A l t e r a t i o n s i n the metabolism of a c a r c i n o g e n by endogenous or exogenous f a c t o r s can produce a s i g n i f i c a n t change i n the a c t u a l dose of i n i t i a t o r . T h i s was demonstrated with 2 - a c e t y l a m i n o f l u o r e n e and p h e n o b a r b i t a l . P h e n o b a r b i t a l a d m i n i s t e r e d a f t e r 2 - a c e t y l a m i n o f l u o r e n e r e s u l t e d i n a s i g n i f i c a n t enhancement of h e p a t i c tumours, whereas si m u l t a n e o u s a d m i n i s t r a t i o n r e s u l t e d i n a d e c r e a s e d i n c i d e n c e of h e p a t i c tumours ( P e r a i n o e t a]_, 1971). I t was l a t e r found t h a t when p h e n o b a r b i t a l was a d m i n i s t e r e d c o n c o m i t a n t l y w i t h 2 - a c e t y l a m i n o f l u o r e n e , the c o v a l e n t b i n d i n g of c a r c i n o g e n was d e c r e a s e d by 80% (M u s h l i n and P e r a i n o , 1974), whereas p h e n o b a r b i t a l a d m i n i s t r a t i o n had no e f f e c t on t h e DNA b i n d i n g of c a r c i n o g e n when i t was f e d a f t e r the c a r c i n o g e n , i . e . , as a promoting agent. The i n i t i a t i o n and promotion stages of h e p a t o c a r c i n o g e n e s i s were f i r s t d emonstrated by P e r a i n o and coworkers (1973). They found t h a t p h e n o b a r b i t a l was a promoter o f l i v e r c a n c e r . In t h e i r s t u d y , the i n i t i a t o r , 2 - a c e t y l a m i n o -f l u orene, was f e d to r a t s f o r t h r e e weeks. T h i s was f o l l o w e d by one week of bas a l d i e t w i t h o u t t he c a r c i n o g e n . A f t e r t h a t , one group was m a i n t a i n e d on b a s a l d i e t , w h i l e the second group was m a i n t a i n e d on the same d i e t c o n t a i n i n g 0.05% p h e n o b a r b i t a l . The i n c i d e n c e of l i v e r neoplasms i n animals f e d p h e n o b a r b i t a l was 1 0 - f o l d t h a t seen i n the c o n t r o l s . These workers ( P e r a i n o e t a l , 1977) were a b l e to show a s i g n i f i c a n t promoting e f f e c t by p h e n o b a r b i t a l even when the i n t e r v a l between the c e s s a t i o n o f 2 - a c e t y l a m i n o f 1 u o r e n e f e e d i n g and t he s t a r t o f p h e n o b a r b i t a l treatment was extended t o 4 months. These i n v e s t i g a t o r s a l s o showed t h a t t he f i n a l y i e l d of neoplasms was i n f l u e n c e d g r e a t l y by the d u r a t i o n of p h e n o b a r b i t a l a d m i n i s t r a t i o n . Weisburger and coworkers (1975) demonstrated t h a t p h e n o b a r b i t a l was c a p a b l e of promoting h e p a t i c tumour f o r m a t i o n induced by the c a r c i n o g e n , d i e t h y l n i t r o s a m i n e . P e r a i n o and coworkers (1973a) showed tha C3H mice had an i n c i d e n c e of spontaneous hepatomas which was 10% i n one y e a r . When thes e mice were f e d a d i e t c o n t a i n i n g 0.05% p h e n o b a r b i t a l f o r one y e a r , t he i n c i d e n c e of hepatoma was 100%. D i c h l o r o d i p h e n y l t r i c h l o r o e t h a n e (DDT) ( P e r a i n o e t aj_, 1975) and b u t y l a t e d h y d r o x y t o l u e n e ( P e r a i n o e t ^1_, 1977a) were shown to e x h i b i t t he same promoting a c t i o n as d i d p h e n o b a r b i t a l . Kimura and coworkers (1976) demonstrated t h a t p o l y c h l o r i n a t e d b i p h e n y l s promoted h e p a t i c neoplasms i n i t i a t e d by 3'-methyl--12-4-dimethylaminoazobenzene. P r e s t o n and coworkers (1981) demonstrated the tumour-promoting a c t i v i t y of p o l y c h l o r i n a t e d b i p h e n y l s u s i n g d i e t h y l n i t r o -samine as the i n i t i a t o r . These workers a l s o demonstrated t h a t the p o l y -c h l o r i n a t e d b i p h e n y l s were c a p a b l e of promoting l i v e r t u m o r i g e n e s i s even when the p o t e n t t o x i n , p o l y c h l o r i n a t e d d i b e n z o f u r a n , was removed from the commer-c i a l b i p h e n y l m i x t u r e used ( A r o c l o r 1254). V. H e p a t i c Enzyme I n d u c t i o n 1) R e l a t i o n s h i p t o Promotion o f H e p a t o c a r c i n o g e n e s i s A l l o f the c h e m i c a l s t h a t have been shown t o possess tumour promoting a c t i v i t y i n the l i v e r share the same b i o l o g i c a l p r o p e r t y of i n d u c i n g t h e h e p a t i c drug m e t a b o l i z i n g enzymes (Conney, 1967; P i t o t , 1978; Snyder and Remmer, 1979). Boutwell (1974) has proposed t h a t tumour promoters act through gene a c t i v a t i o n or the r e g u l a t i o n o f g e n e t i c e x p r e s s i o n . The a b i l i t y t o enhance g e n e t i c e x p r e s s i o n has been demonstrated f o r most tumor promoters ( B o u t w e l l , 1978; P i t o t , 1978). The i n d u c t i o n o f drug m e t a b o l i z i n g enzymes has been shown to i n v o l v e the i n c r e a s e d t r a n s c r i p t i o n and t r a n s l a t i o n of g e n e t i c m a t e r i a l (Nebert and G e l b o i n , 1970; Nebert, 1979). T h e r e f o r e , the two b i o -l o g i c a l responses produced by t h e s e c h e m i c a l s , tumour promotion and enzyme i n -d u c t i o n , i n v o l v e enhanced g e n e t i c e x p r e s s i o n . It appears l i k e l y t h a t the r e -sponse systems f o r h e p a t i c tumour promotion and i n d u c t i o n of h e p a t i c drug m e t a b o l i z i n g enzymes by a given chemical agent are the same, s i n c e both b i o -l o g i c a l responses i n v o l v e enhanced g e n e t i c e x p r e s s i o n . Thus, an u n d e r s t a n d i n g o f the response system f o r the i n d u c t i o n o f drug m e t a b o l i z i n g enzymes c o u l d p r o v i d e important i n f o r m a t i o n on the c o n t r o l of g e n e t i c a c t i v i t y i n the l i v e r ; i n p a r t i c u l a r , an u n d e r s t a n d i n g o f the c o n t r o l mechanisms i n v o l v e d i n hepato-c y t e r e p l i c a t i o n and d i f f e r e n t i a t i o n . It s h o u l d be p o i n t e d out t h a t the se-quences o f DNA and c o n t r o l o f t h e s e genes f o r t h e s e two c h a r a c t i s t i c r e p o n s e s , -13-promotion and i n d u c t i o n , are p r o b a b l y d i f f e r e n t , however. 2) C l a s s e s o f Enzyme Inducers A wide range o f s t r u c t u r a l l y d i v e r s e c h e m i c a l s cause the i n d u c t i o n o f r a t h e p a t i c drug m e t a b o l i z i n g enzymes (Conney, 1967; Snyder and Remmer, 1979). The compounds t h a t produce an i n c r e a s e i n the drug m e t a b o l i z i n g enzymes have been c l a s s i c a l l y d i v i d e d i n t o two groups: p h e n o b a r b i t a l - t y p e o r 3-methyl-c h o l a n t h r e n e - t y p e i n d u c e r s (Conney, 1967). The b i o c h e m i c a l responses e l i c i t e d by t h e s e p r o t o t y p e i n d u c e r s are d r a m a t i c a l l y d i f f e r e n t . P h e n o b a r b i t a l t r e a t -ment r e s u l t s i n the i n c r e a s e o f a wide range of cytochrome P-450 dependent monoxygenase a c t i v i t i e s , whereas 3 - m e t h y l c h o l a n t h r e n e treatment i n c r e a s e s a narrower range o f monoxygenases (Conney and Burns, 1963). 3-Methyl-c h o l a n t h r e n e t r e a t m e n t r e s u l t s i n the p r o d u c t i o n of a cytochrome with an a b s o r p t i o n maximum at 448 nm (cytochrome P-448), whereas p h e n o b a r b i t a l t r e a t -ment induces the f o r m a t i o n of a cytochrome with an a b s o r p t i o n maximum a t 450 nm (cytochrome P - 4 5 0 ) ( A l v a r e s e t aj_, 1967). R e c e n t l y , the major forms o f r a t h e p a t i c cytochrome P-450 induced by t h e s e two compounds were shown to be d i f f e r e n t p r o t e i n s by immunological and b i o c h e m i c a l a n a l y s e s (Ryan e t a l , 1979; Thomas e t ^1_, 1979). H e p a t i c epoxide h y d r o l a s e a c t i v i t y , as determined by the h y d r a t i o n o f s t y r e n e 7,8-oxide, was i n c r e a s e d by p h e n o b a r b i t a l , but not by 3 - m e t h y l c h o l a n t h r e n e ( B r e s n i c k e t aj_, 1977b). These s t u d i e s , along with numerous o t h e r s , c l e a r l y i n d i c a t e the d i s t i n c t p l e i o t r o p i c responses produced by p h e n o b a r b i t a l and 3 - m e t h y l c h o l a n t h r e n e . P r e g n e n o l o n e - 1 6 < X - c a r b o n i t r i l e was shown t o be a new type o f i n d u c e r of d r u g - m e t a b o l i z i n g enzymes (Lu e t aj_, 1972). T h i s c o n c l u s i o n was based on t h e d i f f e r e n c e s i n the magnitude o f s t i m u l a t i o n of s e l e c t e d monooxygenases by maximally e f f e c t i v e doses of p h e n o b a r b i t a l , 3 - m e t h y l c h o l a n t h r e n e and pregneno-lone-16«t-carbonitrile. F o r example, i n the female r a t , pregnenolone-16°<-c a r b o n i t r i l e i n c r e a s e d AHH a c t i v i t y t o 640 per cent of c o n t r o l and -14-benzphetamine N-demethylase a c t i v i t y t o 345 per cent of c o n t r o l . In c o n t r a s t , AHH a c t i v i t y was i n c r e a s e d by p h e n o b a r b i t a l and 3 - m e t h y l c h o l a n t h r e n e t o 400 and 1400 per cent o f c o n t r o l , r e s p e c t i v e l y , whereas, benzphetamine N-demethylase was i n c r e a s e d t o 730 per cent o f c o n t r o l by p h e n o b a r b i t a l and was u n a f f e c t e d by 3 - m e t h y l c h o l a n t h r e n e . R e c e n t l y , the major form of c y t o -chrome P-450 induced by pregnenolone-16°<-carbonitrile was found to be d i f f e r e n t from both the p h e n o b a r b i t a l and 3 - m e t h y l c h o l a n t h r e n e i n d u c i b l e forms, u s i n g b i o c h e m i c a l l y p u r i f i e d cytochrome P-450 along with immunological t e c h n i q u e s ( E l s o u r b a g y and G u z e l i a n , 1980). S e v e r a l o t h e r compounds have been suggested as novel i n d u c e r s of drug m e t a b o l i z i n g enzymes. These i n c l u d e s p i r o n o l a c t o n e ( S t r i p p e t aj_, 1971), t r a n s - s t i l b e n e o x i d e (Schmassmann and Oesch, 1978; S e i d e g a r d e t ^1_, 1979), i s o s a f r o l e ( D i c k e n s e t aj_, 1978), b u t y l a t e d h y d r o x y a n i s o l e (Cha and Martz, 1978) and 2 - a c e t y l a m i n o f l u o r e n e (Astrom and D e p i e r r e , 1981). T h e r e f o r e , t h e r e appears t o be an i n c r e a s i n g number of c h e m i c a l s t h a t e l i c i t t h e i r own c h a r a c -t e r i s t i c b i o c h e m i c a l r e s p o n s e . In a d d i t i o n , the m a j o r i t y of compounds t h a t were a s s i g n e d t o the c l a s s o f p h e n o b a r b i t a l - 1 i k e i n d u c e r s were done so on the b a s i s o f q u a l i t a t i v e a n a l y s e s . These e a r l i e r s t u d i e s c h a r a c t e r i z e d i n d u c e r s on the b a s i s of 1) i n d u c t i o n o f cytochrome P-450 v e r s u s cytochrome P-448 and 2) the spectrum of a s e l e c t e d few cytochrome P-450 dependent monooxygenase a c t i v i t i e s t h a t were i n c r e a s e d . T h e r e f o r e , f o l l o w i n g more d e t a i l e d s t u d i e s o f t h e s e p h e n o b a r b i t a l - 1 i k e i n d u c e r s the number of unique i n d u c e r s of h e p a t i c drug m e t a b o l i z i n g enzymes may be s i g n i f i c a n t l y l a r g e r than the few compounds t h a t have been s u g g e s t e d . However, o n l y t h r e e unique responses have been w e l l a c c e p t e d to d a t e : p h e n o b a r b i t a l , 3 - m e t h y l c h o l a n t h r e n e and pregnenolone-16oc-c a r b o n i t r i l e . 3) t r a n s - S t i l b e n e Oxide and S p i r o n o l a c t o n e t r a n s - S t i l b e n e oxide was d e s c r i b e d by Schmassmann and Oesch (1978) as a -15-unique i n d u c e r o f drug m e t a b o l i z i n g enzymes. These aut h o r s r e p o r t e d t h a t t r a n s - s t i l b e n e o x i d e i n c r e a s e d h e p a t i c microsomal epoxide h y d r o l a s e a c t i v i t y i n a d u l t male r a t s w i thout a s i m u l t a n e o u s augmentation of cytochrome P-450, AHH a c t i v i t y , or a m i n o p y r i n e N-demethylase a c t i v i t y and, t h e r e f o r e , s u g g e s t e d t h a t t r a n s - s t i l b e n e o x i d e was a " s e l e c t i v e " i n d u c e r of epoxide h y d r o l a s e a c t i v i t y . The same l a b o r a t o r y (Bucker e t aj_, 1979) again demonstrated t h e l a c k o f an e f f e c t on AHH a c t i v i t y i n a d u l t male r a t s by t r a n s - s t i l b e n e o x i d e . Oesch and Schmassmann (1979) a l s o r e p o r t e d t h a t h e p a t i c microsomal AHH a c t i v i t y i n a d u l t female r a t s was u n a l t e r e d by t r a n s - s t i l b e n e o x i d e . However, S e i d e g a r d e t aj_. (1979) r e p o r t e d t h a t cytochrome P-450 was s i g n i f i c a n t l y i n c r e a s e d (more than 2 - f o l d ) i n the a d u l t male f o l l o w i n g t r a n s - s t i l b e n e o x i d e . In a d d i t i o n , Mukhtar e t aj_. (1978) found t h a t epoxide h y d r o l a s e a c t i v i t y and cytochrome P-450, aminopyrine N-demethylase and AHH a c t i v i t i e s were a l l i n c r e a s e d i n microsomes o b t a i n e d from t r a n s - s t i l b e n e o x i d e - t r e a t e d immature male and female r a t s . T h i s l a t t e r study suggests t h a t the b i o c h e m i c a l response produced by t r a n s - s t i l b e n e o x i d e i s s i m i l a r to t h a t produced by p h e n o b a r b i t a l . I t i s w e l l known t h a t the basal a c t i v i t i e s o f v a r i o u s r a t h e p a t i c cytochrome P-450 dependent monooxygenases are g r e a t e r i n a d u l t males than i n a d u l t females or immature males (Conney, 1967; El Defrawy E l Masry e t a l , 1974). F u r t h e r , the i n d u c i b i l i t y of t h e s e enzymes i s i n f l u e n c e d by both the age and sex of the r a t s employed (Conney, 1967). B e l l w a r d e t a}_. (1977) have r e p o r t e d t h a t methadone tr e a t m e n t i n c r e a s e d h e p a t i c microsomal epoxide h y d r o l a s e a c t i v i t y w i t h o u t a l t e r i n g AHH a c t i v i t y i n a d u l t male r a t s , whereas, i n a d u l t female r a t s , AHH a c t i v i t y was i n c r e a s e d by methadone. T h e r e f o r e , we h y p o t h e s i z e d t h a t some of the apparent d i s c r e p a n c i e s i n the t r a n s - s t i l b e n e o x i d e s t u d i e s c o u l d have been a r e s u l t of u s i n g d i f f e r e n t groups of r a t s . One o f the aims o f the p r e s e n t study was t o i n v e s t i g a t e the e f f e c t s produced by -16-t r a n s - s t i l b e n e o x i d e i n the r a t and compare them with the e f f e c t s produced by p h e n o b a r b i t a l - t r e a t m e n t . S t r i p p and coworkers (1971) r e p o r t e d t h a t s p i r o n o l a c t o n e i n d u c t i o n d i f f e r e d from the p h e n o b a r b i t a l i n d u c t i o n i n t h a t i t d i d not i n c r e a s e t o t a l r a t h e p a t i c microsomal cytochrome P-450 c o n t e n t . However, the spectrum of cytochrome P-450 dependent monooxygenases t h a t were i n c r e a s e d resembled c l o s e l y t h o s e i n c r e a s e d by p h e n o b a r b i t a l . F e l l e r and G e r a l d (1971) r e p o r t e d t h a t cytochrome P-450 l e v e l s i n mouse l i v e r microsomes were i n c r e a s e d by s p i r o n o l a c t o n e . These a u t h o r s suggested t h a t the i n d u c t i o n responses produced by s p i r o n o l a c t o n e and p h e n o b a r b i t a l were s i m i l a r . A g a i n , c o n f l i c t i n g e v i d e n c e e x i s t s r e g a r d i n g the i n d u c t i o n o f drug m e t a b o l i z i n g enzymes by s p i r o n o l a c t o n e . T h e r e f o r e , one of the o b j e c t i v e s of the p r e s e n t study was to compare t h e i n d u c t i v e e f f e c t s o f s p i r o n o l a c t o n e and p h e n o b a r b i t a l i n the r a t . 4) Mechanisms o f Enzyme I n d u c t i o n I t was soon r e c o g n i z e d t h a t numerous compounds of g r e a t s t r u c t u r a l d i v e r s i t y were c a p a b l e of c a u s i n g the i n d u c t i o n of h e p a t i c drug m e t a b o l i z i n g enzymes. The p r o t o t y p e s f o r the two apparent c l a s s e s of h e p a t i c microsomal enzyme i n d u c e r s , namely, p h e n o b a r b i t a l and 3 - m e t h y l c h o l a n t h r e n e , were shown t o produce t h e i r b i o c h e m i c a l e f f e c t s by de novo s y n t h e s i s of enzyme p r o t e i n . The i n c r e a s e s i n enzyme a c t i v i t i e s were b l o c k e d by i n h i b i t o r s of t r a n s c r i p t i o n and t r a n s l a t i o n (Conney et aj_, 1956; Conney j i t aj_, 1957; O r r e n i u s e t a]_, 1965). The f i r s t s u g g e s t i o n t h a t the i n d u c t i o n of drug m e t a b o l i z i n g enzymes was mediated by a s p e c i f i c b i o l o g i c a l r e c o g n i t i o n s i t e ( r e c e p t o r ) was based on s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s of the i n d u c t i o n o f N-demethylation of an aminoazo dye by a l a r g e number o f p o l y c y c l i c a r o m a t i c hydrocarbons (Arcos e t a l , 1961). More r e c e n t l y , s t u d i e s with the h i g h l y potent p o l y c h l o r i n a t e d a r o m a t i c h y d r o c a r b o n s , the most potent being 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - p -d i o x i n (TCDD) have shown t h a t the p l e i o t r o p i c response induced by TCDD and -17-r e l a t e d a g e n t s , p o l y c y c l i c a r o m a t i c hydrocarbons a n d ^ - n a p h t h o f l a v o n e , i s mediated by a c y t o s o l i c r e c e p t o r p r o t e i n (Poland e t aj_, 1976). Thus, f o r t h e 3 - m e t h y l c h o l a n t h r e n e - t y p e i n d u c e r s , d e f i n i t e s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s can be shown and b i n d i n g to the c y t o s o l i c r e c e p t o r can be c o r r e l a t e d with b i o l o g i c a l a c t i v i t y . However, f o r the l a r g e number of s t r u c t u r a l l y u n r e l a t e d c h e m i c a l s t h a t e l i c i t t he p h e n o b a r b i t a l - t y p e i n d u c t i o n , the mechanism of i n d u c t i o n ( t h e r e c o g n i t i o n or response system) i s as y e t p o o r l y u n d e r s t o o d . An i n t e r e s t i n g model has been proposed to account f o r the i n d u c t i o n by a wide v a r i e t y of s t r u c t u r a l l y d i s s i m i l a r c h e m i c a l s ( M a r s h a l l , 1970; M a r s h a l l and McLean, 1971). The exogenous i n d u c e r i n h i b i t s the metabolism of an endogenous s u b s t r a t e f o r cytochrome P-450. The endogenous s u b s t r a t e then i n c r e a s e s i n c o n c e n t r a t i o n , or i t s d u r a t i o n i n the l i v e r i s extended, and i t produces the i n d u c t i o n r e s p o n s e . C e r t a i n l i n e s o f e v i d e n c e are c o n s i s t e n t with t h i s t h e o r y . R e l a t i v e l y l a r g e doses of p h e n o b a r b i t a l - t y p e i n d u c e r s are r e q u i r e d to e l i c i t t h e b i o l o g i c a l r e s p o n s e . Any o f the h i g h l y p u r i f i e d forms of cytochrome P-450 has been shown t o c a t a l y z e the o x i d a t i o n of a wide number of s u b s t r a t e s , a l t h o u g h the t u r n o v e r numbers vary s i g n i f i c a n t l y (Lu and West, 1980). T h i s c o u l d account f o r the l a c k o f s t r u c t u r a l s i m i l a r i t y between the v a r i o u s i n d u c e r s , which are themselves s u b s t r a t e s f o r cytochrome P-450 (Conney, 1967). A d i r e c t r e c e p t o r mechanism has been suggested by Poland et a l . (1980). However, a more c o m p l i c a t e d p i c t u r e d e v e l o p s i f novel i n d u c e r s of drug m e t a b o l i z i n g enzymes are found. Nebert (1979) has proposed a "sensor system" t h a t i n t e r a c t s w i t h a wide v a r i e t y o f i n d u c i n g c h e m i c a l s , e v o k i n g i n c r e a s e s i n a s p e c i f i c r e c e p t o r f o r each one. The i n d u c e r i s then a b l e t o complex with l a r g e amounts o f the newly-formed c y t o s o l i c r e c e p t o r , which evokes i n c r e a s e s i n s p e c i f i c forms of cytochrome P-450. Thus, m u l t i p l e r e c e p t o r s f o r the i n d u c t i o n o f m u l t i p l e forms o f cytochrome P-450 by a d i v e r s e number of -18-c h e m i c a l s i s a c c o m p l i s h e d . One o f the aims of the p r e s e n t study i s to e s t a b l i s h whether s p i r o n o l a c t o n e and t r a n s - s t i l b e n e oxide produce novel i n d u c t i o n r e s p o n s e s , i . e . , do they d i f f e r from the t h r e e w e l l documented i n d u c t i o n responses e x e m p l i f i e d by p h e n o b a r b i t a l , 3 - m e t h y l c h o l a n t h r e n e and p r e g n e n o l o n e - 1 6 < x - c a r b o n i t r i l e . Are t h e r e j u s t the t h r e e response systems or are t h e r e more? VI. S p e c i f i c Aims Whereas the study of the drug m e t a b o l i z i n g enzymes i n the n u c l e u s may d e t e r m i n e the importance of t h i s s i t e i n the a c t i v a t i o n of c a r c i n o g e n s and t h e i n i t i a t i o n o f c a r c i n o g e n e s i s , the study of the i n d u c t i o n o f drug m e t a b o l i z i n g enzymes, an i n d i c a t o r o f g e n e t i c e x p r e s s i o n , may y i e l d i n s i g h t i n t o the mechanism of tumour promotion i n the l i v e r . The s p e c i f i c aims of t h i s p r o j e c t were as f o l l o w s : 1) t o determine whether the enzymes found i n i s o l a t e d r a t h e p a t i c n u c l e i were of n u c l e a r o r i g i n or simply a r e s u l t of microsomal c o n t a m i n a t i o n 2) t o determine whether the enzymes o f n u c l e a r o r i g i n , i f f o u n d , d i f f e r e d q u a n t i t a t i v e l y from t h o s e o f microsomal o r i g i n i n t h e i r i n h i b i t i o n a c t i v a t i o n and i n d u c t i o n 3) t o study the i n f l u e n c e o f age and sex on the i n d u c t i o n produced by t r a n s -s t i l b e n e o x i d e , s p i r o n o l a c t o n e , p h e n o b a r b i t a l , pregnenolone-16°c-c a r b o n i t r i l e and 3 - m e t h y l c h o l a n t h r e n e i n r a t l i v e r 4) t o compare the i n d u c t i o n responses e l i c i t e d by s p i r o n o l a c t o n e and t r a n s -s t i l b e n e o x i d e with t h o s e e l i c i t e d by p h e n o b a r b i t a l , 3 - m e t h y l c h o l a n t h r e n e and p r e g n e n o l o n e - 1 6 < * - c a r b o n i t r i l e . _19_ MATERIALS AND METHODS I. Chemical s Benzo(a)pyrene, spironolactone, testosterone, 5«-dihydrotestosterone, semicarbazide.HCl, NADPH, NADH, NADP, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, androstenedione, bovine serum albumin, coomassie brilliant blue G, dodecyl sodium sulfate, acrylanide, N,N'-methylene-bis-acrylamide, N,N,N',N'-tetramethye-ethylenediamine and quinine sulfate were obtained from the Sigma Chemical Company (St. Louis, MO). 3-Methylcholanthrene, aniline.HCl, p-aminophenol.HC1 and mercaptoethanol were supplied by the Eastman Organic Chemical Company (Rochester, NY). trans-Stilbene oxide, oC-naphthoflavone and l,2-epoxy-3,3,3-trichloropropane were purchased from the Aldrich Chemical Company, Inc. (Milwaukee, WI). Phenobarbitone sodium was obtained from the British Drug House (Toronto, Ont.). Formaldehyde and bromophenol blue were purchased from the Fisher Scientific Company (Fair Lawn, NJ). Aminopyrine was obtained from Matheson, Coleman and Bell (East Rutherford, NJ). Ethylmorphine.HCl (British Drug House, Toronto, Ont.) and l-<* -acetylmethadol (Mal1inckrodt, Pointe Claire, Que.) were obtained through the Canadian Department of Health and Welfare. Resorufin and ethoxyresorufin were purchased from the Pierce Chemical Company (Rockford, IL). SKF 525-A and pregnenolone-16»<.-carbonitrile were generously provided by Smith, Kline and French Laboratories (Montreal, Que.) and the Upjohn Company (Kalamazoo, Ml), respectively. 3-Hydroxybenzo(a)pyrene was a gift from the NCI Carcinogenesis Research Program, Bethesda, MD. All other reagents and chemicals employed were the best commercial grades available. II. Animals Wistar rats, obtained from the Canadian Breeding Farms Ltd., La Prairie, Quebec, were used throughout the study, unless otherwise stated. They were raised under controlled conditions (22°C, lights on 0600 to 2000 hours) on -20-corncob bedding (Lobund grade, Paxton P r o c e s s i n g L t d . , Paxton, I L ) . P u r i n a l a b o r a t o r y chow and water were a v a i l a b l e at a l l t i m e s , ad l i b . Animals were a l l o w e d to e q u i l i b r a t e f o r at l e a s t one week b e f o r e use. The a n i m a l s d e s i g n a t e d "immature male," "immature female," " a d u l t male," and " a d u l t f e m a l e " r a t s weighed 80-100, 80-100, 200-300, and 180-250g, r e s p e c t i v e l y , at the time they were k i l l e d , u n l e s s o t h e r w i s e s t a t e d . I I I . Treatments The f o l l o w i n g compounds were a d m i n i s t e r e d i n t r a p e r i t o n e a l l y , once per day, w i t h the v e h i c l e i n d i c a t e d i n p a r e n t h e s e s ; p h e n o b a r b i t a l ( s a l i n e ) , 3 - m e t h y l c h o l a n t h r e n e (corn o i l ) , s p i r o n o l a c t o n e (corn o i l ) , t r a n s - s t i l b e n e o x i d e ( c o r n o i l ) and pregnenolone-16«<-carbonitrile (corn o i l ) . The i n j e c t i o n volume was u s u a l l y between 200 and 400/ul. Animals were always s a c r i f i c e d 24 hours a f t e r the l a s t i n j e c t i o n . 1-oi-Acetylmethadol was a d m i n i s t e r e d i n t h e d r i n k i n g water f o r 2 weeks. The a n i m a l s were m a i n t a i n e d on t h i s drug u n t i l t h e time of s a c r i f i c e . No e v i d e n c e of withdrawal symptoms, such as acute l o s s o f weight or wet shakes, was observed at the doses used. IV. P r e p a r a t i o n o f S u b c e l l u l a r F r a c t i o n s 1) L i v e r P r e p a r a t i o n A n i m a l s were stunned by a blow on the head, k i l l e d by d e c a p i t a t i o n and e x s a n g u i n a t e d . The l i v e r s were p e r f u s e d with i c e - c o l d 1.15% (w/v) aqueous KC1, e x c i s e d , b l o t t e d , weighed and p l a c e d i n the i c e - c o l d 1.15% KC1. A l l sub-sequent p r o c e d u r e s were c a r r i e d out at 4°C. In the a d u l t r a t s , both m i c r o -somes and n u c l e i were prepared from the same l i v e r . 2) Microsomal F r a c t i o n T y p i c a l l y , at l e a s t 2 grams o f l i v e r was homogenized l:4(w/v) i n 1.15% KC1, u s i n g a P o t t e r - E l v e j h e m homogenizer with a m o t o r - d r i v e n t e f l o n p e s t l e . The homogenate was then c e n t r i f u g e d at 10,000xg f o r 10 min. F o l l o w i n g c e n t r i -f u g a t i o n , the s u r f a c e l i p i d was removed with Rayswab^ a p p l i c a t o r s and 8 ml -21-o f the s u p e r n a t a n t was s u b s e q u e n t l y c e n t r i f u g e d at 100,000xg f o r 60 min. The r e s u l t a n t microsomal p e l l e t was washed and resuspended i n 4ml of the a p p r o p r i -a t e b u f f e r . 3) N u c l e a r F r a c t i o n H e p a t i c n u c l e i were i s o l a t e d a c c o r d i n g to the method o f B l o b e l and P o t t e r (1966) as m o d i f i e d by Kashnig and Kasper (1969). At l e a s t 5 grams of l i v e r was homogenized l:2(w/v) i n 0.25M s u c r o s e i n 50mM T r i s H C l , 2.5mM KC1, 5mM MgCl2 » pH 7.5 (0.25M sucrose-TKM), u s i n g a P o t t e r - E l v e j h e m homogenizer with a m o t o r - d r i v e n t e f l o n p e s t l e . The homogenate was f i l t e r e d t hrough 6 l a y e r s o f c h e e s e c l o t h and 10ml o f the f i l t r a t e was added to 20ml of 2.3M sucrose-TKM and mixed t h o r o u g h l y . T h i s was u n d e r l a y e r e d w i t h 5ml o f 2.3M sucrose-TKM and c e n t r i f u g e d at 24,000 rpm f o r 60 min i n a Beckman SW-27 r o t o r . The heavy b r o w n i s h - r e d plaque at the top of the c e l l u l o s e n i t r a t e tube was loosened by rimming w i t h a small s p a t u l a and removed. The su p e r n a t a n t was removed by i n v e r t i n g the tube and a l l o w i n g i t to d r a i n t h r o u g h l y . R e s i d u a l s u c r o s e s o l u t i o n was removed by g e n t l y d i r e c t i n g a stream o f d i s t i l l e d water from a p l a s t i c squeeze b o t t l e a g a i n s t the wa l l of the i n v e r t e d t u b e . A f t e r d r a i n i n g , t h e i n s i d e w a l l o f the tube was c a r e f u l l y c l e a n e d with an absorbent t i s s u e and the n u c l e a r p e l l e t was resuspended by Vortex mixing i n IM sucrose-TKM and c e n t r i f u g e d at 4000xg f o r 10 min. The s u p e r n a t a n t was d i s c a r d e d and a f i n a l washing was performed by r e s u s p e n d i n g the n u c l e i i n 0.25M sucrose-TKM and c e n t r i f u g i n g at l,000xg f o r 10 min. The n u c l e i were then resuspended i n the a p p r o p r i a t e b u f f e r . E l e c t r o n microscopy was employed to v e r i f y the presence o f n u c l e i i n the f i n a l washed p e l l e t . The n u c l e i were f i x e d i n g l u t e r -a l d e h y d e , post f i x e d i n osmium t e t r o x i d e , s t a i n e d with l e a d and viewed i n a C a r l Z e i s s EM-10 t r a n s m i s s i o n e l e c t r o n m i c r o s c o p e . V. Enzyme Assays 1) D e t e r m i n a t i o n o f AHH A c t i v i t y AHH a c t i v i t y was determined a c c o r d i n g to the f l u o r o m e t r i c assay of -22-Gei1 en, Goujon and Nebert (1972). The i n c u b a t i o n was c a r r i e d out i n a f i n a l volume o f 1 ml c o n t a i n i n g : 80 nmole benzo( a ) p y r e n e i n 40^*1 acetone; 0.31ml H 20; 0.5ml 50mM T r i s b u f f e r , pH 7.5; 0.37/Amole NADH; 0.38^imole NADPH; 0.6 mg BSA; 3.45 j l m o l e MgC^; and 0.15 ml microsomal s u s p e n s i o n (50 t o 150y*g p r o t e i n ) or n u c l e i (0.5 t o 1.0 mg p r o t e i n ) . In experiments comparing AHH a c t i v i t i e s between the s u b c e l l u l a r f r a c t i o n s , microsomes and n u c l e i were r e -suspended i n 50mM T r i s b u f f e r , pH 7.5, c o n t a i n i n g 3mM M g C ^ In experiments where o n l y the microsomal f r a c t i o n was s t u d i e d , the microsomes were resuspend-ed i n 0.1M phosphate b u f f e r , pH 7.2. A l l subsequent p r o c e d u r e s were c a r r i e d out under dim l i g h t . The i n c u b a t i o n c o n t e n t s , uncapped and minus s u b s t r a t e , were p r e i n c u b a t e d f o r 1 min 15 s e c , at 37°C, i n a shaking w a t e r b a t h . The r e -a c t i o n was i n i t i a t e d by the a d d i t i o n of s u b s t r a t e , and was c a r r i e d out a t 37°C f o r 5 min. The r e a c t i o n was t e r m i n a t e d by the a d d i t i o n of 1ml i c e c o l d a c e t o n e , f o l l o w e d immediately by 3.3 ml hexane. The tubes were then capped and the c o n t e n t s mixed t h o r o u g h l y with a V o r t e x mixer. Tubes i n c u b a t e d w i t h o u t e i t h e r enzyme, s u b s t r a t e , or c o f a c t o r (NADPH, NADH) but added f o l l o w -i n g t e r m i n a t i o n o f the r e a c t i o n , s e r v e d as b l a n k s . F o r each t u b e , a 2 ml a l i q u o t of the acetone-hexane phase was t r a n s f e r r e d i n t o a second tube con-t a i n i n g 4 ml of IN NaOH. The c o n t e n t s were mixed t h o r o u g h l y and the tube was submerged i n t o a m i x t u r e of dry i c e and acetone u n t i l the aqueous phase was s o l i d i f i e d . The o r g a n i c l a y e r was then poured o f f and the tube was s w i r l e d g e n t l y i n a tub o f luke-warm water. The c o n t e n t s of the tube were then t r a n s -f e r r e d to a 12 x 75mm d i s p o s a b l e c u l t u r e tube and the f l u o r e s c e n c e was q u a n t i -t a t e d u s i n g a Turner** model 430 s p e c t r o f l u o r o m e t e r ( a c t i v a t i o n wavelength, 396nm; e m i s s i o n w a v e l e n g t h , 522nm). The predominant a l k a l i - e x t r a c t a b l e m e t a b o l i t e , 3 - h y d r o x y b e n z o ( a ) p y r e n e , has an a c t i v a t i o n peak at 396nm and an e m i s s i o n maximum a t 522 nm. The s t a n d a r d curve of f l u o r e s c e n c e v e r s u s concen-t r a t i o n of a u t h e n t i c 3-hydroxybenzo(a)pyrene was c o n s t r u c t e d f o l l o w i n g c a l i -b r a t i o n of the f l u o r o m e t e r with a q u i n i n e s u l f a t e s t a n d a r d ( O . l g per 1000 ml -23-0.1N H 2 S O 4 ) . A l l subsequent f i u o r o m e t r i c d e t e r m i n a t i o n s were c a r r i e d out on the f l u o r o m e t e r c a l i b r a t e d with the q u i n i n e s t a n d a r d . The f l u o r e s e n c e o f the blank sample was s u b t r a c t e d from the f l u o r e s c e n c e of the e x p e r i m e n t a l sample. Each e x p e r i m e n t a l sample was assayed i n d u p l i c a t e . AHH a c t i v i t y i s e x p r e s s e d throughout as the amount o f enzyme (mg p r o t e i n ) c a t a l y z i n g the f o r m a t i o n per min at 37°C of h y d r o x y l a t e d product c a u s i n g f l u o r e s c e n c e e q u i v a l e n t t o t h a t o f 1 pmole o f a u t h e n t i c 3-hydroxybenzo(a)pyrene. In experiments s t u d y i n g the e f f e c t s of SKF 525-A on the AHH a c t i v i t y i n  v i t r o , the compound was added i n 0.31ml o f H2O. The compound d i d not i n t e r -f e r e w i t h e i t h e r t he e x t r a c t i o n or f l u o r e s c e n c e of a l k a l i m e t a b o l i t e s at a l l c o n c e n t r a t i o n s employed. The IC5Q was d e f i n e d as the c o n c e n t r a t i o n o f SKF 525-A r e q u i r e d to produce 50% i n h i b i t i o n of AHH a c t i v i t y . T h i s v a l u e was de-t e r m i n e d from the s t r a i g h t l i n e p l o t o f % i n h i b i t i o n v e r s u s t he l o g of the SKF 525-A c o n c e n t r a t i o n f o r each enzyme p r e p a r a t i o n . Three c o n c e n t r a t i o n s of SKF 525-A t h a t produced i n h i b i t i o n i n the 30 t o 70% r e g i o n were used to c o n s t r u c t t h e l i n e . The IC5Q was o b t a i n e d from the a b c i s s a at a p o i n t where the l i n e i n t e r s e c t e d t he o r d i n a t e at an i n h i b i t i o n o f 50%. In the s t u d i e s with o ( - n a p h t h o f l a v o n e , the compound was added t o the tube i n 20jt\ a c e t o n e . C o n t r o l tubes r e c e i v e d 2 0 y a l acetone a l o n e . For thes e ex-perim e n t s b e n z o ( a ) p y r e n e (80nmoles) was added i n 20JJ\ a c e t o n e . Theo(-naph-t h o f l a v o n e d i d not i n t e r f e r e with t he a s s a y . 2) D e t e r m i n a t i o n o f Epo x i d e H y d r o l a s e A c t i v i t y Epoxide h y d r o l a s e a c t i v i t y was determined by the method of Oesch, J e r i n a and Daly (1971). The i n c u b a t i o n was c a r r i e d out i n a t o t a l volume of 0.4 ml c o n t a i n i n g : 0.81y*mole ^H - s t y r e n e o x i d e (55 t o 60 x 10-* dpm) i n 20y*l a c e t o n i t r i l e ; 0.10ml 0.5M T r i s b u f f e r , 0.1% Tween 80, pH 9.0; 0.08 ml H 20; and 0.2ml microsomal s u s p e n s i o n (0.5 t o 2.0mg p r o t e i n ) or n u c l e i (0.5 t o 1.0 mg p r o t e i n ) . The n u c l e a r and microsomal p e l l e t s were resuspended i n 0.5M T r i s b u f f e r , 0.1% Tween 80, pH 9.0. The i n c u b a t i o n c o n t e n t s , minus enzyme, were -24-capped and preincubated for 1 min at 3 7 ° C , in a shaking water bath. The reaction was i n i t i a t e d by the addition of enzyme and was carried out at 3 7 ° C for 10 min. The reaction was terminated by the addition of 10 ml petroleum ether ( 3 0 ° - 6 0 ° C fraction) and the contents were shaken thoroughly with a Vortex mixer. The blank tubes received enzyme after the petroleum ether was added. Each tube was submerged in a container of dry ice and acetone un t i l the aqueous phase was s o l i d i f i e d . The solvent was decanted and an additional 10 ml of petroleum ether was added. After several minutes to allow the aqueous phase to thaw, the contents of the tube were again mixed thoroughly. The tube was submerged into the dry ice-acetone and following s o l i d i f i c a t i o n the petroleum ether was decanted. Then 2 ml of ethyl acetate was added, the contents were mixed thoroughly, and after a period of time to allow the two phases to separate completely, a 0 . 4 ml aliquot of the ethyl acetate phase was sampled. The petroleum ether was used to terminate the reaction and to extract the substrate. The product (styrene glycol) was then extracted with ethyl acetate. The 0 .4 ml aliquot was added to 10 ml Biofluor s c i n t i l l a t i o n f l u i d (New England Nuclear, Dorval, Que.) and the radioactivity was quanti-tated in a Searle Mark III l i q u i d s c i n t i l l a t i o n counter. Quench standards were prepared using fixed volumes of -^-toluene (New England Nuclear, Dorval, Que.), Biofluor and ethyl acetate, while varying the amount of acetone, the quenching agent. The v i a l s were then sealed with wax. The counting efficiency for t r i t i u m in the l i q u i d s c i n t i l l a t i o n counter ranged from 46 to 48%. The recovery of ra d i o a c t i v i t y , determined in every experiment was usually 85 to 95%. Experimental samples were assayed in duplicate. The quantitation of product was obtained from the average dpm of the sample tubes following subtraction of the blank. One mole of styrene glycol i s derived from a mole of styrene oxide. Therefore, from the specific a c t i v i t y of the substrate ( 60 ,000 dpm per 0.81yn.mole) the quantity of product can be derived from the dpm of the sample. Epoxide hydrolase a c t i v i t y i s expressed throughout -25-as nmoles s t y r e n e g l y c o l formed per minute o f i n c u b a t i o n at 37°C per mg p r o t e i n . In e xperiments s t u d y i n g the e f f e c t s o f i n v i t r o a d d i t i o n s o f 1,2-epoxy-3 , 3 , 3 - t r i c h l o r o p r o p a n e on epoxide h y d r o l a s e a c t i v i t y , the compound was added i n 20 yu.1 a c e t o n i t r i l e . C o n t r o l tubes r e c e i v e d a c e t o n i t r i l e a l o n e . For t h e s e experiments n u c l e i were i n c u b a t e d with the s u b s t r a t e f o r 60 min. The com-pound, when added t o sample tubes a f t e r the r e a c t i o n was t e r m i n a t e d , or t o blank t u b e s d i d not a l t e r the r e s u l t a n t l e v e l of r a d i o a c t i v i t y o b t a i n e d i n t h e e t h y l a c e t a t e phase. 3) D e t e r m i n a t i o n o f N-Demethylase A c t i v i t i e s Aminopyrine and e t h y l m o r p h i n e N-demethylase a c t i v i t i e s were e s t i m a t e d by formaldehyde f o r m a t i o n a c c o r d i n g to the methods of El Defrawy El Masry, Cohen and Mannering (1974) and Nash (1953). The microsomal and n u c l e a r p e l l e t s were resuspended i n 0.1 M phosphate b u f f e r , pH 7.2. The i n c u b a t i o n was c a r r i e d out i n a f i n a l volume o f 1.5 ml c o n t a i n i n g : 20yumoles aminopyrine or 15yimoles e t h y l m o r p h i n e i n 100/iA H 20; 1.1 ml o f 64.5 mM T r i s b u f f e r , pH 7.5; lOOyul H2O, 6.3yu.moles MgCl2; 11.2yamoles s e m i c a r b a z i d e , 5 u n i t s g l u c o s e - 6 -phosphate dehydrogenase; lOyixmoles g l u c o s e - 6 - p h o s p h a t e ; lynmole NADP: and 0.2 ml microsomal s u s p e n s i o n (about 2.0 mg p r o t e i n ) or n u c l e i (2.0 t o 4.0 mg p r o t e i n ) . The c o n t e n t s o f the i n c u b a t i o n tube were p r e i n c u b a t e d a t 37°C i n a s h a k i n g water bath f o r 1 min 15 sec and the r e a c t i o n was i n i t i a t e d by the a d d i t i o n o f s u b s t r a t e . The r e a c t i o n was t e r m i n a t e d a f t e r 10 min and 60 min f o r microsomes and n u c l e i , r e s p e c t i v e l y , by the a d d i t i o n o f i c e - c o l d 5% (w/v) 1 aqueous z i n c s u l f a t e . A l l steps were c a r r i e d out i n uncapped t u b e s . The t u b e s were shaken on a V o r t e x mixer and were p l a c e d back on i c e . Two b l a n k s , one minus enzyme, the o t h e r l a c k i n g s u b s t r a t e , r e c e i v e d enzyme and s u b s t r a t e , r e s p e c t i v e l y , f o l l o w i n g the z i n c s u l f a t e . I c e - c o l d s a t u r a t e d barium h y d r o x i d e (0.5 ml) was then added to a l l t u b e s , the c o n t e n t s were mixed and the -26-t u b e s were c e n t r i f u g e d at lOOOxg f o r 15 min. To 0.5 ml of the Nash reagent (30g ammonium a c e t a t e and 0.4 ml a c e t y l acetone i n 100 ml H2O) was added 1.5 ml o f the s u p e r n a t a n t . The tubes were i n c u b a t e d f o r 15 min a t 60°C i n a shak-i n g water b a t h . The tubes were a l l o w e d to c o o l and the i n t e n s i t y of the y e l l o w c o l o r produced was q u a n t i t a t e d a t 412 nm on a Coleman model 124 H i t a c h i double-beam g r a t i n g s p e c t r o p h o t o m e t e r . The q u a n t i t y of formaldehyde was dete r m i n e d from the s t a n d a r d c u r v e of absorbance (412 nm) versus formaldehyde c o n c e n t r a t i o n . The absorbance o f the blanks was s u b t r a c t e d from e x p e r i m e n t a l samples. Each enzyme sample was assayed i n d u p l i c a t e . The N-demethylase a c t i v i t i e s are e x p r e s s e d as nmoles formaldehyde formed per min per mg p r o t e i n . 4) D e t e r m i n a t i o n o f A n i l i n e H y d r o x y l a s e A c t i v i t y A n i l i n e h y d r o x y l a s e a c t i v i t y was e s t i m a t e d by p-aminophenol p r o d u c t i o n ( I m a i , I t o and S a t o , 1966). The microsomal and n u c l e a r p e l l e t s were r e s u s -pended i n 0.1 M phosphate b u f f e r , pH 7.2. The i n c u b a t i o n was c a r r i e d out i n a t o t a l volume of 2.0 ml c o n t a i n i n g : 2.5yumole a n i l i n e i n 0.5 ml H2O; 0.25 ml 0.1 M phosphate b u f f e r , pH 7.2; 1.0 ml 0.1M phosphate b u f f e r , pH 7.4; 2.5yu mole NADP: 5yamole g l u c o s e - 6 - p h o s p h a t e ; 2.5 u n i t s glucose-6-phosphate dehydro-genase; 12.5ymmoles MgCl2i and 0.25 ml microsomal s u s p e n s i o n (about 2.5 mg p r o t e i n ) or n u c l e i (2.0 t o 3.0 mg p r o t e i n ) . The i n c u b a t i o n c o n t e n t s , minus s u b s t r a t e , were p r e i n c u b a t e d f o r 1 min 15 sec at 37°C, i n a shak i n g water b a t h . The r e a c t i o n was i n i t i a t e d by the a d d i t i o n of enzyme and was c a r r i e d out at 37°C f o r 15 min and 60 min w i t h microsomes and n u c l e i , r e s p e c t i v e l y . The r e a c t i o n was t e r m i n a t e d by the a d d i t i o n o f 1.0 ml i c e - c o l d 20% (w/v) aqueous t r i c h l o r o a c e t i c a c i d , f o l l o w e d by thorough m i x i n g . A l l s t e p s were c a r r i e d out i n uncapped t u b e s . S u b s t r a t e and enzyme were then added to the b l a n k tubes t h a t were i n c u b a t e d without s u b s t r a t e or enzyme, r e s p e c t i v e l y . The tubes were then c e n t r i f u g e d a t l,000xg f o r 5 min and 1.0 ml of s u p e r n a t a n t was sampled i n t o tubes c o n t a i n i n g 0.5 ml 10% (w/v) aqueous sodium c a r b o n a t e and mixed. One ml of 2% (w/v) phenol i n 0.2N NaOH was added t o each tube and th e tubes were i n c u b a t e d f o r 30 min a t 37°C. The c o l o r produced was q u a n t i -t a t e d at 640 nm. The q u a n t i t y of p-aminophenol was e s t i m a t e d from the s t a n d -ard c u r v e of absorbance (640 nm) versus p-aminophenol c o n c e n t r a t i o n . The absorbance of the blanks was s u b t r a t e d from t he expe r i m e n t a l samples. Each enzyme sample was assayed i n d u p l i c a t e . A n i l i n e h y d r o x y l a s e a c t i v i t y i s e x p r e s s e d throughout as nmoles p-aminophenol formed per min per mg p r o t e i n . 5) D e t e r m i n a t i o n o f T e s t o s t e r o n e A ^ - H y d r o g e n a s e A c t i v i t y T e s t o s t e r o n e A 4 - h y d r o g e n a s e a c t i v i t y was e s t i m a t e d , by measuring i n a spe c t r o p h o t o m e t e r the d e c r e a s e i n o p t i c a l d e n s i t y a t 240 nm which o c c u r r e d w i t h the s a t u r a t i o n o f the 4-5 double bond, a c c o r d i n g to the method of McGuire, J r . and Tomkins (1959). The microsomal and n u c l e a r p e l l e t s were resuspended i n 50 mM t r i s b u f f e r , pH 7.5, c o n t a i n i n g 3 mM M g C ^ The i n c u -b a t i o n was c a r r i e d out i n a f i n a l volume o f 2 ml c o n t a i n i n g : 0.3yumole t e s t o s t e r o n e i n 20yJi methanol; 12.5^imole MgC^; l.Oyjimole NADP; 20ynrtiole g l u c o s e - 6 - p h o s p h a t e ; 1 u n i t g lucose-6-phosphate dehydrogenase; 1.78 ml 0.1 M phosphate b u f f e r , pH 7.2; and 200yuA o f microsomal s u s p e n s i o n (0.4 t o 1.2 mg p r o t e i n ) or n u c l e i (1.3 t o 2.0 mg p r o t e i n ) . The i n c u b a t i o n c o n t e n t s , w i t h o u t s u b s t r a t e , were p r e - i n c u b a t e d f o r 1 min 15 sec at 37°C, i n a shaki n g water b a t h . The r e a c t i o n was s t a r t e d w i t h t he a d d i t i o n of s u b s t r a t e and was c a r r i e d out at 37°C f o r 10 min. The r e a c t i o n was t e r m i n a t e d by the a d d i t i o n of 4 ml di c h l o r o m e t h a n e , the tubes were capped and the c o n t e n t s mixed t h o r o u g h l y . The bla n k s were tubes i n c u b a t e d without enzyme, however enzyme was added a f t e r d i c h l o r o m e t h a n e . The tubes were c e n t r i f u g e d a t l,000xg f o r 15 min, the upper aqueous phase was siphone d o f f and the o r g a n i c phase was t r a n s f e r r e d to a c u v e t t e and scanned i n the s p e c t r o p h o t o m e t e r between 300 and 220 nm. A permanent t r a c e was o b t a i n e d u s i n g a Coleman model 165 H i t a c h i r e c o r d e r . T e s t o s t e r o n e i n d i c h l o r o m e t h a n e y i e l d s an absorbance maximum a t 240 nm. The - 2 8 -e x t r a c t i o n of t e s t o s t e r o n e from the aqueous phase i n t o d i c h l o r o m e t h a n e was g r e a t e r than 95%. Other s t e r o i d s p o s s e s s i n g t h e 4-5 double bond, such as a n d r o s t e n e d i o n e , i n e q u i m o l a r c o n c e n t r a t i o n s , produced i d e n t i c a l absorbance maxima. Equi m o l a r c o n c e n t r a t i o n s of s t e r o i d s s a t u r a t e d at t h e 4-5 p o s i t i o n , such as 5< K-dihydrotestosterone, d i d not absorb at 240 nm. M e t a b o l i s m of t e s t o s t e r o n e at any o t h e r p o s i t i o n w i l l not r e s u l t i n a l o s s of absorbance a t 240 nm, as l o n g as the 4-5 d o u b l e bond remains i n t a c t . The a c t i v i t y i s r e f e r r e d t o as t e s t o s t e r o n e A 4 - h y d r o g e n a s e , s i n c e t o t a l s a t u r a t i o n of the double bond i s b e i n g measured, and the two p r o d u c t s of r e d u c t i o n , 5<*-and 5^ - d i h y d r o t e s t o s t e r o n e were not s e p a r a t e d . A s t a n d a r d c u r v e r e l a t i n g maximum absorbance w i t h t e s t o s t e r e n e c o n c e n t r a t i o n was c o n s t r u c t e d and the d i f f e r e n c e i n t e s t o s t e r o n e c o n c e n t r a t i o n between the enzyme blank (no r e d u c t i o n ) and the sample was r e p r e s e n t a t i v e of the amount of t e s t o s t e r o n e reduced. A l l samples were assayed i n d u p l i c a t e . T e s t o s t e r o n e A 4 - h y d r o g e n a s e i s e x p r e s s e d as nmoles t e s t o s t e r o n e reduced per min per mg p r o t e i n . 6. D e t e r m i n a t i o n o f E t h o x y r e s o r u f i n O-Deethylase A c t i v i t y E t h o x y r e s o r u f i n 0 - d e e t h y l a s e a c t i v i t y was determined a c c o r d i n g to t h e f l u o r o m e t r i c assay of Burke and Mayer (1974). The microsomal p e l l e t s were resuspended i n 0.1 M phosphate b u f f e r , pH 7.2. The i n c u b a t i o n was c a r r i e d out i n a f i n a l volume of 2.15 ml c o n t a i n i n g : 500 pmoles e t h o x y r e s o r u f i n i n 25y<4 1.25% (w/v) aqueous Tween 80; 2 ml 0.1 M phosphate b u f f e r , pH 7.8; NADPH i n 25 jA 0.1 M phosphate b u f f e r , pH 7.8; and 100yuA o f microsomal s u s p e n s i o n (0.8 t o 1.3 mg p r o t e i n except when microsomes from 3 - m e t h y l c h o l a n t h r e n e - t r e a t e d a n i m a l s were used, 8 t o 13 g p r o t e i n ) . The assay was performed i n 12 x 75 mm d i s p o s a b l e c u l t u r e t u b e s . The t u b e s , minus NADPH, were p r e i n c u b a t e d a t 30°C f o r 1 min i n a s h a k i n g water bath and the r e a c t i o n was i n i t i a t e d w i t h a d d i t i o n o f NADPH. The f o r m a t i o n of the f l u o r e s c e n t p r o d u c t , r e s o r u f i n , was -29-f o l l o w e d at an a c t i v a t i o n wavelength o f 510 nm and an e m i s s i o n wavelength of 586 nm. The tube was m a i n t a i n e d a t 30°C i n the f l u o r o m e t e r chamber with a t e m p e r a t u r e c o n t r o l l e d water j a c k e t . The i n i t i a l r e a c t i o n r a t e s were d e t e r -mined from the c h a r t r e c o r d e r t r a c i n g . Each sample was assayed i n d u p l i c a t e . A s t a n d a r d c u r v e was c o n s t r u c t e d u s i n g a u t h e n t i c r e s o r u f i n . The f l u o r o m e t e r was c a l i b r a t e d each time w i t h a s t a n d a r d s o l u t i o n of r e s o r u f i n (5y^M r e s o r u f i n i n e t h a n o l ) . A l l p r o c e d u r e s were c a r r i e d out under dim l i g h t . E t h o x y r e s o r u f i n 0 - d e e t h y l a s e a c t i v i t y i s expressed as pmoles r e s o r u f i n formed per min per mg p r o t e i n . 7) Assay C o n d i t i o n s The f o r m a t i o n o f p r o d u c t , or l o s s o f s u b s t r a t e , was l i n e a r w i t h i n c u b a -t i o n time and p r o t e i n c o n t e n t f o r a l l enzyme assays employed. In a d d i t i o n , s u b s t r a t e and c o f a c t o r c o n c e n t r a t i o n s u t i l i z e d y i e l d e d optimal enzyme a c t i v i t y . The t e s t o s t e r o n eA ^ - h y d r o g e n a s e assay was an e x c e p t i o n . A l t h o u g h the a c t i v i t y was l i n e a r w i t h r e s p e c t to i n c u b a t i o n time and p r o t e i n c o n t e n t , t h e c o n c e n t r a t i o n o f s u b s t r a t e used was below enzyme s a t u r a t i o n . S i n c e the absorbance o f t e s t o s t e r o n e a t 240 nm reached a maximum j u s t beyond a s u b s t r a t e c o n c e n t r a t i o n o f 0.2 nM, l a r g e changes i n t e s t o s t e r o n e c o n c e n t r a t i o n y i e l d e d o n l y s l i g h t d i f f e r e n c e s i n absorbance and, thus a l o s s of assay s e n s i t i v i t y . However, changes i n t e s t o s t e r o n e c o n c e n t r a t i o n c o u l d be measured a c c u r a t e l y at a s u b s t r a t e c o n c e n t r a t i o n below enzyme s a t u r a t i o n (0.15 nM) and, most impor-t a n t l y , the l o s s o f t e s t o s t e r o n e was l i n e a r w i t h i n c u b a t i o n time and p r o t e i n c o n t e n t at t h i s s u b s t r a t e c o n c e n t r a t i o n . Only i n i t i a l v e l o c i t i e s were used. V I . Sodium Dodecyl S u l f a t e - P o l y a c r y l a m i d e Gel E l e c t r o p h o r e s i s Sodium dodecyl s u l f a t e - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s was performed a c c o r d i n g t o the p r o t o c o l of Weber and Osborne (1969). The gel rod was 10 cm l o n g , 0.6 cm i n diameter and c o n s i s t e d o f 7.5% (w/v) a c r y l a m i d e , 0.057% (w/v) ammonium p e r s u l f a t e and 0.11% (v/v) N , N , N ' , N ' - t e t r a m e t h y l - e t h y l e n e d i a m i n e , -30-with a gel buffer of 0.1 M phosphate, pH 7.0, containing 0.1% sodium dodecyl su l fa te . A 3 5 a l i q u o t of microsomal protein (1 ^g/yx\), in 0.1 M phosphate buf fer , pH 7.2, was adjusted to 40yj\ with H2O. To the protein solut ion was added 40JJ\ of a solut ion containing 3.3% (w/v) sodium dodecyl su l fa te ; 0.02% (w/v) bromophenol blue (tracking dye), 16.7% (v/v) mercaptoethanol and 30% (v/v) g l yce ro l , in 0.5 M phosphate buf fer , pH 7.0. This solut ion was then heated for 5 min at 100°C. The solut ion was allowed to cool and a 50y*\ a l iquo t , containing 22yo.g protein was applied to the ge l . Electrophoresis was car r ied out at a constant current of 10 mA per tube for approximately 5 hours at room temperature. This was the approximate time necessary for the blue t racking dye to migrate unt i l 5 mm from the end of the g e l . The proteins were stained overnight with coomassie b r i l l i a n t blue G (0.025% (w/v) in g lac ia l acet ic ac id : isopropyl a lcoho l : water, 100:100:1000) and then destained and stored in isopropyl a lcoho l : g lac ia l acet ic ac id : water, 100:100:1000. The gels were scanned at 550 nm with a G i l fo rd Model 250 spectrophotometer with a gel-scanning attachment. Protein bands were loca l ized in the coomassie blue stained gels by comparison with molecular weight standards (Pharmacia Low Molecular Weight Ca l ib ra t ion K i t ) , phosphorylase B (mol. wt. 94,000), bovine serum albumin (mol. wt. 67,000), ovalbumin (mol. wt. 45,000), carbonic anhydrase (mol. wt. 30,000), t rypsin inh ib i to r (mol. wt. 20,100) and oc - lactalbumin (mol. wt. 14,400). VII. Determination of Proteins Proteins were estimated by the method of Sutherland and coworkers (1949) as modified by Robson, Goll and Temple (1968), with bovine serum albumin as the standard. V I I I . S t a t i s t i c a l Analyses The Student's t - tes t (unpaired sample means, two-tai led) and the two-way analysis of variance (AN0VA) were used to s t a t i s t i c a l l y evaluate the data. -31-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 p<0.05. The e f f e c t s produced by a combin a t i o n of t r e a t m e n t s were a n a l y z e d with the two-way ANOVA (the i n t e r a c -t i o n e f f e c t ) . A s i g n i f i c a n t i n t e r a c t i o n o c c u r s when the e f f e c t s produced by a com b i n a t i o n of t r e a t m e n t s are g r e a t e r or l e s s than the sum of the e f f e c t s pro-duced by each t r e a t m e n t alone ( n o n - a d d i t i v e ) . The i n t e r a c t i o n i s not s i g n i f i -c a nt when a combination of treatments produces an e f f e c t equal to the sum o f the e f f e c t s produced by each t r e a t m e n t a l o n e ( a d d i t i v e ) . -32-RESULTS I . N u c l e a r L o c a l i z a t i o n and Enzymatic P r o f i l e o f Rat H e p a t i c Drug M e t a b o l i z i n g Enzymes 1) E l e c t r o n M i c r o g r a p h s And R e c e n t r i f u g a t i o n o f H e p a t i c N u c l e i E l e c t r o n microscopy was employed i n o r d e r t o 1) v e r i f y the presence o f n u c l e i i n the washed p e l l e t o b t a i n e d f o l l o w i n g d i s c o n t i n u o u s s u c r o s e g r a d i e n t c e n t r i f u g a t i o n of l i v e r homogenate and 2) judge whether h e p a t i c microsomes cosedimented w i t h t h e s e n u c l e i . R e p r e s e n t a t i v e e l e c t r o n micrographs are p r e s e n t e d i n F i g u r e 1. The e l e c t r o n micrographs c l e a r l y i n d i c a t e d the p r e s e n c e of n u c l e i , however the magnitude o f microsomal c o n t a m i n a t i o n c o u l d not be determined with any degree of a c c u r a c y . The o c c l u s i o n o f microsomes by the l a y e r of 2.3 M sucrose-TKM was v e r i f i e d b i o c h e m i c a l l y , u s i n g AHH a c t i v i t y as the marker. The r e s u l t s o f t h e s e c e n t r i f u g a t i o n experiments are shown i n T a b l e I. AHH a c t i v i t y i n i s o l a t e d h e p a t i c n u c l e i from a d u l t male r a t s was found to be 304±42(8) pmoles per min per mg p r o t e i n . There was a p p r o x i m a t e l y 1% of the c o n t r o l n u c l e a r AHH a c t i v i t y i n the f i l m at the bottom of the tube when microsomes a l o n e (an e q u i v a l e n t amount t o t h a t p r e s e n t i n the l i v e r homogenate used t o p r e p a r e n u c l e i ) were s u b j e c t e d to d i s c o n t i n u o u s s u c r o s e g r a d i e n t c e n t r i f u g a -t i o n (3.41+1.5 (8) pmoles per min per mg p r o t e i n ) . In a d d i t i o n , when i s o l a t e d n u c l e i were s u b j e c t e d to f u r t h e r d i s c o n t i n u o u s s u c r o s e g r a d i e n t c e n t r i f u g a t i o n , i n the presence or absence of microsomes, the AHH a c t i v i t y was the same as t h a t of c o n t r o l . T h e r e f o r e , endoplasmic r e t i c u l u m , as microsomes formed upon h o m o g e n i z a t i o n , d i d not account f o r the AHH a c t i v i t y o b s erved i n the n u c l e a r f r a c t i o n . -33-F i g u r e 1. E l e c t r o n micrographs o f i s o l a t e d r a t l i v e r n u c l e i . Samples were f i x e d i n g l u t e r a l d e h y d e , post f i x e d i n osmium t e t r o x i d e , s t a i n e d w i t h l e a d and viewed i n a C a r l Z e i s s EM-10 t r a n s m i s s i o n e l e c t r o n m i c r o s c o p e . The m a g n i f i c a t i o n s are as f o l l o w s : 15.300(A), 18,700(B) and 22.100(C). - 3 4 --35-T a b l e I . R e c e n t r i f u g a t i o n experiments C o n d i t i o n A r y l hydrocarbon h y d r o x y l a s e a c t i v i t y pmoles product/min/mg p r o t e i n * SEM(n) c o n t r o l n u c l e i 3 304 + 42 (8) r e c e n t r i f u g a t i o n b 217 t 27 ( 8 ) n s r e c e n t r i f u g a t i o n + m i c r o s o m e s 0 253 ± 32 ( 8 ) n s microsomes a l o n e ^ 3.41 t 1.5 (8) a N u c l e a r p e l l e t resuspended i n 2 ml 50 mM T r i s HC1, 3 mM M g C ^ s pH 7.5 ^ N u c l e a r p e l l e t resuspended i n 2 ml 0.25 M sucrose-TKM and 8 ml 0.25 M sucrose-TKM was added. T h i s was mixed w i t h 20 ml 2.3 M sucrose-TKM and n u c l e i were i s o l a t e d as d e s c r i b e d i n Methods. c N u c l e a r p e l l e t resuspended i n 2 ml 0.25 M sucrose-TKM and 8 ml o f 10,000 xg s u p e r n a t a n t (from l i v e r homogenized 1:2 i n 0.25 M sucrose-TKM) was added. T h i s was mixed w i t h 20 ml 2.3 M sucrose-TKM and n u c l e i were i s o l a t e d as d e s c r i b e d i n Methods. ^10 ml of 10,000 xg s u p e r n a t a n t (from l i v e r homogenized 1:2 i n 0.25 M sucrose-TKM) was mixed w i t h 20 ml 2.3 M sucrose-TKM and c e n t r i f u g e d as d e s c r i b e d i n Methods. l S N o t s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l ( S t u d e n t ' s t - t e s t , u n p a i r e d sample means, 2 - t a i l e d ) at p<0.05. -36-2) Basal Enzyme L e v e l s : A Comparison o f N u c l e i and Microsomes The s p e c i f i c a c t i v i t i e s of s e v e r a l enzymes of microsomal o r i g i n were det e r m i n e d i n the i s o l a t e d n u c l e i , and compared with t h e i r r e s p e c t i v e a c t i v i t i e s i n the microsomal f r a c t i o n . A d u l t male r a t s were used i n t h e s e s t u d i e s . The r e s u l t s are p r e s e n t e d i n T a b l e I I . AHH, aminopyrine N-demethylase and a n i l i n e h y d r o x y l a s e a c t i v i t i e s i n the i s o l a t e d n u c l e i were a p p r o x i m a t e l y 4% o f the r e s p e c t i v e a c t i v i t i e s observed i n the microsomes. However, n u c l e a r epoxide h y d r o l a s e and t e s t e s t e r o n e A 4 - h y d r o g e n a s e a c t i v i t i e s were about 12.5 and 15% of t h e i r r e s p e c t i v e microsomal a c t i v i t i e s . The r a t i o o f epox i d e h y d r o l a s e to AHH i n h e p a t i c microsomes was 1.6:1, whereas t h i s r a t i o o f a c t i v i t i e s i n h e p a t i c n u c l e i was 4.5:1. The y i e l d of microsomal p r o t e i n was a p p r o x i m a t e l y an o r d e r of magnitude g r e a t e r than n u c l e a r p r o t e i n . The y i e l d of n u c l e a r p r o t e i n per gram of l i v e r averaged 2.7 mg (range 2.4 t o 2.9 mg), whereas the microsomal f r a c t i o n r e p r e s e n t e d , on the av e r a g e , 27.5 mg (range 24 t o 31 mg). When the n u c l e a r and microsomal a c t i v i t i e s were e x p r e s s e d per gram of l i v e r , t h e cytochrome P-450 dependent monooxygenase a c t i v i t i e s i n the i s o l a t e d n u c l e a r f r a c t i o n were about 0.4% o f the r e s p e c t i v e a c t i v i t i e s observed i n the microsomes, whereas th e comparable r a t i o o f t e s t o s t e r o n e A 4 - h y d r o g e n a s e and epoxide h y d r o l a s e i n th e two f r a c t i o n s were 1.7 and 1.3%, r e s p e c t i v e l y ( T a b l e I I I ) . T h e r e f o r e , t h e n u c l e a r enzymes c o n t r i b u t e r e l a t i v e l y l i t t l e to the o v e r a l l metabolism of s u b s t r a t e s by the h e p a t o c y t e . 3) AHH A c t i v i t y , Apparent Km and Vmax The apparent Km and Vmax v a l u e s f o r h e p a t i c microsomal and n u c l e a r AHH a c t i v i t y were determined from the M i c h a e l i s curves of enzyme v e l o c i t y (Vo) ve r s u s s u b s t r a t e c o n c e n t r a t i o n ( [ A o ] ) shown i n F i g u r e 2. The v a l u e s of Km and Vmax, as determined by i n s p e c t i o n of the M i c h a e l i s p l o t or from t he H o f s t e e -37-T a b l e I I . Basal enzyme a c t i v i t i e s ( p e r mg p r o t e i n ) i n h e p a t i c n u c l e i and microsomes. Enzymatic a c t i v i t i e s are expressed as nmoles per minute per mg p r o t e i n . The number i n par e n t h e s e s i n d i c a t e s the sample s i z e . A d u l t male r a t s were used. Enzymatic a c t i v i t y N u c l e i (A) Microsomes (B) (A/B x 100) A n i l i n e h y d r o x y l a s e 0.043 (13) 1.13 (15) 3.8 A r y l hydrocarbon h y d r o x y l a s e 0.14 (11) 3.17 (11) 4.4 Aminopyrine N-demethylase 0.09 (12) 2.45 (30) 3.7 Epox i d e h y d r o l a s e 0.63 (12) 5.03 (16) 12.5 T e s t o s t e r o n e A . 4 _ h y < j r 0 g e n a s e 1.68 (8) 11.4 (9) 14.7 -38-T a b l e I I I . Basal enzyme a c t i v i t i e s ( p e r gram l i v e r ) i n h e p a t i c n u c l e i and microsomes. Enzymatic a c t i v i t i e s are expressed as nmoles per minute per gram wet weight of l i v e r . The number i n par e n t h e s e s i n d i c a t e s the sample s i z e . A d u l t male r a t s were used. Enzymatic a c t i v i t y N u c l e i (A) Microsomes (B) (A/B x 100) A n i l i n e h y d r o x y l a s e 0.136 (13) 33.3 (15) 0.41 A r y l hydrocarbon h y d r o x y l a s e 0.40 (11) 97.0 (ID 0.41 Aminopyrine N-demethylase 0.29 (12) 73.5 (30) 0.39 Epoxide h y d r o l a s e 1.57 (12) 122.1 (16) 1.29 T e s t o s t e r o n e ^5-hydrogenase 46.6 (8) 2702 (9) 1.72 -39-F i g u r e 2. The Michaelis curves of enzyme velocity (Vo) versus substrate concentration ([Ao]) for hepatic nuclear and microsomal aryl hydrocarbon hydroxylase ac t iv i ty . Each point represents the average of duplicate deter-minations. -40-MICROSOMES Benzo(a)pyrene Concentration(pM) NUCLEI -41-p l o t of Vo ver s u s Vo/[Ao] ( F i g u r e 3) are p r e s e n t e d i n T a b l e IV. The apparent Vmax v a l u e s f o r n u c l e a r and microsomal AHH a c t i v i t y were d i f f e r e n t , l i k e l y as a r e s u l t of d i f f e r e n c e s i n the enzyme to p r o t e i n r a t i o between the two f r a c -t i o n s . U n l e s s the enzyme s t u d i e d i s of high p u r i t y , the appa r e n t Vmax v a l u e s are o f l i t t l e meaning. However, the apparent Km v a l u e s , which are independent o f p r o t e i n c o n t e n t and are crude e s t i m a t e s of the enzyme's a f f i n i t y f o r s u b s t r a t e , were s i m i l a r . 4) Sex and Age Dependent Enzyme A c t i v i t i e s The sex and age dependency of h e p a t i c microsomal AHH i s shown i n T a b l e V. The AHH a c t i v i t i e s were s i m i l a r i n the immature r a t , but i n c r e a s e d dramat-i c a l l y upon m a t u r i t y o f t h e male. The a c t i v i t y i n the female remained v i r t u a l l y unchanged. The same p a t t e r n was e x h i b i t e d by t h i s enzyme i n the h e p a t i c n u c l e i . Microsomal t e s t o s t e r o n e A 4 - h y d r o g e n a s e a c t i v i t y was s i g n i f i c a n t l y g r e a t e r i n the a d u l t female than the a d u l t male ( T a b l e V I ) . The t e s t o s t e r o n e A 4 - h y d r o g e n a s e a c t i v i t y i n i s o l a t e d n u c l e i e x h i b i t e d the same sex d i f f e r e n c e . 5) I n h i b i t i o n And A c t i v a t i o n o f AHH A c t i v i t y The a b i l i t y of SKF 525-A, added d i r e c t l y to the i n c u b a t i o n tube, t o i n h i b i t h e p a t i c AHH a c t i v i t y was s t u d i e d . The r e s u l t s of thes e experiments a r e shown i n F i g u r e 4. The SKF 525-A produced a c o n c e n t r a t i o n - d e p e n d e n t i n h i b i t i o n of AHH a c t i v i t y . The i n h i b i t i o n o f AHH a c t i v i t y by SKF 525-A was i d e n t i c a l i n both n u c l e a r and microsomal f r a c t i o n s o b t a i n e d from c o n t r o l , p h e n o b a r b i t a l - o r 3 - m e t h y l c h o l a n t h r e n e - t r e a t e d r a t s . The e f f e c t s of<*-naphthof1avone added to the i n c u b a t i o n t u b e , on h e p a t i c v. AHH a c t i v i t y , were i n v e s t i g a t e d . The f i n d i n g s are summarized i n T a b l e V I I . The e f f e c t s seen i n the microsomal f r a c t i o n were i n d i s t i n g u i s h a b l e from t h o s e -42-F i g u r e 3. The H o f s t e e p l o t of enzyme v e l o c i t y (Vo) versus enzyme v e l o c i t y o v e r s u b s t r a t e c o n c e n t r a t i o n (Vo/[Ao]) f o r h e p a t i c n u c l e a r and microsomal a hydrocarbon h y d r o x y l a s e a c t i v i t y . Each p o i n t r e p r e s e n t s t he average of d u p l i c a t e d e t e r m i n a t i o n s . - 4 3 -MICROSOMES Vo nM product/min/mg/pM Benzo(a)pyrene NUCLEI Vo nM product/min/mg/pM Benzo(a)pyrene - 4 4 -T a b l e IV. Apparent Km and Vmax v a l u e s f o r h e p a t i c microsomal and n u c l e a r a r y l hydrocarbon h y d r o x y l a s e a c t i v i t y . A d u l t male r a t s were used i n t h e s e e x p e r i m e n t s . Method and Source o f Enzyme I n s p e c t i o n of M i c h a e l i s curve-microsomes n u c l e i Km / A M 6.0 7.5 Vmax nM product/min/mg p r o t e i n 2160 148 H o f s t e e p l o t -microsomes n u c l e i 8.7 10.2 2471 152 -45-T a b l e V. The sex and age dependency o f h e p a t i c a r y l hydrocarbon hy-d r o x y l a s e a c t i v i t y . A nimals A r y l hydrocarbon h y d r o x y l a s e a c t i v i t y pmoles/min/mg p r o t e i n 1 SEM(n) Immature-mal e female Microsomes N u c l e i 750 t 26 (8) 54.7 + 6 (8) 580 t 26 (8) 24.0 t 1.3 (8) A d u l t -mal e female 3086 t 503 (4) 541 t 91 (4) 209 t 19 (4) 17.5 t 4.6 (4) -46-T a b l e V I . H e p a t i c t e s t o s t e r o n e A 7-hydrogenase a c t i v i t y i n a d u l t male and female r a t s . Animal T e s t o s t e r o n e A 4 - h y d r o g e n a s e a c t i v i t y nmoles t e s t o s t e r o n e reduced/min/mg p r o t e i n ! SEM(n) Microsomes N u c l e i A d u l t male 11.4 ± 1.2 (9) 1.68 + 0.3 (8) A d u l t female 40.1 t 2.2 (5) 4.01 t 0.3 (5) -47-F i g u r e 4 . Inhibition of rat hepatic aryl hydrocarbon hydroxylase (AHH) activity by SKF 525-A in vitro. Phenobarbital treated (80 mg/kg/day ip for 4 consecutive days), 3-methylcholanthrene treated (20 mg/kg/day ip for 2 days), or control rats (220-300 g) were employed. SKF 525-A was added to the tubes prior to incubation. Each point represents the average of 4 to 6 experiments. All standard errors were less than 10% of the means. -48-• MICROSOMES vNUCLEI log concentration(M) SKF525-A -49-T a b l e V I I . The e f f e c t s of c x-naphthoflavone i n v i t r o on h e p a t i c a r y l hy-d r o c a r b o n h y d r o x y l a s e a c t i v i t y . The «*-naphthoflavone (10" 5M) was added i n 20 jt\ a c e t o n e . C o n t r o l tubes r e c e i v e d 20 jA a c e t o n e . In t h e s e experiments 80 nmoles benzo(a)pyrene was added i n 20 jA a c e t o n e . A d u l t r a t s were used. Male Female Treatment Microsomes N u c l e i Microsomes N u c l e i None +21* 3 ( 1 8 ) a + 1 7 ± 2(15) no e f f e c t no e f f e c t P h e n o b a r b i t a l +24* 4(8) +23 ± 3(4) +13* 3(4) +15 1 3(4) 3 - M e t h y l c h o l a n t h r e n e -20* 3(4) - 2 0 * 5(4) -26 * 7(4) -32 ± 1(4) a E x p r e s s e d as % s t i m u l a t i o n (+) or % i n h i b i t i o n (-) + SEM(n). The c o n t r o l a c t i v i t i e s i n the u n t r e a t e d groups were: female microsomes, 404 + 3 3 ( 4 ) ; female n u c l e i , 31.6 + 3.1(4); male microsomes, 4743 + 60(18); male n u c l e i , 163 + 8 ( 1 5 ) ; i n the p h e n o b a r b i t a l groups, female microsomes, 1870 + 7 6 ( 4 ) ; female n u c l e i , 54.6 + 6.1(4); male microsomes, 2172 + 220(4); male n u c l e i , 120 + 1 0 ( 4 ) ; and i n the 3 - m e t h y l c h o l a n t h r e n e groups; female microsomes, 6315 + 580(4); female n u c l e i , 409 + 4 4 ( 4 ) ; male microsomes, 7897 + 5 2 0 ( 4 ) ; male n u c l e i , 380 +43(4) pmoles/min/mg + SEM(n). -50-i n the n u c l e a r f r a c t i o n . In the 3 - m e t h y l c h o l a n t h r e n e t r e a t e d r a t s , AHH a c t i v i t y was i n h i b i t e d by<x.-naphthoflavone, whereas i n the c o n t r o l male and t h e p h e n o b a r b i t a l - t r e a t e d male and female groups the compound s t i m u l a t e d AHH a c t i v i t y . T h e o c-naphthoflavone had no e f f e c t on AHH i n c o n t r o l female r a t s . A s i n g l e c o n c e n t r a t i o n o f o < - n a p t h t h o f l a v o n e (lO'^M) was employed i n a l l t h e s e s t u d i e s . 6) A l t e r a t i o n s In H e p a t i c AHH A c t i v i t y F o l l o w i n g Chemical A d m i n i s t r a t i o n The e f f e c t s of v a r i o u s agents on h e p a t i c AHH a c t i v i t y f o l l o w i n g t h e i r i n t r a p e r i t o n e a l a d m i n i s t r a t i o n were i n v e s t i g a t e d . A d e c r e a s e i n h e p a t i c AHH a c t i v i t y was produced f o l l o w i n g a d m i n i s t r a t i o n of t r a n s - s t i l b e n e oxide t o a d u l t male r a t s . These r e s u l t s are p r e s e n t e d i n F i g u r e 5. F o l l o w i n g t r a n s -s t i l b e n e oxide t r e a t m e n t , a 50% d e c r e a s e i n both n u c l e a r and microsomal AHH a c t i v i t y was n o t e d . The e f f e c t s o f p h e n o b a r b i t a l t r e a t m e n t i n the immature male and female r a t , as w e l l as i n the a d u l t male r a t were s t u d i e d . The r e s u l t s of t h e s e e x p e r i m e n t s are shown i n T a b l e V I I I . In the immature female r a t , pheno-b a r b i t a l t r e a t m e n t i n c r e a s e d h e p a t i c microsomal AHH by 500%. The i n c r e a s e i n n u c l e a r AHH produced by p h e n o b a r b i t a l was o n l y 215%. S i m i l a r l y , i n the immature male r a t , the i n c r e a s e s observed i n h e p a t i c microsomal AHH a c t i v i t y were s i g n i f i c a n t l y g r e a t e r than t h a t noted f o r n u c l e a r AHH a c t i v i t y f o l l o w i n g p h e n o b a r b i t a l t r e a t m e n t . In the a d u l t male r a t , h e p a t i c AHH a c t i v i t y was u n a f f e c t e d by p h e n o b a r b i t a l t r e a t m e n t , r e g a r d l e s s o f the source of enzyme. A d u l t female r a t s were a d m i n i s t e r e d p h e n o b a r b i t a l f o r v a r y i n g l e n g t h s o f t i m e t o determine whether 1) t h e i n c r e a s e i n n u c l e a r AHH a c t i v i t y f o l l o w i n g 4 days o f p h e n o b a r b i t a l was maximum by t h i s time p o i n t , o r 2) f u r t h e r t r e a t m e n t w i t h the drug would r e s u l t i n the same p e r c e n t i n d u c t i o n as observed with microsomal AHH. The r e s u l t s o b t a i n e d from the time c o u r s e study are seen i n -51-F i g u r e 5. The effects of trans-stilbene oxide on hepatic aryl hydrocarbon hydroxylase (AHH) activity. Adult male rats were treated with transstilbene oxide (t-so) in corn oil (400 mg/kg/day ip for 3 days) and aryl hydrocarbon hydroxylase levels were determined 24 hours after the last injection. Controls received corn oil alone. Asterisks indicate significant difference from control at p<0.05 (Student's t-test, 2-tail). -52-AHH ACTIVITY pmoles 3-OHBP formed/min/mg protein iSEM(n) O O « O 3 O i | . . I I I I I H H I I | NV.V.V.V.Vq » , ' , ' ' t , ' ' ' , ' , t < ' , ' » ' » y « * ro o o z c n o L o o o o o 3 § H T O O to o m in i o -53-TABLE V I I I . The e f f e c t s of p h e n o b a r b i t a l t r e a t m e n t on h e p a t i c a r y l hydrocarbon h y d r o x y l a s e a c t i v i t y . Rats were t r e a t e d with p h e n o b a r b i t a l i n s a l i n e at 80 mg/kg/day i p f o r 4 c o n s e c u t i v e days. C o n t r o l r a t s r e c e i v e d s a l i n e a l o n e . S u b c e l l u l a r f r a c t i o n A r y l hydrocarbon h y d r o x y l a s e a c t i v i t y 3 C o n t r o l T r e a t e d % I n c r e a s e Immature male microsomes n u c l e i 648 + 70(4) 57.1 + 3(12) 1532 + 130 ( 4 ) * 85.9 ± 5 ( 1 2 ) * 140 50 Immature female microsomes n u c l e i 202 + 40(4) 19.5 + 5(4) 1193 + 4 0 ( 4 ) * 61.6 + 4 ( 4 ) * 500 215 A d u l t male -microsomes n u c l e i 4810 + 300(16) 312 + 25(8) 4400 + 270(16) 322 ± 8(8) a E x p r e s s e d as pmoles/min/mg p r o t e i n + SEM(n) * I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l at p < 0.05 (St u d e n t ' s t - t e s t ) -54-F i g u r e 6. The i n c r e a s e i n microsomal AHH was g r e a t e r at a l l time p o i n t s and the i n c r e a s e i n n u c l e a r AHH was c l e a r l y maximum a f t e r 4 days of t r e a t m e n t . In c o n t r a s t , a g r e a t e r p e r c e n t i n c r e a s e o f n u c l e a r AHH than o f microsomal AHH was observed f o l l o w i n g 3 - m e t h y l c h o l a n t h r e n e treatment of immature male r a t s . The r e s u l t s are p r e s e n t e d i n T a b l e IX. 7) I n h i b i t i o n and I n d u c t i o n o f Epoxide H y d r o l a s e A c t i v i t y The i n h i b i t i o n of h e p a t i c e p o x i d e h y d r o l a s e a c t i v i t y was s t u d i e d with i n  v i t r o a d d i t i o n s o f l , 2 - e p o x y - 3 , 3 , 3 - t r i c h l o r o p r o p a n e . The i n h i b i t i o n of n u c l e a r and microsomal epoxide h y d r o l a s e a c t i v i t y i s shown i n F i g u r e 7. N u c l e a r and microsomal epoxide h y d r o l a s e were s i m i l a r l y i n h i b i t e d by I , 2 - e p o x y - 3 , 3 , 3 - t r i c h l o r o p r o p a n e . The e f f e c t s of p r e t r e a t m e n t with a c e t y l m e t h a d o l , p h e n o b a r b i t a l and t r a n s -s t i l b e n e o x i d e on h e p a t i c epoxide h y d r o l a s e a c t i v i t y were s t u d i e d ( T a b l e X ) . A l l t h r e e agents produced s i g n i f i c a n t i n c r e a s e s i n n u c l e a r and microsomal e p o x i d e h y d r o l a s e a c t i v i t y . However, the i n c r e a s e s were much g r e a t e r i n t h e microsomes than i n the n u c l e i . The d i f f e r e n c e i n the magnitude of i n d u c t i o n was about 3 - f o l d . I I . I n d u c t i o n o f Rat H e p a t i c Drug M e t a b o l i z i n g Enzymes 1) The P h e n o b a r b i t a l Response Some of the b i o c h e m i c a l responses produced by p h e n o b a r b i t a l a d m i n i s t r a -t i o n (80 mg/kg/day i p f o r 4 c o n s e c u t i v e days) were e v a l u a t e d i n the immature male, a d u l t male and a d u l t female r a t . More s p e c i f i c a l l y , the e f f e c t s of p h e n o b a r b i t a l on the a c t i v i t i e s o f c e r t a i n h e p a t i c microsomal enzymes were q u a n t i t a t e d . Microsomal a r y l hydrocarbon h y d r o x y l a s e a c t i v i t y was i n c r e a s e d s i g n i f i c a n t l y i n the immature males and a d u l t females ( F i g u r e 8 ) . However, i n t h e a d u l t males, p h e n o b a r b i t a l t r e a t m e n t d i d not a l t e r t h i s enzyme a c t i v i t y - 5 5 -F i g u r e 6 . The ti m e course o f a r y l hydrocarbon h y d r o x y l a s e (AHH) i n d u c t i o n a f t e r p h e n o b a r b i t a l (Pb) t r e a t m e n t . A d u l t female r a t s were a d m i n i s t e r e d Pb i n s a l i n e at 80 mg/kg/day i p f o r v a r y i n g l e n g t h s o f t i m e . C o n t r o l s r e c e i v e d s a l i n e a l o n e . AHH a c t i v i t y i s e x p r e s s e d as per cent of c o n t r o l . The c o n t r o l l e v e l s o f AHH a r e i n pmoles/min/mg p r o t e i n + SEM(n). The a s t e r i s k i n d i -c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l , p < 0.05. -56-Q. 0 cr ro c o l AHH ACTIVITY /o of control ±s.e.m.(n) g S o>4 o _L cn o o -57-TABLE IX. The e f f e c t s of 3 - m e t h y l c h o l a n t h r e n e t r e a t m e n t on h e p a t i c a r y l hydrocarbon h y d r o x y l a s e a c t i v i t y . Immature male r a t s were t r e a t e d w i t h 3 - m e t h y l c h o l a n t h r e n e i n c o r n o i l at 20 mg/kg/day i p f o r 2 days. C o n t r o l s r e c e i v e d c o r n o i l a l o n e . S u b c e l l u l a r f r a c t i o n A r y l hydrocarbon h y d r o x y l a s e a c t i v i t y 9 C o n t r o l T r e a t e d % I n c r e a s e microsomes 680 + 36(12) 7195 + 560(8) * 950 n u c l e i 73 + 11(4) 1140 + 110(4) * 1450 E x p r e s s e d as 3-hydroxybenzo(a)pyrene formed per min per mg p r o t e i n + SEM(n). * I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l at p < 0.05 (Student's t - t e s t ) . - 5 8 -F i g u r e 7. I n h i b i t i o n o f r a t h e p a t i c e p o x i d e h y d r o l a s e by 1,2-epoxy-3,3,3-t r i c h l o r o p r o p a n e (TCPO) i n v i t r o . Microsomes and n u c l e i from a d u l t male r a t s were employed. The TCPO was added i n 2 0 o f a c e t o n i t r i l e b e f o r e i n c u b a t i o n . C o n t r o l tubes r e c e i v e d a c e t o n i t r i l e a l o n e . For t h e s e experiments n u c l e i were i n c u b a t e d with the s u b s t r a t e f o r 60 min. Each p o i n t r e p r e s e n t s the average of f o u r e x p e r i m e n t s . A l l s t a n d a r d e r r o r s were < 1 0 % of the means. -60-TABLE X. The e f f e c t s of a c e t y l m e t h a d o l , p h e n o b a r b i t a l and t r a n s - s t i l b e n e o x i d e on h e p a t i c epoxide h y d r o l a s e a c t i v i t y i n a d u l t male r a t s . A c e t y l m e t h a d o l , average dose of 25 mg/kg/day, was a d m i n i s t e r e d f o r 2 weeks v i a the d r i n k i n g water. P h e n o b a r b i t a l (80 mg/kg/day f o r 4 days) and t r a n s - s t i l b e n e o x i d e (400 mg/kg/day f o r 3 days) were given i p . Treatment and s u b c e l l u l a r f r a c t i o n Epoxide h y d r o l a s e a c t i v i t y 3 C o n t r o l T r e a t e d % I n c r e a s e A c e t y l m e t h a d o l -microsomes 4.0 + 0.2(8) 12.8 + 0 . 6 ( 8 ) * 220 n u c l e i 0.69 + 0.07(8) 0.95 + 0.1(8)* 45 P h e n o b a r b i t a l -microsomes n u c l e i t r a n s - S t i l b e n e Oxide -microsomes n u c l e i 7.3 + 0.5(12) 15.4 0.55 + 0.05(8) 0.73 6.15 + 0.4(8) 22.5 0.51 + 0.03(4) 1.03 + 0.8(12)* 115 + 0.04(8)* 35 + 2 . 3 ( 7 ) * 265 + 0.08(4)* 100 E x p r e s s e d as nmoles s t y r e n e g l y c o l formed/min/mg + SEM(n) * I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l at p <0.05 -61-F i g u r e 8. The effects of phenobarbital (Pb) treatment (80 mg/kg/day for 4 days, ip) on hepatic microsomal aryl hydrocarbon hydroxylase (AHH) activity in immature male, adult male and adult female rats. AHH act ivi ty is expressed as pmoles 3-hydroxybenzo(a)pyrene formed/min/mg protein + SEM(n). The asterisk indicates signif icant difference from control , P < 0.05. IMMATURE MALE 6000 2000-1 4000 i CM 1000-2000 (4) control Pb ADULT MALE FEMALE -63-from c o n t r o l . Groups of r a t s were c a s t r a t e d at 21 t o 24 days of age. When they were about 200 t o 250 g, h e p a t i c microsomes were prepared and AHH a c t i v i t y was e s t i m a t e d . The basal l e v e l of AHH i n t h e s e r a t s and the e f f e c t o f p h e n o b a r b i t a l t reatment are p r e s e n t e d i n T a b l e XI i n comparison with the p r e v i o u s d a t a . The basal l e v e l of AHH i n the c a s t r a t e d r a t s was about 35 p e r c e n t of t h a t observed in the a d u l t male, however, they were s i g n i f i c a n t l y h i g h e r than the f e m a l e . As was observed i n the a d u l t male, p h e n o b a r b i t a l t r e a t m e n t d i d not a l t e r the AHH a c t i v i t y i n the c a s t r a t e d male. Aminopyrine N-demethylase ( F i g u r e 9) and epoxide h y d r o l a s e ( F i g u r e 10) a c t i v i t i e s were i n c r e a s e d by p h e n o b a r b i t a l i n the immature male, a d u l t male and a d u l t female r a t . The p h e n o b a r b i t a l response was f u r t h e r c h a r a c t e r i z e d i n the a d u l t female r a t . The r e s u l t s of t h e s e s t u d i e s are p r e s e n t e d i n T a b l e X I I . P h e n o b a r b i t a l was found to i n c r e a s e the l i v e r weight t o body weight r a t i o , AHH, aminopyrine N-demethylase, e t h y l m o r p h i n e N-demethylase and epoxide h y d r o l a s e a c t i v i t i e s . E t h o x y r e s o r u f i n o - d e e t h y l a s e a c t i v i t y was u n a f f e c t e d by p h e n o b a r b i t a l t r e a t m e n t . 2) The t r a n s - S t i l b e n e Oxide Response t r a n s - S t i l b e n e o x i d e t r e a t m e n t i n c r e a s e d epoxide h y d r o l a s e a c t i v i t y i n the immature male, a d u l t female and a d u l t male r a t s ( F i g u r e 11). AHH a c t -i v i t y , however, was i n c r e a s e d o n l y i n the a d u l t females and immature males. At 200 mg/kg/day f o r 3 days, AHH a c t i v i t y was i n c r e a s e d i n a d u l t female r a t s by 110 per cent over c o n t r o l . At 400 mg/kg/day f o r 3 d a y s , AHH a c t i v i t y was i n c r e a s e d i n immature male r a t s by 85 per c e n t . In c o n t r a s t , AHH a c t i v i t y was d e c r e a s e d t o 65 and 50 per cent of c o n t r o l i n a d u l t males f o l l o w i n g 3 c o n s ecu-t i v e days of t r a n s - s t i l b e n e o x i d e a t 200 and 400 mg/kg/day, r e s p e c t i v e l y . Groups of r a t s were c a s t r a t e d at 21 t o 24 days of age and used when they weighed 200-250 g. The basal l e v e l of AHH i n t h e s e r a t s and the e f f e c t o f -64-T a b l e XI. The e f f e c t s o f p h e n o b a r b i t a l t r e a t m e n t (80 mg/kg/day f o r 4 days, i p ) on h e p a t i c microsomal a r y l hydrocarbon h y d r o x y l a s e (AHH) a c t i v i t y . Animal Treatment AHH a c t i v i t y pmoles/min/mg p r o t e i n + SEM(n) A d u l t Female S a l i n e 374 + 52 (12) P h e n o b a r b i t a l 1324 + 98 ( 1 2 ) * Immature Male A d u l t Male C a s t r a t e d M a l e 3 S a l i n e P h e n o b a r b i t a l S a l i n e P h e n o b a r b i t a l S a l i n e P h e n o b a r b i t a l 646 + 76 (4) 1531 + 134 ( 4 ) * 4807 + 292 (16) 4397 + 271 (16) 1657 + 318 (8) 1844 + 242 (8) °Male r a t s were c a s t r a t e d between 21 and 24 days o f age. They were t r e a t e d and k i l l e d when they weighed 200 - 250 g. i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l , P<0.05. -65-F i g u r e 9. The e f f e c t s o f p h e n o b a r b i t a l (Pb) t r e a t m e n t (80 mg/kg/day f o r 4 d a y s , i p ) on h e p a t i c microsomal a m i n o p y r i n e N-demethylase a c t i v i t y i n immature male, a d u l t male and a d u l t female r a t s . Aminopyrine N-demethylase a c t i v i t y i s e x p r e s s e d as nmoles formaldehyde formed/min/mg p r o t e i n + SEM(n). The a s t e r i s k i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l , P<0.05. I M M A T U R E M A L E A D U L T M A L E 1.5T F E M A L E 1 . 0 H 0 . 5 H 0 4 control Pb -67-F i g u r e 10. The e f f e c t s o f p h e n o b a r b i t a l (Pb) t r e a t m e n t (80 mg/kg/day f o r 4 days, i p ) on h e p a t i c microsomal epoxide h y d r o l a s e a c t i v i t y i n immature male, a d u l t male and a d u l t female r a t s . E p o x i d e h y d r o l a s e a c t i v i t y i s e x p r e s s e d as nmoles s t y r e n e g l y c o l formed/min/mg p r o t e i n ± SEM(n). The a s t e r i s k i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l , P<0.05. IMMATURE MALE 20-1 20 T a b l e X I I . The e f f e c t s o f p h e n o b a r b i t a l treatment (80 mg/kg/day f o r 4 days, i p ) on l i v e r weight and h e p a t i c microsomal enzyme a c t i v i t i e s i n a d u l t female r a t s . cytochrome P-450 dependent monooxygenase a c t i v i t i e s e poxide Treatment l i v e r weight (g) a r y l hydrocarbon aminopyrine e t h y l m o r p h i n e e t h o x y r e s o r u f i n h y d r o l a s e 100 g body weight h y d r o x y l a s e N-demethylase N-demethylase o - d e e t h y l a s e a c t i v i t y (pmoles/min/mg) (nmoles/min/mg) (nmoles/min/mg) (pmoles/min/mg) (nmoles/min/mg) s a l i n e 4.51 ± 0.14 a 272 ± 38 1.37 ± 0.05 0.75 ± 0.06 60 ± 10 3.95 ± 0.60 p h e n o b a r b i t a l 5.65 ± 0.13* 1364 ± 42* 5.02 ± 0.21* 2.39 + 0.12* 88 ± 2 10.49 ± 0.53* a V a l u e s r e p r e s e n t the mean + SEM o f 4 ani m a l s * l n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l at P<0.05 -70-F i g u r e 11. The e f f e c t s o f t r a n s - s t i l b e n e o x i d e ( t - s o ) t r e a t m e n t (3 con-s e c u t i v e days, i p ) on h e p a t i c microsomal a r y l hydrocarbon h y d r o x y l a s e (AHH) and e p o x i d e h y d r o l a s e (EH) a c t i v i t i e s i n the r a t . A d u l t female r a t s r e c e i v e d 200 mg t-so/kg/day (panel a ) . Immature male r a t s r e c e i v e d 400 mg t-SO/kg/day (panel b ) . AHH a c t i v i t y i s e x p r e s s e d as pmoles 3-hydroxybenzo(a)pyrene formed/min/mg p r o t e i n + SEM(n), and EH a c t i v i t y as nmoles s t y r e n e g l y c o l formed/min/mg p r o t e i n + SEM(n), except i n panel c where enzyme a c t i v i t i e s a r e e x p r e s s e d as per c e n t o f c o n t r o l . The a s t e r i s k i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l , P<0.05. -71-K M A U AHH lOOOl 500-1 (4) I control t-SO b IMMATURE MALE AHH 2000-1 1000-1 7*3 m i control t-SO CH fa (4) J hi. control t-SO EH 20H (8) control t-SO ADULT MALE AHH ( X ol control) control=3577 pmolM/mn/mg -139(16) .200 100 0-f 3 \ * (4) EH U of control) control: 6.44 nmoU/min/mg 10.16(16) ' control * -5 (7) 400 300 200-1 100f 0-* (4) 200 400 0 DOSE OF t -SO ("fl/kg/doy Ip) 200 (7) -control 400 -72-t r a n s - s t i l b e n e o x i d e t r e a t m e n t are p r e s e n t e d i n T a b l e X I I I . The basal l e v e l o f AHH i n the c a s t r a t e d r a t s was about 40 per cent of t h a t observed i n the normal a d u l t male. Treatment with t r a n s - s t i l b e n e o x i d e , at 400 mg/kg/day f o r 3 days, was found to d e c r e a s e the AHH a c t i v i t y i n the c a s t r a t e d males by 35 p e r c e n t . At the same dose, t r a n s - s t i l b e n e o x i d e was found t o d e c r e a s e AHH a c t i v i t y i n the a d u l t male r a t by 50 per c e n t . Aminopyrine N-demethylase a c t i v i t y was a l s o determined i n a d u l t male and a d u l t female r a t s ( T a b l e XIV). At 200 mg/kg/day f o r 3 days, a dose which d e c r e a s e d AHH by 35 per cent i n the a d u l t males, aminopyrine N-demethylase a c t i v i t y as i n c r e a s e d by 45 per c e n t . In the a d u l t female, t r a n s - s t i l b e n e o x i d e at 200 mg/kg/ day e l e v a t e d aminopyrine N-demethylase by 110 per c e n t . A d u l t female r a t s were used to f u r t h e r c h a r a c t e r i z e the t r a n s - s t i l b e n e o x i d e r e s p o n s e . The r e s u l t s o f t h e s e experiments are p r e s e n t e d i n T a b l e XV. At a dose o f 200 mg/kg/day f o r 4 days, t r a n s - s t i l b e n e oxide i n c r e a s e d AHH, a m i n o p y r i n e N-demethylase, e t h y l m o r p h i n e N-demethylase and epoxide h y d r o l a s e a c t i v i t i e s . The l i v e r t o body weight r a t i o was a l s o i n c r e a s e d . However, e t h o x y r e s o r u f i n o - d e e t h y l a s e a c t i v i t y was not s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . 3) S t u d i e s w i t h A c e t y l m e t h a d o l The e f f e c t s o f a c e t y l m e t h a d o l , i n the d r i n k i n g water f o r 2 weeks, on AHH and e p o x i d e h y d r o l a s e a c t i v i t i e s i n a d u l t male and a d u l t female r a t s are i l l u s t r a t e d i n F i g u r e 12. A c e t y l m e t h a d o l was found t o i n c r e a s e epoxide hydro-l a s e i n both male and female r a t s . In the a d u l t female, AHH a c t i v i t y was e l e -v a t e d to about 300 per cent of c o n t r o l . A c e t y l m e t h a d o l i n the male e i t h e r had no e f f e c t or produced a s l i g h t but s i g n f i c a n t d e c r e a s e i n AHH a c t i v i t y . In t h e f e m a l e , AHH 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 at a dose t h a t was at l e a s t 20 times l e s s than t h a t r e q u i r e d to produce a s i g n i f i c a n t i n c r e a s e o f e p o x i d e h y d r o l a s e a c t i v i t y . -73-T a b l e X I I I . The e f f e c t s o f t r a n s - s t i l b e n e o x i d e ( t - s o ) t r e a t m e n t (3 c o n s e c u t i v e days, i p ) on h e p a t i c microsomal a r y l hydrocarbon h y d r o x y l a s e a c t i v i t y . Animal Treatment A r y l hydrocarbon h y d r o x y l a s e a c t i v i t y pmole/min/mg p r o t e i n + SEM(n)  A d u l t Female corn o i l 337 + 17 (4) t - s o (200) 697 + 40 ( 4 ) * Immature Male corn o i l 900 + 106 (8) t - s o (400) 1669 + 114 ( 8 ) * A d u l t Male cor n o i l 4245 + 263 (4) t - s o (200) 2652 + 182 ( 4 ) * A d u l t Male cor n o i l 3231 + 182 (8) t - s o (400) 1599 + 116 ( 7 ) * C a s t r a t e d Male cor n o i l 1544 + 244 (8) t - s o (400) 988 + 127 ( 8 ) * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l , P<0.05. -74-T a b l e XIV. The e f f e c t s o f t r a n s - s t i l b e n e o x i d e ( t - s o ) t r e a t m e n t (3 c o n s e c u t i v e days, i p ) on h e p a t i c microsomal aminopyrine N-demethylase a c t i v i t y i n a d u l t male and a d u l t female r a t s . Animal Treatment A r y l hydrocarbon h y d r o x y l a s e a c t i v i t y (mg/kg/day) pmole/min/mg p r o t e i n + SEM (n) A d u l t Female c o r n o i l 0.62 + 0.03 (8) t - s o (200) 1.30 + 0.03 ( 8 ) * A d u l t Male corn o i l 2.47 + 0.16 (8) t - s o (400) 3.52 + 0.10 ( 8 ) * A d u l t Male corn o i l 1.33 + 0.07 (7) t - s o (200) 1.78 + 0.10 ( 6 ) * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l , P<0.05. T a b l e XV. The e f f e c t s of t r a n s - s t i l b e n e o xide treatment (200 mg/kg/day f o r 4 days, i p ) on l i v e r weight and h e p a t i c microsomal enzyme a c t i v i t i e s i n a a d u l t female r a t s . cytochrome P-450 dependent monooxygenase a c t i v i t i e s e poxide Treatment l i v e r weight (g) a r y l hydrocarbon aminopyrine ethylmorphine e t h o x y r e s o r u f i n h y d r o l a s e 100 g body weight h y d r o x y l a s e N-demethylase N-demethylase o - d e e t h y l a s e a c t i v i t y (pmoles/min/mg) (nmoles/min/mg) (nmoles/min/mg) (pmoles/min/mg) (nmoles/min/mg) c o r n o i l 4.18+ 0.09 a 222 + 10 0.64 + 0.09 0.43 + 0.04 60 1 10 2.88 +• 0.40 t r a n s - s t i l b e n e o x i d e 5 . 5 5 ± 0.11* 569 +• 33* 2.46 ± 0.16* 0.75 +• 0.55* 62 1 9 2 1 . 3 + 1.4* a V a l u e s r e p r e s e n t the mean + SEM o f 4 a n i m a l s * I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l at P<0.05 -76-F i g u r e 12. The e f f e c t s o f a c e t y l m e t h a d o l (LAMM) tre a t m e n t ( i n the d r i n k -i n g water f o r 2 weeks) on h e p a t i c microsomal a r y l hydrocarbon h y d r o x y l a s e (AHH) and epox i d e h y d r o l a s e (EH) a c t i v i t i e s i n a d u l t male and a d u l t female r a t s . The dose o f LAAM r e p r e s e n t s the average d a i l y i n t a k e per animal o v e r 2 weeks. AHH and EH a c t i v i t i e s a r e e x p r e s s e d as per cent of c o n t r o l . The con-t r o l l e v e l s of AHH and EH a r e i n pmoles/min/mg p r o t e i n ± SEM and nmoles/ min/mg p r o t e i n + SEM, r e s p e c t i v e l y . The a s t e r i s k i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l , P<0.05. -77-AHH 500H 300-1 100 female control*298pmoles/min/mg±34.3(16) male control* 4974 pmolet/min/mg 2248(12) I (4) * T * / .^r-4—v rt) (4) I FEMALE (4) (4)2 + control "j 2 MALE (4) T" ° - 1 ° - 3 1-0 3.0 7.0 10.0 20.0 dote of L A A M (mg/kg /day ) EH 300 200H FEMALE (12) 1 0 0 t c o n t r o l female control' 3.87nmole/min/mg ±0.24(20) male controU 5.49 nmole/min/mg 10.32(24) o-f , , , , p-1 0 3.0 7.0 10.0 20.0 30.0 dose of LAAM (mg/kg/day) -78-4) The S p i r o n o l a c t o n e Response The e f f e c t s of s p i r o n o l a c t o n e (100 mg/kg/day i p f o r 4 days) on s e v e r a l enzymatic a c t i v i t i e s determined i n a d u l t males are shown i n T a b l e XVI. At t h i s dose o f s p i r o n o l a c t o n e , AHH a c t i v i t y was d e c r e a s e d s i g n i f i c a n t l y , w h i l e both a m i n o p y r i n e N-demethylase and epoxide h y d r o l a s e a c t i v i t i e s were s i g n i f -i c a n t l y i n c r e a s e d . The responses o b t a i n e d i n the a d u l t female r a t f o l l o w i n g s p i r o n o l a c t o n e t r e a t m e n t (100 mg/kg/day f o r 4 days) are summarized i n T a b l e XVII. S p i r o n o l -a c t o n e was found t o produce an i n c r e a s e i n AHH, aminopyrine N-demethylase, e t h y l m o r p h i n e N-demethylase, and epoxide h y d r o l a s e a c t i v i t i e s . S p i r o n o l a c t o n e a l s o produced a s i g n i f i c a n t i n c r e a s e i n t h e l i v e r weight t o body weight r a t i o . However, e t h o x y r e s o r u f i n o - d e e t h y l a s e a c t i v i t y was not s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . 5) The 3 - M e t h y l c h o l a n t h r e n e Response The a d m i n i s t r a t i o n o f 3 - m e t h y l c h o l a n t h r e n e (20 mg/kg/day f o r 2 days) t o a d u l t male r a t s produced a 2 - f o l d i n c r e a s e i n AHH a c t i v i t y . I n c r e a s e s i n AHH a c t i v i t y o f 950 and 1250 per cent were observed i n immature male and a d u l t female r a t s r e s p e c t i v e l y . These r e s u l t s are p r e s e n t e d i n F i g u r e 13. Aminopyrine N-demethylase and epoxide h y d r o l a s e a c t i v i t i e s i n a d u l t male r a t s were not a f f e c t e d by 3 - m e t h y l c h o l a n t h r e n e t r e a t m e n t ( F i g u r e 14). Again a modest c h a r a c t e r i z a t i o n o f the 3 - m e t h y l c h o l a n t h r e n e response was c a r r i e d out i n a d u l t female r a t s . These r e s u l t s are shown i n T a b l e X V I I I . Treatment w i t h 3 - m e t h y l c h o l a n t h r e n e (20 mg/kg/day f o r 4 days) i n c r e a s e d AHH and e t h o x y r e s o r u f i n o - d e e t h y l a s e a c t i v i t i e s . E t h y l m o r p h i n e N-demethylase, a m i n o p y r i n e N-demethylase and epoxide h y d r o l a s e a c t i v i t i e s were u n a f f e c t e d . The l i v e r to body weight r a t i o was i n c r e a s e d by 3 - m e t h y l c h o l a n t h r e n e t r e a t m e n t . Table XVI. The e f f e c t s o f s p i r o n o l a c t o n e treatment (100 mg/kg/day f o r 4 c o n s e c u t i v e days, i p ) on the a c t i v i t i e s of some h e p a t i c microsomal enzymes i n the a d u l t male r a t . Treatment cytochrome P-450 dependent monooxygenase a c t i v i t i e s a r y l hydrocarbon aminopyrine h y d r o x y l a s e N-demethylase (pmoles/min/mg) (nmoles/min/mg)  epoxide h y d r o l a s e a c t i v i t y (nmoles/min/mg) c o r n o i l s p i ronol actone 2 9 8 4 t 9 4 a 1 7 2 5 t 146* 3.63 + 0.08 4.02 ± 0.07* 5.66 + 0.22 7.87 ± 0.33* a V a l u e s r e p r e s e n t the mean + SEM of 4 a n i m a l s i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l , PO.05 Table XVII. The effects of spironolactone treatment (100 mg/kg/day for 4 days, ip) on liver weight and hepatic microsomal enzyme activities in adult female rats. cytochrome P-450 dependent monooxygenase activities epoxide Treatment liver weight (g) aryl hydrocarbon aminopyrine ethylmorphine ethoxyresorufin hydrolase 100 g body weight hydroxylase N-demethylase N-demethylase o-deethylase activity (pmoles/min/mg) (nmoles/min/mg) (nmoles/min/mg) (pmoles/min/mg) (nmoles/min/mg) saline 4.34 ± 0.07a 486 * 160 1.48 + 0.05 0.76 1 0.01 60 - 10 3.70 + 0.16 spironolactone 5.09 + 0.15* 1160 + 108* 3.20 + 0.23* 1.89 ± 0.19* 46 ± 5 6.94 t 0.43* aValues represent the mean + SEM of 4 animals *Indicates significant difference from control, P<0.05. -81-Figure 13. The e f f e c t s of 3 - m e t h y l c h o l a n t h r e n e (3-MC) tr e a t m e n t (20 mg/kg/day f o r 2 c o n s e c u t i v e days, i p ) on h e p a t i c microsomal a r y l hydrocarbon h y d r o x y l a s e (AHH) a c t i v i t y . AHH a c t i v i t y i s e x p r e s s e d as pmoles 3-hydroxy-b e n z o ( a ) p r y r e n e formed/min/mg p r o t e i n ± SEM ( n ) . The a s t e r i s k i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l a t P<0.05. I M M A T U R E M A L E 10000 5 0 0 0 (8) CONTROL 3-MC A D U L T M A L E 10000H 5000 0^ (9) CONTROL 3-MC F E M A L E 100004 5000-1 OJ f4i • • n CONTROL 3-MC -83-F i g u r e 14. The e f f e c t s o f 3 - m e t h y l c h o l a n t h r e n e (3-MC) tre a t m e n t (20 mg/kg/day f o r 2 days, i p ) on h e p a t i c microsomal epoxide h y d r o l a s e (EH) and a m i n o p y r i n e N-demethylase (Apy) a c t i v i t i e s . EH (panel a) and Apy (panel b) a c t i v i t i e s a re e x p r e s s e d as nmoles product/min/mg p r o t e i n ± SEM(n). A d u l t male r a t s were used. ADULT MALE (8) k(8); CONT 3-MC CONT 3-MC Table XVIII. The e f f e c t s o f 3 - m e t h y l c h o l a n t h r e n e treatment (20 mg/kg/day f o r 4 days, i p ) on l i v e r weight and h e p a t i c microsomal enzyme a c t i v i t i e s i n a d u l t female r a t s . cytochrome P-450 dependent monooxygenase a c t i v i t i e s e poxide Treatment l i v e r w eight (g) a r y l hydrocarbon aminopyrine e t h y l m o r p h i n e e t h o x y r e s o r u f i n h y d r o l a s e 100 g body weight h y d r o x y l a s e N-demethylase N-demethylase o - d e e t h y l a s e a c t i v i t y (pmoles/min/mg) (nmoles/min/mg) (nmoles/min/mg) (pmoles/min/mg) (nmoles/min/mg) c o r n o i l 4.05 ± 0.13 a 404 + 33 0.66 ± 0.04 0.36 ± 0.01 60 + 10 4.03 + 0.5 3-methyl-c h o l a n t h r e n e 4.91 t 0.38* 6315 + 580* 0.51 ± 0.06 0.23 t 0.01 9020 + 420* 4.16 ± 0 . 6 a V a l u e s r e p r e s e n t the mean + SEM o f 4 a n i m a l s * I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l at P<0.05. -86-6) The P r e g n e n o 1 o n e - 1 6 o ( - C a r b o n i t r i l e Response The e f f e c t s of p r e g n e n o l o n e - 1 6 < x - c a r b o n i t r i l e a d m i n i s t r a t i o n on c e r t a i n cytochrome P-450 dependent monooxygenase a c t i v i t i e s i n l i v e r microsomes from a d u l t male r a t s are shown i n T a b l e XIX. AHH a c t i v i t y i n microsomes from p r e g n e n o l o n e - 1 6 « c - c a r b o n i t r i l e - t r e a t e d r a t s was the same as c o n t r o l . However, am i n o p y r i n e N-demethylase a c t i v i t y was i n c r e a s e d f o l l o w i n g pregnenolone-16<*-c a r b o n i t r i l e a d m i n i s t r a t i o n . The b i o c h e m i c a l response to pregnenolone-16«<-carbonitrile a d m i n i s t r a t i o n was a l s o s t u d i e d i n the a d u l t female r a t ( T a b l e XX). The l i v e r to body weight r a t i o , AHH, aminopyrine N-demethylase, e t h y l m o r p h i n e N-demethylase and epoxide h y d r o l a s e a c t i v i t i e s were a l l s i g n i f i c a n t l y i n c r e a s e d f o l l o w i n g a d m i n i s t r a t i o n of pregnenolone-16«><rcarbonitrile. However, e t h o x y r e s o r u f i n O - d e e t h y l a s e a c t i v i t y was u n a f f e c t e d . 7) In V i t r o I n h i b i t i o n o f H e p a t i c Microsomal AHH A c t i v i t y by SKF 525-A The i n h i b i t i o n of h e p a t i c microsomal AHH a c t i v i t y was s t u d i e d by the a d d i t i o n o f SKF 525-A t o the i n c u b a t i o n t u b e . The i n h i b i t i o n of AHH a c t i v i t y was determined i n h e p a t i c microsomes o b t a i n e d from r a t s of v a r y i n g age, sex and drug t r e a t m e n t . The i n h i b i t i o n o f h e p a t i c AHH a c t i v i t y by SKF 525-A, i n microsomes o b t a i n e d from u n t r e a t e d male and female r a t s , i s shown i n F i g u r e 15. A p l o t o f per c e n t i n h i b i t i o n o f AHH a c t i v i t y v ersus - l o g SKF 525-A c o n c e n t r a t i o n y i e l d e d a s i g m o i d a l r e l a t i o n s h i p w i t h complete i n h i b i t i o n of AHH a c t i v i t y noted at 10" 2M SKF 525-A. The per cent i n h i b i t i o n of AHH a c t i v i t y was l i n e a r between 30 and 70 per cent i n h i b i t i o n ( r > 0.95). The per cent i n h i b i -t i o n of AHH a c t i v i t y by SKF 525-A was determined over a wide range of p r o t e i n c o n t e n t ( w i t h i n the l i n e a r range of enzyme a c t i v i t y versus p r o t e i n c o n t e n t ; 0.03 t o 0.3 mg p r o t e i n ) and was found to be independent o f p r o t e i n c o n t e n t . -87-Table XIX. The e f f e c t s o f pregnenolone-16<x-carbonitri1e a d m i n s t r a t i on (40 mg/kg/day f o r 4 days, i p ) on h e p a t i c microsomal a r y l hydrocarbon h y d r o x y l a s e and aminopyrine N-demethylase a c t i v i t i e s i n a d u l t male r a t s . Treatment A r y l hydrocarbon Aminopyrine N-demethylase h y d r o x y l a s e a c t i v i t y a c t i v i t y pmoles/min/mg SEM(n) nmoles/min/mg SEM(n) corn o i l 3645 + 91 (4) 1.86 ± 0.12 (4) p r e g n e n o l o n e - 1 6 < x - c a r b o n i t r i l e 3903 + 96 (4) 3.43 + 0.10 ( 4 ) * * I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l at P<0.05. Table XX. The e f f e c t s o f p r e g n e n o l o n e - 1 6 < * - c a r b o n i t r i l e (40 mg/kg/day f o r 4 days , i p ) on h e p a t i c microsomal enzyme a c t i v i t i e s and l i v e r weight i n a d u l t female r a t s . cytochrome P-450 dependent monooxygenase a c t i v i t i e s epoxide Treatment l i v e r weight (g) a r y l hydrocarbon aminopyrine e t h y l m o r p h i n e e t h o x y r e s o r u f i n h y d r o l a s e 100 g body weight h y d r o x y l a s e N-demethylase N-demethylase o - d e e t h y l a s e a c t i v i t y (pmoles/min/mg) (nmoles/min/mg) (nmoles/min/mg) (pmoles/min/mg) (nmoles/min/mg) c o r n o i l 4.51 + 0.12 a 522 ± 5 0.86 + 0.05 0.40 ± 0.03 43 + 3 4.25 ± 0.52 pregnenolone-16°c-carbonitrile 6.18 i 0.08* 3729 + 89* 3.07 ± 0.10* 2.61 ± 0.03* 34 ± 3 10.52 ± 0 . 3 5 * a V a l u e s r e p r e s e n t the mean + SEM o f 4 an i m a l s * I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l at P<0.05. -89-Figure 15. I n h i b i t i o n o f a d u l t male and female r a t h e p a t i c microsomal a r y l h ydrocarbon h y d r o x y l a s e (AHH) a c t i v i t y by SKF 525-A. The ben z o ( a ) p y r e n e c o n c e n t r a t i o n was 8 x 1 0 " 5 M. Each each p o i n t r e p r e s e n t s the average of d u p l i c a t e d e t e r m i n a t i o n s . SKF 525-A Concentration(M) -91-However, as t y p i f i e d i n F i g u r e 15, the AHH a c t i v i t y i n h e p a t i c microsomes from male r a t s was more s e n s i t i v e t o the i n h i b i t o r y e f f e c t s o f SKF 525-A than female r a t s . The c o n c e n t r a t i o n r e q u i r e d t o produce 50% i n h i b i t i o n of AHH a c t i v i t y ( IC50) was c a l c u l a t e d f o r the i n h i b i t i o n of AHH a c t i v i t y i n h e p a t i c microsomes o b t a i n e d from u n t r e a t e d a d u l t male, a d u l t female, immature male, immature female and c a s t r a t e d male r a t s . The basal l e v e l s of AHH and t h e IC50 v a l u e s are l i s t e d i n T a b l e XXI. The IC50 v a l u e s o b t a i n e d f o r the a d u l t female and the immature r a t s were s i m i l a r . The basal a c t i v i t i e s o f AHH were a l s o s i m i l a r i n t h e s e a n i m a l s . However, the basal l e v e l of AHH was s i g n i f i c a n t l y g r e a t e r i n the a d u l t male than the a d u l t female. A l s o , the IC50 v a l u e f o r the a d u l t male was s i g n i f i c a n t l y lower than t h a t o b t a i n e d f o r t h e a d u l t f e m a l e . The AHH a c t i v i t y i n the c a s t r a t e d male was a p p r o x i m a t e l y 40% o f the a c t i v i t y observed i n the a d u l t male, and was s i g n i f i c a n t l y g r e a t e r than t h a t seen i n the a d u l t female. The IC50 v a l u e o b t a i n e d f o r the c a s t r a t e d male was i n d i s t i n g u i s h a b l e from t h a t o b t a i n e d f o r the a d u l t male. The i n h i b i t i o n o f h e p a t i c microsomal AHH a c t i v i t y by SKF 525-A was a l s o s t u d i e d i n a d u l t p s e u d o h e r m a p h r o d i t i c r a t s and t h e i r normal King-Holtzman male and female l i t t e r m a t e s . These r e s u l t s are shown i n T a b l e XXII. The basal a c t i v i t y o f AHH was about 4 f o l d g r e a t e r i n the normal male than the normal f e m a l e . The basal l e v e l o f AHH i n the pseudo male was i n d i s t i n g u i s h a b l e from t h a t observed i n the normal f e m a l e . The IC50 v a l u e o b t a i n e d i n the normal female was s i g n i f i c a n t l y g r e a t e r than i n the normal male, but was i n d i s t i n -g u i s h a b l e from the v a l u e o b t a i n e d i n the pseudo male. The s e n s i t i v i t y to the i n h i b i t o r y e f f e c t of SKF 525-A was a l s o s t u d i e d i n h e p a t i c microsomal s u s p e n s i o n s o b t a i n e d from r a t s t r e a t e d with v a r i o u s agents -92-T a b l e XXI. The age and sex dependency of h e p a t i c microsomal a r y l hydro-c a r b o n h y d r o x y l a s e a c t i v i t y and c a l c u l a t e d IC5Q v a l u e s . Animal A r y l hydrocarbon IC5Q v a l u e 3 h y d r o x y l a s e a c t i v i t y M x 10 + SEM(n=4) pmoles/min/mg + SEM(n=4)  A d u l t m a l e b 4060 * 125 1.11 - 0.07 A d u l t f e m a l e 0 707 ± 22* 2.88 ± 0.08* Immature m a l e 0 714+ 23* 3.15 ± 0.18* Immature f e m a l e 0 656 ± 27* 3.56 + 0.30* C a s t r a t e d m a l e d 1560 + 158* 1.12 + 0.15 a D e f i n e d as the c o n c e n t r a t i o n o f SKF 525-A r e q u i r e d to produce 50 p e r cent i n h i b i t i o n of AHH a c t i v i t y . b A d u l t r a t s were 60 days o f age ( f e m a l e , 200-250 g; male, 250-300 g ) . cImmature r a t s were 16 days o f age (female and male, 40-50 g ) . ^ C a s t r a t e d at 16 days o f age, enzyme a c t i v i t y and IC50 v a l u e d e t e r -mined a t 60 days of age. I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from a d u l t male at P<0.05. -93-T a b l e X X I I . The basal a r y l hydrocarbon h y d r o x y l a s e a c t i v i t y and the c a l -c u l a t e d IC50 v a l u e s f o r SKF 525-A i n h e p a t i c microsomes p r e -pared from a d u l t p s e u d o h e r m a p h r o d i t i c (pseudo) r a t s and t h e i r male and female l i t t e r m a t e s ( K i n g - H o l t z m a n ) . Animal A r y l hydrocarbon IC5Q v a l u e 3 h y d r o x y l a s e a c t i v i t y M x 10 + SEM(n=4) pmoles/min/mg + SEM(n=4)  normal male 1695 + 133 1.52 + 0.05 normal female 475 ± 24* 4.54 ± 0.50* pseudo male 407 + 70* 3.54 + 0.70* " D e f i n e d as the c o n c e n t r a t i o n o f SKF 525-A r e q u i r e d to produce 50 p e r c e n t i n h i b i t i o n of AHH a c t i v i t y . * I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from normal males at P<0.05. -94-that increased AHH a c t i v i t y . The i n h i b i t i o n of hepatic AHH a c t i v i t y by SKF 525-A, in microsomes obtained from untreated, phenobarbital- or 3-methylcholanthrene-treated adult female rats, i s shown in Figure 16. The IC50 values obtained in rats treated with various agents that increased AHH a c t i v i t y are given in Figure 17. In the adult female rats, phenobarbital, spironolactone, trans-stilbene oxide and pregnenolone-l&K-carbonitrile treatment were found to increase the s e n s i t i v i t y of microsomal AHH a c t i v i t y to the inhibitory effects of SKF 525-A. A l l of these treatments resulted in a decrease in the IC5Q value in the adult female, similar to the value observed in control adult males. In microsomes obtained from male and female rats treated with 3-methylcholanthrene a marked decrease in the s e n s i t i v i t y of hepatic AHH a c t i v i t y to the inhibitory action of SKF 525-A was noted. 8) Nuclear Versus Microsomal Induction The effects of the various compounds on hepatic microsomal and nuclear AHH a c t i v i t y was studied in adult female rats (Table XXIII). A l l of the agents under investigation produced significant increases in both microsomal and nuclear AHH a c t i v i t y . Phenobarbital, spironolactone, and trans-stilbene oxide increased microsomal AHH to a greater extent than nuclear AHH a c t i v i t y . Conversely, 3-methylcholanthrene treatment resulted in a r e l a t i v e l y greater increase in nuclear AHH a c t i v i t y . Pregnenolone-16«>c-carbonitrile appeared to increase both nuclear and microsomal AHH a c t i v i t y to the same extent. 9) Dose- and Time- Effect Studies The time- and dose- response relationships for the drug-induced increases in l i v e r weight and microsomal enzyme a c t i v i t i e s were studied in the adult -95-F i g u r e 16. I n h i b i t i o n , by SKF 525-A, o f a r y l hydrocarbon h y d r o x y l a s e (AHH) a c t i v i t y i n h e p a t i c microsomes from p h e n o b a r b i t a l - and 3-methyl-chol a n t h r e n e - t r e a t e d a d u l t female r a t s . The b e n z o ( a ) p y r e n e c o n c e n t r a t i o n - w a s 8 x 1 0 ~ 5 M. Each p o i n t r e p r e s e n t s the average of d u p l i c a t e d e t e r m i n a t i o n s . SKF 525-A Concentration(M) -97-F i g u r e 17. The s e n s i t i v i t y o f r a t h e p a t i c microsomal a r y l hydrocarbon hy-d r o x y l a s e a c t i v i t y to the i n h i b i t o r y e f f e c t s of SKF 525-A i n v i t r o . H e p a t i c microsomes were from a d u l t r a t s t r e a t e d f o r 4 c o n s e c u t i v e days i p with pheno-b a r b i t a l (Pb, 80 mg/kg/day), s p i r o n o l a c t o n e ( s p i r , 100 mg/kg/day), t r a n s -s t i l b e n e o x i d e ( t - s o , 200 mg/kg/day), pregnenolone-16«x~carbonitrile (PCN, 40 mg/kg/day), o r 3 - m e t h y l c h o l a n t h r e n e (3-MC, 20 mg/kg/day). C o n t r o l r a t s r e -c e i v e d c o r n o i l . The IC50 v a l u e r e p r e s e n t s the c o n c e n t r a t i o n o f SKF 525-A r e q u i r e d to produce 50 per cent i n h i b i t i o n o f h e p a t i c microsomal AHH a c t i v i t y . The b e n z o ( a ) p y r e n e c o n c e n t r a t i o n was 8.0 x 1 0 " 5 M. -99-T a b l e X X I I I . Microsomal v e r s u s n u c l e a r i n d u c t i o n o f a r y l hydrocarbon h y d r o x y l a s e (AHH) a c t i v i t y . T r e a t m e n t 3 S u b c e l l u l a r AHH a c t i v i t y 1 3 f r a c t i o n c o n t r o l t r e a t e d pmoles/min/mg i n c r e a s e P h e n o b a r b i t a l (80 mg/kg/day) microsomes 310 ±21 n u c l e i 1185 ±35* 285 14.8+1.1 31.6+2.7* 115 Spi r o n o l a c t o n e (100 mg/kg/day) microsomes 291 +16 1312 +133* 350 n u c l e i 17.9±1.2 62.8+3.6* 250 t r a n s - S t i l b e n e o x i d e (200 mg/kg/day) microsomes n u c l e i 291+16 17.9+1.2 659 ±19* 29.8+3.1* 126 67 3-Methylchol anthrene (20 mg/kg/day) microsomes 581+26 n u c l e i 9.8+1.0 7764 +104* 1235 207 +7* 2015 Pregnenolone-16«<-carbonitrile microsomes 310 51 3227 153* 970 (40 mg/kg/day) n u c l e i 17.8 1.5 181 14* 920 a A l l agents were a d m i n i s t e r e d to a d u l t female r a t s i p f o r 4 da y s . C o n t r o l animals r e c e i v e d t he a p p r o p r i a t e v e h i c l e . ^AHH a c t i v i t y i s e x p r e s s e d as the mean + SEM of 4 a n i m a l s . * I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l at P<0.05. - 1 0 0 -female r a t . These s t u d i e s were o f importance i n o r d e r to 1) f u r t h e r c h a r a c -t e r i z e the d r u g - i n d u c e d responses and 2) determine the time and dose r e q u i r e d f o r maximal r e s p o n s e . P h e n o b a r b i t a l was a d m i n i s t e r e d to a d u l t female r a t s at 80 mg/kg/day f o r 1 , 2 , 4 and 7 c o n s e c u t i v e days. The t i m e - c o u r s e of the p l e i o t r o p i c response i s shown i n F i g u r e 18. The cytochrome P -450 dependent monoxygenase a c t i v i t i e s , AHH, a m i n o p y r i n e N-demethylase and e t h y l m o r p h i n e N-demethylase were i n c r e a s e d m a x i m a l l y a f t e r 4 days o f p h e n o b a r b i t a l . However, the l i v e r weight/body weight r a t i o and epoxide h y d r o l a s e a c t i v i t i e s were i n c r e a s e d f u r t h e r f o l l o w i n g 7 days of t r e a t m e n t , not e x h i b i t i n g maximal e f f e c t s a f t e r 4 days. AHH and epo x i d e h y d r o l a s e a c t i v i t i e s were determined i n a d u l t female r a t s a d m i n i s t e r e d v a r i e d doses of p h e n o b a r b i t a l , t r a n s - s t i l b e n e o x i d e and pregneno-l o n e - 1 6 o < . - c a r b o n i t r i l e . The r e s u l t s of t h e s e experiments are shown i n F i g u r e 1 9 . The i n c r e a s e s i n a r y l hydrocarbon h y d r o x y l a s e a c t i v i t y f o l l o w i n g 4 days o f t r e a t m e n t with p h e n o b a r b i t a l , t r a n s - s t i l b e n e o xide or pregnenolone - 1 6 -c a r b o n i t r i l e appeared to e x h i b i t maxima at 80 mg/kg/day, 50 mg/kg/day and 40 mg/kg/day r e s p e c t i v e l y . However, the maximal responses produced by the 3 com-pounds were d i f f e r e n t , w i t h pregnenolone - 1 6 o<r-carbonitrile » p h e n o b a r b i t a l > t r a n s - s t i l b e n e o x i d e . Epoxide h y d r o l a s e a c t i v i t y was i n c r e a s e d i n a dose dependent manner by the agents s t u d i e d . However, i t appeared t h a t l a r g e r doses were r e q u i r e d to produce maximal i n d u c t i o n of t h i s a c t i v i t y . In the l i m i t e d dose range, i t d i d not appear t h a t maximal i n c r e a s e s o f epoxide hydro-l a s e a c t i v i t y were o b t a i n e d w i t h any of the compounds s t u d i e d . At a dose o f 50 mg/kg/day, t r a n s - s t i l b e n e o x i d e produced a maximal i n c r e a s e of AHH a c t i v -i t y , whereas t h i s same dose produced an i n c r e a s e i n epoxide h y d r o l a s e a c t i v i t y t h a t was at a p o i n t on the dose-response c u r v e well below the maximum. In o t h e r words, the ED50 f o r the i n c r e a s e i n AHH a c t i v i t y was s u b s t a n t i a l l y lower than t h a t f o r the i n c r e a s e i n epoxide h y d r o l a s e a c t i v i t y . The i n c r e a s e s -101-F i g u r e 18. The t i m e - c o u r s e o f the p h e n o b a r b i t a l response. P h e n o b a r b i t a l (Pb, 80 mg/kg/day i p ) f o r 1,2,4 and 7 c o n s e c u t i v e days was a d m i n i s t e r e d t o a d u l t female r a t s . C o n t r o l r a t s r e c e i v e d s a l i n e . L i v e r weight and h e p a t i c microsomal enzyme a c t i v i t i e s were deter m i n e d . -102-1500 1000 A R Y L H Y D R O C A R B O N H Y D R O X Y L A S E 500 / 3.0J 2.0H 1.0 A M I N O P Y R I N E N - D E M E T H Y L A S E • I / 1.0-i °s E T H Y L M O R P H I N E N - D E M E T H Y L A S E 6 II c w CO + 1 1. o B 60 II c If w CO •fl a o 6-1 5J L IVER W T / B O D Y W T / 15^ 10-E P O X I D E H Y D R O L A S E / u 2 4 Days off Phenobarbital -103-F i g u r e 19. A d o s e - r e s p o n s e study of the i n c r e a s e i n a r y l hydrocarbon h y d r o x y l a s e a c t i v i t y , epoxide h y d r o l a s e a c t i v i t y and l i v e r weight by pheno-b a r b i t a l ( P b ) , t r a n s - s t i l b e n e o x i d e ( t - s o ) and pregnenolone-16«-carbonitrile (PCN). A d u l t female r a t s were t r e a t e d with t h e s e compounds f o r 4 days. The a s t e r i s k i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l , P<0.05. -104-Aryl Hydrocarbon Hydroxylase 1000 DOSE (mg/kg/day) -105-i n l i v e r weight produced by t h e s e agents i s a l s o shown i n F i g u r e 19. The i n c r e a s e s i n l i v e r weight by pregnenolone-16<x.-carbonitrile, p h e n o b a r b i t a l o r t r a n s - s t i l b e n e o x i d e t r e a t m e n t a l s o d i d not appear to reach maxima, s i m i l a r to t h a t seen w i t h epoxide h y d r o l a s e a c t i v i t y . The o r d e r of potency of the compounds f o r a l l t h r e e b i o c h e m i c a l r e s p o n s e s , from h i g h e s t t o l o w e s t , was pregnenolone-16o<-carbonitrile > p h e n o b a r b i t a l > t r a n s - s t i l b e n e o x i d e . 10) A d d i t i o n Experiments A d d i t i o n experiments are o f t e n u s e f u l p h a r m a c o l o g i c a l l y i n d e t e r m i n i n g whether two compounds act by the same or by d i f f e r e n t mechanisms. In the p r e s e n t e x p e r i m e n t s , the e f f e c t s o f i n d u c e r s a l o n e or i n combination were examined. 10a. P h e n o b a r b i t a l and S p i r o n o l a c t o n e P h e n o b a r b i t a l , alone or i n c o m b i n a t i o n w i t h s p i r o n o l a c t o n e was a d m i n i s -t e r e d t o groups of a d u l t female r a t s . The e f f e c t s of t h e s e t r e a t m e n t s on s e v e r a l microsomal cytochrome P-450 dependent monooxygenases are given i n F i g u r e 20. P h e n o b a r b i t a l , a t 80 mg/kg/day i p f o r 4 days, i n c r e a s e d amino-p y r i n e N-demethylase, e t h y l m o r p h i n e N-demethylase and AHH a c t i v i t i e s . S p i r o -n o l a c t o n e , a t 100 mg/kg/day i p f o r 4 days a l s o i n c r e a s e d t h e s e a c t i v i t i e s . These a c t i v i t i e s were i n c r e a s e d s l i g h t l y f u r t h e r by 200 mg/kg/day s p i r o n o -l a c t o n e . The i n c r e a s e i n e t h y l m o r p h i n e N-demethylase a c t i v i t y by s p i r o n o -l a c t o n e appeared to be g r e a t e r than t h a t seen f o l l o w i n g p h e n o b a r b i t a l . The i n c r e a s e s i n the o t h e r a c t i v i t i e s were s i m i l a r . When p h e n o b a r b i t a l and s p i r o n o l a c t o n e were a d m i n i s t e r e d t o g e t h e r , the r e s u l t a n t a c t i v i t i e s were s i g n i f i c a n t l y l e s s than a d d i t i v e (Two-way AN0VA, i n t e r a c t i o n e f f e c t s i g n i f i -c a n t , P<0.05), i . e . , an a d d i t i v e e f f e c t was not produced. The aminopyrine N-demethylase a c t i v i t y i n l i v e r microsomes from r a t s t r e a t e d with the -106-F i g u r e 20. The ef fects of phenobarbital (Pb) and spironolactone (S) alone or in combination on hepatic microsomal cytochrome P-450 dependent mono-oxygenase a c t i v i t i e s . Phenobarbital was administered at 80 mg/kg/day ip for 4 days. Spironolactone was administered for 4 consecutive days at 100 or 200 mg/kg/day i p . The aster isk indicates s ign i f i can t di f ference from control at P<0.05. The double aster isk indicates a s ign i f i can t interact ion ef fect (Two-way AN0VA, P<0.05). Aminopyrine N-Demethyla$e Ethylmorphine N-Demethylase Aryl Hydrocarbon Hydroxylase -108-c o m b i n a t i o n o f drugs was the same as t h a t seen with p h e n o b a r b i t a l a l o n e . The e t h y l m o r p h i n e N-demethylase a c t i v i t y i n microsomes from r a t s t r e a t e d with the co m b i n a t i o n of drugs was the same as seen with s p i r o n o l a c t o n e a l o n e , a l s o . The e f f e c t s of p h e n o b a r b i t a l and s p i r o n o l a c t o n e alone or i n co m b i n a t i o n on h e p a t i c microsomal epoxide h y d r o l a s e a c t i v i t y are i l l u s t r a t e d i n F i g u r e 21. Treatment, f o r 4 days, with e i t h e r p h e n o b a r b i t a l or s p i r o n o l a c t o n e caused a 100 p e r cent i n c r e a s e o f epoxide h y d r o l a s e a c t i v i t y . D o u b l i n g the dose of s p i r o n o l a c t o n e produced o n l y a m i n i m a l l y g r e a t e r e f f e c t . An a d d i t i v e e f f e c t was observed when p h e n o b a r b i t a l and s p i r o n o l a c t o n e were a d m i n i s t e r e d t o g e t h e r (Two-way ANOVA, i n t e r a c t i o n e f f e c t not s i g n i f i c a n t , P<0.05). When p h e n o b a r b i t a l was a d m i n i s t e r e d f o r 7 day s , epoxide h y d r o l a s e was e l e v a t e d t o a much g r e a t e r l e v e l than observed f o l l o w i n g 4 days of t r e a t m e n t . The a d m i n i s t r a t i o n of s p i r o n o l a c t o n e , at 200 mg/kg/day f o r 7 days d i d not i n c r e a s e e p o x i d e h y d r o l a s e a c t i v i t y past t h a t observed f o l l o w i n g 4 days of tr e a t m e n t . The combined a d m i n i s t r a t i o n o f s p i r o n o l a c t o n e and p h e n o b a r b i t a l f o r one week r e s u l t e d i n an epoxide h y d r o l a s e a c t i v i t y t h a t was comparable to the l e v e l o b t a i n e d with 7 days of p h e n o b a r b i t a l a l o n e . 10b. P h e n o b a r b i t a l and t r a n s - S t i l b e n e Oxide The e f f e c t s o f p h e n o b a r b i t a l and t r a n s - s t i l b e n e o x i d e , a l o n e or i n c o m b i n a t i o n , on the a c t i v i t i e s of c e r t a i n h e p a t i c microsomal enzymes i n female r a t s are p r e s e n t e d i n F i g u r e 22. H e p a t i c microsomal aminopyrine N-demethylase a c t i v i t i e s were comparable i n r a t s t r e a t e d with e i t h e r p h e n o b a r b i t a l , t r a n s -s t i l b e n e o x i d e , or a combi n a t i o n o f the two. V i r t u a l l y t h e same was observed w i t h e t h y l m o r p h i n e N-demethylase a c t i v i t y . Treatment with p h e n o b a r b i t a l r e s u l t e d i n a h i g h e r AHH a c t i v i t y than observed f o l l o w i n g t r a n s - s t i l b e n e o x i d e t r e a t m e n t . Combined treatment d i d not r e s u l t i n a d d i t i v e e f f e c t s , and the AHH a c t i v i t y was the same as observed with p h e n o b a r b i t a l t r e a t m e n t a l o n e . Epoxide -109-F i g u r e 21. The e f f e c t s o f p h e n o b a r b i t a l (Pb) and s p i r o n o l a c t o n e (s) alone o r i n co m b i n a t i o n on h e p a t i c microsomal epoxide h y d r o l a s e a c t i v i t y . Phenobar-b i t a l was a d m i n i s t e r e d at 80 mg/kg/day i p . S p i r o n o l a c t o n e was a d m i n i s t e r e d at 100 o r 200 mg/kg/day i p . These drugs were a d m i n i s t e r e d once a day f o r 4 days (panel A) or f o r 7 days (panel B ) . The a s t e r i s k i n d i c a t e s s i g n i f i c a n t d i f f e r -ence from c o n t r o l a t P<0.05. The double a s t e r i s k i n d i c a t e s a s i g n i f i c a n t i n t e r a c t i o n e f f e c t (Two-way AN0VA, P<0.05). Epoxide Hydrolase - I l l -F i g u r e 22. The e f f e c t s o f p h e n o b a r b i t a l (Pb) and t r a n s - s t i l b e n e o x i d e ( t - s o ) a l o n e or i n combi n a t i o n on h e p a t i c microsomal enzyme a c t i v i t i e s . P h e n o b a r b i t a l was a d m i n i s t e r e d at 80 mg/kg/day i p f o r 4 da y s , except when ep o x i d e h y d r o l a s e a c t i v i t i e s were determined i t was given f o r 7 days. t r a n s -S t i l b e n e o x i d e was a d m i n i s t e r e d at 200 mg/kg/day i p f o r 4 da y s . The a s t e r i s k i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l a t P<0.05. The double a s t e r i s k i n d i c a t e s a s i g n i f i c a n t i n t e r a c t i o n e f f e c t (Two-way ANOVA, P<0.05). -112 -Aminopyrine N-Demethylase Ethylmorphine N-Demethyla •4* jj* *f**f* i 2 o> .1 1 E^  M JB O E c cont Pb t-SO Pb + t-SO Aryl Hydrocarbon Hydroxyl cont Pb t-SO Pb Epoxide Hydrolase t-SO ase T cont Pb t-SO Pb t-SO cont Pb t-SO Pb + t-SO -113-h y d r o l a s e a c t i v i t y was i n c r e a s e d to a g r e a t e r e x t e n t by t r a n s - s t i l b e n e o x i d e than by p h e n o b a r b i t a l , however the combined e f f e c t s of the two compounds were not a d d i t i v e . In f a c t , t he epoxide h y d r o l a s e a c t i v i t y observed f o l l o w i n g c o - a d m i n i s t r a t i o n of p h e n o b a r b i t a l and t r a n s - s t i l b e n e oxide was i n d i s t i n g u i s h a b l e from t h a t seen with t r a n s - s t i l b e n e o x i d e a l o n e . 10c. P h e n o b a r b i t a l and P r e g n e n o l o n e - 1 6 < * ~ C a r b o n i t r i l e P h e n o b a r b i t a l and p r e g n e n o l o n e - 1 6 * < - c a r b o n i t r i l e were a d m i n i s t e r e d alone or t o g e t h e r to groups of a d u l t female r a t s and t h e i r e f f e c t s on some cytochrome P-450 dependent monooxygenases were determined ( F i g u r e 2 3 ) . Aminopyrine N-demethylase a c t i v i t y was i n c r e a s e d to the same extent by p h e n o b a r b i t a l or pregnenolone-16«<-carbonitrile. Treatment of r a t s with both compounds d i d not y i e l d a d d i t i v e e f f e c t s . E t h y l m o r p h i n e N-demethylase and AHH a c t i v i t i e s were i n c r e a s e d s i g n i f i c a n t l y by both compounds; however t h e s e a c t i v i t i e s were i n c r e a s e d to a g r e a t e r e x t e n t by pregnenolone-16ot-c a r b o n i t r i l e . The AHH and e t h y l m o r p h i n e N-demethylase a c t i v i t i e s i n r a t s t r e a t e d w i t h both compounds s i m u l t a n e o u s l y were i n d i s t i n g u i s h a b l e from t h o s e o b s e r v e d i n r a t s t r e a t e d with pregnenolone-16<<-carbonitrile a l o n e . The e f f e c t s o f t h e s e compounds on h e p a t i c microsomal epoxide h y d r o l a s e a c t i v i t y are p r e s e n t e d i n F i g u r e 24. A f t e r 4 days t r e a t m e n t , both compounds s i g n i f i c a n t l y i n c r e a s e d epoxide h y d r o l a s e a c t i v i t y . T h i s a c t i v i t y was i n c r e a s e d s u b s t a n t i a l l y f u r t h e r f o l l o w i n g 7 days o f t r e a t m e n t . A d d i t i v e e f f e c t s were o b t a i n e d when t h e s e compounds were a d m i n i s t e r e d t o g e t h e r f o r 4 da y s . However, when thes e compounds were a d m i n i s t e r e d t o g e t h e r f o r 7 da y s , a d d i t i o n was not o b s e r v e d . The epoxide h y d r o l a s e a c t i v i t y i n r a t s t r e a t e d w i t h both compounds f o r 7 days was o n l y s l i g h t l y h i g h e r than t he a c t i v i t i e s o b s e r v e d f o l l o w i n g 7 days of treatment with e i t h e r compound a l o n e . -114-F i g u r e 23. The e f f e c t s of p h e n o b a r b i t a l (Pb) and pregnenolone-16*-c a r b o n i t r i l e (PCN) a l o n e or i n combi n a t i o n on h e p a t i c microsomal cytochrome P-450 dependent monooxygenase a c t i v i t i e s . P h e n o b a r b i t a l was a d m i n i s t e r e d at 80 mg/kg/day i p f o r 4 days. Pregnenolone-16°<^carbonitrile was a d m i n i s t e r e d . f o r 4 c o n s e c u t i v e days at 40 mg/kg/day i p . The a s t e r i s k i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l a t P<0.05. The double a s t e r i s k i n d i c a t e s a s i g n i f i -c a n t i n t e r a c t i o n e f f e c t (Two-way ANOVA, P<0.05). -116-F i g u r e 24. The e f f e c t s o f p h e n o b a r b i t a l (Pb) and pregnenolone-16<*<-c a r b o n i t r i l e (PCN) a l o n e or i n c o m b i n a t i o n on h e p a t i c microsomal e p o x i d e h y d r o l a s e a c t i v i t y . P h e n o b a r b i t a l (80 mg/kg/day i p ) and pregnenolone-l&*-c a r b o n i t r i l e (40 mg/kg/day i p ) were a d m i n i s t e r e d f o r 4 days (panel A) or f o r 7 days (panel B ) . The a s t e r i s k i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l at P<0.05. The double a s t e r i s k i n d i c a t e s a s i g n i f i c a n t i n t e r a c t i o n e f f e c t (Two-way ANOVA, P<0.05). Epoxide Hydrolase PCN PCN -118-l O d . P r e g n e n o l o n e - 1 6 < * - C a r b o n i t r i l e and 3 - M e t h y l c h o l a n t h r e n e P r e g n e n o l o n e - 1 6 < x - c a r b o n i t r i l e and 3 - m e t h y l c h o l a n t h r e n e were a d m i n i s t e r e d a l o n e or i n c o m b i n a t i o n t o a d u l t female r a t s . H e p a t i c microsomal AHH and e t h y l m o r p h i n e N-demethylase a c t i v i t i e s were determined ( F i g u r e 25). Adminis-t e r e d a l o n e , each o f the agents i n c r e a s e d AHH a c t i v i t y , w i t h a g r e a t e r e f f e c t produced by 3 - m e t h y l c h o l a n t h r e n e . The AHH a c t i v i t y i n microsomes from r a t s a d m i n i s t e r e d both agents was the same as i n microsomes from r a t s t r e a t e d with 3 - m e t h y l c h o l a n t h r e n e a l o n e . E t h y l m o r p h i n e N-demethylase a c t i v i t y was i n -c r e a s e d s i g n i f i c a n t l y by pregnenolone-16«^-carbonitrile, w h i l e 3-methyl-c h o l anthrene treatment had no e f f e c t . The e t h y l m o r p h i n e N-demethylase a c t i v i t y i n microsomes from r a t s a d m i n i s t e r e d the two compounds s i m u l t a n e o u s l y was i n d i s t i n g u i s h a b l e from t h a t observed i n microsomes from r a t s t r e a t e d w i t h pregnenolone-16«-carbonitri1e a l o n e . 11). Sodium Dodecyl S u l f a t e - P o l y a c r y l a m i d e Gel E l e c t r o p h o r e s i s H e p a t i c microsomes were prepa r e d from u n t r e a t e d , as w e l l as pheno-b a r b i t a l - , s p i r o n o l a c t o n e - , t r a n s - s t i l b e n e o x i d e - , 3 - m e t h y l c h o l a n t h r e n e - and pregnenolone-16<X[-carbonitrile-treated a d u l t female r a t s . The v a r i o u s m i c r o -somal p r e p a r a t i o n s were s u b j e c t e d t o S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s . The s e p a r a t e d p r o t e i n bands were v i s u a l i z e d f o l l o w i n g s t a i n i n g (Coomasie B l u e ) . Photographs and d e n s i t o m e t e r t r a c i n g s of the v a r i o u s g e l s were used t o compare the band p a t t e r n s . The r e s u l t s o b t a i n e d from h e p a t i c microsomes prepared from a d u l t female r a t s t r e a t e d f o r 4 days with e i t h e r p h e n o b a r b i t a l (80 mg/kg/day), t r a n s -s t i l b e n e o x i d e (200 mg/kg/day), s p i r o n o l a c t o n e (200 mg/kg/day) or 3-methyl-c h o l a n t h r e n e (20 mg/kg/day) are g i v e n i n F i g u r e 26. Microsomes from corn o i l c o n t r o l s were a l s o p r e p a r e d . There were 4 d i s t i n c t bands p r e s e n t i n a l l of the g e l s ; band A (93,000 m o l e c u l a r w e i g h t ) , band B (76,000 m o l e c u l a r w e i g h t ) , -119-F i g u r e 25. The e f f e c t s o f p r e g n e n o l o n e - l & X c - c a r b o n i t r i l e (PCN) and 3 - m e t h y l c h o l a n t h r e n e (3-MC) al o n e or i n combination on h e p a t i c microsomal cytochrome P-450 dependent monooxygenase a c t i v i t i e s . Pregnenolone-16°<-c a r b o n i t r i l e (40 mg/kg/day i p ) and 3 - m e t h y l c h o l a n t h r e n e (20 mg/kg/day i p ) were a d m i n i s t e r e d f o r 4 and 2 da y s , r e s p e c t i v e l y . The a s t e r i s k i n d i c a t e s s i g n i f i -c a n t d i f f e r e n c e from c o n t r o l a t P<0.05. The double a s t e r i s k i n d i c a t e s a s i g n i f i c a n t i n t e r a c t i o n e f f e c t (Two-way ANOVA, P<0.05). Aryl Hydrocarbon Hydroxylase cont PCN 3-MC PCN 3-MC Ethylmorphine N-Demethylase cont PCN 3-MC PCN + 3-MC -121-F i g u r e 26. S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of l i v e r microsomes from a d u l t female r a t s t r e a t e d f o r 4 days with e i t h e r p h e n o b a r b i t a l (Pb, 80 mg/kg/day), t r a n s - s t i l b e n e o x i d e ( t - s o , 200 mg/kg/day), s p i r o n o l a c t o n e (SPIR, 200 mg/kg/day) o r 3 - m e t h y l c h o l a n t h r e n e (3-MC, 20 mg/kg/day). The c o n t r o l r e c e i ved c o r n o i l . -123-band C (61,000 m o l e c u l a r weight) and band E (50,000 m o l e c u l a r w e i g h t ) . A r e g i o n o f high i n t e n s i t y s t a i n i n g ( r e g i o n D) appeared to c o n s i s t of s e v e r a l bands. A c c o r d i n g t o the d e n s i t o m e t e r t r a c i n g s , c o n t r o l microsomes c o n t a i n e d 2 predominant bands i n t h i s r e g i o n (53,000 and 56,000 m o l e c u l a r w e i g h t ) . Microsomes from p h e n o b a r b i t a l - and t r a n s - s t i l b e n e o x i d e - t r e a t e d r a t s c o n t a i n e d a major band in t h i s r e g i o n (53,000 m o l e c u l a r w e i g h t ) . S e v e r a l p r o t e i n s t a i n i n g bands were p r e s e n t i n microsomes from r a t s t r e a t e d with s p i r o n o l a c t o n e (55,000 and 56,000 m o l e c u l a r weight) and t h e r e was a major band w i t h 3 - m e t h y l c h o l a n t h r e n e (55,000 m o l e c u l a r w e i g h t ) . The c h a r a c t e r i s t i c f e a t u r e s noted i n the v a r i o u s gel p a t t e r n s were: p h e n o b a r b i t a l and t r a n s - s t i l b e n e o x i d e i n c r e a s e d band s t a i n i n g at 50,000 and 53,000 m o l e c u l a r weight; 3 - m e t h y l c h o l a n t h r e n e i n c r e a s e d band s t a i n i n g a t 55,000 m o l e c u l a r w e i g h t ; and s p i r o n o l a c t o n e r e s u l t e d i n a r e l a t i v e l o s s of s t a i n i n g i n r e g i o n D w i t h 2 apparent bands p r e s e n t at 55,000 and 56,000 m o l e c u l a r weight. Some g e l s were all o w e d to d e s t a i n f o r l o n g e r p e r i o d s of t i m e . These g e l s ( F i g u r e 27) c l e a r l y i n d i c a t e d t h e 2 major bands present i n microsomes from p h e n o b a r b i t a l - and t r a n s - s t i l b e n e o x i d e - t r e a t e d r a t s (50,000 and 53,000 m o l e c u l a r weight) and the s i n g l e major band pres e n t i n microsomes from 3 - m e t h y l c h o l a n t h r e n e - t r e a t e d r a t s (55,000 m o l e c u l a r w e i g h t ) . H e p a t i c microsomes were prepared from a d u l t female r a t s t r e a t e d f o r 7 days w i t h e i t h e r p h e n o b a r b i t a l (80 mg/kg/day), t r a n s - s t i l b e n e o x i d e (200 mg/ kg/day), s p i r o n o l a c t o n e (200 mg/kg/day) o r 3 - m e t h y l c h o l a n t h r e n e (20 mg/kg/ d a y ) . Microsomes from corn o i l c o n t r o l s were a l s o p r e p a r e d . The p r o t e i n band p a t t e r n s o b t a i n e d with t h e s e microsomal p r e p a r a t i o n s are shown i n F i g u r e 28. The band p a t t e r n s appeared to be the same as observed f o l l o w i n g 4 days of t r e a t m e n t with t h e s e a g e n t s . The same type of experiment was c a r r i e d out f o l l o w i n g treatment f o r 7 days w i t h e i t h e r p h e n o b a r b i t a l (80 mg/kg/day), p r e g n e n o l o n e - 1 6 < * - c a r b o n i t r i l e -124-F i g u r e 27. S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f l i v e r microsomes from a d u l t female r a t s t r e a t e d f o r 4 days with e i t h e r p h e n o b a r b i t a l (Pb, 80 mg/kg/day), t r a n s - s t i l b e n e o x i d e ( t - s o , 200 mg/kg/day), or 3-m e t h y l c h o l a n -t h r e n e (3-MC, 20 mg/kg/day). The c o n t r o l r e c e i v e d corn o i l . - 1 2 5 -r 1 Mi - — control P b t-SO 3-MC -126-F i g u r e 28. S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f l i v e r microsomes from a d u l t female r a t s t r e a t e d f o r 7 days with e i t h e r p h e n o b a r b i t a l (Pb, 80 mg/kg/day), t r a n s - s t i l b e n e o x i d e ( t - s o , 200 mg/kg/day), s p i r o n o l a c t o n e (SPIR, 200 mg/kg/day) o r 3 - m e t h y l c h o l a n t h r e n e (3-MC, 20 mg/kg/day). The c o n t r o l r e c e i v e d c o r n o i l . -128-(40 mg/kg/day) or 3 - m e t h y l c h o l a n t h r e n e (20 mg/kg/day) ( F i g u r e 2 9 ) . In mic r o -somes from p h e n o b a r b i t a l t r e a t e d r a t s , a h i g h l y s i g n i f i c a n t i n c r e a s e i n s t a i n i n g was found i n two p r o t e i n bands (50,000 and 53,000 m o l e c u l a r w e i g h t ) . The microsomes from the 3 - m e t h y l c h o l a n t h r e n e - t r e a t e d animals e x h i b i t e d a major band at 55,000 m o l e c u l a r weight. Microsomes from pregnenolone-16<*-c a r b o n i t r i l e - t r e a t e d r a t s e x h i b i t e d 2 major p r o t e i n s t a i n i n g bands at 50,000 and 55,000 m o l e c u l a r weight. -129-F i g u r e 29. S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of l i v e r microsomes from a d u l t female r a t s t r e a t e d f o r 7 days with e i t h e r p h e n o b a r b i t a l (Pb, 80 mg/kg/day), pregneno1one-16«~carbonitrile (PCN, 40 mg/kg/day) or 3-methyl-c h o l a n t h r e n e (3-MC, 20 mg/kg/day). The c o n t r o l r e c e i v e d corn o i l . -130--131-DISCUSSION I. N u c l e a r L o c a l i z a t i o n o f Rat H e p a t i c Drug M e t a b o l i z i n g  Enzymes 1) The Q u e s t i o n o f Microsomal C o n t a m i n a t i o n The l e v e l s o f cytochrome P-450 and r e l a t e d monooxygenase a c t i v i t i e s i n n u c l e a r p r e p a r a t i o n s from r a t l i v e r a r e q u i t e low i n comparison to t h o s e o b s e r v e d i n the microsomal f r a c t i o n . For example, AHH and N-demethylase a c t i v i t i e s i n the n u c l e a r membrane were 7 and 11 per c e n t o f the s p e c i f i c a c t i v i t i e s o b t a i n e d f o r the same enzymes i n the microsomal membrane, r e s p e c t i v e l y (Kasper, 1971). T h e r e f o r e , the r e l a t i v e l y low l e v e l s found i n n u c l e a r p r e p a r a t i o n s c o u l d s i m p l y be due t o microsomal c o n t a m i n a t i o n . In the p r e s e n t s t u d y , e l e c t r o n m i c r o g r a p h s v e r i f i e d the p r e s e n c e o f n u c l e i i n the p e l l e t o b t a i n e d f o l l o w i n g d i s c o n t i n u o u s s u c r o s e g r a d i e n t c e n t r i f u g a t i o n . However, the degree o f microsomal c o n t a m i n a t i o n c o u l d not be determined q u a n t i t a t i v e l y by t h i s method. The o c c l u s i o n o f microsomes by the l a y e r o f 2.3M s u c r o s e was v e r i f i e d b i o c h e m i c a l l y , u s i n g AHH a c t i v i t y as the marker. C e n t r i f u g a t i o n o f microsomes a l o n e y i e l d e d a f i l m a t the bottom o f the tube t h a t e x h i b i t e d AHH a c t i v i t y t h a t was about 1 per c e n t o f t h a t o b s e r v e d i n i s o l a t e d h e p a t i c n u c l e i . A f t e r s u b j e c t i n g the n u c l e i to f u r t h e r d i s c o n t i n u o u s s u c r o s e - g r a d i e n t c e n t r i f u g a t i o n , i n the p r e s e n c e o r absence o f l i v e r microsomes, the r e s u l t i n g a c t i v i t i e s o f AHH were not d i f f e r e n t from c o n t r o l . These e x p e r i -ments demonstrated t h a t the endoplasmic r e t i c u l u m , i n the form o f microsomes g e n e r a t e d by h o m o g e n i z a t i o n , was not r e s p o n s i b l e f o r the o b s e r v e d AHH a c t i v i t y i n the n u c l e a r p e l l e t . -132-A second p o t e n t i a l form o f endoplasmic r e t i c u l u m c o n t a m i n a t i o n e x i s t s which p r e s e n t s a more d i f f i c u l t problem to r e s o l v e . S i n c e the endoplasmic r e t i c u l u m i s c o n t i n u o u s w i t h the o u t e r n u c l e a r membrane a t c e r t a i n p o i n t s , i t i s p o s s i b l e t h a t i s o l a t e d n u c l e i and p u r i f i e d n u c l e a r e n v e l o p e p r e p a r a t i o n s c o u l d be c o n t a m i n a t e d w i t h fragments o r " t a g s " o f the endoplasmic r e t i c u l u m . Small " t a g s " o f endoplasmic r e t i c u l u m , not v i s i b l e by e l e c t r o n m i c r o s c o p y , c o u l d remain a t t a c h e d to the e n v e l o p e o f the i s o l a t e d n u c l e i f o l l o w i n g c e n t r i f u g a t i o n and c o u l d a c c o u n t f o r a l l o f the enzyme a c t i v i t y d e t e c t e d i n t h i s f r a c t i o n . I t was reasoned t h a t i f endoplasmic r e t i c u l u m " t a g s " a c c o u n t e d f o r a l l o f t h e o b s e r v e d n u c l e a r a c t i v i t y , the n u c l e a r / m i c r o s o m a l r a t i o s f o r a l l enzyme a c t i v i t i e s measured would be e x p e c t e d to be the same. The basal a c t i v i t i e s o f t h r e e cytochrome P-450 dependent 4 monooxygenases, p l u s t e s t o s t e r o n e A - hydrogenase and e p o x i d e h y d r o l a s e were de t e r m i n e d i n h e p a t i c n u c l e i and microsomes. A n i l i n e h y d r o x y l a s e , a m i n o p y r i n e N-demethylase, and AHH a c t i v i t i e s were p r e s e n t i n the i s o l a t e d n u c l e i a t about 4% o f t h e i r r e s p e c t i v e microsomal a c t i v i t i e s . However, the e p o x i d e h y d r o l a s e and 4 t e s t o s t e r o n e A - hydrogenase a c t i v i t i e s were about 12.5 and 15 per c e n t o f t h e i r r e s p e c t i v e microsomal a c t i v i t i e s . The r a t i o o f e p o x i d e h y d r o l a s e to AHH i n h e p a t i c microsomes was 1.6:1, whereas t h i s r a t i o o f a c t i v i t i e s i n h e p a t i c n u c l e i was 4.5:1. One o f the c o n c l u s i o n s t h a t can be reached from t h e s e experiments i s t h a t the a c t i v i t i e s o b s e r v e d i n the n u c l e a r f r a c t i o n a r e due to enzymes o f n u c l e a r o r i g i n and a r e not s i m p l y a r e s u l t o f endoplasmic r e t i c u l u m c o n t a m i n a t i o n . T h i s assumes t h a t the enzyme a c t i v i t i e s a r e e q u a l l y - 133 -d i s t r i b i t t e d t h r o u g h o u t the e n d o p l a s m i c r e t i c u l u m . A l t e r n a t i v e l y , t h e s e " t a g s " o f endoplasmic r e t i c u l u m might have a d i f f e r e n t d i s t r i b u t i o n o f t h e enzymes measured as compared t o the t o t a l e n d o plasmic r e t i c u l u m i s o l a t e d from h e p a t o c y t e s . The microsomal f r a c t i o n has been found by one group to c o n s i s t o f a m i x t u r e o f smooth and rough e n d o p l a s m i c r e t i c u l u m which e x h i b i t d i f f e r e n t enzyme d i s t r i b u t i o n s ( S e i d e g a r d e t al _ , 1978). These workers showed t h a t the d i s t r i b u t i o n o f AHH and e p o x i d e h y d r o l a s e a c t i v i t i e s t h r o u g h o u t t h e endoplasmic r e t i c u l u m was h i g h l y heterogenous. T h i s was demonstrated i n p r e p a r a t i o n s from c o n t r o l and i n d u c e d r a t s . The y i e l d o f n u c l e a r p r o t e i n per gram o f l i v e r was about 2.7 mg, whereas t h e microsomal f r a c t i o n r e p r e s e n t e d about 28 mg o f p r o t e i n per gram o f l i v e r . When the n u c l e a r and microsomal a c t i v i t i e s were e x p r e s s e d per gram o f l i v e r , the a c t i v i t i e s o f monooxygenases i n the i s o l a t e d n u c l e a r f r a c t i o n were about 0.4 per c e n t o f the r e s p e c t i v e a c t i v i t i e s o b s e r v e d i n the microsomes, 4 whereas the comparable r a t i o o f t e s t o s t e r o n e A - hydrogenase and e p o x i d e h y d r o l a s e i n the two f r a c t i o n s were 1.7 and 1.3 per c e n t , r e s p e c t i v e l y . T h e r e f o r e , l o o k i n g a t t o t a l c e l l u l a r m e t a b o l i s m , the n u c l e a r f r a c t i o n would c o n t r i b u t e r e l a t i v e l y l i t t l e t o t h e o v e r a l l m e t abolism o f s u b s t r a t e s by the h e p a t o c y t e . However, because o f t h e i r p r o x i m i t y to the n u c l e a r DNA, t h e s e " n u c l e a r enzymes" may assume t o x i c o l o g i c a l s i g n i f i c a n c e , whether they a r e p r e s e n t on endoplasmic r e t i c u l u m as " t a g s " o r on the n u c l e a r membrane i t s e l f . -134-2) A Comparison o f N u c l e a r and Microsomal Drug M e t a b o l i z i n g  Enzymes The s e l e c t i v e m a n i p u l a t i o n o f n u c l e a r enzymes would be a u s e f u l t o o l t o e v a l u a t e the c o n t r i b u t i o n o f t h e s e enzymes towards the c o v a l e n t b i n d i n g o f c a r c i n o g e n s to n u c l e a r DNA and, u l t i m a t e l y , t h e i r r o l e i n t h e i n i t i a t i o n o f n e o p l a s i a . The age and sex dependency, as w e l l as the e f f e c t s o f i n d u c e r s , i n h i b i t o r s and a c t i v a t o r s o f h e p a t i c drug m e t a b o l i z i n g enzymes were i n v e s t i g a t e d . In the p r e s e n t e x p e r i m e n t s , many s i m i l a r i t i e s were o b s e r v e d between the enzymes o f microsomal and n u c l e a r o r i g i n s . The a p p a r e n t Km v a l u e s d e t e r m i n e d f o r both n u c l e a r and microsomal AHH were s i m i l a r . AHH a c t i v i t y was shown to be age and sex dependent i n r a t h e p a t i c microsomes. The same t r e n d s were found w i t h AHH 4 l e v e l s i n i s o l a t e d r a t h e p a t i c n u c l e i . T e s t o s t e r o n e A -hydrogenase was found t o be 4 to 5 f o l d h i g h e r i n the a d u l t female than t h e a d u l t male i n both n u c l e i : and microsomes. SKF 525-A produced a c o n c e n t r a t i o n dependent i n h i b i t i o n o f microsomal and n u c l e a r AHH. The i n h i b i t i o n o f n u c l e a r and microsomal AHH by SKF 525-A was d e t e r m i n e d i n h e p a t i c p r e p a r a t i o n s d e r i v e d from c o n t r o l , p h e n o b a r b i t a l - , and 3 - m e t h y l c h o l a n t h r e n e - t r e a t e d r a t s . In each c a s e t h e i n h i b i t i o n was i d e n t i c a l i n both n u c l e a r and microsomal f r a c t i o n s . The e f f e c t s o f i n v i t r o a d d i t i o n s o f < x - n a p h t h o f l a v o n e on both n u c l e a r and microsomal AHH were the same. In the 3-m e t h y l c h o l a n t h r e n e - t r e a t e d r a t s , AHH was i n h i b i t e d by cx> n a p h t h o f l a v o n e , whereas i n the c o n t r o l male and the p h e n o b a r b i t a l -t r e a t e d male and f e m a l e groups t h e compound s t i m u l a t e d AHH. <x-N a p h t h o f l a v o n e had no e f f e c t on AHH i n c o n t r o l female r a t s . -135-A d m i n i s t r a t i o n o f t r a n s - s t i l b e n e o x i d e t o a d u l t male r a t s r e s u l t e d i n a b o u t a 50 per c e n t d e c r e a s e i n h e p a t i c microsomal AHH. The same e f f e c t was o b s e r v e d w i t h n u c l e a r AHH. N u c l e a r and microsomal e p o x i d e h y d r o l a s e were s i m i l a r l y i n h i b i t e d by 1,2-epoxy-3,3,3-t r i c h l o r o p r o p a n e . The i n h i b i t i o n o f AHH a c t i v i t y by SKF 525-A, the e f f e c t s o f o c-naphthoflavone on AHH a c t i v i t y and the a p p a r e n t Km v a l u e o f AHH were the same i n the n u c l e a r and microsomal f r a c t i o n s . These s t u d i e s s t r o n g l y s u g g e s t t h a t the c o n s t i t u t i v e f o r m ( s ) o f AHH a r e the same i n the two f r a c t i o n s . O nly the r e l a t i v e q u a n t i t i e s o f t h e s e enzymes d i f f e r i n the two s u b c e l l u l a r f r a c t i o n s . S i m i l a r e f f e c t s o f SKF 525-A and<<-naphthoflavone on h e p a t i c n u c l e a r and microsomal AHH d e r i v e d from p h e n o b a r b i t a l - and 3-m e t h y l c h o l a n t h r e n e - t r e a t e d r a t s s u g g e s t t h a t the i n d u c e d forms o f AHH a r e a l s o s i m i l a r i n the two f r a c t i o n s . The c o n s t i t u t i v e f o r m ( s ) o f e p o x i d e h y d r o l a s e a l s o appear to be s i m i l a r based on the i n d i s t i n g u i s h a b l e i n h i b i t i o n o f e p o x i d e h y d r o l a s e i n n u c l e a r and microsomal f r a c t i o n s . The s i m i l a r i t i e s found between microsomal and n u c l e a r AHH and e p o x i d e h y d r o l a s e s t r o n g l y s u g g e s t t h a t the enzymes o f n u c l e a r and microsomal o r i g i n a r e the same p r o t e i n s . Only the q u a n t i t y o f enzyme d i f f e r s between the two f r a c t i o n s . These d a t a a r e i n a c c o r d w i t h the s t u d y o f Thomas e t al_ (1979) who demonstrated t h a t n u c l e a r and microsomal cytochrome P-448 as w e l l as e p o x i d e h y d r o l a s e were i m m u n o l o g i c a l l y i d e n t i c a l . C e r t a i n d i f f e r e n c e s were o b s e r v e d between t h e enzymes o f microsomal and n u c l e a r o r i g i n s . B e s i d e s the r a t i o o f e p o x i d e h y d r o l a s e to AHH i n the i s o l a t e d n u c l e i being about 3 times g r e a t e r -136-than the same r a t i o o f enzyme a c t i v i t i e s found i n the microsomes (4.5 v e r s u s 1.6), s e v e r a l o t h e r d i s s i m i l a r i t i e s were o b s e r v e d . P h e n o b a r b i t a l a d m i n i s t e r e d t o immature male and female r a t s produced s i g n i f i c a n t i n c r e a s e s i n h e p a t i c microsomal and n u c l e a r AHH a c t i v i t y . However, the i n c r e a s e s o b s e r v e d i n the microsomal f r a c t i o n were s u b s t a n t i a l l y g r e a t e r than t h o s e seen i n the n u c l e a r f r a c t i o n . The s m a l l e r i n c r e a s e i n n u c l e a r enzyme a c t i v i t y f o l l o w i n g 4 days o f p h e n o b a r b i t a l t r e a t m e n t c o u l d have been a r e s u l t o f a d e c r e a s e d r a t e o f i n c o r p o r a t i o n o f new enzyme i n t o n u c l e a r membrane r e l a t i v e t o the r a t e o f i n c o r p o r a t i o n i n t o the endoplasmic r e t i c u l u m . However, a slower r a t e o f i n d u c t i o n o f n u c l e a r AHH v e r s u s microsomal AHH was not s u p p o r t e d by the experiment d e m o n s t r a t i n g the time c o u r s e AHH i n d u c t i o n i n the f e m a l e r a t f o l l o w i n g d a i l y a d m i n i s t r a t i o n o f p h e n o b a r b i t a l . The i n d u c t i o n o f microsomal AHH was g r e a t e r a t a l l time p o i n t s between one and s i x days o f p h e n o b a r b i t a l t r e a t m e n t , and the i n d u c t i o n was d e f i n i t e l y maximum i n both f r a c t i o n s a f t e r 4 days. O p p o s i t e t o t h a t o b s e r v e d w i t h p h e n o b a r b i t a l , 3 - m e t h y l c h o l a n t h r e n e i n c r e a s e d n u c l e a r AHH a c t i v i t y i n immature male r a t s by a g r e a t e r p e r c e n t a g e than i n the microsomes. A l s o the i n c r e a s e s i n h e p a t i c n u c l e a r e p o x i d e h y d r o l a s e a c t i v i t y o b s e r v e d f o l l o w i n g p h e n o b a r b i t a l , a c e t y l m e t h a d o l and t r a n s - s t i l b e n e o x i d e t r e a t m e n t were about 3 f o l d lower than the i n c r e a s e s o b s e r v e d i n microsomal e p o x i d e h y d r o l a s e a c t i v i t y . These d i f f e r e n c e s s u g g e s t t h a t endoplasmic r e t i c u l u m c o n t a m i n a t i o n , i n the form o f small " t a g s " o f endoplasmic r e t i c u l u m c o n t i n u o u s w i t h the o u t e r n u c l e a r membrane, does not a c c o u n t f o r a l l o f the enzymatic a c t i v i t y measured i n i s o l a t e d h e p a t i c n u c l e i . -137-However, t h e s e segments o f membrane c o u l d d i f f e r from the o v e r a l l e ndoplasmic r e t i c u l u m (microsomes) i n the r e l a t i v e q u a n t i t i e s o f i n d u c e d as w e l l as c o n s t i t u t i v e enzymes. In summary, the enzyme a c t i v i t i e s o b s e r v e d i n i s o l a t e d r a t h e p a t i c n u c l e i a r e not a r e s u l t o f c o n t a m i n a t i o n by microsomes formed upon ho m o g e n i z a t i o n . The i s s u e o f endoplasmic r e t i c u l u m " t a g s " as a p o t e n t i a l form o f c o n t a m i n a t i o n cannot be r u l e d o u t i n our s t u d i e s u t i l i z i n g n u c l e a r f r a c t i o n s , s i n c e t h e s e " t a g s " may d i f f e r from the o v e r a l l e n d oplasmic r e t i c u l u m (microsomes) i n the r e l a t i v e d i s t r i b u t i o n o f c o n s t i t u t i v e o r i n duced enzyme a c t i v i t i e s . I t a p p e a r s t h a t both o f t h e s e forms o f enzymes a r e the same i n the two f r a c t i o n s and t h e y s i m p l y d i f f e r i n r e l a t i v e amounts p r e s e n t . In s u p p o r t o f t h i s c o n c l u s i o n , B r e s n i c k and coworkers (1980) demonstrated the p r e s e n c e o f cytochrome P-450 i n p u r i f i e d n u c l e o l i o b t a i n e d from the l i v e r s o f 3 - m e t h y l c h o l a n t h r e n e - t r e a t e d r a t s . U s i n g m o n o s p e c i f i c a n t i b o d y t o microsomal cytochrome P-448 and e p o x i d e h y d r o l a s e , t h e y found t h a t t h e n u c l e o l a r and microsomal enzymes were i m m u n o l o g i c a l l y i d e n t i c a l . Whether " n u c l e a r " a c t i v i t i e s e x i s t i n the n u c l e o p l a s m , or the n u c l e a r e n v e l o p e o r as endoplasmic r e t i c u l u m " t a g s " c o n t i n u o u s w i t h the n u c l e a r e n v e l o p e ( i n d i s c e r n a b l e i n e l e c t r o n m i c r o g r a p h s ) , they r e p r e s e n t a c t i v i t i e s i n c l o s e p r o x i m i t y t o the n u c l e a r DNA. I f t h e s e n u c l e a r enzymes c o u l d be s e l e c t i v e l y m a n i p u l a t e d then t h e y c o u l d be s t u d i e d i n i s o l a t e d h e p a t o c y t e s o r i n v i v o t o d e t e r m i n e t h e i r i n v o l v e m e n t s i n t h e i n i t i a t i o n o f n e o p l a s i a . U n f o r t u n a t e l y , t h a t p a r t i c u l a r goal o f our s t u d i e s was not a c h i e v e d . -138-I I . The I n d u c t i o n o f Rat H e p a t i c Drug M e t a b o l i z i n g Enzymes 1) Sex and Age Dependence o f the I n d u c t i o n o f Rat H e p a t i c  Microsomal Drug M e t a b o l i z i n g Enzymes In the p r e s e n t s t u d y , the i n d u c t i o n r e s p o n s e s produced by t r a n s - s t i l b e n e o x i d e and s p i r o n o l a c t o n e were compared t o the t h r e e unique i n d u c t i o n r e s p o n s e s t h a t have been w e l l documented. 3-M e t h y l c h o l a n t h r e n e i s t h e p r o t o t y p e f o r a group o f s t r u c t u r a l l y r e l a t e d compounds t h a t e l i c i t a c h a r a c t e r i s t i c p l e i o t r o p i c r e s p o n s e . P h e n o b a r b i t a l produces i t s own c h a r a c t e r i s t i c b i o -c h e m i c a l r e s p o n s e and pregnenolone-16°<-carbonitrile e l i c i t s the t h i r d unique i n d u c t i o n r e s p o n s e . F i r t l y , t he e f f e c t s o f age and sex on the i n d u c t i o n r e s p o n s e s produced by t h e s e agents were i n v e s t i g a t e d . T h i s was prompted by the a p p a r e n t d i s c r e p a n c y i n r e s u l t s between the s t u d i e s o f Schmassmann and Oesch (1978) and Mukhtar and coworkers (1978). Schmassmann and Oesch demonstrated t h a t t r a n s - s t i l b e n e o x i d e i n c r e a s e d e p o x i d e h y d r o l a s e a c t i v i t y w i t h o u t a s i m u l t a n e o u s i n c r e a s e i n cytochrome P-450 and r e l a t e d monooxygenase a c t i v i t i e s . Based on t h e s e o b s e r v a t i o n s , t h e s e workers suggested t h a t t r a n s -s t i l b e n e o x i d e was a u n i q u e , " s e l e c t i v e " i n d u c e r o f h e p a t i c microsomal e p o x i d e h y d r o l a s e . However, Mukhtar and coworkers : (1978) found t h a t t r a n s - s t i l b e n e o x i d e i n c r e a s e d e p o x i d e h y d r o l a s e a c t i v i t y , as w e l l as cytochrome P-450 and r e l a t e d monooxygenase a c t i v i t i e s . We reasoned t h a t r a t s o f d i f f e r i n g age and sex used i n t h e s e s t u d i e s c o u l d be t h e s o u r c e o f t h e a p p a r e n t d i s c r e p a n c y . Schmassmann and Oesch employed a d u l t male r a t s , whereas Mukhtar and coworkers c a r r i e d out t h e i r e x p eriments i n immature males and -139-f e m a l e s . I t i s w e l l known t h a t the i n d u c t i b i l i t y o f h e p a t i c microsomal enzymes i s i n f l u e n c e d by both the age and sex o f the r a t s employed (Conney, 1967). B e l l ward and coworkers (1977) have r e p o r t e d t h a t methadone t r e a t m e n t i n c r e a s e d h e p a t i c microsomal e p o x i d e h y d r o l a s e a c t i v i t y w i t h o u t a l t e r i n g AHH a c t i v i t y i n a d u l t male r a t s . In a d u l t female r a t s , however, AHH a c t i v i t y was i n c r e a s e d by methadone. T h e r e f o r e , the e f f e c t s o f age and sex on the i n d u c t i o n r e s p o n s e produced by t r a n s - s t i l b e n e o x i d e were i n v e s t i g a t e d . In a d d i t i o n , t h e s e s t u d i e s were extended to i n c l u d e p h e n o b a r b i t a l , 3 - m e t h y l c h o l a n t h r e n e , s p i r o n o l a c t o n e , p r e g n e n o l e n e -1 6 < x - c a r b o n i t r i l e and a c e t y l m e t h a d o l , a l o n g e r a c t i n g congener o f methadone ( B e l l ward e t al_, 1977a). In t h e p r e s e n t s t u d y t r a n s - s t i l b e n e o x i d e was f o u n d t o i n c r e a s e AHH a c t i v i t y i n the immature male and a d u l t female r a t s . In the a d u l t male r a t , however, t h i s compound produced a dose-dependent d e c r e a s e i n AHH a c t i v i t y . E p o x i d e h y d r o l a s a c t i v i t y was i n c r e a s e d i n a l l r a t s s t u d i e d f o l l o w i n g t r a n s - s t i l b e n e o x i d e t r e a t m e n t . Aminopyrine N-demethylase a c t i v i t y was i n c r e a s e d by t r a n s - s t i l b e n e o x i d e i n a d u l t female and a d u l t male r a t s . T h e r e f o r e , t r a n s -s t i l b e n e o x i d e was not a " s e l e c t i v e " i n d u c e r o f e p o x i d e h y d r o l a s e . T h i s was s u p p o r t e d by the s t u d y o f S e i d e g a r d and coworkers (1979), who found t h a t t o t a l microsomal cytochrome P-450 was i n c r e a s e d by t r a n s - s t i l b e n e o x i d e i n a d u l t male r a t s . The e f f e c t s produced by t r a n s - s t i l b e n e o x i d e were s i m i l a r t o t h o s e o b s e r v e d f o l l o w i n g p h e n o b a r b i t a l t r e a t m e n t . P h e n o b a r b i t a l was found t o i n c r e a s e e p o x i d e h y d r o l a s e and a m i n o p y r i n e N-demethylase a c t i v i t i e s i n a l l r a t s s t u d i e d . However, AHH a c t i v i t y was i n c r e a s e d o n l y i n the immature male and a d u l t f e m a l e , but not -140-i n t he a d u l t male. In c o n t r a s t , 3 - m e t h y l c h o l a n t h r e n e was found to i n c r e a s e AHH a c t i v i t y i n a l l r a t s s t u d i e d , but was w i t h o u t e f f e c t on e p o x i d e h y d r o l a s e and a m i n o p y r i n e N-demethylase a c t i v i t i e s . T h e r e f o r e , t r a n s - s t i l b e n e o x i d e appeared t o be p h e n o b a r b i t a l - 1 i k e i n i t s p a t t e r n o f i n d u c t i o n . P r e g n e n o l o n e - 1 6 < x - c a r b o n i t r i l e was found to produce a s i g n i f i c a n t i n c r e a s e i n AHH a c t i v i t y i n t h e ~ a d u l t female r a t ; however t h i s a c t i v i t y was not i n c r e a s e d by pregnenolone-! 6<*-c a r b o n i t r i l e i n the a d u l t male. Aminopyrine N-demethylase a c t i v i t y was i n c r e a s e d i n both the male and female by t h i s agent. S p i r o n o l a c t o n e t r e a t m e n t r e s u l t e d i n an i n c r e a s e d AHH a c t i v i t y i n a d u l t female r a t s and a d e c r e a s e d AHH a c t i v i t y i n a d u l t male r a t s . A m i n o p y r i n e N-demethylase and e p o x i d e h y d r o l a s e a c t i v i t i e s were i n c r e a s e d by s p i r o n o l a c t e n e i n both male and female r a t s . A c e t y l m e t h a d o l was f o u n d t o i n c r e a s e e p o x i d e h y d r o l a s e a c t i v i t y i n a d u l t male and female r a t s ; however AHH a c t i v i t y was found to be i n c r e a s e d o n l y i n , a d u l t f e m a l e s . In a d u l t males, a c e t y l m e t h a d o l , a t h i g h e r d o s e s , was a l s o found to produce a d e c r e a s e i n AHH a c t i v i t y . In summary, o n l y 3 - m e t h y l c h o l a n t h r e n e produced a s i g n i f i c a n t i n c r e a s e i n AHH a c t i v i t y i n the a d u l t male r a t . A l l the o t h e r a g e n t s s t u d i e d e i t h e r had no e f f e c t o r d e c r e a s e d t h e AHH a c t i v i t y from c o n t r o l , even though e v e r y one o f t h e s e agents i n c r e a s e d AHH a c t i v i t y i n t h e female r a t . However, a l l but one o f the agents i n c r e a s e d e p o x i d e h y d r o l a s e and/or a m i n o p y r i n e N-demethylase a c t i v i t i e s i n a l l r a t s s t u d i e d . The e x c e p t i o n was 3-m e t h y l c h o l a n t h r e n e , which d i d not a l t e r t h e s e a c t i v i t i e s i n male -141T o r f e m a l e r a t s . I t i s w e l l e s t a b l i s h e d t h a t t h e r e i s a sex d i f f e r e n c e i n the ba s a l a c t i v i t i e s o f c e r t a i n cytochrome P-450 dependent monooxygenases (Conney, 1967; E l Defrawy E l Masry e t _ a l _ , 1974; Framer e t al _ , 1979). The b a s a l l e v e l s o f AHH and a m i n o p y r i n e N-demethylase a r e g r e a t e r i n the a d u l t male than the a d u l t f e m a l e . T h e r e i s no a p p a r e n t sex d i f f e r e n c e i n drug m e t a b o l i z i n g enzyme a c t i v i t y i n the immature r a t (Henderson, 1971; E l Defrawy E l Masry e t a l _ , 1974). However, as male r a t s mature, enzyme a c t i v i t i e s i n c r e a s e , c o i n c i d e n t a l w i t h the i n c r e a s e i n serum t e s t o s t e r o n e l e v e l s ( F o r e s t , 1979). T e s t o s t e r o n e , i n p a r t , i s n e c e s s a r y t o m a i n t a i n t h e sex d i f f e r e n c e . C a s t r a t i o n o f male r a t s r e s u l t s i n a d e c r e a s e i n enzyme l e v e l s , whereas t e s t o s t e r o n e a d m i n i s t r a t i o n to c a s t r a t e d males o r immature f e m a l e s r e s u l t s i n enzyme a c t i v i t i e s o b s e r v e d i n the a d u l t male ( E l Defrawy E l Masry and Mannering, 1974; Gurtoo and P a r k e r , 1977). Ovariectomy o f a d u l t f emale r a t s has no e f f e c t on h e p a t i c drug m e t a b o l i z i n g enzymes (Gurtoo and P a r k e r , 1977; G o n t o v n i c k e t al _ , 1979). The i n c r e a s e s i n cytochrome P-450 dependent monooxygenase a c t i v i t i e s by t e s t o s t e r o n e does not appear to i n v o l v e a s i g n i f i c a n t change i n t o t a l cytochrome P-450 c o n t e n t (Kramer e t al_, 1979). In f a c t , t h e r e does not appear to be a sex d i f f e r e n c e i n t o t a l cytochrome P-450 ( S t r i p p et_ a l , 1973), even though the d i f f e r e n c e i n cytochrome P-450 dependent enzyme a c t i v i t y between males and f e m a l e s i s many f o l d . The i n c r e a s e i n cytochrome P-450 dependent oxygenase a c t i v i t i e s by t e s t o s t e r o n e was shown t o be m e d i a t e d by c e n t r a l hormones (Kramer e t aj_, 1979). From the r e s u l t s o f t h e s e p r e v i o u s s t u d i e s , -142 -t e s t o s t e r o n e i n the r a t may be r e g a r d e d as an endogenous i n d u c e r o f monooxygenase a c t i v i t y . The i n c r e a s e s i n cytochrome P-450 dependent monooxygenases produced by t e s t o s t e r o n e upon m a t u r i t y o f the male r a t a r e l i k e l y due to t h e i n d u c t i o n o f a s p e c i f i c a n d r o g e n - i n d u c e d form o f cytochrome P-450. The i n d u c t i o n o f the drug m e t a b o l i z i n g enzymes by t e s t o s t e r o n e has been su g g e s t e d t o be m e diated by a h e p a t i c c y t o s o l i c androgen r e c e p t e r , s i m i l a r to the mechanism i n v o k e d i n the r e g u l a t i o n o f androgen a c t i o n i n p r o s t a t e (Brown e t al_, 1976; S k e t t and G u s t a f s s o n , 1979; Sonawe e t al_, 1979). In the p r e s e n t s t u d y , t h e sex d i f f e r e n c e i n AHH a c t i v i t y was o f the o r d e r o f 6 to 8 f o l d . T h e r e f o r e , t e s t o s t e r o n e i n the W i s t a r r a t can be r e g a r d e d as a v e r y e f f e c t i v e i n d u c e r o f AHH a c t i v i t y . In the a d u l t male r a t , AHH a c t i v i t y was not i n c r e a s e d f u r t h e r by. p h e n o b a r b i t a l o r p r e g n e n o l o n e - 1 6 < * - c a r o n i t r i l e , even though t h e y were e f f e c t i v e i n d u c e r s i n the female r a t . The p r e s e n c e o f t e s t o s t e r o n e and i t s subsequent e f f e c t s on AHH a c t i v i t y appears t o p r e v e n t the f u r t h e r i n c r e a s e i n a p p a r e n t AHH a c t i v i t y by t h e s e a g e n t s . 3 - M e t h y l c h o l a n t h r e n e was c a p a b l e o f i n c r e a s i n g AHH a c t i v i t y i n t h e a d u l t male r a t s , however the a b s o l u t e i n c r e a s e s appeared t o be s m a l l e r i n the a d u l t male than i n the immature male o r f e m a l e . '.The AHH a c t i v i t y i n the 3 - m e t h y l c h o l a n t h r e n e - t r e a t e d a d u l t male appears to be l e s s than t h a t p r e d i c t e d based upon the a b s o l u t e i n c r e a s e i n AHH a c t i v i t y produced by t h i s compound i n the f e m a l e . Warren and B e l l w a r d (1978) found t h a t m a x i m a l l y e f f e c t i v e doeses o f 3 - m e t h y l c h o l a n t h r e n e y i e l d e d s i m i l a r l e v e l s o f AHH a c t i v i t y i n the male and f e m a l e , even though the b a s a l l e v e l s i n the male were s u b s t a n t i a l l y h i g h e r than the f e m a l e . T h e r e f o r e , the -143-e f f e c t s o f 3 - m e t h y l c h o l a n t h r e n e i n t h e male appear to be i n f l u e n c e d by the p r e s e n c e o f t e s t o s t e r o n e as w e l l . S p i r o n o l a c t o n e , a c e t y l m e t h a d o l and t r a n s - s t i l b e n e o x i d e were a l l f ound t o produce i n c r e a s e s i n AHH a c t i v i t y i n the female. However, t h e s e a g e n t s were found t o d e c r e a s e AHH a c t i v i t y i n the male. S p i r o n o l a c t o n e i s known to p o s s e s s a n t i - a n d r o g e n i c a c t i v i t y (Bonne and Raynaud, 1974) and a c e t y l m e t h a d o l i s known to d e c r e a s e serum t e s t o s t e r o n e l e v e l s (Mendelson e t a]_, 1976). These a c t i o n s o f s p i r o n o l a c t o n e and a c e t y l m e t h a d o l c o u l d a c c o u n t f o r the d e c r e a s e i n AHH a c t i v i t y i n the a d u l t male. R e c e n t l y , some t r a n s - s t i l b e n e compounds were shown to p o s s e s s e s t r o g e n i c a c t i v i t y (Pento e t a l , 1981). These compounds a r e r e l a t e d s t r u c t u r a l l y t o the s y n t h e t i c e s t r o g e n d i e t h y l s t i l b e s t r o l . E s t r o g e n i s a well-known p h a r m a c o l o g i c a l and p h y s i o l o g i c a l a n t a g o n i s t o f t e s t o s t e r o n e . E s t r o g e n a d m i n i s t e r e d t o normal male r a t s d e c r e a s e s cytochrome P-450 dependent enzyme a c t i v i t i e s t o female l e v e l s ( E l Defrawy El Masry and Mannering, 1974; Sweeney and C o l e , 1980). T h e r e f o r e , t h e d e c r e a s e seen i n the AHH l e v e l s i n the a d u l t male f o l l o w i n g t r a n s -s t i l b e n e o x i d e t r e a t m e n t c o u l d be the r e s u l t o f the e s t r o g e n i c a c t i o n s o f t r a n s - s t i l b e n e o x i d e . S t u d i e s were c a r r i e d o u t i n a d u l t r a t s t h a t were c a s t r a t e d a t 21 t o 24 days o f age. The AHH a c t i v i t i e s i n t h e s e r a t s were d e c r e a s e d s i g n i f i c a n t l y from c o n t r o l . However, t h e l e v e l s o f AHH were s t i l l 2 t o 3 f o l d h i g h e r than t h o s e found i n the a d u l t f e m a l e . In a d d i t i o n , p h e n o b a r b i t a l and t r a n s - s t i l b e n e o x i d e had the same e f f e c t on AHH a c t i v i t i e s i n the c a s t r a t e d male r a t s as i n the normal males. The c a s t r a t e d r a t s were a p p a r e n t l y not female w i t h -144-r e s p e c t t o t h e i r AHH a c t i v i t i e s . A p o s s i b l e e x p l a n a t i o n i s t h a t serum androgen l e v e l s were s t i l l h i g h e r i n the c a s t r a t e d males than the f e m a l e s and t h a t the androgen r e s p o n s e system had i n c r e a s e d i t s s e n s i t i v i t y towards t h e r e s i d u a l c i r c u l a t i n g androgens. Thus, o n l y a p a r t i a l i n h i b i t i o n o f the t e s t o s t e r o n e - i n d u c e d AHH a c t i v i t y was produced. T h i s c o u l d be t e s t e d w i t h the use o f a n t i - a n d r o g e n i c a g e n t s , such as f l u t a m i d e . T h i s p a r t i a l i n h i b i t i o n o f the t e s t o s t e r o n e induced AHH a c t i v i t y would a c c o u n t f o r the same e f f e c t s o b s e r v e d w i t h p h e n o b a r b i t a l and t r a n s - s t i l b e n e o x i d e i n t h e c a s t r a t e d and normal male r a t s . F u l l i n h i b i t i o n o f the t e s t o s t e r o n e e f f e c t , which can be a c h i e v e d w i t h e s t r o g e n t r e a t m e n t o f a d u l t male r a t s , s h o u l d r e s u l t i n AHH l e v e l s o b s e r v e d i n the fe m a l e . I t would be p r e d i c t e d t h a t AHH a c t i v i t y i n t h e s e a n i m a l s would be i n c r e a s e d by p h e n o b a r b i t a l and t r a n s - s t i l b e n e o x i d e t r e a t m e n t . The i n t e r a c t i o n between t e s t o s t e r o n e and the v a r i o u s exogenous i n d u c e r s o f cytochrom P-450 dependent monooxygenases can be e x p l a i n e d on the b a s i s o f r e l a t i v e c o n t r i b u t i o n s o f d i f f e r e n t forms o f cytochrome P-450 towards t h e o x i d a t i o n o f v a r i o u s s u b s t r a t e s , as w i l l be d i s c u s s e d i n more d e t a i l i n S e c t i o n II 4. -145-2) Q u a l i t a t i v e Comparison o f the B i o c h e m i c a l Responses Produced  by P h e n o b a r b i t a l , T r a n s - S t i l b e n e Oxide, S p i r o n o l a c t o n e , 3- M e t h y l c h o l a n t h r e n e and Pregnenolone-16°<-Carbonitrile The a d u l t female o r immature r a t i s the p r e f e r r e d system t o q u a n t i t a t e t h e i n d u c t i o n o f drug m e t a b o l i s m i n t h i s s p e c i e s . The degree o f i n d u c t i o n i s g e n e r a l l y g r e a t e r and the p o t e n t i a l c o m p l i c a -t i o n o f an i n t e r a c t i o n w i t h the androgen dependent enzymes i s a v o i d e d . A d u l t female r a t s were used i n the p r e s e n t s t u d y to compare the b i o c h e m i c a l e f f e c t s e l i c i t e d by p h e n o b a r b i t a l , 3-m e t h y l c h o l a n t h r e n e , pregnenolone-16»<-carbonitrile, s p i r o n o l a c t o n e and t r a n s - s t i l b e n e o x i d e . F o l l o w i n g 4 days t r e a t m e n t , a l l o f t h e s e agents s i g n i f i c a n t l y i n c r e a s e d l i v e r w eight as i n d i c a t e d by the i n c r e a s e i n the l i v e r weight/body weight r a t i o . P h e n o b a r b i t a l , pregnenolone-16 c x - c a r b o n i t r i l e , t r a n s - s t i l b e n e o x i d e and s p i r o n o l a c t o n e were a l l found to i n d u c e the same p a t t e r n o f microsomal enzyme a c t i v i t i e s . However, the p a t t e r n o f enzymes i n c r e a s e d by 3 - m e t h y l c h o l a n t h r e n e was c l e a r l y d i f f e r e n t from the r e s t . 3 - M e t h y l c h o l a n t h r e n e produced a 20 f o l d i n c r e a s e i n the cytochrome P-450 dependent monooxygenase a c t i v i t y , e t h o x y r e s o r u f i n 0 - d e e t h y l a s e . At doses t h a t produced i n c r e a s e s i n o t h e r cytochrome P-450 dependent monooxygenases, the o t h e r i n d u c e r s s t u d i e d d i d not s i g n i f i c a n t l y a l t e r t h i s a c t i v i t y . A l l o f t h e i n d u c e r s , w i t h t h e e x c e p t i o n o f 3 - m e t h y l c h o l a n t h r e n e , i n c r e a s e d ethylmorphone N-demethylase, a m i n o p y r i n e N-demethylase and e p o x i d e h y d r o l a s e a c t i v i t i e s . A l l o f the i n d u c e r s s t u d i e d i n c r e a s e d AHH a c t i v i t y . T h e r e f o r e , based on the spectrum o f enzyme a c t i v i t i e s i n c r e a s e d , the 3 - m e t h y l c h o l a n t h r e n e r e s p o n s e can -146-be c l e a r l y d i s t i n g u i s h e d from the r e s p o n s e s produced by t h e o t h e r a g e n t s . I t has p r e v i o u s l y been shown t h a t p h e n o b a r b i t a l and 3-m e t h y l c h o l a n t h r e n e induce the f o r m a t i o n o f d i s t i n c t s p e c i e s o f cytochrome P-450 i n the r a t (Ryan e t al_, 1979). B e s i d e s the s p e c t r a l , immunolgical and e l e c t r o p h o r e t i c a n a l y s e s t h a t i n d i c a t e two d i s t i n c t forms o f cytochrome P-450, the two hemeproteins have a l s o been shown t o d i f f e r i n t h e i r c a t a l y t i c a c t i v i t i e s towards a v a r i e t y o f s u b s t r a t e s . However, based o n l y on t h e spectrum o f enzyme a c t i v i t i e s i n c r e a s e d , the i n d u c t i o n r e s p o n s e s produced by s p i r o n o l a c t o n e , t r a n s - s t i l b e n e o x i d e , p h e n o b a r b i t a l and pregnenolone-!6°<-carbonitrile a r e i n d i s t i n g u i s h a b l e . T h i s i s the t y p e o f s t u d y which has l e d to the large- number o f compounds t h a t a r e termed p h e n o b a r b i t a l - 1 i k e i n d u c e r s . 3) A More D e t a i l e d A n a l y s i s o f the P l e i o t r o p i c Responses E l i c i t e d  by These Agents F u r t h e r c h a r a c t e r i z a t i o n o f the i n d u c t i o n r e s p o n s e s was pursued from a number o f d i r e c t i o n s . F i r s t l y , s t u d i e s o f the i n h i b i t i o n and a c t i v a t i o n o f AHH a c t i v i t y i n microsomes from a n i m a l s o f v a r y i n g age, sex and t r e a t m e n t have shown t h a t d i f f e r e n t forms o f AHH e x i s t (Goujon e t al_, 1972; Wiebel and G e l b o i n , 1975). In the p r e s e n t s t u d y i n h i b i t i o n o f AHH a c t i v i t y by SKF 525-A was det e r m i n e d i n microsomes from r a t s o f v a r y i n g age, sex and t r e a t m e n t . The aim o f t h e s e experiments was t o det e r m i n e whether the forms o f AHH i n d u c e d by the v a r i o u s agents c o u l d be d i f f e r -e n t i a t e d by i n h i b i t i o n w i t h SKF 525-A. I f so, i t was hoped t h a t the forms o f microsomal AHH i n a d u l t male and a d u l t female r a t s -147-c o u l d a l s o be d i s t i n g u i s h e d . SKF 525-A was found t o produce a c o n c e n t r a t i o n - d e p e n d e n t i n h i b i t i o n o f AHH a c t i v i t y . In a l l c a s e s s t u d i e d , SKF 525-A was c a p a b l e o f complete i n h i b i t i o n o f AHH a c t i v i t y . The i n h i b i t i o n o f AHH a c t i v i t y by SKF 525-A was not i n f l u e n c e d by p r o t e i n c o n t e n t . T h e r e f o r e , changes i n the amounts o f the same form o f AHH would not c o n t r i b u t e t o a d i f f e r e n c e i n s e n s i t i v i t y t o i n h i b i t i o n by SKF 525-A. Changes i n s e n s i t i v i t y would most l i k e l y be a t t r i b u t a b l e t o forms o f AHH w i t h d i f f e r e n t a f f i n i t i e s o r c a t a l y t i c a c t i v i t i e s f o r t h e s u b s t r a t e and/or i n h i b i t o r . The i n d i c a t o r o f s e n s i t i v i t y t o the i n h i b i t o r y e f f e c t o f SKF 525-A, the IC50, was d e t e r m i n e d i n microsomes from r a t s o f v a r y i n g age and sex. Microsomes from a d u l t f emale and immature r a t s e x h i b i t e d s i m i l a r s e n s i t i v i t y t o SKF 525-A. However, microsomes from a d u l t male r a t s were s i g n i f i c a n t l y more s e n s i t i v e t o the i n h i b i t o r y e f f e c t o f SKF 525-A. Microsomes from androgen-i n s e n s i t i v e , p s e u d o h e r m a p h r o d i t i c male r a t s ( B u l l o c k e t al_, 1971) showed the same s e n s i t i v i t y t o SKF 525-A as the f e m a l e s . T h e r e -f o r e , i t appears t h a t the i n c r e a s e i n AHH a c t i v i t y a t t r i b u t a b l e t o t e s t o s t e r o n e i s l i k e l y due to a d i f f e r e n t form o f AHH than t h a t which predominates i n the a d u l t f emale and immature r a t . Wiebel and G e l b o i n (1975) demonstrated that<X-naphthoflavone i n h i b i t e d h e p a t i c microsomal AHH a c t i v i t y from a d u l t f emale r a t s , whereas the same compound enhanced AHH a c t i v i t y i n microsomes from a d u l t male r a t s . On t h i s b a s i s , t h e y c o n c l u d e d t h a t the predominant forms o f AHH i n a d u l t male and female r a t s were d i f f e r e n t . Microsomes from c a s t r a t e d male r a t s show the same s e n s i t i v i t y to SKF 525-A as do normal male r a t s . The l e v e l o f AHH -148-i n the c a s t r a t e d male r a t was 2 t o 3 f o l d h i g h e r than t h e f e m a l e . The m a l e - l i k e s e n s i t i v i t y t o SKF 525-A s u p p o r t s the s u g g e s t i o n t h a t the androgen-sensi.ti.ve AHH i s o n l y p a r t i a l l y reduced i n t h e s e c a s t r a t e d males. Microsomes from p h e n o b a r b i t a l - , pregnenolone-! 6°c-c a r b o n i t r i l e - , t r a n s - s t i l b e n e o x i d e - and s p i r o n o l a c t o n e - t r e a t e d f e m a l e r a t s e x h i b i t e d s i m i l a r i n c r e a s e s i n s e n s i t i v i t y t o the i n h i b i t o r y e f f e c t s o f SKF 525-A compared t o u n t r e a t e d f e m a l e s . However, microsomes from male and female r a t s t r e a t e d w i t h 3-m e t h y l c h o l a n t h r e n e d i s p l a y e d a marked d e c r e a s e i n s e n s i t i v i t y t o SKF 525-A. T h e r e f o r e , t h e form o f AHH i n d u c e d by 3 - m e t h y l c h o l a n t h r e n e i s c l e a r l y d i f f e r e n t from t h e forms i n d u c e d by t h e o t h e r a g e n t s . In the p r e s e n t s t u d y , the r e s p o n s e produced by p h e n o b a r b i t a l was d i f f e r e n t from t h a t by 3-methylcho!anthrene on the b a s i s o f s u b c e l l u l a r d i s t r i b u t i o n o f newly formed AHH. The per c e n t i n c r e a s e o f AHH a c t i v i t y i n the n u c l e i was g r e a t e r than t h e microsomes f o l l o w i n g 3 - m e t h y l c h o l a n t h r e n e , whereas the o p p o s i t e was o b s e r v e d f o l l o w i n g p h e n o b a r b i t a l . Pregnenolone-16©<.-carbonitrile t r e a t m e n t r e s u l t e d i n i n c r e a s e d AHH a c t i v i t y i n n u c l e i and microsomes t o the same e x t e n t . S p i r o n o l a c t o n e and t r a n s - s t i l b e n e o x i d e were s i m i l a r t o p h e n o b a r b i t a l i n t h a t t h e y i n c r e a s e d microsomal AHH to a g r e a t e r e x t e n t than n u c l e a r AHH. T h e r e f o r e , 3 - m e t h y l c h o l a n t h r e n e , p h e n o b a r b i t a l and pregnenolone-!6°<.-carbonitrile d i f f e r e d i n t h e i r i n d u c t i o n r e s p o n s e s w i t h r e s p e c t t o s u b c e l l u l a r d i s t r i b u t i o n . However, t r a n s - s t i l b e n e o x i d e and s p i r o n o l a c t o n e were a g a i n i n d i s t i n g u i s h a b l e from p h e n o b a r b i t a l . In t h e o r y , e q u i v a l e n t maximal e f f e c t s s h o u l d r e s u l t i f two -149-compounds produce t h e i r a c t i o n s by the same mechanism. In t h e p r e s e n t s t u d y , i t was reasoned t h a t i f two compounds i n d u c e d the same p l e i o t r o p i c r e s p o n s e , then the cytochrome P-450 dependent monooxygenases i n c r e a s e d s h o u l d e x h i b i t the same maximum o b t a i n a b l e l e v e l s . Experiments were c a r r i e d out t o determine the m a x i m a l l y e f f e c t i v e dose o f p h e n o b a r b i t a l . A t 80 mg/kg/day, a l l o f the cytochrome P-450 dependent monooxygenases s t u d i e d were i n c r e a s e d m a x i m a l l y f o l l o w i n g 4 days o f t r e a t m e n t . A t m a x i m a l l y e f f e c t i v e doses t r a n s - s t i l b e n e o x i d e i n c r e a s e d AHH to a l e v e l markedly lower than t h a t a t t a i n a b l e w i t h p h e n o b a r b i t a l . On the o t h e r hand, pregnenolone-16°<.-carbonitrile i n c r e a s e d AHH t o a l e v e l t h a t was s i g n i f i c a n t l y h i g h e r . t h a n t h a t seen w i t h p h e n o b a r b i t a l . The d i f f e r e n c e i n maximal e f f e c t i v e n e s s between pregnenolone-!6°< - c a r b o n i t r i l e and p h e n o b a r b i t a l l e d Lu e t al_ (1972) t o c o n c l u d e t h a t pregnenolone-!6«(-carbonitrile was a novel i n d u c e r o f drug m e t a b o l i z i n g enzymes. In the p r e s e n t s t u d y , the d i f f e r e n c e i n t h e maximum AHH a c t i v i t y o b s e r v e d f o l l o w i n g p h e n o b a r b i t a l and t r a n s - s t i l b e n e o x i d e p r o v i d e s the f i r s t e v i d e n c e t h a t t h e s e two ' i n d u c e r s e l i c i t unique r e s p o n s e s . These r e s u l t s s u g g e s t t h a t the cytochrome P-450 induced by t r a n s - s t i l b e n e o x i d e d i f f e r s from t h a t i n d u c e d by p h e n o b a r b i t a l . The o t h e r cytochrome P-450 dependent monooxygenases were i n c r e a s e d by both compounds t o a s i m i l a r maximum l e v e l . S p i r o n o l a c t o n e produced maximal i n c r e a s e s o f cytochrome P-450 dependent monooxygenases a t 200 mg/kg/day. The i n c r e a s e s were comparable to t h o s e o b s e r v e d f o l l o w i n g m a x i m a l l y e f f e c t i v e doses o f p h e n o b a r b i t a l . T h e r e f o r e , s p i r o n o l a c t o n e and p h e n o b a r b i t a l a r e s i m i l a r w i t h r e s p e c t to the maximal l e v e l s o f monooxygenases i n d u c e d , s u g g e s t i n g t h e y induce - 1 5 0 -the same form o f cytochrome P-450. I f two compounds i n d u c e d i s t i n c t forms o f cytochrome P-450, then t r e a t m e n t w i t h the two compounds s i m u l t a n e o u s l y s h o u l d produce r e s p o n s e s t h a t a r e the summation o f the two i n d i v i d u a l r e s p o n s e s . S p i r o n o l a c t o n e and p h e n o b a r b i t a l a d m i n i s t e r e d c o n c o m i t a n t l y y i e l d e d monooxygenase a c t i v i t i e s t h a t were i n d i s -t i n g u i s h a b l e from the maximal e f f e c t s produced by p h e n o b a r b i t a l a l o n e . T h i s s u g g e s t s t h a t t h e two compounds in d u c e the same form o f hemeprotein. However, c o m b i n a t i o n s o f i n d u c e r s t h a t a r e known to e l i c i t t he f o r m a t i o n o f d i s t i n c t hemeproteins produce monooxygenase l e v e l s t h a t a r e c l e a r l y l e s s than a d d i t i v e . For example, p h e n o b a r b i t a l and p r e g n e n o l o n e - 1 6 < * . - c a r b o n i t r i l e a d m i n i s t e r e d s i m u l t a n e o u s l y y i e l d AHH l e v e l s t h a t a r e the same as the l e v e l s o b s e r v e d f o l l o w i n g p r e g n e n o l o n e - ! 6 o < - c a r b o n i t r i l e a l o n e . T h e r e f o r e , a s i g n i f i c a n t i n t e r a c t i o n i s t a k i n g p l a c e , t h a t i s , t h e e f f e c t s o f the i n d u c e r s a r e not independent o f each o t h e r . The i n d u c t i o n by one compound appears t o i n t e r f e r e w i t h the second. The i n t e r a c t i o n between the v a r i o u s exogenous i n d u c e r s o f cytochrome P-450 dependent monooxygenases can be e x p l a i n e d on the b a s i s o f r e l a t i v e c o n t r i b u t i o n s o f d i f f e r e n t forms o f cytochrome P-450 towards the o x i d a t i o n o f v a r i o u s s u b s t r a t e s , as w i l l be d i s c u s s e d i n more d e t a i l i n S e c t i o n I I 4. The i n d u c t i o n o f epoxide h y d r o l a s e a c t i v i t y by p h e n o b a r b i t a l , t r a n s - s t i l b e n e o x i d e and pregnenolone-16»<-carbonitrile r e q u i r e d s i g n i f i c a n t l y g r e a t e r doses than t h a t r e q u i r e d t o i n c r e a s e mono-oxygenase a c t i v i t y . The same was o b s e r v e d w i t h a c e t y l m e t h a d o l . F o l l o w i n g 4 days o f p h e n o b a r b i t a l a t 80 mg/kg/day, epox i d e -151-hydrolase was not increased maximally and could be increased further following 7 days of treatment. Seidegard et^al_ (1979) suggested that phenobarbital and trans-stilbene oxide were different inducers since the latter was a more effective inducer of epoxide hydrolase activity. However, they did not establish the maximally effective dose of phenobarbital for the induction of epoxide hydrolase activity with a proper dose-response study. In fact, the suggestion was based on a dose of phenobarbital that produced a maximum increase of cytochrome P-450 and not epoxide hydrolase. In the present study, it also appeared that higher doses were required to increase liver weight. Therefore, the dose-response curves do not seem to overlap for the various responses induced by a parti-cular compound. This is an indication that distinct gene products are responsive to different doses of inducer. Consequently, doses of agents that are effective in inducing cytochrome P-450 may not be adequate to e l ic i t other effects such as tumour promotion. Amobarbital and diphenylhydantoin were ineffective as promoters of liver tumorigenesis even though these agents were inducers of hepatic drug metabolizing enzymes (Peraino et al_, 1975). However, toxicity precluded the testing of higher doses, so the possibility s t i l l exists that these compounds are tumour promotors, as has been found with all other inducers tested. Trans-Stilbene oxide, phenobarbital and pregnenolone-!6e<-carbonitrile appeared to increase epoxide hydrolase activity to a similar level. Phenobarbital treatment for 7 days increased epoxide hydrolase activity to a level similar to that seen following trans-stilbene oxide. Simultaneous administration of -152-t h e s e compounds d i d not produce a f u r t h e r i n c r e a s e i n e p o x i d e h y d r o l a s e a c t i v i t y than t h a t seen w i t h t r a n s - s t i l b e n e o x i d e a l o n e . P r e g n e n o l o n e - ! 6 < * - c a r b o n i t r i l e and p h e n o b a r b i t a l a d m i n i s t e r e d s i m u l t a n e o u s l y f o r one week r e s u l t e d i n an e p o x i d e h y d r o l a s e a c t i v i t y t h a t was s i m i l a r t o t h a t seen w i t h e i t h e r compound a l o n e . The maximum e f f e c t on epoxide h y d r o l a s e a c t i v i t y was not c l e a r l y e s t a b l i s h e d i n t h e p r e s e n t e x p e r i m e n t , but t h e d a t a i n d i c a t e the p o s s i b i l i t y o f s i m i l a r maximal e f f e c t s . S p i r o n o l a c t o n e , however, i s c l e a r l y l e s s e f f i c a c e o u s than p h e n o b a r b i t a l . A dose o f 200 mg/ kg/day f o r 4 days appears to e l i c i t t he maximal e f f e c t , which i s s i g n i f i c a n t l y lower than t h a t seen f o l l o w i n g p h e n o b a r b i t a l , t r a n s -s t i l b e n e o x i d e and pregnenolone-16«*-carbonitrile t r e a t m e n t . The p l e i o t r o p i c r e s p o n s e s i n d u c e d by p h e n o b a r b i t a l , 3-m e t h y l c h o l a n t h r e n e , pregnenol one-16°*-carbonitri1e, t r a n s - s t i 1 bene o x i d e and s p i r o n o l a c t o n e were a l s o s t u d i e d by a n a l y s i s o f the SDS-p o l y a c r y l a m i d e g e l e l e c t r o p h o r e t i c p a t t e r n s o f microsomes p r e p a r e d from female r a t s t r e a t e d w i t h t h e s e a g e n t s . The gel p a t t e r n s o b t a i n e d i n microsomes from t r a n s - s t i l b e n e - o x i d e - and p h e n o b a r b i t a l - t r e a t e d r a t s were s i m i l a r , w i t h d r a m a t i c i n c r e a s e s i n band s t a i n i n g a p p a r e n t a t 50,000 and 53,000 m o l e c u l a r weight. 3 - M e t h y l c h o l a n t h r e n e t r e a t m e n t r e s u l t e d i n a s u b s t a n t i a l i n c r e a s e i n band s t a i n i n g a t 55,000 m o l e c u l a r weight. The i n c r e a s e i n p r o t e i n a t 53,000 and 55,000 m o l e c u l a r weight a r e i n the range f o r the major forms o f cytochrome P-450 in d u c e d by p h e n o b a r b i t a l and 3 - m e t h y l c h o l a n t h r e n e , r e s p e c t i v e l y (Lu and West, 1980). E p o x i d e h y d r o l a s e has been r e p o r t e d to be a p r o t e i n o f 49,000 to 50,000 m o l e c u l a r weight (Lu and Miwa, 1980). The i n c r e a s e s i n band -153-in tens i ty at 50 ,000 molecular weight could be large ly due to induction of epoxide hydrolase. Microsomes from pregnenolone-!6*x - ca rbon i t r i l e - t rea ted rats exhibi ted a band pattern that was d i s t i n c t from that seen with phenobarbital and 3-methylcholanthrene. Increases at 50 ,000 and 55 ,000 molecular weight were seen. The major form of cytochrome P-450 induced by pregnenolone-16** - ca rbon i t r i l e has a reported molecular weight of 51 ,000 (Elshourbagy and Guzel ian, 1 9 8 0 ) . Spironolactone c l ea r l y d i f f e r s from the other inducers in i t s band pattern. There does not appear to be any s ign i f i can t increase in band in tens i t y , however two peaks at 55 ,000 and 56 ,000 molecular weight were i d e n t i f i e d . The lack of a s ign i f i can t band sta in ing in the 50 ,000 to 58 ,000 molecular weight region indicates that to ta l cytochrome P-450 i s not s i g n i f i c a n t l y a l te red . This would support the f indings of Str ipp et_ al_ (1971) that to ta l cytochrome P-450 was not s i g n i f i -cant ly a l tered by spironolactone at doses that produced maximal increases in cer ta in cytochrome P-450 dependent monooxygenases. The di f ference in the protein band patterns produced by phenobarbital and spironolactone suggests that the two p le io t rop ic responses are d i f fe rent from one another. -154-4) R e l a t i v e C o n t r i b u t i o n o f D i f f e r e n t Forms o f Cytochrome P-450  to t h e O x i d a t i o n o f S u b s t r a t e s A number o f f i n d i n g s i n the p r e s e n t s t u d y s u g g e s t e d t h a t t h e m e t a b o l i s m o f s u b s t r a t e by one form o f cytochrome P-450 was i n f l u e n c e d by the p r e s e n c e o f a second form. For example, i n the a d u l t male r a t , pregnenolone-16°<.-carbonitrile d i d not i n c r e a s e AHH a c t i v i t y , whereas t h i s a gent was an e f f e c t i v e i n d u c e r o f AHH a c t i v i t y i n t h e a d u l t f e m a l e . The p r e s e n c e o f a t e s t o s t e r o n e dependent cytochrome P-450 appeared t o mask the a b i l i t y o f the p r e g n e n o l o n e - 1 6 < X - c a r b o n i t r i l e i n d u c e d cytochrome P-450 t o o x i d i z e t he s u b s t r a t e , b e n z o ( a ) p y r e n e . The i n d u c t i o n o f cytochrome P-450 by pregnenolone-16«<-carbonitrile d i d not appear t o be a l t e r e d s i n c e a m i n o p y r i n e 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 i n t h e a d u l t male. S e c o n d l y , the s i m u l t a n e o u s a d m i n i s t r a t i o n o f two compounds t h a t a r e known to in d u c e d i f f e r e n t forms o f cytochrome P-450 d i d not r e s u l t i n a d d i t i v e e f f e c t s . S i m u l t a n e o u s a d m i n i s t r a t i o n o f 3 - m e t h y l c h o l a n t h r e n e and pregnenolone-16©c-carbonitrile to female r a t s r e s u l t e d i n the same AHH a c t i v i t i e s o b s e r v e d f o l l o w i n g 3 - m e t h y l c h o l a n t h r e n e t r e a t m e n t a l o n e . However, the pregnenolone-16 «*-carbonitrile induced form o f cytochrome P-450 was p r e s e n t s i n c e ethylmorphone N-demethylase a c t i v i t y was e l e v a t e d . R e c e n t l y , Harada and Omura (1981) demonstrated t h a t the a b i l i t y o f one form o f cytochrome P-450 t o o x i d i z e s u b s t r a t e was s i g n i f i c a n t l y i n f l u e n c e d by t h e p r e s e n c e o f a second form o f cytochrome P-450. They produced maximal i n d u c t i o n i n r a t s w i t h p h e n o b a r b i t a l and then a d m i n i s t e r e d a s i n g l e does o f 3--155-m e t h y l c h o l a n t h r e n e . Twenty-four hours l a t e r , a t a p o i n t when t h e l e v e l o f t h e p h e n o b a r b i t a l i n d u c e d form o f cytochrome P-450 had not changed, a l l o f the b e n z o ( a ) p y r e n e was o x i d i z e d i n v i t r o by the 3 - m e t h y l c h o l a n t h r e n e i n d u c e d form o f cytochrome P-450. T h i s was accompanied by a complete l o s s o f t h e o x i d a t i o n o f s u b s t r a t e by t he p h e n o b a r b i t a l form. However the p h e n o b a r b i t a l form r e t a i n e d t he a b i l i t y t o p r e f e r e n t i a l l y o x i d i z e benzphetamine. The 3 - m e t h y l c h o l a n t h r e n e form d i d not c o n t r i b u t e t o the o x i d a t i o n o f t h i s s u b s t r a t e . They c o n c l u d e d t h a t the e l e c t r o n f l o w from NADPH to v a r i o u s forms o f cytochrome P-450 and t h e i r r e s u l t a n t o x i d a t i v e a b i l i t i e s were dependent on the i n d u c e d s t a t e o f the a n i m a l . T h e r e f o r e , i n a n i m a l s t r e a t e d s i m u l t a n e o u s l y w i t h p h e n o b a r b i t a l and 3 - m e t h y l c h o l a n t h r e n e , t he o x i d a t i o n o f b e n z o ( a ) p y r e n e c o u l d be e x c l u s i v e l y c a t a l y z e d by the 3 - m e t h y l c h o l a n t h r e n e i n d u c e d form, thus y i e l d i n g n o n - a d d i t i v e e f f e c t s . From the p r e s e n t s t u d y , we sugg e s t t h a t t he t e s t o s t e r o n e -i n d u c e d cytochrome P-450 may be the predominant form o x i d i z i n g b e n z o ( a ) p y r e n e i n t h e a d u l t male. Even though p h e n o b a r b i t a l and p r e g n e n o l o n e - ! 6 < * . - c a r b o n i t r i l e i n d u c e t h e i r r e s p e c t i v e forms o f cytochrome P-450, i t does not c o n t r i b u t e t o the metabolism o f b e n z o ( a ) p y r e n e . T h e r e f o r e an i n c r e a s e i n a c t i v i t y i s not o b s e r v e d . In a d d i t i o n , t he l a c k o f a d d i t i v e e f f e c t s w i t h the s i m u l t a n e o u s a d m i n i s t r a t i o n o f i n d u c e r s can be e x p l a i n e d on the b a s i s o f one form o f cytochrome P-450 e x c l u s i v e l y c a t a l y z i n g t he o x i d a t i o n o f a p a r t i c u l a r s u b s t r a t e . The s i m u l t a n e o u s a d m i n i s t r a t i o n o f 3 - m e t h y l c h o l a n t h r e n e and pregnenolone-16°<-c a r b o n i t r i l e r e s u l t s i n the l e v e l o f AHH produced by t r e a t m e n t -156-o f 3 - m e t h y l c h o l a n t h r e n e a l o n e , s u g g e s t i n g t h a t the 3-m e t h y l c h o l a n t h r e n e i n d u c e d cytochrome P-450 i s e x c l u s i v e l y c a t a l y z i n g the o x i d a t i o n o f b e n z o ( a ) p y r e n e i n t h i s c a s e . T h e r e f o r e , the o x i d a t i o n o f s u b s t r a t e i s not s i m p l y the sum o f a c t i v i t i e s o f a l l the forms o f cytochrome P-450 p r e s e n t i n the microsomes. Depending on the s u b s t r a t e and the induced s t a t e o f the a n i m a l , o x i d a t i o n may be c a r r i e d out e x c l u s i v e l y by one form o f cytochrome P-450 a l o n e . Other major forms o f cytochrome P-450 may be p r e s e n t but do not c o n t r i b u t e t o the o x i d a t i o n o f s u b s t r a t e when the p r e f e r e n t i a l form i s a v a i l a b l e , even though t h e y may o x i d i z e t h e s u b s t r a t e under o t h e r c i r c u m s t a n c e s . 5) I n d u c t i o n Mechanism C e r t a i n r e s u l t s o b t a i n e d i n the p r e s e n t s t u d y i n d i c a t e t h a t s p i r o n o l a c t o n e and t r a n s - s t i l b e n e o x i d e a r e unique i n d u c e r s o f h e p a t i c microsomal enzymes. Pol a n d et_ al_ (1976) demonstrated t h a t the i n d u c t i o n r e s p o n s e produced by 3 - m e t h y l c h o l a n t h r e n e and s t r u c t u r a l l y r e l a t e d compounds was mediated by a c y t o s o l i c r e c e p t o r . I t appears t h a t t h e a c t i o n o f t e s t o s t e r o n e i n the l i v e r may a l s o be mediated by a c y t o s o l i c r e c e p t o r . The i n d u c t i o n o f an androgen-dependent p r o t e i n i n r a t l i v e r was shown t o be mediated by an androgen r e c e p t o r (Roy e t al_, 1974), and a s i m i l a r r e c e p t o r mechanism has been s u g g e s t e d f o r the androgen mediated a c t i o n on the l i v e r microsomal enzymes (Brown e t al_, 1976). The e f f e c t s produced by t h e v a r i o u s i n d u c e r s o f drug m e t a b o l i z i n g enzymes a r e a r e s u l t o f an i n c r e a s e d e x p r e s s i o n a t c e r t a i n gene l o c i . These a c t i o n s a r e " h o r m o n e - l i k e " i n n a t u r e and the p r e s e n t -157-c o n e e p t s o f s t e r o i d hormone a c t i o n i n v o l v e a r e c o g n i t i o n s i t e o r r e c e p t o r which m e d i a t e s the a c t i o n s o f t h e hormone i n the n u c l e u s ( B a x t e r and Funder, 1979). The a l t e r e d e x p r e s s i o n o f DNA i s mediated by the hormone-receptor complex. I t seems r e a s o n a b l e , t h e r e f o r e , t o s u g g e s t t h a t the i n d u c t i o n o f drug m e t a b o l i z i n g enzymes by p r e g n e n o l o n e - 1 6 < * - c a r b o n i t r l l e , s p i r o n o l a c t o n e , t r a n s - s t i l b e n e o x i d e and p h e n o b a r b i t a l i s mediated by a c y t o s o l i c r e c e p t o r . However, s i n c e each o f t h e s e i n d u c t i o n r e s p o n s e s i s u n i q u e , then a d i f f e r e n t r e c e p t o r f o r each o f t h e s e compounds s h o u l d e x i s t . S i n c e the p l e i o t r o p i c r e s p o n s e s produced by t h e s e compounds a l s o i n c l u d e l i v e r growth and p o s s i b l y tumour promotion, then the proposed r e c e p t o r - m e d i a t e d e f f e c t s i n v o l v e much more than s i m p l y t h e i n d u c t i o n o f drug m e t a b o l i z i n g enzymes. However, a g r e a t d e a l remains t o be u n d e r s t o o d r e g a r d i n g the h e p a t i c r e s p o n s e system b e f o r e such c a u s e - a n d - e f f e c t r e l a t i o n s h i p s w i l l be worked o u t . In o r d e r t o c o n f i r m the c o n c l u s i o n t h a t s p i r o n o l a c t o n e and t r a n s - s t i l b e n e o x i d e i n d u c e d i f f e r e n t forms o f cytochrome P-450 the f o l l o w i n g e x p e r i m e n t s s h o u l d be c a r r i e d o u t . The major forms o f cytochrome P-450 from s p i r o n o l a c t o n e - and t r a n s - s t i l b e n e o x i d e -t r e a t e d r a t s s h o u l d be i s o l a t e d , p u r i f i e d and compared t o the major forms o f cytochrome P-450 in d u c e d by p h e n o b a r b i t a l , 3-m e t h y l c h o l a n t h r e n e and pregnenolone-16°<-carbonitrile. These e x p e r i m e n t s s h o u l d p r o v i d e more d i r e c t e v i d e n c e t h a t the p l e i o t r o p i c r e s p o n s e s induced by s p i r o n o l a c t o n e and t r a n s - s t i l b e n e o x i d e a r e i n d e e d d i f f e r e n t . -158-SUMMARY The study o f the n u c l e a r l o c a l i z a t i o n and i n d u c t i o n o f r a t h e p a t i c drug m e t a b o l i z i n g enzymes l e d t o the f o l l o w i n g c o n c l u s i o n s : 1. The enzyme a c t i v i t i e s i n i s o l a t e d r a t h e p a t i c n u c l e i were not due t o c o n t a m i n a t i o n by microsomes formed upon ho m o g e n i z a t i o n . 2. The r e l a t i v e d i s t r i b u t i o n o f enzymes between the microsomes and n u c l e i were s i g n i f i c a n t l y d i f f e r e n t . In a d d i t i o n , the n u c l e i and microsomes d i f f e r e d i n t h e i r q u a n t i t a t i v e i n d u c i b i l i t y by exogenous a g e n t s . These r e s u l t s t e n d t o s u p p o r t t he s u g g e s t i o n t h a t drug m e t a b o l i z i n g enzymes e x i s t i n i s o l a t e d n u c l e i and a r e not p r e s e n t as segments o f endoplasmic r e t i c u l u m c o n t i n u o u s w i t h t h e n u c l e a r membrane. However, i n l i g h t o f the e v i d e n c e t h a t the t o p o l o g y o f the enzymes t h r o u g h o u t t he endoplasmic r e t i c u l u m i s heter o g e n e o u s , t h e s e r e s u l t s can be i n t e r p r e t e d d i f f e r e n t l y . 3. In g e n e r a l , t he p r o p e r t i e s o f the enzymes o f n u c l e a r and microsomal o r i g i n s appeared t o be s i m i l a r , w i t h d i f f e r e n c e s noted o n l y i n the r e l a t i v e amounts p r e s e n t i n the two f r a c t i o n s . 4. Whether t h e enzymes i n i s o l a t e d n u c l e i a r e p r e s e n t i n the n u c l e o p l a s m and/or n u c l e a r membrane, o r as " t a g s " o f endoplasmic r e t i c u l u m , t h e y r e p r e s e n t enzymes i n c l o s e p r o x i m i t y t o the n u c l e a r DNA. T h e i r s e l e c t i v e m a n i p u l a t i o n would be a u s e f u l t o o l t o d e t e r m i n e t h e i r t o x i c o l o g i c a l s i g n i f i c a n c e . -159-5. Multiple forms of AHH were distinguishable in the rat by their di f ferent ia l sensi t iv i ty to the inhibitory effects of SKF 525-A. The studies with SKF 525-A suggest that testosterone induces a form of AHH that di f fers from the predominant form present in the female. These studies also indicate that the predominant forms of AHH induced by phenobarbital and 3-methylcholanthrene are different from each other and, from the predominant form of AHH present in the female. 6. Microsomal epoxide hydrolase act iv i ty appeared to exhibit a similar maximum following treatment with phenobarbital, trans-sti lbene oxide and pregnenolone-16«<-carbonitrile. The induction of epoxide hydrolase act iv i ty by these agents required s igni f icant ly greater doses than required for the induction of cytochrome P-450 dependent monooxygenase act iv i ty . This demonstrates that the induction of separate gene products does not necessarily exhibit overlapping dose-response curves. 7. Maximally effective doses of trans-sti lbene oxide and phenobarbital yielded s igni f icant ly different AHH ac t i v i t i es . These data suggested that trans-sti lbene oxide was a different inducer of drug metabolizing enzymes. 8. Microsomes from phenobarbital- and spironolactone-treated rats were compared by SDS-polyacrylamide gel electrophoresis. The electrophoretic patterns were dramatically dif ferent. These data suggested that spironolactone was a different inducer of drug metabolizing enzymes. - 1 6 0 -The r e s u l t s o b t a i n e d i n the s t u d y o f combined t r e a t m e n t o f i n d u c e r s and the s t u d y o f age and sex dependency o f i n d u c t i o n s u p p o r t the c o n c e p t t h a t t h e c a t a l y t i c a c t i v i t y o f any p a r t i c u l a r form o f cytochrome P-450 dependent on the i n d u c e d s t a t e o f the animal and the s u b s t r a t e b e i n g o x i d i z e d . -161-REFERENCES A l e x a n d r e s , K., Brookes, P., K i n g , H.W.S., Osborne, M.R. and Thompson, M.H. Comparison o f the m e t a b o l i s m o f be n z o ( a ) p y r e n e and b i n d i n g to DNA caused by r a t l i v e r n u c l e i and microsomes. Chem.-Biol. I n t e r a c t i o n s 12: 269-277 (1976). A l v a r e s , A.P., S c h i l l i n g s * G., L e v i n , W. and Kuntzman, R. S t u d i e s on the i n d u c t i o n o f CO-binding pigment i n l i v e r microsomes by p h e n o b a r b i t a l and 3 - m e t h y l c h o l a n t h r e n e . Biochem. B i o p h y s . Res. Commun. 29: 521-526, 1967. Ames, B.N., D u r s t o n , W.E., Yamasaki, E. and Lee, F.D. C a r c i n o g e n s and mutagens: A s i m p l e t e s t system combining l i v e r homogenates f o r a c t i v a t i o n and b a c t e r i a f o r d e t e c t i o n . Proc. N a t l . Acad. S c i . U.S. 70: 2881-2885, 1973. A r c o s , J . C , Conney, A.H. and Buu-Hoi, N.P. I n d u c t i o n o f microsomal enzyme s y n t h e s i s by p o l y c y c l i c a r o m a t i c hydrocarbons o f d i f f e r e n t m o l e c u l a r s i z e s . J . B i o l . Chem. 236: 1291-1296, 1961. Armuth, V. and Berenblum, I. Promotion o f mammary c a r c i n o g e n e s i s and leukemogenic a c t i o n by p h o r b o l i n v i r g i n female W i s t a r r a t s . Cancer Research 34: 2704-2707, 1974. Astrom, A. and D e P i e r r e , J.W. C h a r a c t e r i z a t i o n o f the i n d u c t i o n o f dru g -m e t a b o l i z i n g enzymes by 2 - a c e t y l a m i n o f l u o r e n e . B i o c h i m . B i o p h y s . A c t a 673: 225-233, 1981. Aune, T., Dybing, E. and N e l s o n , S.D. Mutagenic a c t i v a t i o n o f 2, 4-diamino-a n i s o l e and 2 - a m i n o f l u o r e n e by i s o l a t e d r a t l i v e r n u c l e i and microsomes. Chem.-Biol. I n t e r a c t i o n s 3J_: 35-49, 1980. B a x t e r , J.D. and Funder, J.W. Hormone r e c e p t o r s . N. E n g l . J . Med. 301: 1149-1161 , 1979. B e l l w a r d , G.D., G o n t o v n i c k , L. and O t t e n , M. I n d u c t i o n o f h e p a t i c a r y l h y drocarbon h y d r o x y l a s e and ep o x i d e hydrase i n W i s t a r r a t s p r e t r e a t e d w i t h o r a l methadone h y d r o c h l o r i d e . Drug Metab. D i s p o s . 5_: 211-218, 1977. B e l l w a r d , G.D., L i , M., Lopaschuk, G.D. and Warren, P. The e f f e c t o f d-**-a c e t y l m e t h a d o l h y d r o c h l o r i d e on r a t h e p a t i c a r y l hydrocarbon h y d r o x y l a s e .and e p o x i d e h y d r a s e . Res. Commun. Chem. Path. Pharmacol. ]8: 415-422, 1977a. Berenblum, I. and Haran-Ghera, N. A q u a n t i t a t i v e s t u d y o f the s y s t e m i c i n i t i a t i n g a c t i o n o f urethane ( e t h y l c a r b a m a t e ) i n mouse s k i n c a r c i n o -g e n e s i s . B r i t . J . Cancer ]_]_: 77-90, 1957. Berenblum, I. and Shubik, P. A new, q u a n t i t a t i v e approach to the st u d y o f the s t a g e s o f chemical c a r c i n o g e n e s i s i n the mouse's s k i n . B r i t . J . Cancer 1_: 383-391 , 1947. -162-Berenblum, I. and Shubik, P. The p e r s i s t e n c e o f l a t e n t tumour c e l l s i n d u c e d i n the mouse's s k i n by a s i n g l e a p p l i c a t i o n o f 9, 10 -d i m e t h y l - 1, 2 - benzanthracene. B r i t . J . Cancer 3; 384-386, 1949. B i e l s c h o w s k y , F. Tumours o f t h y r o i d produced by 2 - a c e t y l - a m i n o - f l u o r e n e and a l l y l - t h i o u r e a . Br. J . ExptT.. Path. 25; 90-95, 1944. B l o b e l , G. and P o t t e r , V.R. N u c l e i from r a t l i v e r : I s o l a t i o n method t h a t combines p u r i t y w i t h high y i e l d . S c i e n c e 154: 1662-1665, 1966. Bonne, C. and Raynaud, E.P. Mode o f s p i r o n o l a c t o n e a n t i - a n d r o g e n i c a c t i o n : I n h i b i t i o n o f a n d r o s t a n o l e n e b i n d i n g to r a t p r o s t a t e androgen r e c e p t o r . MoT. C e l l . E n d o c r i n o l . 2: 59-67, 1974. B o u t w e l l , R.K. The f u n c t i o n and mechanism o f promoters o f c a r c i n o g e n e s i s . CRC C r i t . Rev. T o x i c o l . 2: 419-443, 1974. B o u t w e l l , R.K. The b i o c h e m i s t r y o f p r e n e o p l a s i a i n mouse s k i n . Cancer Research 36: 2631-2635, 1976. B o u t w e l l , R.K. B i o c h e m i c a l mechanism o f tumor promotion. In: C a r c i n o g e n e s i s , V o l . 2, Mechanisms o f Tumor Promotion a n d ' C o c a r c i n o g e n e s i s ( S l a g a , T . J . , S i v a k , A., B o u t w e l l , R.K. e d s . ) , pp. 49-58, Raven P r e s s , New York, 1978. B o v e r i , T. Zur Frage Der Entstehung M a l i g n e r Tumoren. J e n a : Gustau F i s c h e r , 1914. B r e s n i c k , E., Vaught, J.B., Chuang, A.H.L., Stoming, T.A., Bockman, D. and Mukhtar, H. N u c l e a r a r y l h ydrocarbon h y d r o x y l a s e and i n t e r a c t i o n o f p o l y c y c l i c hydrocarbons w i t h n u c l e a r components. A r c h . Biochem. B i o p h y s . 181_: 257-269, 1977. B r e s n i c k , E., Stoming, T.A., Vaught, J.B.', Thakker, D.R. and J e r i n a , D.M. N u c l e a r m e t a b o l i s m o f b e n z o ( a ) p y r e n e and o f (.+ ) - t r a n s - 7, 8 - d i h y -d r o x y - 7, 8, - d i h y d r o b e n z o ( a ) p y r e n e . Comparative c h r o m a t o g r a p h i c a n a l y s i s o f a l k y l a t e d DNA. A r c h . Biochem. B i o p h y s . 183: 31-37, 1977a. B r e s n i c k , E., Mukhtar, H. Stoming, T.A., D a n s e t t e , P.M. and J e r i n a , D.M. E f f e c t o f p h e n o b a r b i t a l and 3-methylcholaimthrene a d m i n i s t r a t i o n on epoxide hydrase l e v e l s i n l i v e r microsomes. Biochem. Pharmacol. 26: 891-892, 1977b. B r e s n i c k , E., Hassuk, B., L i b e r a t o r , P., L e v i n , W. and Thomas, P. N u c l e o l a r cytochrome P-450. Mol. Pharmacol. 18; 550-552, 1980. Brookes, P., and Lawley, P.D. E v i d e n c e f o r the b i n d i n g o f p o l y n u c l e a r a r o m a t i c hydrocarbons t o t h e n u c l e i c a c i d s o f mouse s k i n : R e l a t i o n between c a r c i n o g e n i c power o f hydrocarbons and t h e i r b i n d i n g to de-o x y r i b o n u c l e i c a c i d . Nature 202_: 781-784, 1964. -163-Brown, T.R., Green, F.E. and Bardon, C.W. Androgen r e c e p t o r dependent and independent a c t i v i t i e s o f t e s t o s t e r o n e on h e p a t i c microsomal drug metabolism. E n d o c r i n o l . 99; 1353-1362, 1976. Bucker, M., G o l a n , M., Schmassmann, H.U., G l a t t , P. S t a s i e c k i , P. and Oesch, F. The e p o x i d e h y d r a t a s e i n d u c e r tra-n's-jstilbene o x i d e s h i f t s t h e m e t a b o l i c e p o x i d a t i o n o f b e n z o ( a ) p y r e n e from the bay- to the K-r e g i o n and reduces i t s m u t a g e n i c i t y . M o l e c u l a r Pharmacol. 16: 656-666, 1979. B u l l o c k , L.P., B a r d i n , C.W., Gram, T.E., S c h r o e d e r , D.H'. and G i l l e t t e , J.R. H e p a t i c e t h y l m o r p h i n e and A 4 - s t e r o i d r e d u c t a s e i n the a n d r o g e n - i n s e n -s i t i v e p s e u d o n e r m a p h r o d i t i c r a t . E n d o c r i n o l . 88: 1521-1523, 1971. Burke, M.D. and Mayer, R.T. E t h o x y r e s o r u f i n : D i r e c t f l u o r o m e t r i c assay o f a microsomal o - d e a l k y l a t i o n which i s p r e f e r e n t i a l l y i n d u c i b l e by 3-m e t h y l c h o l a n t h r e n e . Drug Metab. D i s p o s . 2: 583-588, 1974. B u t l i n , H.T. Cancer o f the scrotum i n chimney-sweeps and o t h e r s . I I . Why f o r e i g n sweeps do n o t s u f f e r from s c r o t a l c a n c e r . B r i t . Med. J . 2: 1-6, 1892. Cha, Y-N. and M a r t z , F. E f f e c t o f 2(3) - t e r t b u t y l - 4 - h y d r o x y a n i s o l e (BHA) a d m i n i s t r a t i o n on h e p a t i c e p o x i d e h y d r a t a s e and o t h e r enzymes. Fed. Proc. 37: 596, 1978. Chu, E.H.Y., T r o s k o , J . E . and Chang, C. M u t a t i o n a l approaches t o the s t u d y o f c a r c i n o g e n e s i s . J . T o x i c o l . Env. H e l a t h 2: 1317-1334, 1977. C l a y s o n , D.B. Chemical c a r c i n o g e n e s i s , 467 pp. B o s t o n : L i t t l e , Brown and Co., 1962. Clemmesen, J . On the e t i o l o g y o f some human c a n c e r s . J . N a t l . Cancer I n s t . j_2: 1-21, 1951. Conney, A.H. P h a r m a c o l o g i c a l i m p l i c a t i o n s o f microsomal enzyme i n d u c t i o n . Pharmacol. Reviews 19 ( 3 ) : 317-366, 1967. Conney, A.H., M i l l e r , E.C. and M i l l e r , J.A. The metabolism o f m e t h y l a t e d aminoazo dyes. V. E v i d e n c e f o r i n d u c t i o n o f enzyme s y n t h e s i s i n the r a t by 3-methycholanthrene. Cancer Research 1_6: 450-459, 1956. Conney, A.H., M i l l e r , E.C. and M i l l e r , J.A. S u b s t r a t e - i n d u c e d s y n t h e s i s and o t h e r p r o p e r t i e s o f benzpyrene h y d r o x y l a s e i n r a t l i v e r . J . B i o l . Chem. 228: 753-766, 1957. Conney, A.H. and Burns, J . J . Induced s y n t h e s i s o f o x i d a t i v e enzymes i n l i v e r microsomes by p o l y c y c l i c hydrocarbons and d r u g s . Advance. Enzyme R e g u l a t i o n 1_: 189-214, 1963. -164-Cook, J.W., Hewett, C L . and H i e g e r , I. The i s o l a t i o n o f a c a n c e r - p r o d u c i n g hydrocarbon from c o a l t a r . P a r t s I , II and I I I . J . Chem. Soc. 395-405, 1933. D a n s e t t e , P.M., A l e x a n d r o v , K., A z e r a d , R. and F r a y s s i n e t , CH. The e f f e c t o f some mixed f u n c t i o n o x i d a s e i n d u c e r s on a r y l hydrocarbon h y d r o x y l a s e and e p o x i d e hydrase i n n u c l e i and microsomes from r a t l i v e r and l u n g . The e f f e c t o f c i g a r e t t e smoke. Europ. J . Cancer 15/. 915-922, 1979. D i c k e n s , M., B r i d g e s , J.W., El combe, C R . and N e t t e r , K.J. A novel haemo-p r o t e i n i n d u c e d by i s o s a f r o l e p r e t r e a t m e n t i n the r a t . Biochem. B i o p h y s . Res. Commun. 80: 89-96, 1978. D o l l , R. and V o d o p i j a , I. Eds. Host environment i n t e r e a c t i o n s i n the e t i o l o g y o f c a n c e r i n man, IARC S c i e n t i f i c P u b l i c a t i o n No. 7, 464 pp. Lyen. 1973. El Defrawy El Masry, S., Cohen, G.M. and Mannering, G.J. Sex-dependent d i f -f e r e n c e s i n drug metabolism i n the r a t . I. Temporal changes i n the microsomal d r u g - m e t a b o l i z i n g system o f the l i v e r d u r i n g sexual matura-t i o n . Drug Metab. D i s p o s . 2: 267-278, 1974. El Defrawy El Masrey, S. and Mannering, G.J. Sex-dependent d i f f e r e n c e s i n drug m e t a b o l i s m i n the r a t . I I . Q u a l i t a t i v e changes produced by c a s t r a t i o n and the a d m i n i s t r a t i o n o f s t e r o i d hormones and p h e n o b a r b i t a l . Drug Metab. D i s p o s . 2: 279-284, 1974. E l s h o u r b a g y , N.A. and G u z e l i a n , P.S. S e p a r a t i o n , p u r i f i c a t i o n , and c h a r a c -t e r i z a t i o n o f a novel form o f h e p a t i c cytochrome P-450 from r a t s t r e a t e d w i t h pregnenolone-16«<-carbonitri1e. J . B i o l . Chem. 255: 1279-1285, 1980. F a h l , W.E., J e f c o a t e , C.R. and Kasper, C B . C h a r a c t e r i s t i c s o f b e n z o ( a ) p y r e n e m e t a b o l i s m and cytochrome P-450 h e t e r o g e n e i t y i n r a t l i v e r n u c l e a r e n v e l o p e and comparison t o microsomal membrane. J . B i o l . Chem. 253: 3106-3113, 1978. F a r b e r , E. H y p e r p l a s t i c l i v e r n o d u l e s . Methods Cancer Research ]_: 345-375, 1973. F a r b e r , E. and Magee, P.N. The p r o b a b l e a l k y l a t i o n o f l i v e r r i b o n u c l e i c a c i d by the h e p a t i c c a r c i n o g e n s d i m e t h y l n i t r o s a m i n e and e t h i o n i n e . Biochem. J . 76: 58P, 1960. F e l l e r , D.R, and G e r a l d , M.C I n t e r a c t i o n s o f s p i r o n o l a c t o n e w i t h h e p a t i c microsomal d r u g - m e t a b o l i z i n q enzyme systems. Biochem. Pharmacol. 20: 1991-2000, 1971. F i r m i n g e r , H.I. and Reuber, M.D. I n f l u e n c e o f a d r e n o c o r t i c a l , a n d r o g e n i c , and a n a b o l i c hormones on t h e development o f carcinoma and c i r r h o s i s o f the l i v e r i n A X C r a t s f e d N - 2 - f l u o r e n y l d i a c e t a m i d e . J . N a t l . Cancer I n s t . 27: 559-572, 1961 -165-F o r e s t , M.G. Plasma androgens ( t e s t o s t e r e n e and 4 - a n d r o s t e n e d i o n e ) and 17-hydroxy p r o g e s t e r o n e i n the n e o n a t a l , p r e p u b e r t a l and p e r i p u b e r t a l p e r i o d s i n the human and the r a t : D i f f e r e n c e s between s p e c i e s . J . S t e r o i d Biochem. JT-: 543-548, 1979. G e l b o i n , H.V. A microsome-dependent b i n d i n g o f b e n z o ( a ) p y r e n e t o DNA. Cancer Research 29: 1272-1276, 1969. G i e l e n , J . E . , Goujon, F.M. and Nebert, D.W. G e n e t i c r e g u l a t i o n o f a r y l h ydrocarbon h y d r o x y l a s e i n d u c t i o n . J . B i o l . Chem. 247: 1125-1137, 1972. G o n t o v n i c k , L.S., R o e l o f s , L. and B e l l w a r d , G.D. The e f f e c t s o f gonadectomy on the h e p a t i c a c t i v i t i e s o f a r y l hydrocarbon h y d r o x y l a s e , e p o x i d e hy-d r a t a s e , and g l u t a t h i o n e S - t r a n s f e r a s e i n W i s t a r r a t s p r e t r e a t e d w i t h o r a l methadone H C l . Can. J . P h y s i o l . Pharmacol. 57_: 286-290, 1979. Goujon, F.M., N e b e r t , D.W. and G i e l e n , J . E . G e n e t i c e x p r e s s i o n o f a r y l hydro-carbon h y d r o x y l a s e i n d u c t i o n . IV. I n t e r a c t i o n o f v a r i o u s compounds w i t h d i f f e r e n t forms o f cytochrome P-450 and the e f f e c t on b e n z o ( a ) -pyrene m e t a b o l i s m i n v i t r o . Mol. Pharmacol. 8: 667-680, 1972. G r o v e r , P.L. and Sims, P. Enzyme c a t a l y z e d r e a c t i o n s o f p o l y c y c l i c hydro-carbons w i t h d e o x y r i b o n u c l e i c a c i d and p r o t e i n In V i t r o . Biochem. J . 110: 159-160, 1969. Gurtoo, H.L. and P a r k e r , N.B. Sex-dependent r e g u l a t i o n o f b e n z o ( a ) p y r e n e and zoxazolamine m e t a b o l i s m i n r a t t i s s u e s . Drug Metab. D i s p o s . 5_: 474-481, 1977. Haddow, A. and Kon, G.A.R. C h e m i s t r y o f c a r c i n o g e n i c compounds. B r i t . Med. B u l l . 4: 314-326, 1947. H a e n s z e l , W. and K u r i h a r a , M, S t u d i e s o f . J a p a n e s e m i g r a n t s . I. M o r t a l i t y from c a n c e r and o t h e r d i s e a s e s among Japanese i n the U n i t e d S t a t e s . J . N a t l . Cancer I n s t . 40: 43-68, 1968. Harada, N. and Omura, T. S e l e c t i v e i n d u c t i o n o f two d i f f e r e n t m o l e c u l a r s p e c i e s o f cytochrome P-450 by p h e n o b a r b i t a l and 3 - m e t h y l c h o l a n t h r e n e . J . Biochem. 89: 237-248, 1981. H e i d e l b e r g e r , C. S t u d i e s on the m o l e c u l a r mechanism o f hydrocarbon c a r c i n -ogensis. J . C e l l u l a r Comp. P h y s i o l . 64: (Suppl."1 ): 129-148, 1964. H e i d e l b e r g e r , C. Chemical c a r c i n o g e n e s i s . Ann. Rev. Biochem. 44: 79-121, 1975. — Henderson, P.T. M e t a b o l i s m o f drugs i n r a t l i v e r d u r i n g the p e r i n a t a l p e r i o d . Biochem. Pharmacol. 20: 1225-1232,1971. -166-H i c k s , R.M., W a k e f i e l d , J . S . J , and Chowariec, J . C o - c a r c i n o g e n i c a c t i o n o f s a c c h a r i n i n the che m i c a l i n d u c t i o n o f b l a d d e r c a n c e r . Nature 243: 347-349, 1973. H i c k s , R.M., W a k e f i e l d , J . S . J , and Chowaniec, J . E v a l u a t i o n o f a new model to d e t e c h b l a d d e r c a r c i n o g e n s o r c o - c o r c i n o g e n s ; R e s u l t s o b t a i n e d w i t h s a c c h a r i n , c y c l a m a t e and cyclophosphamide. Chem.-B i o l . I n t e r a c t i o n s ]_]_. 225-233, 1975. H i e g e r , I. LVI11. The s p e c t r a o f c a n c e r - p r o d u c i n g t a r s and o i l s o f r e l a t e d s u b s t a n c e s . Biochem. J . 2M 505-511, 1930. H i g g i n s o n , J . In: Environment and c a n c e r , 24th Symp. Fundam. Cancer Res., 69-92. B a l t i m o r e : W i l l i a m s and Wi1 k i n s . 1972. H i g g i n s o n , J . and M u i r , C.S. In: Cancer M e d i c i n e , ed. J . F . H o l l a n d , E. F r e i 111, 241-306. P h i l a d e l p h i a : Lea and F e b i g e r . 1973. Hueper, W.C, W i l e y , F.H. and Wolfe, H.D. Expe r i m e n t a l p r o d u c t i o n o f b l a d d e r tumors i n dogs by a d m i n i s t r a t i o n o f bet a n a p h t h y l a m i n e . J . Ind. Hyg. T o x i c o l . " 2 0 : 46-84, 1938. Imai, Y., I t o , A. and S a t o , R. Ev i d e n c e 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 t y p e s o f v e s i c l e s i n the h e p a t i c microsomal f r a c t i o n . J . Biochem. 60: 417-428, 1966. J e n n e t t e , K.W., B o r n s t e i n , W., Chuang, A.H.L. and B r e s n i c k , E. S t e r e o -s p e c i f i c i t y o f me t a b o l i s m o f be n z o ( a ) p y r e n e (BP) t o (+) t r a n s -7, 8 - d i h y d r o x y -7, 8 - d i h y d r o - BP by r a t l i v e r n u c l e a r enzymes. Biochem. Pharmacol. 28: 338-339,1979. J e r n s t r o m , B., V a d i , H. and O r r e n i u s , S. Formation i n i s o l a t e d r a t l i v e r microsomes and n u c l e i o f be n z o ( a ) p y r e n e m e t a b o l i t e s t h a t b i n d to DNA. Cancer Research 36: 4007-4113,1976. J e r n s t r o m , B., V a d i , H, and O r r e n i u s , S. Formation o f DNA-binding p r o d u c t s from i s o l a t e d benzo(a) pyrene m e t a b o l i t e s i n r a t l i v e r n u c l e i . Chem.-B i o l . I n t e r a c t i o n s 20: 311-321,1978. Kas h n i g , D.M. and Kasper, C.E. I s o l a t i o n , morphology, and c o m p o s i t i o n o f the n u c l e a r membrane from r a t l i v e r . J . B i o l . Chem. 244: 3786-3792, 1969. Kasper, C.B. B i o c h e m i c a l d i s t i n c t i o n s between the n u c l e a r and microsomal membranes from r a t h e p a t o c y t e s . J . B i o l . Chem. 246: 577-581, 1971 Kennaway, E.L. and H i e g e r , I. C a r c i n o g e n i c s u b s t a n c e s and t h e i r f l u o r e s c e n c e s p e c t r a . B r i t . Med. J . 1: 1044-1046, 1930. Kimura, N.T., Kanematsu, T. and Baba, T. P o l y c h l o r i n a t e d b i p h e n y l ( s ) as a promotor i n e x p e r i m e n t a l h e p a t o c a r c i n o g e n e s i s i n r a t s . Z. K r e b s f o r s c h . 87: 257-266, 1976. -167-K i t i g a w a , T. and Sugano, H. Enhancement o f azo-dye h e p a t o - c a r c i n o g e n e s i s w i t h d i e t a r y p h e n o b a r b i t a l i n r a t s . Gann 68: 255-256, 1977. Kramer, R.E., G r e i n e r , J.W., Rumbaugh, R . C , Sweeney, T.D. and C o l b y , H.D. Requirements: o f the p i t u i t a r y g l and f o r gonadal hormone e f f e c t s on h e p a t i c drug metabolism i n r a t s . J . Pharmacol. Exp. Ther. 208: 19-26, 1979'. L o t l i k a r , P.D., Enomoto, M., M i l l e r , E.C. and M i l l e r , J.A. The e f f e c t s o f a d r e n a l e c t o m y , hypophysectomy, and c a s t r a t i o n on the u r i n a r y m e t a b o l i t e s o f 2 - a c e t y l a m i n o f l u o r e n e i n the r a t . Cancer Research 24: 1835-1842, 1964. Lu, A.Y.H., Somogyi, A., West, S., Kuntzman, R. and Conney, A.H. Pregnenolone-\ 1 6 < - c a r b o n i t r i l e : A new t y p e o f i n d u c e r o f d r u g - m e t a b o l i z i n g enzymes. A r c h . Biochem. B i o p h y s . 152: 457-462, 1972. Lu, A.Y.H. and West, S.B. M u l t i p l i c i t y o f mammalian microsomal cytochromes P-450. Pharmacol. Rev. 31_: 277-295, 1980. Lu, A.Y.H. and Miwa, G.T. M o l e c u l a r p r o p e r t i e s and b i o l o g i c a l f u n c t i o n s o f microsomal epoxide hydrase. Ann. Rev. Pharmacol. T o x i c o l . 20: 513-531, 1980. Magee, P.N. and F a r b e r , E. T o x i c l i v e r i n j u r y and c a r c i n o g e n e s i s . M e t h y l a -ti o n o f r a t l i v e r n u c l e i c a c i d s by d i m e t h y l n i t r o s a m i n e In V i v o . Biochem. J . 83:: 114-124, 1962. M a r r o q u i n , R.F. and F a r b e r , E. The a p p a r e n t b i n d i n g o f r a d i o a c t i v e 2 - a c e t y l -a m i n o f l u o r e n e t o r a t - l i v e r r i b o n u c l e i c a c i d In V i v o . Biochim. B i o p h y s . A c t a . 55_: 403-405, 1962. M a r s h a l l , W.J. The c o n t r o l o f drug metabolism. PhD T h e s i s , U n i v e r s i t y o f London, 1970. M a r s h a l l , W.J. and McLean, A.E.M. A r e q u i r e m e n t f o r d i e t a r y l i p i d s f o r i n -d u c t i o n o f cytochrome P-450 by p h e n o b a r b i t o n e i n r a t l i v e r microsomal f r a c t i o n . Biochem. J . 122; 569-573, 1971. McCarr, J . , C h o i , E., Yamasaki, E. and Ames, B.N. The - d e t e c t i o n o f c a r c i n -ogens as mutagens i n the S a l m o n e l l a microsome t e s t : A s s a y o f 300 chem-i c a l s . P r o c. N a t l . Acad. S c i . U.S. 72; 5135-5139, 1975. McGuire, J r . , J.S. and Tomkins, G.M. The e f f e c t s o f t h y r o x i n a d m i n i s t r a t i o n on the enzymic r e d u c t i o n o f A - 3 - k e t o s t e r o i d s . J . B i o l . Chem. 234: 791-794, 1959. Mendelson, J.H., I n t u r r i s i , C.E., R e n a u l t , P. and Senay, E. E f f e c t s o f ace-t y l m e t h a d o l on plasma t e s t o s t e r o n e . C l i n . Pharmacol. T h e r . 1_9: 371-374, 1976. -168-M i l l e r , E.C. Some c u r r e n t p e r s p e c t i v e s on chemical c a r c i n o g e n e s i s i n humans and e x p e r i m e n t a l a n i m a l s : P r e s i d e n t i a l a d d r e s s . Cancer Research 38_: 1479-1496, 1978. M i l l e r , E.C. and M i l l e r , J.A. The p r e s e n c e and s i g n i f i c a n c e o f bound aminoazo dyes i n the l i v e r s o f r a t s f e d p-dimethylaminoazobenzene. Cancer Res. ]_: 468-480, 1947. M i l l e r , E.C. and M i l l e r , J.A. In V i v o c o m b i n a t i o n s between c a r c i n o g e n s and t i s s u e c o n s t i t u e n t s and t h e i r p o s s i b l e r o l e i n c a r c i n o g e n e s i s . Cancer Research 12: 547-556, 1952. M i l l e r , E.C. and M i l l e r , J.A. In: Chemical Mutagens ( H o l l a e n d e r , A.P., ed.) pp. 83-119, Plenum P r e s s , New York, 1971. M i l l e r , J.A. C a r c i n o g e n e s i s by c h e m i c a l s : An o v e r v i e w - G.H.A. Clowes memorial l e c t u r e . Cancer Research 30: 559-576, 1970. Mukhtar, H., Elmamlouk, T.H. and Bend, J.R. t r a n s - S t i T b e n e o x i d e : An i n d u c e r o f r a t h e p a t i c microsomal and n u c l e a r e p o x i d e h y d r o l a s e and m i x e d - f u n c t i o n o x i d a s e a c t i v i t i e s . Chem.-Biol. I n t e r a c t i o n s 22/ 125-137, 1978. Mukhtar, H., Elmamlouk, T.H., P h i l p o t , R.M. and Bend, J.R. Rat h e p a t i c n u c l e a r cytochrome P-450 and e p o x i d e hydrase i n membranes p r e p a r e d by two methods: S i m i l a r i t i e s w i t h the microsomal enzymes. Mol. Pharmacol. 1_5: 192-196, 1979. Mukhtar, H., Elmamlouk, T.H. and Bend, J.R. Epoxide hydrase and mixed-f u n c t i o n o x i d a s e a c t i v i t i e s o f r a t l i v e r n u c l e a r membranes. A r c h . Biochem. B i o p h y s . 192: 10-21, 1979a. M u s h l i n , P.S. and P e r a i n o , C. E f f e c t s o f d i e t a r y p h e n o b a r b i t a l on the b i n d i n g o f 2 - a c e t y l a m i n o f l u o r e n e t o r a t l i v e r n u c l e a r DNA. Proc. Soc. Exp. B i o l . Med. 145: 859-862, 1974. Nash, T. The c o l o r i m e t r i c e s t i m a t i o n o f formaldehyde by means o f the Hantzsh r e a c t i o n . Biochem. J . 5J5: 416-421 , 1953. N e b e r t , D.W. M u l t i p l e forms o f i n d u c i b l e d r u g - m e t a b o l i z i n g enzymes: A r e a s o n a b l e mechanism by which any organism can cope w i t h a d v e r s i t y . Mol. Cell... Biochem. 27: 27-46, 1979. N e b e r t , D.W. and G e l b g i n , H.V. The r o l e o f r i b o n u c l e i c a c i d and p r o t e i n s y n t h e s i s i n microsomal a r y l hydrocarbon h y d r o x y l a s e i n d u c t i o n i n c e l l c u l t u r e . J . B i o l . Chem. 245: 160-168, 1970. Oesch, F., J e r i n a , D.M. and D a l y , J.W. A r a d i o m e t r i c a s s a y f o r h e p a t i c e p o x i d e hydrase a c t i v i t y w i t h (7- 3H) s t y r e n e o x i d e . Biochem. Bi o p h y s . A c t a 227: 685-691, 1971. -169-Oesch, F. and Schmassmann, H. S p e c i e s and organ s p e c i f i c i t y o f the t r a n s - s t i l b e n e o x i d e i n d u c e d e f f e c t s on e p o x i d e h y d r a t a s e and b e n z o ( a ) p y r e n e monooxygenase a c t i v i t y i n r o d e n t s . Biochem. Pharmacol. 28: 171-176, 1979. O r r e n i u s , S., E r i c s s o n , J . L . E . and E r h s t e r , 1. P h e n o b a r b i t a l - i n d u c e d s y n t h e s i s o f the microsomal drug - m e t a b o l i z i n g enzyme system and p r o l i f e r a t i o n o f endoplasmic membranes. J . C e l l . B i o l . 28: 181-198, 1966. Pento, J.T., M a g a r i a n , R.A., W r i g h t , R.J., K i n g , M.M. and Benjamin, E . J . N o n s t e r o i d a l e s t r o g e n s and a n t i e s t r o g e n s : B i o l o g i c a l a c t i v i t y o f c y c l o p r o p y l a n a l o g s o f s t i l b e n e and s t i l b e n e d i o l . J . Pharm. S c i . 70: 399-402, 1981. P e r a i n o , C., F r y , R.J.M. and S t a f f e l d t , E. Reducti o n and enhancement by p h e n o b a r b i t a l o f h e p a t o c a r c i n o g e n e s i s i n d u c e d i n the r a t by 2-a c e t y l a m i n o - f l u o r e n e . Cancer Research 31_: 1506-1512, 1971. P e r a i n o , C , F r y , R.J.M., S t a f f e l d t , E. and K i s i e l e s k i , W.E. E f f e c t s o f v a r y i n g t he exposure t o p h e n o b a r b i t a l on i t s enhancement o f 2-a c e t y l a m i n o f l u o r e n e - i n d u c e d h e p a t i c t u m o r i g e n e s i s i n the r a t . Cancer Res. 33: 2701-2705, 1973. P e r a i n o , C., F r y , R.J.M. and S t a f f e l d t , E. Enhancement o f spontaneous h e p a t i c t u m o r i g e n e s i s i n C3H mice by d i e t a r y p h e n o b a r b i t a l . J . N a t l . Cancer I n s t . 5J_: 1349-1350, 1973a. P e r a i n o , C., F r y , R.J.M., S t a f f e l d t , E. and C h r i s t o p h e r , J.P. Comparative e n h a n c i n g e f f e c t o f p h e n o b a r b i t a l , a m o b a r b i t a l , d i p h e n y l h y d a n t o i n , and d i c h l o r o p h e n y l t r i c h l o r o e t h a n e on 2 - a c e t y l a m i n o f l u o r e n e - i n d u c e d h e p a t i c t u m o r i g e n e s i s i n t h e r a t . Cancer Research 35: 2884-2890, 1975. P e r a i n o , C , F r y , R.J. and S t a d t f e l d t , E. E f f e c t s o f v a r y i n g the o n s e t and d u r a t i o n o f exposure t o p h e n o b a r b i t a l on i t s enhancement o f 2-a c e t y l a m i n o - f l u o r e n e - i n d u c e d h e p a t i c t u m o r i g e n e s i s . Cancer Res. 37: 3623-3627, 1977. P e r a i n o , C , F r y , R.J.M., S t a f f e l d t , E. and C h r i s t o p h e r , J.P. Enhancing e f f e c t s o f p h e n o b a r b i t a l and b u t y l a t e d h y d r o x y t o l u e n e on 2 - a c e t y l a m i n o -f l u o r e n e - i n d u c e d h e o a t i c t u m o r i g e n e s i s i n the r a t . Food Cosmet. T o x i c o l . 1_5: 93-96, 1977a. P e z z u t o , J.N., L e a , M.A. and Yang, C.S. B i n d i n g o f m e t a b o l i c a l l y a c t i v a t e d b e n z o ( a ) p y r e n e to n u c l e a r macromolecules. Cancer Research 36: 3647-3653, 1976. P e z z u t o , J.M., Lea, M.A. and Yang, C.S. The r o l e o f microsomes and n u c l e a r e n v e l o p e i n the m e t a b o l i c a c t i v a t i o n o f be n z o ( a ) p y r e n e l e a d i n g to b i n d i n g w i t h n u c l e a r macromolecules. Cancer Research 37: 3427-3433, 1977. — -170-P e z z u t o , J.M. Yang, C.S., Yang, S.K., McCourt, D.W. and G e l b o i n , H.V. M e t a b o l i s m o f b e n z o ( a ) p y r e n e and ( - ) - t r a n s - 7 , 8 - d i h y d r o x y - 7 , 8-d i h y d r o b e n z o ( a ) p y r e n e by r a t l i v e r microsomes. Cancer Research 38: 1241-1245, 1978. P i t o t , H.C. Drugs as promoters o f c a r c i n o g e n e s i s . I n : The I n d u c t i o n o f Drug M e t a b o l i s m ( E s t a b r o o k , R.W., L i n d e n l a u b , E., e d s . ) , pp. 471-483, F.K. S c h a t t a u e r V e r l a g , Stuttgard-New York, 1978. P i t o t , H.C. and S i r i c a , A. The s t a g e s o f i n i t i a t i o n and promotion i n h e o a t o c a r c i n o g e n e s i s . B i o c h i m . B i o p h y s . A c t a 605: 191-215, 1980. P o l a n d , A., G l o v e r , E. and Kende, A.S. S t e r e b s p e c i f i T e , : h i g h a f f i n i t y b i n d i n g o f 2, 3, 7, 8 - t e t r a c h l o r o d i b e n z o - p - d i o x i n by h e p a t i c c y t o s o l . J . B i o l . Chem. 251: 4936-4946, 1976. P o l a n d , A., Mak, I . , G l o v e r , E., Boatman, R.J. and Kende, A.S. 1,4-B i s ( 2 - ( 3 , 5 - d i c h l o r o p y r i d y l o x y ) ) - b e n z e n e , a p o t e n t p h e n o b a r b i t a l - 1 i k e i n d u c e r o f microsomal monoxygenase a c t i v i t y . Mol. Pharmacol. 1_8: 571-580, 1980. P o t t , P. C h i r c u r g i c a l o b s e r v a t i o n s r e l a t i v e t o the c a n c e r o f the scrotum. London, 1775. R e p r i n t e d i n N a t l . Cancer I n s t . Monograph, 10: 7-13, 1963. P r e s t o n , B.D., Van M i l l e r , J.P., Moore, R.W. and A l l e n , J.R. Promoting e f f e c t s o f p o l y c h l o r i n a t e d b i p h e n y l s ( A r o c h l o r 1254) and p o l y c h l o r i n a t e d d i b e n z o f u r a n - f r e e A r o c h l o r 1254 on d i e t h y l n i t r o s a m i n e - i n d u c e d tumorigene-s i s i n the r a t . J . N a t l . Cancer I n s t . 66: 509-515, 1981. Redmond, D.E., J r . Tobacco and c a n c e r : The f i r s t c l i n i c a l r e p o r t , 1761. Mew E n g l . J . Med. 282: 18-23, 1970. Rehn, L. B l a s e n g e s c h w u l s t e b e i f u s h s i n a r b e i t e r n . A r c h . K l i n . C h i r . 50_: 588-600, 1895. Reuber, M.D. E f f e c t o f age and t e s t o s t e r o n e on the i n d u c t i o n o f h y p e r p l a s -t i c n o d u l e s , c a r c i n o m a s , and c i r r h o s i s o f the l i v e r i n r a t s i n g e s t i n g N - 2 - f l u o r e n y l d i a c e t a m i d e . Eur. J . Cancer 12; 137-141, 1976. Robson, R.M., G o l l , D.E. and Temple, M.L. D e t e r m i n a t i o n o f p r o t e i n s i n t r i s b u f f e r by the b i u r e t r e a c t i o n . A n a l . Biochem. 24: 339-341 , 1968. Rogan, E.G. and C a v a l i e r i , E. 3 - M e t h y l c h o l a n t h r e n e - i n d u c i b l e b i n d i n g o f a r o m a t i c hydrocarbons to DNA i n p u r i f i e d r a t l i v e r n u c l e i . Biochem. Bi o p h y s . Res. Commun. 58: 1119-1126, 1974. -171-Roy, R.K., M i l i n , B.S. and McMinn, D.A. Androgen r e c e p t o r i n r a t l i v e r : Hormonal and developmental r e g u l a t i o n o f the c y t o p l a s m i c r e c e p t o r and i t s c o r r e l a t i o n w i t h the androgen-dependent s y n t h e s i s o f e*. -2u- g l o b u l i n . Biochem. B i o p h y s . A c t a . 354: 213-232, 1974. Ryan, D.E., Thomas, P.E., K o r z e n i o w s k i , D. and L e v i n . W. S e p a r a t i o n and c h a r a c t e r i z a t i o n o f h i g h l y p u r i f i e d forms o f l i v e r microsomal cytochrome P-450 from r a t s t r e a t e d w i t h p o l y c h l o r i n a t e d b i p h e n y l s , p h e n o b a r b i t a l , and 3 - m e t h y l c h o l a n t h r e n e . J . B i o l . Chem. 254: 1365-1374, 1979. Schmassmann, H. and Oesch, F. T r a n s - s t i l b e n e o x i d e : A s e l e c t i v e i n d u c e r o f r a t l i v e r e p o x i d e h y c r a t a s e . M o l e c u l a r Pharmacol. 14-: 834-847, 1978. S e i d e g a r d , J . , Moron, M.S., E r i k s s o n , L.C. and D e P i e r r e , J.W. The t o p o l o g y o f epoxide h y d r a t a s e and benzo(a)phrene monooxygenase i n the en d o p l a s m i c r e t i c u l u m o f r a t l i v e r . B i o c h i m . B i o p h y s . A c t a 543: 29-40, 1978. S e i d e g a r d , J . , M o r g e n s t e r n , R., D e P i e r r e , J.W. and E r n s t e r , L. T r a n s - s t i l -bene o x i d e : A new type o f i n d u c e r o f d r u g - m e t a b o l i z i n g enzymes. Bio c h i m . B i o p h y s . A c t a 586: 10-21, 1979. Sims, P., Gro v e r , P.L., S w a i s l a n d , A., P a l , K. and Hewer, A. M e t a b o l i c ac-t i v a t i o n o f b e n z o ( a ) p y r e n e p r o c e e d s by a d i o l - e p o x i d e . Nature 252: 326-328, 1974. S k e t t , P. and G u s t a f s s o n , J.A. I m p r i n t i n g o f enzyme systems o f x e n o b i o t i c and s t e r o i d m e t a b o l i s m . Reviews i n B i o c h e m i c a l T o x i c o l o g y 1_: 27-52, 1979. Snyder, R. and Remmer, H. C l a s s e s o f h e p a t i c microsomal mixed f u n c t i o n o x i d a s e i n d u c e r s . Pharmacol. Ther. 7_: 203-244, 1979. Sonowane, B.R., Y a f f e , S . J . and S h a p i r o , B.H. H e p a t i c drug m e t a b o l i s m i n p s e u d o h e r m a p h r o d i t i c r a t s . Drug metab. D i s p o s . 7_: 444-445, 1979. S t a t i s t i c s Canada, V i t a l S t a t i s t i c s , Volume I I I , M o r t a l i t y , Summary l i s t o f c a u s e s , 1978. S t e k o l , J.A., Mooly, U. and P e r r y , J . The i n c o r p o r a t i o n o f the carbon o f the e t h y l group o f e t h i o n irie i n t o l i v e r n u c l e i c a c i d s and the e f f e c t o f e t h i o n i n e f e e d i n g on the c o n t e n t o f n u c l e i c a c i d s i n r a t l i v e r . J . B i o l . Chem. 235: PC59-PC60, 1960. S t r i p p , B . , Hamrick, M.E., Zampaglione, N.G. and G i l l e t t e , J.R. The e f f e c t o f s p i r o n o l a c t o n e on drug metabolism by h e p a t i c microsomes. J.' Phar-macol. Exp. Ther. 176: 766-771, 1971. S t r i p p , B., Menard, R.H., Zampaglione, N.G., Hamrick, M.E. and G i l l e t t e , J.R. E f f e c t o f s t e r o i d s on drug metabolism i n male and female r a t s . Drug Metab. Di s p o s , 1_: 216-221 , 1973. -172-S u t h e r l a n d , E.W., C o r i , C.F., Haynes, R. and O l s e n , N.S. P u r i f i c a t i o n o f the h y p e r g l y c e m i c - g l y c o g e n o l y t i c f a c t o r from i n s u l i n and from g a s t r i c mucosa. J . B i o l . Chem. 180: 825-837, 1949. Sweeney, G.C. and C o l e , F.M. E f f e c t s o f e t h y n y l e s t r a d i o l on l i v e r m i c r o -somal mixed f u n c t i o n oxygenase a c t i v i t y i n male r a t s . Lab. I n v e s t . 42: 231-235, 1980. Thomas, P.E., K o r z e n i o w s k i , D., B r e s n i c k , E., B o r n s t e i n , W.A., Kasper, C.B., F a h l , W.E., J e f c o a t e , C R . and L e v i n , W. H e p a t i c cytochrome P-448 and e p o x i d e h y d r a s e : Enzymes o f n u c l e a r o r i g i n a r e i m m u n o l o g i c a l l y i d e n t i c a l w i t h t h o s e o f microsomal o r i g i n . A r c h . Biochem. B i o p h y s . 192: 22-26, 1979. Thomas, P.E., K o r z e n i o w s k i , . D . , Ryan, D. and Levin,W. P r e p a r a t i o n o f mono-s p e c i f i e d a n t i b o d i e s a g a i n s t two forms o f r a t l i v e r cytochrome P-450 and q u a n t i t a t i o n o f these a n t i g e n s i n microsomes. A r c h . Biochem. B i o p h y s . 192: 524-532, 1979. T o m a t i s , L., Agthe, C , B a r t s c h , H., H u f f , J . , Montesano, R., S a r a c c i , R., Walker, E. and W i l b o u r n , J . E v a l u a t i o n o f the c a r c i n o g e n i c i t y o f c h e m i c a l s . A r e v i e w o f the monograph program o f the I n t e r n a t i o n a l Agency f o r r e s e a r c h on Cancer (1971 to 1977). Cancer Research 36_: 877-885, 1978. T s u t s u i , H. Uber das K u n s t l i c h e r z e u g t e c a n c r o i d b e i d e r maus. Gann, 12/ 17-21, 1918. V i v i a n i , A., L u t z , W.K. and S c h l a t t e r , C. Time c o u r s e o f the i n d u c t i o n o f a r y l hydrocarbon h y d r o x y l a s e i n r a t l i v e r n u c l e i and microsomes by p h e n o b a r b i t a l , 3 - m e t h y l c h o l a n t h r e n e , 2, 3, 7, 8 - t e t r a c h l o r o d i b e n z o - p -d i o x i n , d i e l d r i n and o t h e r i n d u c e r s . Biochem. Pharmacol. 27_: 2103-2108, 1978. Vizethum, W. and Goerz, G. I n d u c t i o n o f the h e p a t i c microsomal and n u c l e a r cytochrome P-450 system by h e x a c h l o r o b e n z e n e , p e n t a c h l o r o p h e n o l and t r i c h l o r o p h e n o l . Chem.-Biol. I n t e r a c t i o n s 28: 291-299, 1979. Warren, P.M. and B e l l w a r d , G.D. I n d u c t i o n o f a r y l hydrocarbon h y d r o x y l a s e by 3 - m e t h y l c h o l a n t h r e n e i n l i v e r , l u n g and k i d n e y o f gonadectomized and sham-operated W i s t a r r a t s . Biochem. Pharmacol. 27_: 2537-2541 , 1978. Weber, K. and Osborn, M. The r e l i a b i l i t y o f m o l e c u l a r w e i g h t d e t e r m i n a t i o n s by d o d e c y l s u l f a t e - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s . J . B i o l . Chem. 244: 4406-4412, 1969. W e i n s t e i n , I.B., J e f f r e y , A.M., J e n n e t t e , K.W., B l o b s t e i n , S.H., Harvey, R.G., H a r r i s , C , A u t r u p , H., K a s a i , H. and N a k a n i s h i , K. B e n z o ( a ) -pyrene d i o l e p o x i d e s as i n t e r m e d i a t e s i n n u c l e i c a c i d b i n d i n g In V i t r o and In V i v o . S c i e n c e 193: 592-595, 1976. -173-Weisburger, J.H., Madison, R.M., Ward, J.M., V i g n e r a , C. and W e i s b u r g e r , E.F. M o d i f i c a t i o n o f d i e t h y l n i t r o s a m i n e l i v e r c a r c i n o g e n e s i s w i t h p h e n o b a r b i t a l but not w i t h immunosuppression. J . N a t l . Cancer I n s t . 54: 1185-1188, 1975. W e i s b u r g e r , J.H. and Weisburger, E.K. P a r t V. Pharmacodynamics o f c a r c i n -o g e n i c azo dyes, a r o m a t i c amines, and n i t r o s a m i n e s . C l i n . Pharmacol. Ther. 4: 110-129, 1963. Wheeler, G.P. and S k i p p e r , H.E. S t u d i e s w i t h mustards. I I I . In Vivo f i x a t i o n o f C ^ from n i t r o g e n mustard -C' H3 i n n u c l e i c a c i d f r a c t i o n s o f animal t i s s u e s . A r c h . Biochem. B i o p h y s . 72; 465-475, 1957. W i e b e l , F . J . and G e l b o i n , H.V. A r y l hydrocarbon ( b e n z o ( a ) p y r e n e ) h y d r o x y l a s e s i n l i v e r from r a t s o f d i f f e r e n t age, sex and n u t r i t i o n a l s t a t u s . D i s -t i n c t i o n o f two t y p e s by 7 , 8 - b e n z o f l a v o n e . Biochem. Pharmacol. 24: 1511-1515, 1975. Wood, A.W., L e v i n , W., Lu, A.Y.H., Y a g i , H., Hernandez, 0., J e r i n a , D.M. and Conney, A.H. M e t a b o l i s m o f b e n z o ( a ) p y r e n e and b e n z o ( a ) p y r e n e d e r i v a t i v e s t o mutagenic p r o d u c t s by h i g h l y p u r i f i e d h e p a t i c m i c r o -somal enzymes. J . B i o l . Chem. 25]_: 4882-4890, 1976. Yamagiwa, K. and Ichikawa, K. E x p e r i m e n t e l l e s t u d i e uber d i e pathogenese d e r e p i t h e l i a l g e s c h w u l s t e . M i t t . Med. Fak. T o k i o , 15; 295-344, 1915. Y o s h i d a , T. Uber d i e s e r i e n weise v e r f o l g u n g d e r veranderungen d e r l e b e r d e r e x p e r i m e n t e l l e n hepatomerzeugung durch o - a m i n a z o t o l u o l . T r a n s . Japan P a t h o l . Soc. 23: 636-638, 1933. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0095513/manifest

Comment

Related Items