Drug Metabolism Concepts (ACS symposium series ; 44)

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.fw001 Drug Metabolism Concepts Publication Date: June 1, 1...

Author: Donald M. Jerina

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Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.fw001

Drug Metabolism Concepts


Drug Metabolism Concepts Donald M . Jerina, EDITOR The National Institutes of Health


A symposium co-sponsored by the Division of Medicinal Chemistry and the Division of Analytical Chemistry at the 172nd Meeting of the American Chemical Society, San Francisco, Calif., August 31,

ACS

1976

SYMPOSIUM

SERIES

AMERICAN CHEMICAL SOCIETY WASHINGTON, D. C. 1977

44


Library of Congress CIP Data Drug metabolism concepts. (ACS symposium series; 44 ISSN 0097-6156) Includes bibliographical references and index. 1. Drug metabolism—Congresses. 2. Cytochrome P-450 —Congresses. 3. Benzpyrene—Metabolism—Congresses. 4. Carcinogenesis—Congresses. I. Jerina, Donald M., 1940- . II. American Chemical Society. Division of Analytical Chemistry. III. American Chemical Society. Division of Medicinal Chemistry. IV. Series: American Chemical Society. Symposium series; 44. RM301.D76 ISBN 0-8412-0370-9

615'.7 ACSMC8

77-2279 44 1-196

Copyright © 1977 American Chemical Society All Rights Reserved. No part of this book may be reproduced or transmitted in any form or by any means—graphic, electronic, including photocopying, recording, taping, or information storage and retrieval systems—without written permission from the American Chemical Society. PRINTED IN THE UNITED STATES OF AMERICA

ACS Symposium Series


Robert F. Gould, Editor

Advisory Board Donald G. Crosby Jeremiah P. Freeman E. Desmond Goddard Robert A. Hofstader John L. Margrave Nina I. McClelland John B. Pfeiffer Joseph V. Rodricks Alan C. Sartorelli Raymond B. Seymour Roy L. Whistler Aaron Wold


FOREWORD The ACS SYMPOSIUM S E R I E S was founded in 1974 to provide a medium for publishing symposia quickly in book form. The format of the S E R I E S parallels that of the continuing ADVANCES IN CHEMISTRY S E R I E S except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. As a further means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book. Papers published in the ACS SYMPOSIUM S E R I E S are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.

PREFACE he metabolism of drugs and other environmental chemicals has attracted the attention and concern of scientists whose specialties range from analytical and physical chemistry to toxicology, biology, and ecology. The efforts of enzymologists, biochemists, and pharmacologists have established that the basic components of microsomal drug metabolism consist of a membrane or lipid environment, reductases for electron transport, and a family of heme-containing terminal oxidases at which half of the oxygen molecule is reduced to water and the other half is incorporated into substrate. The mechanism by which dioxygen undergoes two-electron reduction to form water and an "active oxygen' species has fascinated inorganic and organic chemists alike. Rates and pathways of metabolism are critically important to medicinal chemistry, pharmacology, and clinical medicine. Chemically reactive intermediates are of concern to toxicologists and to biologists studying carcinogenicity and provide a special fascination for the chemist in terms of structure-activity relationships. Thus, the metabolism of drugs and other xenobiotics not only effects detoxication by conversion to polar, readily excretable compounds but, in many instances, forms reactive intermediates which are responsible for toxic or carcinogenic effects. The concept that chemical carcinogens induce neoplasia via conversion to reactive metabolites which cause chemical modification of critical cellular molecules has made the field of drug metabolism very relevant to the effects of drugs and environmental pollutants on man and his world.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.pr001

A

This volume is the outgrowth of a symposium titled "Recent A d vances in the Study of Drug Metabolism." For this symposium, an effort was made to assemble scientists who would represent a broad spectrum of research in drug metabolism. Hopefully, this interdisciplinary collection will prove of value. The National Institutes of Health Bethesda, Md. December 1976

DONALD M. JERINA

ix

1 Cytochrome P-450—Its Role in Oxygen Activation for Drug Metabolism R. W. ESTABROOK and J. WERRINGLOER

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

Department of Biochemistry, University of Texas Health Science Center, Dallas, TX 75235

At t h i s time many of our colleagues are directing their a t t e n t i o n (1,2) to e v a l u a t i n g the e x i s t e n c e of oxygen on the p l a n e t Mars - an effort d i c t a t e d by the d e s i r e to l e a r n whether any life form, as we know it, e x i s t s on that p l a n e t . What is it t h a t conf e r s on oxygen s p e c i a l p r o p e r t i e s t h a t make it so critical f o r the f u n c t i o n i n g of cellular metabolism. Much has been written on the "fitness of oxygen" (3) p o i n t i n g to the capability to metabolically reduce atmospheric oxygen to hydrogen peroxide or water by two or f o u r e l e c t r o n t r a n s f e r processes, r e s p e c t i v e l y . More important to the t o p i c of this symposium is the ability to enzymatically " a c t i v a t e " molecular oxygen, p e r m i t t i n g the i n c o r p o r a t i o n of one atom of oxygen i n t o an organic s u b s t r a t e molecule concomitant w i t h the r e d u c t i o n of the other atom of oxygen to water. The enzyme systems r e s p o n s i b l e f o r c a t a l y z i n g these types of r e a c t i o n s (Figure 1) are termed mixed-function oxidases, hydroxylases or oxygenases. In many i n s t a n c e s , the i n t r o d u c t i o n of a h y d r o x y l group to the hydrophobic s u b s t r a t e molecule provides a site f o r subsequent conjugation w i t h h y d r o p h i l i c compounds thereby i n c r e a s i n g the solubility of the product f o r its t r a n s p o r t and e x c r e t i o n from the organism. C e n t r a l to the f u n c t i o n i n g of many mixed-function o x i d a t i o n r e a c t i o n s i s a f a m i l y of hemoproteins g e n e r a l l y classified as cytochromes P-450. The present symposium is directeded to f u r t h e r our understanding of how t h i s unique hemoprotein p a r t i c i p a t e s in "oxygen activation" and " s u b s t r a t e h y d r o x y l a t i o n " . Studies w i t h the perfused r a t l i v e r by Thurman and Scholz (4j 5^) as w e l l as S i e s , et a l . (6,7) i n d i c a t e t h a t approximately 60 percent of c e l l u l a r r e s p i r a t i o n by t h i s organ i s s e n s i t i v e to i n h i b i t i o n by Antimycin A - a chemical w e l l recognized as a powerf u l i n h i b i t o r of the m i t o c h o n d r i a l r e s p i r a t o r y c h a i n . Of the r e maining 40 percent of c e l l u l a r r e s p i r a t i o n approximately h a l f i s s e n s i t i v e to the i n h i b i t o r sodium cyanide. A s i g n i f i c a n t p o r t i o n

Supported i n p a r t by a r e s e a r c h grant from the N a t i o n a l I n s t i t u t e s of Health (NIGMS - 16488).

1


2

DRUG M E T A B O L I S M CONCEPTS

of the antimycin A i n s e n s i t i v e r e s p i r a t i o n of the l i v e r c e l l i s presumed to occur by o x i d a t i v e enzymes a s s o c i a t e d w i t h the endoplasmic r e t i c u l u m , i . e . the microsomal f r a c t i o n . A c o n s i d e r a t i o n of microsomal e l e c t r o n t r a n s f e r r e a c t i o n s (Figure 2) r e v e a l s the f u n c t i o n i n g of a t l e a s t three f l a v o p r o t e i n s , two hemoproteins, and an i r o n c o n t a i n i n g p r o t e i n . The f l a v o p r o t e i n s f u n c t i o n as reduced p y r i d i n e n u c l e o t i d e dehydrogenases and they p a r t i c i p a t e i n the t r a n s f e r of reducing e q u i v a l e n t s to cytochrome 1>5 from NADH, v i a the f l a v o p r o t e i n ( f p i ) , NADH-cytochrome b$ reductase, or from NADPH by (fp2>, NADPH-cytochrome P-450 reductase ( 8 ) . Reduced cytochrome b$ may i n t e r a c t w i t h a r e c e n t l y i s o l a t e d i r o n cont a i n i n g p r o t e i n (9) i n the cyanide s e n s i t i v e d e s a t u r a t i o n of f a t t y acyl-Coenzyme A compounds (10,11). The reduced f l a v o p r o t e i n , f p 2 , can a l s o undergo o x i d a t i o n by f e r r i c cytochrome P-450 i n r e a c t i o n s to be described below, o r , by an as y e t p o o r l y understood r e a c t i o n , t h i s reduced f l a v o p r o t e i n has been p o s t u l a t e d (12,13) to r e a c t w i t h molecular oxygen g i v i n g r i s e to a superoxide anion f o r the i n i t i a t i o n of l i p i d p e r o x i d a t i o n or heme degradation. A t h i r d f l a v o p r o t e i n , f p 3 , p a r t i c i p a t e s i n the o x i d a t i o n of t e r t i a r y amines g i v i n g r i s e to N-oxides as described by Z i e g l e r et a l . (14,15). Of i n t e r e s t are recent r e s u l t s reported by Z i e g l e r et a l . (16) t h a t t h i s f l a v o p r o t e i n , f p 3 , may a l s o f u n c t i o n i n an oxygen dependent o x i d a t i o n of s u l f h y d r y l groups f o r the format i o n of d i s u l f i d e bonds. Cytochrome P-450 has many i n t e r e s t i n g p r o p e r t i e s that serve as a challenge to the biochemist concerned w i t h understanding the f u n c t i o n of t h i s hemoprotein as i t p a r t i c i p a t e s i n a broad spectrum of o x i d a t i v e r e a c t i o n s . I t s n a t u r a l environment i n most mammalian t i s s u e s i s the membrane s t r u c t u r e c a l l e d the endoplasmic r e t i c u l u m ; t h e r e f o r e , considerable i n t e r e s t i s d i r e c t e d to understanding the i n f l u e n c e imposed by a presumed r e s t r i c t e d m o b i l i t y of p r o t e i n molecules i n a m i l e u of l i p i d as t h i s pigment i n t e r a c t s w i t h oxygen, s u b s t r a t e molecules, and the f l a v o p r o t e i n e l e c t r o n donor. F u r t h e r , i t r e q u i r e s the s k i l l and perseverance of groups such as those l e d by Coon e t a l . (17-19) as w e l l as Lu and L e v i n (20-22) to i s o l a t e and p u r i f y v a r i o u s forms of t h i s hemoprotein f o r p h y s i c a l and chemical c h a r a c t e r i z a t i o n . Such s t u d i e s have revealed that the f a m i l y of cytochromes P-450 have molecular weights i n the range of 46,000 to 52,000 and d i s p l a y a great p r o p e n s i t y to aggregate because of t h e i r hydrophobic p r o p e r t i e s . F u r t h e r , cytochrome P-450 i s r e a d i l y i n d u c i b l e upon treatment of an animal w i t h v a r i o u s drugs or p o l y c y c l i c hydrocarbons. Although t h i s property i s p o o r l y understood i t appears to be r e s p o n s i b l e i n p a r t f o r the long observed phenomenon of drug t o l e r a n c e (23«24). As w i l l be discussed by Dr. Coon during t h i s symposium (25), cytochrome P-450 can e x i s t i n m u l t i p l e forms w i t h an ever i n c r e a s i n g r o s t e r of new p r o t e i n s i d e n t i f i e d as p u r i f i c a t i o n methodology becomes more s o p h i s t i c a t e d and w i d e l y used.


1.

ESTABROOK A N D WERRINGLOER

Cytochrome

P-450 in Oxygen Activation

3

I n i t i a l i n t e r e s t i n microsomal mixed-function o x i d a t i o n r e a c t i o n s occured i n the 1950's; such s t u d i e s were focused on three g e n e r a l areas of b i o m e d i c a l importance. Brodie and h i s c o l l a b o r a t o r s (26,27) recognized the p o t e n t i a l r o l e of h y d r o x y l a t i o n r e a c t i o n s i n the o x i d a t i v e t r a n s f o r m a t i o n of many drugs - hence t h i s r e a c t i o n system was looked upon as a general mechanism f o r d e t o x i f i c a t i o n of f o r e i g n chemicals. In c o n t r a s t , the M i l l e r s and t h e i r colleagues (28,29) were concerned w i t h the o x i d a t i v e conversion of p r e c a r c i n o g e n i c chemicals, such as p o l y c y c l i c hydrocarbons, to carcinogens - r e a c t i o n s of great p o t e n t i a l harm to the maintenance of the v i a b i l i t y of the organism. A t h i r d group, the s t e r o i d e n d o c r i n o l o g i s t s , were a c t i v e l y studying (30, 31) the o x i d a t i v e processes r e s p o n s i b l e f o r the enzymatic conv e r s i o n of c h o l e s t e r o l to g l u c o c o r t i c o i d s and m i n e r a l o c o r t i c o i d s products of c r i t i c a l importance f o r the maintenance of homeos t a s i s of the organism. We now know t h a t the general f a m i l y of hemoproteins, cytochromes P-450, p l a y p i v i t o l r o l e s i n d i r e c t i n g the o p e r a t i o n of each of these, as w e l l as other s i m i l a r r e a c t i o n s . Thus t h i s c l a s s of hemoproteins has a d u a l i t y of f u n c t i o n (Figure 3) jL.e_., i t may serve as e i t h e r a panacea or a plague f o r the c e l l . Proposed C y c l i c F u n c t i o n of Cytochrome P-450. How do we c u r r e n t l y v i s u a l i z e the f u n c t i o n of t h i s unique hemoprotein, cytochrome P-450? One p o s t u l a t e d scheme (32) i l l u s t r a t i n g the c y c l i c p a t t e r n of r e d u c t i o n and oxygenation of cytochrome P-450 as i t i n t e r a c t s w i t h s u b s t r a t e molecules, e l e c t r o n donors, and oxygen i s shown i n F i g u r e 4. B r i e f l y , these r e a c t i o n s may be summarized as f o l l o w s : ^ A. The f e r r i c hemoprotein (Fe'") can i n t e r a c t w i t h a molec u l e of s u b s t r a t e (R) r e s u l t i n g i n a complex (Fe ^»R) analogous to an enzyme - s u b s t r a t e complex. This i n t e r a c t i o n can be d i r e c t l y measured s i n c e the s u b s t r a t e molecule appears to be c l o s e l y a s s o c i a t e d w i t h the heme moiety of cytochrome P-450 r e s u l t i n g i n a s p e c t r a l p e r t u r b a t i o n measurable by e i t h e r o p t i c a l absorbance spectrophotometry or e l e c t r o n paramagnetic resonance spectrometry (33-36). The o r i e n t a t i o n of the s u b s t r a t e molecule as i t s i t s i n p r o x i m i t y of the heme i r o n , the presumed b i n d i n g s i t e f o r oxygen, remains a conjecture although the r o l e of s t e e r i n g groups on the s u b s t r a t e molecule (37) or the i n f l u e n c e of the hydrophobic environment o f the heme 738) may be considered as p o s s i b l e d i r e c t ing f o r c e s . B. The s u b s t r a t e complex of f e r r i c - cytochrome P-450 (Fe 3*R) undergoes r e d u c t i o n to a f e r r o u s cytochrome P-450 s u b s t r a t e complex (Fe ^»R) by e l e c t r o n s o r i g i n a t i n g from NADPH and t r a n s f e r r e d by the f l a v o p r o t e i n (fp2)> NADPH-cytochrome P-450 r e ductase. The q u e s t i o n of a stimultaneous two e l e c t r o n t r a n s f e r to cytochrome P-450, as suggested by Coon et a l . (39,40), or the e x i s t e n c e of two d i s c r e t e one e l e c t r o n donating s t e p s , as demons t r a t e d f o r the p u r i f i e d b a c t e r i a l cytochrome P-450 by Tyson e t +

+

+

DRUG M E T A B O L I S M

4

CONCEPTS

oxygenase 2 e ~ + Substrate + 0

Product +

2 M

(hydrophobic)

F

a

H 0 2

(hydroxylated)

Figure 1. General representation of microsomal-catalyzed oxygenase or mixed-function oxidation reactions

Conjugation i t Excretion

F A T T Y ACID DESATURATION


,NADH fp

—•fpi

>b /

3

/

N A D P H — * ffPp p2a, ^ f

-

•* X ,

x

2

V...

•

5

x

P-450

*0

2

MIXED FUNCTION OXIDATION

\

LIPID LI PI PEROXIDATION

N-OXIDATION

Figure 2. Schematic of microsomal electron transport reactions

0 ,e~ — • 2

A.

Figure 3. The duality of cytochrome P-450-catalyzed reactions

A c t i v e Drug

Inactive

Drug

0 ,e 2

B.

Precarcinogen

•

Carcinogen

CO

I 02

Figure 4. Schematic of the proposed cyclic function of cytochrome P-450. The substrate molecule is designated R. The valence state of the heme iron of cytochrome P-450 is indicated.

CO

1.


Cytochrome


a l . (41) as w e l l as Peterson (42), remains as a p o i n t o f uncert a i n t y . F o r the present d i s c u s s i o n the scheme presented i s based on the premise t h a t two separate e l e c t r o n donating r e a c t i o n s o c cur. C. Reduced cytochrome P-450 ( F e * R ) can r e a c t w i t h carbon monoxide t o form a d e r i v a t i v e which i s r e a d i l y i d e n t i f i a b l e s p e c t r o p h o t o m e t r i c a l l y by an absorbance band maximum a t about 450 nm - hence the o r i g i n (43) o f the name cytochrome P-450. A l t e r n a t i v e l y , reduced cytochrome P-450 can r e a c t w i t h oxygen t o form a complex termed (44) oxycytochrome P-450 (Fe 2»02*R). Knowledge o f the chemistry o f oxycytochrome P-450 should provide the needed c l u e t o evaluate the f i r s t step o f "oxygen a c t i v a t i o n " f o r h y d r o x y l a t i o n r e a c t i o n s ; t h e r e f o r e , much e f f o r t has been d i r e c t e d to understanding the parameters t h a t i n f l u e n c e the g e n e r a t i o n and subsequent u t i l i z a t i o n o f t h i s i n t e r m e d i a t e i n cytochrome P-450 c a t a l y z e d r e a c t i o n s . D. Oxycytochrome P-450 (Fe 2»02*R) can presumably d i s s o c i a t e to g i v e a superoxide a n i o n (02~) concomitant w i t h the r e g e n e r a t i o n of the f e r r i c hemoprotein. The r e s u l t a n t hydrogen p e r o x i d e formed by d i s m u t a t i o n o f the superoxide a n i o n may be a measure o f t h i s a b o r t i v e "uncoupling" (45,46) o f cytochrome P-450 f u n c t i o n , .i.e.., a r e a c t i o n which d i v e r t s the t e r n a r y complex o f oxygen, hemoprot e i n , and s u b s t r a t e from i t s r o l e i n oxygen a c t i v a t i o n and subsequent s u b s t r a t e h y d r o x y l a t i o n . A l t e r n a t i v e l y , the complex o f oxycytochrome P-450 may undergo f u r t h e r r e d u c t i o n t o form the equival e n t o f a p e r o x i d e a n i o n d e r i v a t i v e o f the s u b s t r a t e bound hemoprot e i n . Studies (47-50) w i t h the membrane bound cytochrome P-450 of l i v e r microsomes suggests t h a t a d o n a t i o n o f a proposed second e l e c t r o n occurs v i a cytochrome b$ (51). T h i s c o n c l u s i o n i s the b a s i s f o r e x p l a i n i n g the s y n e r g i s t i c T e f f e c t observed d u r i n g the concomitant o x i d a t i o n o f NADPH and NADH by t h i s system (52). E. The proposed p e r o x i d e a n i o n complex o f cytochrome P-450 may undergo p r o t o n a t i o n and d i s s o c i a t e as hydrogen peroxide o r i t may rearrange t o form an oxene d e r i v a t i v e (53) concomitant w i t h the r e l e a s e of water. The e x i s t e n c e o f oxygen as an oxenoid species i s c o n j e c t u r e although s t u d i e s w i t h o r g a n i c p e r o x i d e supported h y d r o x y l a t i o n r e a c t i o n s (54-58) mediated by cytochrome P-450 demonstrate the e x i s t e n c e o f an EPR species s i m i l a r t o Complex I of peroxidase as an i n t e r m e d i a t e i n the r e a c t i o n (59). C l e a r l y , more r e s e a r c h i s needed t o f i r m l y e s t a b l i s h the presence of t h i s form o f oxygen as " a c t i v e oxygen". F. L e a s t understood i s the mechanism o f d i s s o c i a t i o n o f the hydroxylated product and the r e s t o r a t i o n o f the low s p i n form of f e r r i c cytochrome P-450. I n some i n s t a n c e s (60) epoxide i n t e r mediates e x i s t , such as occurs i n those r e a c t i o n s i n v o l v i n g p o l y c y c l i c hydrocarbons (61). I n other c a s e s , product adducts r e s u l t (62-65) which impede the f u r t h e r f u n c t i o n o f cytochrome P-450 as i t p a r t i c i p a t e s i n s u b s t r a t e h y d r o x y l a t i o n r e a c t i o n s . +2

+


5

+


6


Hydrogen Peroxide Formation, The generation o f hydrogen peroxide during NADPH o x i d a t i o n by l i v e r microsomes has been r e cognized f o r a number o f years (66,67). L i t t l e a t t e n t i o n was p a i d to t h i s phenomenon - i n l a r g e p a r t because o f the a s s o c i a t i o n of c a t a l a s e w i t h the microsomal f r a c t i o n r e s u l t i n g i n the breakdown o f hydrogen peroxide as r a p i d l y as i t was formed. Recent s t u d i e s (54-58) on the p e r o x i d a t i c f u n c t i o n o f cytochrome P-450 as w e l l as t h e coupled r e a c t i o n o f hydrogen peroxide w i t h c a t a l a s e f o r the o x i d a t i o n o f a l c o h o l s (68-70) during NADPH o x i d a t i o n by l i v e r microsomes has s t i m u l a t e d a f u r t h e r c o n s i d e r a t i o n o f the r e a c t i o n s i n v o l v e d i n hydrogen peroxide formation. I n a d d i t i o n , an understanding o f the mechanism o f hydrogen peroxide generation may provide a needed c l u e f o r b e t t e r exami n i n g the p r o p e r t i e s o f " a c t i v a t e d oxygen" proposed t o be a necessary intermediate i n cytochrome P-450 c a t a l y z e d r e a c t i o n s (71). As i l l u s t r a t e d i n F i g u r e 5, the a d d i t i o n o f NADPH t o a suspension o f r a t l i v e r microsomes incubated i n the presence o f sodium a z i d e t o i n h i b i t a d v e n t i t i o u s c a t a l a s e , r e s u l t s i n a s t o i c h i o m e t r i c r e d u c t i o n o f oxygen. Approximately 50 percent o f the oxygen reduced can be accounted f o r as hydrogen peroxide generated during the r e a c t i o n * Repeated a d d i t i o n s of NADPH r e s u l t s i n t h e stepwise formation of equal i n c r e ments o f hydrogen peroxide i n d i c a t i n g the a b i l i t y t o a d d i t i v e l y form t h i s product. The f a i l u r e t o observe a s t o i c h i o m e t r i c amount o f hydrogen peroxide formed t o the amount o f oxygen reduced o r NADPH o x i d i z e d has been a t t r i b u t e d (72) t o the p r e sence o f "endogenous s u b s t r a t e s " a s s o c i a t e d w i t h the microsomal f r a c t i o n which can undergo mixed f u n c t i o n o x i d a t i o n r e a c t i o n s . The nature of the proposed "endogenous s u b s t r a t e s " remains t o be b e t t e r d e f i n e d . A number o f p o s s i b i l i t i e s e x i s t t o e x p l a i n the source o f hydrogen peroxide. As proposed i n the scheme presented i n F i g u r e 4 hydrogen peroxide may a r i s e e i t h e r from d i s s o c i a t i o n o f oxycytochrome P-450 o r from the two e l e c t r o n reduced form o f the t e r n a r y complex o f oxygen, s u b s t r a t e , and cytochrome P-450. A l t e r n a t i v e l y one must consider t h a t hydrogen peroxide format i o n may n o t i n v o l v e cytochrome P-450, such as by the autox i d a t i o n o f reduced f l a v o p r o t e i n s . I n order t o f i r m l y e s t a b l i s h the r o l e o f cytochrome P-450 i n hydrogen peroxide gene r a t i o n a s e r i e s o f experiments were c a r r i e d out t o examine the i n f l u e n c e o f i n h i b i t o r s o f cytochrome P-450 f u n c t i o n , such as carbon monoxide o r metyrapone, as w e l l as determine the i n f l u e n c e o f temperature, pH, and the e f f e c t o f v a r i o u s l e v e l s o f NADPH on the r e a c t i o n . I n each i n s t a n c e comparat i v e s t u d i e s were c a r r i e d out t o determine the changes observed i n the r a t e o f N-demethylation o f ethylmorphine - a s u b s t r a t e recognized t o undergo o x i d a t i v e t r a n s f o r m a t i o n c a t a l y z e d by cytochrome P-450.

1.

ESTABROOK A N D W E R R I N G L O E R

Cytochrome


7

n moles/min/mg

9.3

if ^no

u u n MM n U g


2

73liMNADPHQuid. 72 M Og utillztd M

min. 2 mg

Pb

liver m i c r o t o m e t / m l

Figure 5. The formation of hydrogen peroxide during NADPH oxidation. Liver microsomes from phenobarbital-treated rats were incubated at 2 mg of protein per ml in a reaction medium containing 50mM tris-chloride buffer, pH 7.5, 150mM KCl, 10 mM MgCl and ImM NaN . At times equal to 0 and 10 min, aliquots of NADPH were added to initiate the reaction. In the experiment shown in dashes, NADPH was added only at 0 time. Samples were removed at the points indicated and after addition to trichloroacetic acid the concentration of hydrogen peroxide formed was determined colorometrically with potassium thiocyanate and ferrous ammonium sulfate (S3). Oxygen use was measured in a comparable series of experiments polarographically, and NADPH oxidation was determined spectrophotometrically. 2>

s

y


8


CONCEPTS

Incubation o f l i v e r microsomes i n a v e s s e l designed t o m a i n t a i n f i x e d r a t i o s o f carbon monoxide t o oxygen r e s u l t s i n an e q u i v a l e n t i n c r e a s e i n i n h i b i t i o n o f both hydrogen peroxide formation and ethylmorphine N-demethylation as the concentrat i o n o f carbon monoxide i n c r e a s e s r e l a t i v e t o oxygen as shown i n F i g u r e 6. The o b s e r v a t i o n t h a t hydrogen peroxide formation i s i n h i b i t e d by carbon monoxide i s strong presumptive evidence f o r the r o l e o f cytochrome P-450 i n t h i s r e a c t i o n (73). The f a c t t h a t t h e extent o f i n h i b i t i o n i s e f f e c t i v e l y i d e n t i c a l t o t h a t observed f o r the metabolism o f ethylmorphine r e i n f o r c e s t h i s c o n c l u s i o n . However, t o date no photochemical a c t i o n spectrum s t u d i e s (74,75) have been c a r r i e d out t o c o n f i r m the r o l e o f c y t o chrome P-450 i n the formation o f hydrogen peroxide a s has been done f o r other s u b s t r a t e s o x i d a t i v e l y metabolized by t h i s hemop r o t e i n . I t i s o f i n t e r e s t t o note t h a t t h e degree o f carbon monoxide i n h i b i t i o n i s independent o f the presence o f NADH where a s y n e r g i s t i c (47-50) e f f e c t on product formation d u r i n g t h e NADPH supported r e a c t i o n may occur (see below). Studies w i t h t h e i n h i b i t o r metyrapone ( 2 - m e t h y l - l , 2 - b i s ( 3 p y r i d y l ) - l - p r o p a n o n e ) are more e q u i v o c a l s i n c e hydrogen peroxide formation i s maximally i n h i b i t e d about 50 percent by t h i s compound whereas the metabolism o f ethylmorphine i s g r e a t e r than 90 percent i n h i b i t e d (Figure 7 ) . One p o s s i b l e e x p l a n a t i o n f o r t h i s d i f f e r e n c e r e l a t e s t o the o b s e r v a t i o n t h a t o n l y 50 percent o f t h e cytochrome P-450 a s s o c i a t e d w i t h l i v e r microsomes 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 r e a c t s w i t h metyrapone (76) t o form a s p e c t r a l l y i d e n t i f i a b l e complex. This would imply t h a t a l l forms o f c y t o chrome P-450 present i n l i v e r microsomes can p a r t i c i p a t e i n hydrogen peroxide formation whereas a unique metyrapone b i n d i n g form f u n c t i o n s i n ethylmorphine N-demethylation r e a c t i o n s . C l e a r l y more experiments w i l l be r e q u i r e d t o support t h i s hypothesis. An examination o f the i n f l u e n c e o f v a r y i n g suboptimal steady s t a t e l e v e l s o f NADPH shows (Figure 8) t h a t the apparent Km's f o r NADPH r e q u i r e d t o support the generation o f hydrogen peroxide and t h e N-demethylation o f ethylmorphine a r e i d e n t i c a l . L i k e w i s e s t u d i e s on the i n f l u e n c e o f temperature (Figure 9) on the r a t e s o f t h e two r e a c t i o n s shows an e q u i v a l e n t c a l c u l a t e d energy o f a c t i v a t i o n o f approximately 20 k i l o c a l o r i e s per degree. Thus a wide range o f d i f f e r e n t f a c t o r s have been examined t o e s t a b l i s h the s i m i l a r i t y o f r e a c t i o n s i n which c y t o chrome P-450 serves as the pigment i n t e r a c t i n g w i t h oxygen f o r the formation o f hydrogen peroxide. The q u e s t i o n remains unanswered, however, as t o the mechanism o f hydrogen peroxide gene r a t i o n , i..je. by d i s m u t a t i o n o f t h e superoxide anion d i s s o c i a t i n g from oxycytochrome P-450 o r by p r o t o n a t i o n of the proposed two e l e c t r o n reduced s t a t e e q u i v a l e n t t o the peroxide anion complex of cytochrome P-450.


Cytochrome

PDnn. IPT PRODUCT

IOC

HCHO

u n "2 2

• • • A

U

>


§

AnniTinMC ADDITIONS o o A

80

P-450 in Oxygen

EM EM EM

CONTROL , ACTIVITY - i , -i)

( n m o l e s

m i n

NADPH NADPH NADPH+ NADH

13.3 10.7 23.5

EM

NADPH NADPH NADPH+ NADH NADPH+ NADH

9.2 10.0 10.2 10.0

1

1

EM

Activation

m q

60-

o 8

4 0

O

20

i

1

1

1

1

1

>

RATIO

C

%

Figure 6. The inhibition by carbon monoxide of the generation of hydrogen peroxide and the N-demethylation of ethyl morphine. A series of experiments was carried out as described in Figure 5 in a reaction vessel designed to permit equilibration with various gas mixtures of carbon monoxide, oxygen, and nitrogen. The concentration of oxygen was maintained at 20% for all experiments. The reaction mixture was supplemented by adding 5mM sodium isocitrate, 0.5 units of isocitrate dehydrogenase per ml, 2mM of 5'AMP, and 5/AM rotenone. The reaction was initiated by adding 200fiM NADPH and 200[xM NADPH where indicated. Samples were removed every 30 sec for the initial 5 min of the reaction and analyzed for hydrogen peroxide (S3) or formaldehyde (84;.

10


CONCEPTS

EM(HCHO)

1412.

10-

H

E


I

2°2

8-

Figure 7. The effect of metyrapone 6on the rate of hydrogen peroxide for-1 e mation as compared with the rate of 4'N-demethylation of ethylmorphine. Liver microsomes from phenobarbital< treated rats were incubated at 1 me of 2protein per ml in the reaction medium described in Figure 5 supplemented with an NADPH generating system (cf. Figure 6). Varying concentrations of metyrapone were added as indicated. The initial rates of product formation were determined as described in Figure 6.

0 .02

.1

.5 2

0 .02

METYRAPONE

.1

.5

2

(mM)

80-

Figure 8. The influence of varying g 60steady state concentrations of NADPH ^> on the rate of formation of hydrogen ^ peroxide and the N-demethylation of § ethylmorphine. 2 40Liver microsomes from phenobarbitaU &. treated rats were incubated in a reaction medium as described in Figure 5 supplemented with 5mM sodium isocitrate and 2 0 0.5 units of isocitrate dehydrogenase per ml. Where indicated, 5mM ethylmorphine was present. Varying concentrations of NADPH were added to initiate the reaction, and the rate of hydrogen peroxide or formaldehyde formed was determined as described in Figure 6.

• X

// if il §[

In m I HCHO o-«o

• ' ' • | 5

| 10 >)M

NADPH

H 0 2

I 15

2


1. E S T A B R O O K A N D W E R R I N G L O E R

Cytochrome


11

The I n f l u e n c e of Substrates o f Cytochrome P-450 on t h e Generation o f Hydrogen Peroxide. Presumably oxycytochrome P-450 serves a t a p i v i t o l p o i n t i n the c y c l i c f u n c t i o n o f cytochrome P-450 ( c f . F i g u r e 4 ) . T h i s t e r n a r y complex o f oxygen, s u b s t r a t e and cytochrome P-450 can undergo d i s s o c i a t i o n t o g i v e r i s e t o hydrogen peroxide r e g e n e r a t i n g t h e complex o f f e r r i c cytochrome P-450 w i t h s u b s t r a t e o r i t can proceed, by a mechanism as y e t unknown, t o a c t i v a t e oxygen f o r i n s e r t i o n i n t o the s u b s t r a t e r e s u l t i n g i n the formation o f a hydroxylated product and t h e low s p i n uncomplexed form o f f e r r i c cytochrome P-450. Therefore i t was o f i n t e r e s t t o c a r r y out experiments t o evaluate the i n f l u ence o f v a r i o u s s u b s t r a t e s o f cytochrome P-450, known t o undergo enzymatic h y d r o x y l a t i o n , and t o determine how such s u b s t r a t e s i n f l u e n c e t h e r a t e o f hydrogen peroxide formation. As i l l u s t r a ted i n F i g u r e 10, the presence o f ethylmorphine markedly a t tenuated t h e extent of hydrogen peroxide formed when a l i m i t i n g amount o f NADPH i s added t o i n i t i a t e the r e a c t i o n ( c f . F i g u r e 5 ) . In a d d i t i o n a s m a l l b u t r e p r o d u c i b l e i n h i b i t i o n o f t h e i n i t i a l r a t e o f hydrogen peroxide formation was observed. The decrease i n the extent o f hydrogen peroxide formation i n the presence o f ethylmorphine can be a t t r i b u t e d i n l a r g e p a r t t o t h e s t i m u l a t i o n of NADPH o x i d a t i o n observed i n t h e presence o f t h i s s u b s t r a t e , I.e. a n e a r l y a d d i t i v e e f f e c t o f NADPH o x i d a t i o n occurs when a Ndemethylation r e a c t i o n f u n c t i o n s concomitant w i t h hydrogen p e r oxide formation. Of i n t e r e s t i s the o b s e r v a t i o n t h a t a slow but p e r c e p t i b l e r a t e o f N-demethylation o f ethylmorphine cont i n u e s a f t e r the t o t a l o x i d a t i o n o f NADPH. T h i s slow r e a c t i o n ( o c c u r r i n g a f t e r 4 minutes i n t h e experiments shown i n F i g u r e 10) appears r e l a t e d t o a s t i m u l a t i o n i n the r a t e o f u t i l i z a t i o n of hydrogen peroxide i n the presence o f sodium a z i d e . As d i s cussed below, the a b i l i t y t o support the o x i d a t i v e metabolism o f a v a r i e t y o f s u b s t r a t e s by hydrogen peroxide, i n the absence o f reducing e q u i v a l e n t s generated from NADPH, d i r e c t l y demonstrates the p e r o x i d a t i c f u n c t i o n o f cytochrome P-450. A f u r t h e r s e r i e s o f experiments were c a r r i e d o u t t o examine the e f f e c t o f other s u b s t r a t e s on t h e r a t e o f formation o f hydrogen peroxide as summarized i n F i g u r e 11. I n t h i s case NADPH c o n c e n t r a t i o n was maintained by the a d d i t i o n o f sodium i s o c i t r a t e and i s o c i t r a t e dehydrogenase. I n agreement w i t h t h e experimental r e s u l t s d e s c r i b e d i n F i g u r e 10, the presence o f ethylmorphine r e s u l t e d i n approximately a 20 percent i n h i b i t i o n o f the r a t e o f hydrogen peroxide f o r m a t i o n . I n c o n t r a s t , s u b s t r a t e s o f c y t o chrome P-450 such as benzphetamine and h e x o b a r b i t a l caused a marked s t i m u l a t i o n i n the r a t e o f hydrogen peroxide f o r m a t i o n . U l l r i c h and D i e h l (45), Hildebrandt e t a l (46) and Werringloer e t a l (70) have d e s c r i b e d t h e a b i l i t y o f v a r i o u s compounds t o serve as "uncouplers" o f cytochrome P-450 f u n c t i o n , chemicals which s t i m u l a t e the r a t e o f NADPH o x i d a t i o n and oxygen u t i l i z a t i o n w i t h out a corresponding i n c r e a s e i n the r a t e o f s u b s t r a t e h y d r o x y l a t i o n . There appear t o be two c l a s s e s o f such "uncouplers"; those

12


CONCEPTS

100 50 •X.

V o Z> Q O

or o_

V

HCHO

1

1

10 5

20 Kcal udegree" jimole" X

H 0 2

2

21 Kcal xdegree" jtmole"

1


1

Figure 9. The effect of varying temperatures on the rate of hydrogen peroxide formation and the rate of Ndemetnylation of ethylmorphine. A series of experiments similar to those described in Figure 6 were carried out at the temperatures indicated.

27°

37° 3.2

33

17°

3.4

3.5, 3.6 J-.IO 5

80—EM 60-

40-

Figure 10. The influence of ethylmorphine on the rate and extent of hydrogen peroxide formation in the presence of a limiting concentration of NADPH. A series of experiments similar to those described in Figure 5 were carried out in the presence or absence of 5mM ethylmorphine or ImM sodium azide. The amount of hydrogen peroxide or formaldehyde formed * were determined as described in Figure 6. An NADPH generating system was omitted during this series of experiments. The reactions were initiated by addition of 145uM NADPH.

20•

+EM

+ Az 80-Az 60-

40-

20-

10 TIME (minutes)

1.


Cytochrome


13


compounds t h a t r e s u l t i n an i n c r e a s e d r a t e o f f o r m a t i o n o f hydrogen peroxide where t h e i n c r e a s e d r a t e o f u t i l i z a t i o n o f oxygen i s accompanied by an e q u i v a l e n t i n c r e a s e i n the r a t e o f NADPH o x i d a t i o n (such as h e x o b a r b i t a l o r benzphetamine) and a second type o f "uncoupling" where non-metabolized s u b s t r a t e s such as p e r f l u o r i nated, a l i p h a t i c f l u o r o c a r b o n s (45) o r compounds l i k e halothane (Werringloer, J and Estabrook, R.W., unpublished r e s u l t s ) r e s u l t i n a f a i l u r e t o see any a d d i t i o n a l hydrogen peroxide formed upon s t i m u l a t i o n o f oxygen u t i l i z a t i o n , l*e., two moles o f NADPH a r e u t i l i z e d f o r each a d d i t i o n a l mole o f oxygen reduced o f f s e t t i n g the s t o i c h i o m e t r y o f 1 mole o f oxygen u t i l i z e d p e r mole o f NADPH o x i d i z e d . The d e t a i l s o f how "uncouplers" d i v e r t the f u n c t i o n o f cytochrome P-450 i n a presumed a b o r t i v e r e a c t i o n r e s u l t i n g i n t h e d i s s i p a t i o n o f the oxycytochrome P-450 t e r n a r y complex remains as a c h a l l e n g e f o r f u r t h e r experimental examination. E l e c t r o n paramagnetic resonance s t u d i e s . The above d i s c u s s i o n centers on the p o t e n t i a l r o l e o f a superoxide anion as an i n t e r m e d i a t e i n the formation o f hydrogen peroxide d u r i n g NADPH o x i d a t i o n by l i v e r microsomes. Since the superoxide anion i s a f r e e r a d i c a l i t should be d e t e c t a b l e by e l e c t r o n p a r a magnetic resonance spectroscopy (77). When l i v e r microsomes are incubated i n an oxygen s a t u r a t e d b u f f e r i n the presence of sodium a z i d e and a sample i s r a p i d l y f r o z e n i n l i q u i d n i t r o g e n soon a f t e r i n i t i a t i o n o f the r e a c t i o n by the a d d i t i o n of NADPH, examination by EPR spectroscopy r e v e a l s t h e f o r m a t i o n of a s i g n a l a t about g • 2.0 as shown i n F i g u r e 12. The gene r a t i o n o f t h i s new EPR s i g n a l , however, does n o t appear t o be r e l a t e d t o hydrogen peroxide f o r m a t i o n o r t h e g e n e r a t i o n o f a f r e e r a d i c a l o f the superoxide anion type. The same EPR s i g n a l a t g » 2.0 i s obtained i f NADH i s employed r a t h e r than NADPH and t h e s i g n a l i s u n a l t e r e d i f sodium a z i d e i s omitted from the medium when reduced p y r i d i n e n u c l e o t i d e i s added. F u r t h e r the s i g n a l remains when the f r o z e n sample of microsomes i s warmed from the temperature o f l i q u i d n i t r o g e n t o -10° and i t s power s a t u r a t i o n c h a r a c t e r i s t i c s resemble those d e s c r i b e d f o r a s i m i l a r f r e e r a d i c a l s i g n a l d e s c r i b e d by Iyanagi and Mason (78) which they a t t r i b u t e d t o a f l a v i n f r e e r a d i c a l generated during t h e f u n c t i o n o f the microsomal f l a v o p r o t e i n , NADPH-cytochrome c reductase. Thus no p o s i t i v e evidence f o r the presence o f the superoxide anion c o u l d be obtained by examining l i v e r microsomes by EPR spectroscopy. A number o f exp l a n a t i o n s c o u l d be o f f e r e d t o r a t i o n a l i z e t h i s n e g a t i v e r e s u l t , such as the presence of an a c t i v e superoxide dismutase a s s o c i a t e d w i t h the microsomal f r a c t i o n ; evenso, t h i s approach to d e f i n e the presence o f the superoxide anion does remain t o be f u r t h e r e x p l o r e d . NADH Synergism. A number o f years ago (47,48) i t was r e cognized t h a t NADH o x i d a t i o n by l i v e r microsomes concomitant


20-


E

16 Figure 11. The "uncoupling effect" of various sub-c strates on the rate of gen- E eration of hydrogen peroxide during NADPH 12oxidation by rat liver microsomes. A series of experiments were carried out using liver micro- 8somes from phenobarbitaU CM treated rats incubated in I a reaction mixture as described in Figure 7 containing an o 4NADPH generating system.£ c Where indicated 5mM ethylmorphine (EM), ImM benzphetamine (BPh), or 2mM hexobarbital (Hx) were added to the reaction mixture.

E M

B

P

h

Hx

ImM

-

AZIDE

NADPH

+ NADPH

2600

2800

3000

3200

3400 H

(Oersted)

Figure 12. Changes in the electron paramagnetic resonance signals of liver microsomes associated with the initiation of NADPH oxidation by liver microsomes. Liver microsomes from phenobarbital-treated rats were suspended at 10 mg protein per ml in an oxygenated reaction medium containing 50mM tris-chbride buffer, pH 7.5, 150mU KCl, lOmU MgCL, and ImM sodium azide. An aliquot was removed, placed in a calibrated EPR tube, and rapidly frozen in liquid nitrogen (upper curve). NADPH (final concentration, 200fiM) was then added to the suspension and an aliquot removed and frozen within 15 sec (lower curve). First derivative spectra were obtained with an E-4 Varian EPR with the samples maintained at the temperature of liquid nitrogen.


1.

ESTABROOK AND WERRINGLOER

Cytochrome


15

w i t h t h e o x i d a t i o n o f l i m i t i n g c o n c e n t r a t i o n s o f NADPH r e s u l t e d i n a marked enhancement i n the o x i d a t i v e metabolism o f s u b s t r a t e s of cytochrome P-450 such as aminopyrine and ethylmorphine. As shown i n F i g u r e 13 the i n i t i a l r a t e o f N-demethylation o f e t h y l morphine by r a t l i v e r microsomes i s n e a r l y doubled when NADH i s added i n the presence o f NADPH. T h i s s t i m u l a t i o n o f a c t i v i t y i s r e f l e c t e d not o n l y i n the r a t e but a l s o t h e extent o f product formed. T h i s observed e f f e c t o f NADH g r e a t l y exceeds an a d d i t i v e e f f e c t o f the a c t i o n of the two forms o f reduced p y r i d i n e n u c l e o t i d e s s i n c e the N-demethylation of ethylmorphine i s r e l a t i v e l y slow i n the presence of NADH a l o n e . This type o f experiment t o gether w i t h measurements on changes i n the extent of steady s t a t e r e d u c t i o n o f cytochrome b$ (51) have served as one o f the foundat i o n s f o r the development of the c y c l i c scheme o f cytochrome P-450 f u n c t i o n as d e s c r i b e d i n F i g u r e 4. I t i s proposed t h a t NADH serves t o donate, v i a the f l a v o p r o t e i n reductase and c y t o chrome b^, the e l e c t r o n r e q u i r e d t o reduce oxycytochrome P-450 to an i n t e r m e d i a t e w i t h an o x i d a t i o n s t a t e e q u i v a l e n t t o a p e r oxide a n i o n form. Of i n t e r e s t was the q u e s t i o n whether a s i m i l a r NADH synergism would be observed d u r i n g t h e g e n e r a t i o n o f hydrogen p e r o x i d e a s s o c i a t e d w i t h the o x i d a t i o n o f NADPH. I n t h i s way i t may be p o s s i b l e t o g a i n some i n s i g h t i n t o which form o f the oxygen comp l e x o f cytochrome P-450 serves as t h e source o f hydrogen p e r oxide. As shown i n F i g u r e 14, NADH does not have a s i g n i f i c a n t syne r g i s t i c a f f e c t on the g e n e r a t i o n o f hydrogen p e r o x i d e d u r i n g NADPH o x i d a t i o n . A s m a l l i n c r e a s e i n the i n i t i a l r a t e o f hydrogen peroxide f o r m a t i o n i s observed i n the presence o f NADH and NADPH, r e l a t i v e t o the r a t e observed when NADPH alone i s used as the donor o f r e d u c i n g e q u i v a l e n t s , b u t t h i s i n c r e a s e i s a p p r o x i mately equal t o the r a t e observed when NADH i s used t o support the r e a c t i o n . The extent o f hydrogen peroxide f o r m a t i o n observed does i n c r e a s e measurably b u t t h i s may be a t t r i b u t e d i n l a r g e p a r t to a " s p a r i n g e f f e c t " of r e d u c i n g e q u i v a l e n t s from NADPH used t o support the mixed f u n c t i o n o x i d a t i o n o f "endogenous s u b s t r a t e s " as d e s c r i b e d i n an e a r l i e r s e c t i o n o f t h i s paper. On the b a s i s o f these r e s u l t s i t i s concluded t h a t the same type o f NADH synerg i s t i c e f f e c t c h a r a c t e r i s t i c o f h y d r o x y l a t i o n r e a c t i o n s mediated by cytochrome P-450 does not f u n c t i o n f o r the g e n e r a t i o n o f hydrogen p e r o x i d e . T h i s o b s e r v a t i o n may r e s u l t from an a l t e r a t i o n o f the r o l e f o r a needed second e l e c t r o n t o form the p e r o x i d e a n i o n form o f cytochrome P-450 o r from t h e dominant r o l e o f t h e d i s s o c i a t i o n o f oxycytochrome P-450 g i v i n g r i s e t o the superoxide a n i o n . The l a t t e r h y p o t h e s i s would p r e c l u d e the need f o r donation o f a second e l e c t r o n i n the f o r m a t i o n o f hydrogen p e r o x i d e . The p e r o x i d a t i c f u n c t i o n o f cytochrome P-450. Hrycay and O'Brien (54-56) have d e s c r i b e d experiments which demonstrate t h e


CONCEPTS

72pM NAOPH + 96JIM NADH

A" 4-


4-

2

Figure 13.

4

6 8 TIME (minutes)

10

12

The synergistic effect of NADH on the NADPH-dependent N-demethylation of ethylmorphine. Liver microsomes from phenoharhital-treated rats were incubated at 1 mg of protein per ml in the presence of 5mM ethylmorphine. NADPH and NADH were added in the concentrations indicated to initiate the reaction. Samples were removed and the amount of formaldehyde formed determined by the Nash reagent (84). Sodium azide and an NADPH generating system were omitted from the reaction medium.


1.


Cytochrome


17

TIME (minutes)

Figure 14. The effect of varying concentrations of NADH on the rate and extent of formation of hydrogen peroxide associated with the oxidation of a limiting concentration of NADPH by liver microsomes. A series of experiments similar to those described in Figure 5 were carried out in the presence of NADPH and NADH as indicated.


18


CONCEPTS

a b i l i t y o f cytochrome P-450 o f l i v e r microsomes t o serve as a peroxidase. About the same t i m e , Kadlubar e t a l (57) demonstrated t h e a b i l i t y o f o r g a n i c hydroperoxides t o support N-demethylat i o n r e a c t i o n s when l i v e r microsomes a r e added t o the r e a c t i o n mixture. R e c e n t l y c o n s i d e r a b l e i n t e r e s t has centered on the mechanism o f these peroxide c a t a l y z e d r e a c t i o n s as they i n v o l v e cytochrome P-450 s i n c e such r e a c t i o n s may p r o v i d e a d i f f e r e n t means of e v a l u a t i n g the presence and nature o f " a c t i v e oxygen". When l i v e r microsomes i n t e r a c t w i t h ethylmorphine and hydrogen p e r o x i d e , i n the presence o f sodium a z i d e t o i n h i b i t contamina t i n g c a t a l a s e , one observes (Figure 15) a s t o i c h i o m e t r i c u t i l i z a t i o n o f hydrogen p e r o x i d e concomitant w i t h the f o r m a t i o n o f formaldehyde - the product o f N-demethylation o f ethylmorphine (58). T h i s r e a c t i o n i s n o t dependent on the presence o f oxygen and i s not i n h i b i t e d by carbon monoxide. R e l a t i v e l y h i g h l e v e l s o f hydrogen p e r o x i d e a r e r e q u i r e d t o o b t a i n maximal r a t e s o f the Ndemethylation r e a c t i o n and an apparent Km o f approximately 20 mM f o r hydrogen p e r o x i d e has been determined (58). Under o p t i m a l cond i t i o n s r a t e s o f N-demethylation a r e observed i n the presence o f hydrogen peroxide which a r e g r e a t e r than 50 f o l d the r a t e o b t a i n e d when NADPH supports the o x i d a t i v e t r a n s f o r m a t i o n of ethylmorphine. Both o p t i c a l (Figure 16) and e l e c t r o n paramagnetic resonance (Figure 17) spectroscopy s t u d i e s (59) r e v e a l e d changes i n the o x i d a t i o n p r o p e r t i e s of microsomal cytochrome P-450 upon a d d i t i o n o f o r g a n i c hydroperoxides. T r a n s i e n t changes i n the o p t i c a l s p e c t r a were observed upon a d d i t i o n o f cumene hydroperoxide and these s p e c t r a l changes d i f f e r e d from those r e p o r t e d (44) f o r oxycytochrome P-450 observed d u r i n g the a e r o b i c steady s t a t e o x i d a t i o n o f NADPH by l i v e r microsomes. F u r t h e r , e l e c t r o n paramagnetic resonance s t u d i e s (Figure 17) r e v e a l e d the f o r m a t i o n of EPR s i g n a l s i n the a r e a o f g * 2.0 d i f f e r e n t from those d e s c r i b e d i n F i g u r e 12. Indeed, t h e unique nature o f the EPR s i g n a l s observed a t about g • 2.0 when cumene hydroperoxide r e a c t s w i t h microsomal cytochrome P-450 resemble t h e s i g n a l s observed when hydrogen peroxide r e a c t s w i t h metmyoglobin o r cytochrome c peroxidase (79, 80). From these r e s u l t s i t was concluded (59) t h a t cytochrome P-450 may o b t a i n h i g h e r v a l e n c e s t a t e s o f the heme i r o n i n a manner analogous t o t h a t proposed by Yamazaki e t a l (81) f o r p e r o x i dases. T h i s would suggest t h a t the e q u i v a l e n t o f aiTôxene" form of oxygen might be formed d u r i n g t h e a c t i v a t i o n o f oxygen by cytochrome P-450 as i t f u n c t i o n s i n h y d r o x y l a t i o n r e a c t i o n s . I t i s o f i n t e r e s t t o note t h a t the EPR s i g n a l observed when p e r o x i des i n t e r a c t w i t h l i v e r microsomal cytochrome P-450 i s v e r y s i m i l a r t o t h a t r e p o r t e d by Vanneste e t a l (82) f o r the complex o f oxygen w i t h reduced cytochrome P-450 o b t a i n e d i n r a p i d f r e e z e quenching experiments. Concluding Remarks. The experimental r e s u l t s d e s c r i b e d i n the preceeding s e c t i o n s have been presented t o p r o v i d e some background as t o the present s t a t u s of our understanding o f " a c t i v e

1.


R A ITI IUOH -C H -Q RM 2

2

3

too

!{

g g 4

.

A

2

2

u t i l i 2 e
,

3.3

nmoles

J HCH0

o ? g .. liberated

It

^ £ ^

(plus

I A '

xmin '

40-

J*

s

20

i i ,

ft X " 4

8

PB microsomes/ml,

Figure 15.

5mM

ETHYLMORPHINE) H 0 * 1 utilized I 65 2

2mg

19

* i|

b e r a t e d

0.4 °*

,

• • 8

Cytochrome

12 I mM

2

16 20 TIME (minutes) Azide :

A o a

aerob ARGON CARBON MONOXIDE

The hydrogen peroxide-dependent N-demethylation of ethylmorphine as catalyzed by liver microsomes. Liver microsomes from phenobarbital-treated rats were diluted to 2 mg protein per ml in a reaction mixture similar to that described in Figure 5. Where indicated, 5mM ethylmorphine was added. The reaction was initiated by adding lOOuM. hydrogen peroxide. Special reaction vessels were used to permit equilibration with various gas mixtures and to permit sampling the reaction at the times indicated. The changes in the concentrations of hydrogen peroxide used or formaldehyde formed were determined colorometrically.

20


Z

1

41


-^%Sh

442-500nm -0.005

^^^^^^

I

\vj W V422

-0.06-

400

579

^

-0.000

|j

—0.005

g

--OJOIO

8
iM P-450) 50>iM CumtntOOH

450

500

550

600

WAVELENGTH (not)

Figure 16.

Spectrophotometric measurement of changes occurring during the oxidation of cumene hydroperoxide by rat liver microsomes. Liver microsomes from phenobarbital-treated animals were diluted to a protein concentration of 3 mg per ml in a reaction mixture containing 50mM tris-chloride buffer, pH 7.5, 150mM KCl, and 5mM MgCl . After recording a baseline of equal light absorbance, 50fiM cumene hydroperoxide was added to the contents of the sample cuvette, and the change of absorbance with time was determined by repetitive scanning at 2 nm per sec. (Insert) The kinetics of formation and decay of the absorbance at 442 nm at different concentrations of cumene hydroperoxide. 2



1.

Cytochrome


21

Biochemical and Biophysical Research Communications

Figure 17. Changes in the EPR spectra associated with the interaction of cumene hydroperoxide and liver microsomes. Experiments were carried out using liver microsomes from phenobarbital-treated rabbits. (A) no additions; (B) after adding 1.5mM cumene hydroperoxide; (C) after adding 46mM cyclohexane followed by cumene hydroperoxide; (D) 50/AM metmyoglobin mixed with 1.5mM cumene hydroperoxide; (E) a baseline obtained in the absence of liver microsomes

HO

Oxonium ion

+.p..

H0 2

fOxene

> e

2 Oxygen w

—

H

+

H0

H 0

2

2

Perhydroxyl radical

H+

HO*

2

Hydrogen peroxide

pK *4.5

7*

pK *ll.8

0

0

+

H +H0 Superoxide ion

2

Hydroperoxide Hy ion

I +

H +0

2

Peroxide ion

Hydroxy I radical

^ H M+

2

0 Water

I PK 7 S

0

H++OH" Hydroxyl ion

Figure 18. The possible various states of oxygen during its stepwise reduction to water


22


CONCEPTS

oxygen" formed d u r i n g cytochrome P-450 c a t a l y z e d r e a c t i o n s . Oxygen may e x i s t i n a number o f o x i d a t i o n s t a t e s as i l l u s t r a t e d i n F i g u r e 18. As yet i t i s not p o s s i b l e t o a s s i g n a s p e c i f i c f u n c t i o n i n h y d r o x y l a t i o n r e a c t i o n s f o r the superoxide a n i o n , peroxide a n i o n , oxene o r t h e i r protonated forms. The a b i l i t y to observe the formation of hydrogen peroxide during NADPH o x i d a t i o n by l i v e r microsomes and to a s s i g n a r o l e f o r cytochrome P-450 i n the generation of hydrogen peroxide lends credence to the scheme p r o posed i n F i g u r e 4. F u r t h e r the experimental observations obtained during the p e r o x i d a t i c f u n c t i o n of cytochrome P-450 a l l p o i n t to the c e n t r a l r o l e f o r a peroxide anion complex of t h i s pigment p l a y i n g a c e n t r a l and p i v i t o l r o l e i n the a c t i v a t i o n of oxygen. New approaches and more experiments w i l l be r e q u i r e d to b e t t e r e s t a b l i s h the v a l i d i t y of the c u r r e n t hypotheses on the proposed intermediates formed during cytochrome P-450 f u n c t i o n . Oxygen i s c e n t r a l to the maintalliances of the l i f e of higher organisms as we now understand i t . In a d d i t i o n to r e a c t i o n s i n the c e l l where oxygen i s reduced to water concomitant w i t h the c o n s e r v a t i o n of energy i n the form of ATP, as c a t a l y z e d by the m i t o c h o n d r i a l r e s p i r a t o r y c h a i n , oxygen p l a y s a key r o l e i n the s y n t h e s i s and degradation of a wide d i v e r s i t y of n a t u r a l compounds, such as s t e r o i d s , as w e l l as f o r e i g n chemical agents. For these l a t t e r r e a c t i o n s cytochrome P-450 p l a y s a c r i t i c a l r o l e s e r v i n g t o a c t i v a t e oxygen f o r i n t e r a c t i o n w i t h these organic s u b s t r a t e s . Undoubtably the f u t u r e w i l l provide many new s u r p r i s e s as we delve deeper to g a i n a f u l l e r understanding of t h i s important enzyme system.

Literature Cited 1.

2.

3.

4. 5. 6. 7.

N i e r , A.O., Hanson, W.B., S e i f f , A., McElroy, M.F., Spencer, N.W., Duckett, R.J., Knight, T.C.D., and Cook, W.S., Science (1976) 193, 786-788. K l e i n , H.P., Horowitz, N.H., L e v i n , G.V., Oyama, V . I . , Lederberg, J., R i c h , A., Hubbard, J.S., Hobby, G.L., S t r a a t , P.A., Berdahl, B.J., C a r l e , G.C., Brown, F.C., and Johnson, R.D., Science (1976) 194, 99-105. George, P., in "Oxidases and R e l a t e d Redox Systems", e d i t e d by King, T.E., Mason, H.S., and M o r r i s o n , M., V o l . 1, pgs. 3-32, John Wiley and Sons, Inc., New York, 1965. Thurman, R.G. and Scholz, R., Eur. J . Biochem. (1969) 10, 459-467. Thurman, R.G. and Scholz, R., Eur. J . Biochem. (1973) 38, 73-78. S i e s , H. and Brauser, B., Eur. J . Biochem. (1970) 15, 531540. Brauser, B., S i e s , H., and Bucher, Th., FEBS L e t t e r s (1969) 2, 170-176.


1.


Cytochrome P-450 in Oxygen Activation

23

8. Masters, B.S.S., Baron, J., T a y l o r , W.E., Isaacson, E . I . , and L o S p a l l u t o , J., J. Biol. Chem. (1971) 246, 4143-4150. 9. S t r i t t m a t t e r , P., Spatz, L., Corcoran, D., Rogers, M.J., Setlow, B., and R e d l i n , R., Proc. Nat'l. Acad.Sci.(USA) (1974) 71, 4565-4569. 10. Holloway, P.W., Biochemistry (1971) 10, 1556-1560. 11. Oshino, N., Imai, Y., and Sato, R., J. Biochem. (Tokyo) (1971) 69, 155-168. 12. K e l l o g g , E.W. and Fridovich, I . , J. Biol. Chem. (1975) 250, 8812-8817. 13. Masters, B.S.S. and Schacter, B.A., Annals o f Clinical Research (1976), Vol. 8, s u p p l . 17, 18-27. 14. Z i e g l e r , D.M. and Mitchell C.H., A r c h i v e s Biochem. Biophys. (1972) 150, 116-125. 15. Kadlubar, F.F. and Ziegler, D.M., A r c h i v e s Biochem. Biophys. (1974) 162, 83-92. 16. Z i e g l e r , D.M., Hyslop, R.M., and Poulsen, L.L., HoppeS e y l e r ' s Z. Physiol. Chem. (1976) 357, 1067. 17. Coon, M.J. and Lu, A.Y.H., in "Microsomes and Drug O x i d a t i o n s " , e d i t e d by Gillette, J.R., Conney, A.H., Cosmides, G.J., Estabrook, R.W., F o u t s , J.R., and Mannering, G.J., pgs. 151166, Academic P r e s s , New York (1969). 18. van der Hoeven, T.A., and Coon, M.J., J. Biol. Chem. (1974) 249, 6302-6310. 19. Haugen, D.A., van der Hoeven, T.A., and Coon, M.J., J. Biol. Chem. (1975) 250, 3567-3570. 20. L u , A.Y.H. and L e v i n , W., Biochem. Biophys. Res. Comm. (1972) 46, 1334-1339. 21. L u , A.Y.H., L e v i n , W., and Kuntzman, R., Biochem. Biophys. Res. Comm. (1974) 60, 266-272. 22. Ryan, D., L u , A.Y.H., West, S., and L e v i n , W., J. Biol. Chem. 250, 2157-2163. 23. Remmer, H. and Merker, H.J., Annals o f the New York Acad. Sci. (1965) 123, 79-97. 24. Conney, A.H., Pharmacol. Rev. (1967) 19, 317-366. 25. Coon, M.J., V e r m i l i o n , J.L., Vatsis, K.P., French, J.S., Dean, W.L. and Haugen, D.A., T h i s volume. 26. B r o d i e , B.B., Science (1955) 121, 603. 27. Cooper, J.R. and B r o d i e , B.B., J. Pharmacol. exp. Ther. (1955) 114, 409-417. 28. Conney, A.H., Miller, E.C., and Miller, J.A., J. Biol. Chem. (1957) 228, 753-766. 29. Miller, E.C., Miller, J.A., Brown, R.R., and MacDonald, J.C., Cancer Res. (1958) 18, 469-477. 30. M u e l l e r , G.C. and Rumney, G., J. Amer. Chem. Soc. (1957) 79, 1004-1005. 31. Ryan, K.J. and Engel, L.L., J. Biol. Chem. (1957) 225, 103114. 32. Estabrook, R.W., Schenkman, J.B., Cammer, W., Remmer, H., Cooper, D.Y., Narasimhulu, S., and Rosenthal, O. in

24

33.

34. 35. 36.


37. 38.

39. 40. 41. 42. 43. 44.

45. 46. 47. 48. 49. 50. 51. 52.


CONCEPTS

"Biological and Chemical Aspects o f Oxygenases" e d i t e d by K. Bloch and O. H a y a i s h i , pgs. 153-170, Maruzen Co. Ltd., Tokyo (1966). Remmer, H., Schenkman, J. B., Estabrook, R.W., Sasame, H., Gillette, J., Narasimhulu, S., Cooper, D.Y., and R o s e n t h a l , 0., Molec. Pharmacol. (1966) 2, 187-190. Schenkman, J.B., Remmer, H., and Estabrook, R.W., Molec. Pharmacol. (1967) 3, 113-123. Cammer, W., Schenkman, J.B., and Estabrook, R.W., Biochem. Biophys. Res. Comm. (1968) 23, 264-268. Estabrook, R.W., Baron, J., Peterson, J. and Ishimura, Y. in "Biological Hydroxylation Mechanisms", e d i t e d by Boyd, G.S. and S m e l l i e , R.M.S., pgs. 159-186, Academic P r e s s , London (1972). Estabrook, R.W., M a r t i n e z - Z e d i l l o , G., Young S., Peterson, J.A., and McCarhty, J., J. S t e r o i d Biochem. (1975) 6, 419-425. Narasimhulu, S., in "Proceedings o f the T h i r d I n t e r n a t i o n a l Symposium on Microsomes and Drug O x i d a t i o n s " , e d i t e d by Ullrich, V., Roots, I., H i l d e b r a n d t , A.G., Estabrook, R.W., and Conney, A., Pergamon P r e s s , Oxford, in press (1977). B a l l o u , D.P., Veeger, C., van der Hoeven, T.A., and Coon, M.J., FEBS L e t t e r s (1974) 38, 337-340. Guengerich, F.P., Ballou, D.P., and Coon, M.J., J. Biol. Chem. (1975) 250, 7405-7414. Tyson, C.A., Lipscomb, J.D. and Gunsalus, I.C., J. Biol. Chem. (1972) 247, 5777-5784. P e t e r s o n , J.A., A r c h i v e s Biochem. Biophys. (1971) 144, 678693. Omura, T. and Sato, R., J. Biol. Chem. (1964) 239, 2370-2378. Estabrook, R.W., H i l d e b r a n d t , A.G., Baron, J., N e t t e r , K . J . , and Leibman, K., Biochem. Biophys. Res. Comm. (1971) 42, 132139. Ullrich, V. and Diehl, H., Eur. J. Biochem. (1971) 20, 509412. H i l d e b r a n d t , A.G., Speck, M., and Roots, I., Biochem. Biophys. Res. Commun. (1093) 54, 968-975. Cohen, B.S. and Estabrook, R.W., Arch. Biochem. Biophys. (1971) 143, 46-53. Cohen, B.S. and Estabrook, R.W., Arch. Biochem. Biophys. (1971) 143, 54-65. C o r r e i a , M.A. and Mannering, G., M o l . Pharmacol. (1973) 9, 455-469. C o r r e i a , M.A. and Mannering, G.J., M o l . Pharmacol. (1973) 9, 470-485. H i l d e b r a n d t , A. and Estabrook, R.W., A r c h i v e s Biochem. Biophys. (1971) 143, 66-79. Peterson, J.A., Ishimura, Y., Baron, J., and Estabrook, R.W., in "Oxidases and R e l a t e d Redox Systems" e d i t e d by K i n g , T.E., Mason, H.S., and M o r r i s o n , M., pgs. 565-581, U n i v e r s i t y Park P r e s s , B a l t i m o r e , Md. (1973).


1.


Cytochrome P-450 in Oxygen Activation

25

53. Ullrich, V. and Staudinger, Hj., in 'Microsomes and Drug O x i d a t i o n s " edited by Gillette, J.R., Conney, A.H., Cosmides, G.J., Estabrook, R.W., Fouts, J.R., and Mannering, G.J., pg. 199-217, Academic P r e s s , New York (1969). 54. Hrycay, E.G. and O'Brien, P.J., A r c h i v e s Biochem. Biophys. (1972) 153, 480-494. 55. Hrycay, E.G. and O'Brien, P.J., A r c h i v e s Biochem. Biophys. (1973), 157, 7-22. 56. Hrycay, E.G. and O'Brien, P.J., Archives Biochem. Biophys. (1974), 160, 230-245. 57. Kadlubar, F.F., Morton, K.C., and Ziegler, D.M., Biochem. Biophys. Res. Comm. (1973) 54, 1255-1261. 58. W e r r i n g l o e r , J. in "Proceedings o f the T h i r d I n t e r n a t i o n a l Symposium on Microsomes and Drug O x i d a t i o n s " , e d i t e d by Ullrich, V., Roots, I., H i l d e b r a n d t , A.G., Estabrook, R.W., and Conney, A., Pergamon P r e s s , Oxford, in press (1977). 59. Rahimtula, A.D., O'Brien, P.J., Hrycay, E.G., Peterson, J.A., and Estabrook, R.W., Biochem. Biophys. Res. Commun. (1974) 60, 695-702. 60. Thakker, D.R., Y a g i , H., L u , A.Y.H., L e v i n , W., Conney, A.H., and J e r i n a , D.M., Proc. N a t ' l . Acad. Sci. (USA), (1976) 73, 3381-3385. 61. H e i d e l b e r g e r , C., Ann. Rev. Biochemistry (1975) 44, 79-121. 62. P h i l p o t , R.M. and Hodgson, E., Mol. Pharm. (1972) 8, 204-214. 63. F r a n k l i n , M., X e n o b i o t i c a (1971) 1, 581-591. 64. Schenkman, J.B., Wilson, B.J., and Cinti, D.L., Biochem. Pharmacol. (1972) 21, 2373-2383. 65. W e r r i n g l o e r , J. and Estabrook, R.W., Life Sciences (1973) 13, 1319-1330. 66. Gillette, J.R., B r o d i e , B.B., and LaDu, B.N., J. Pharm. Exp. Therap. (1957) 119, 532-540. 67. Orme-Johnson, W.H. and Z i e g l e r , D.M., Biochem. Biophys. Res. Comm. (1965) 21, 78-85. 68. Oshino, N., Oshino, R., and Chance, B., Biochem. J. (1973) 131, 555-563. 69. I s s e l b a c h e r , K.J. and C a r t e r , E.A., Biochem. Biophys. Res. Comm. (1970) 39, 530-537. 70. W e r r i n g l o e r , J., Chacos, N., Estabrook, R.W., Roots, I., and H i l d e b r a n d t , A.G. in " A l c o h o l and Aldehyde M e t a b o l i z i n g Systems" e d i t e d by Thurman, R.G., W i l l i a m s o n , J.R., D r o t t , H. and Chance, B., Academic P r e s s , New York (1977) in p r e s s . 71. Estabrook, R.W. and W e r r i n g l o e r , J. in "Proceedings o f the T h i r d I n t e r n a t i o n a l Symposium on Microsomes and Drug O x i d a t i o n s " e d i t e d by Ullrich, V., Roots, I., H i l d e b r a n d t , A.G., Estabrook, R.W. and Conney, A., Pergamon P r e s s , Oxford, in press (1977). 72. W e r r i n g l o e r , J. and Estabrook, R.W., in p r e p a r a t i o n . 73. Estabrook, R.W., F r a n k l i n , M.R., and H i l d e b r a n d t , A.G., Annals New York Acad. Sci. (1970) 174, 218-232.

26 74. 75. 76. 77. 78. 79. 80.


81.

82. 83. 84.


CONCEPTS

Estabrook, R.W., Cooper, D.Y. and Rosenthal, O., Biochem. Zeit. (1963) 338, 741-755. Cooper, D.Y., L e v i n , S., Narasimhulu, S., Rosenthal, O. and Estabrook, R.W., Science (1965) 147, 400-402. W e r r i n g l o e r , J. and Estabrook, R.W., A r c h i v e s Biochem. Biophys. (1975) 167, 270-286. Knowles, P.F., Gibson, J.F., P i c k , F.M. and Bray, R.C. Biochem. J. (1969) 111, 53-58. I y a n a g i , T. and Mason, H.S., Biochemistry (1973) 12, 22972308. K i n g , N.K. and Winfield, M.P., J. Biol. Chem. (1963) 238, 1520-1528. Yonetani, T. and Schleyer, H., J. Biol. Chem. (1967) 242, 1974-1979. Yamazaki, I., Nakajima, R., M i y o s h i , K., Makino, R., and Tamura, M., in "Oxidases and R e l a t e d Redox Systems" e d i t e d by K i n g , T.E., Mason, H.S., and M o r r i s o n , M., Vol. 1, pg. 407-418, U n i v e r s i t y Park P r e s s , B a l t i m o r e , Md. (1973). Vanneste, M.Y., Vanneste, W.H., and Mason, H.S., Biochem. Biophys. A c t a (1972) 267, 268-274. H i l d e b r a n d t , A.G. and Roots, I., A r c h i v e s Biochem. Biophys. (1975) 171, 385-397. Nash, T., Biochem. J. (1953) 55, 416-421.

2 Synthetic Models for the Reaction Stages of Cytochrome P-450 JAMES P. COLLMAN and THOMAS N. SORRELL


Department of Chemistry, Stanford University, Stanford, CA 94305

Metalloenzymes often exhibit primary coordination spheres which have no counterparts among structurally-characterized, s y n t h e t i c complexes. The p h y s i c a l and c h e m i c a l p r o p e r t i e s o f t h e s e m e t a l c e n t e r s may a l s o be u n u s u a l and may not have a n a l o g i e s t o the corresponding p r o p e r t i e s of s y n t h e t i c coordination compounds. The relationship between protein s t r u c t u r e and t h e s e uncommon p h y s i c a l and c h e m i c a l properties is essential to understanding the f u n c t i o n s of metal p r o t e i n active sites. In this paper I will summarize t h e work o f our group in c h a r a c t e r i z i n g model complexes f o r t h e i r o n c e n t e r i n t h r e e o f the five r e c o g n i z e d r e a c t i o n s t a g e s o f the hemoprotein cytochrome P-450 f a m i l y (1, 2). T h i s work is f a r from complete so t h a t the p r e s e n t paper is a p r o g r e s s report. Many o t h e r l a b o r a t o r i e s have a l s o c o n t r i b u t e d t o m o d e l l i n g t h e s e P-450 s t a g e s , but t h e p r e s e n t a c c o u n t will not attempt t o r e v i e w the c o n t r i b u t i o n s o f o t h e r groups in any d e p t h . Consider a simplified reaction c y c l e f o r the soluble, bacterial camphor h y d r o x y l a s e , P-450 , from Pseudomonas p u t i d a (3). Using the h i g h l y purified soluble P-450 system, G u n s a l u s , et al. have been a b l e t o reassemble components o f t h e enzyme system i n vitro and t h u s t o o b s e r v e and t o c h a r a c t e r i z e f o u r s t a b l e i n t e r m e d i a t e s i n the P-450 c y c l e as w e l l as the i n a c t i v e f e r r o u s c a r b o n y l s t a g e (4). These s t u d i e s have r e s u l t e d i n the e l u c i d a t i o n o f t h e r e a c t i o n sequence shown i n F i g u r e 1. I t s h o u l d be emphasized t h a t t h e b a c t e r i a l P - 4 5 0 c y c l e shown i n F i g u r e 1 d i f f e r s i n some degree from t h a t o f t h e membrane bound m i c r o s o m a l c y c l e deduced by Coon (5^, 6). The e l e c t r o n t r a n s p o r t components w h i c h a r e d i f f e r e n t i n the P - 4 5 0 and P - 4 5 0 i systems a r e o m i t t e d h e r e as t h e s e a r e f a r beyond the p r e s e n t scope cam

cam

cam

c a m

c a m

m

27

28


of modelling* Because o f t h e i r p r i o r r e c o g n i t i o n , the stages i n t h e P - 4 5 0 c y c l e ( F i g u r e 1) s e r v e d a s t a r g e t s f o r t h e m o d e l l i n g s t u d i e s t o be d e s c r i b e d herein. F i g u r e 2 shows a s i m p l i f i e d v e r s i o n o f t h e r e a c t i o n c y c l e along with the d i s t i n c t i v e properties a s s o c i a t e d w i t h each stage which a r e necessary t o p r o v i d e f o r a s s e s s i n g t h e v a l u e o f any s y n t h e t i c model « It i s c l e a r that there are strong s i m i l a r i t i e s between r e c o g n i z e d r e a c t i o n s t a g e s o f t h e P " 4 5 0 and t h e P - 4 5 0 i r e a c t i o n c y c l e s . B e f o r e d i s c u s s i n g models f o r t h e s e s t a g e s o f cytochrome P-450, i t i s a p p r o p r i a t e t o r e v i e w t h e s t r u c t u r a l c h a r a c t e r i s t i c s o f low and h i g h s p i n i r o n porphyrins. These r e l a t i o n s h i p s which were f i r s t p r e d i c t e d by Hoard (7) a r e now w e l l e s t a b l i s h e d and have no e x c e p t i o n s among s t r u c t u r a l l y c h a r a c t e r i z e d synthetic porphyrins. The s i t u a t i o n i s summarized i n T a b l e I . F e r r o u s i r o n has s i x d e l e c t r o n s . I n t h e c a m

c a m


m

Table I Structural Characteristics of I r o n i n Hemoproteins Biological Example

Oxidation and S p i n S t a t e S=2

5

2.09

d

6

S=0

6

2.00

3 +

d

5

S=5/2

5

2.07

3 +

d

5

S=l/2

6

1.99

Mb0 ,Hb0 ,HbCO

Pe

2 +

P

Pe

3 +

^ ) Fe . . . Pe

2

P

450°2' B 450 ( A ,

2

P

s u

450

C O

s t r a t e

F

o

Fe-N A

6

2 +

5 Q

Expected

d

Pe

Mb, Hb, P ^

Coord. No.

e

Radius o f p o r p h y r i n c o r e

-2.01,

d i a m a g n e t i c (S=0) s t a t e , i r o n ( I I ) p o r p h y r i n s p o s s e s s two a x i a l l i g a n d s a f f o r d i n g o v e r a l l s i x - c o o r d i n a t i o n . Low s p i n i r o n ( I I ) has a c o v a l e n t r a d i u s w h i c h f i t s without s t r e s s i n t o the porphyrin core. Welle s t a b l i s h e d examples a r e d i a m a g n e t i c f e r r o u s c a r b o n y l d e r i v a t i v e s s i m i l a r t o s t a g e D i n F i g u r e 1. H i g h s p i n i r o n ( I I ) has f o u r u n p a i r e d e l e c t r o n s (S=2) and a c o v a l e n t r a d i u s t o o l a r g e t o be accommodated by t h e porphyrin core. Such h i g h s p i n f e r r o u s complexes have a s i n g l e a x i a l l i g a n d ( o v e r a l l 5 - c o o r d i n a t e ) and i r o n i s d i s p l a c e d out o f the plane o f the porphyrin r i n g a s , f o r example, i n deoxymyoglobin. The f e r r i c s t a t e

2.

C O L L M A N A N D SORRELL

Reaction

Stages of Cytochrome

P-450

29

with f i v e d electrons i s s t r u c t u r a l l y s i m i l a r to the f e r r o u s s t a t e . Low s p i n f e r r i c p o r p h y r i n s have one u n p a i r e d e l e c t r o n (S=%), two a x i a l l i g a n d s , and i n p l a n e i r o n . High s p i n f e r r i c p o r p h y r i n s have f i v e u n p a i r e d e l e c t r o n s , (S=5/2), a s i n g l e a x i a l l i g a n d , and i r o n o u t o f t h e p o r p h y r i n p l a n e . Low S p i n F e r r i c Stage A. C o n s i d e r t h e r e s t i n g s t a g e o f cytochrome P - 4 5 0 , s t a g e A, F i g u r e 2. The heme i r o n i s i n t h e l o w - s p i n f e r r i c s t a t e and must, t h e r e f o r e , have two a x i a l l i g a n d s . Mason was t h e f i r s t t o r e c o g n i z e t h a t t h e a x i a l l i g a t i o n i n P-450 i s u n c o n v e n t i o n a l and t o s u g g e s t s u l f u r c o o r d i n a t i o n (£,9.) • The e s r s p e c t r a o f t h i s low s p i n form have u n u s u a l rhombic g v a l u e s which a r e s i m i l a r t o t h e g v a l u e s a f f o r d e d by a d d i t i o n o f t h i o l s t o methemog l o b i n and m y o g l o b i n ( 1 0 r i i ) • On t h e b a s i s o f model complexes g e n e r a t e d i n situ by o u r group (12) and t h a t o f my c o l l e a g u e , Holm (13), i t seems c e r t a i n t h a t s t a g e A has one a x i a l t h i o l a t e l i g a n d and a n o t h e r u n s p e c i f i e d a x i a l ligand. The t h i o l a t e l i g a n d u n d o u b t e d l y r e s u l t s from the m e r c a p t i d e a n i o n o f c y s t e i n e . Solutions of t h i o l a t e complexes o f f e r r i c p o r p h y r i n s a r e i n t r i n s i c a l l y u n s t a b l e , s p o n t a n e o u s l y a f f o r d i n g d i s u l f i d e and f e r r o u s p o r p h y r i n s as shown i n e q . 1. The scope and


c a m

2RS-Pe

II3:

( P ) (B) + 2B

(P r e p r e s e n t s base)•

>

R-S-S-R + 2 B F e

1 1

(P) (B) (1)

any p o r p h y r i n a t o group and B any a x i a l

d e t a i l e d mechanism o f t h i s redox r e a c t i o n i s a t present unclear. The p r o t e i n and/or l i p i d membrane o f cytochrome P-450 must somehow s e r v e t o i n h i b i t t h i s redox p r o c e s s . S y n t h e t i c models o f f e r a c l u e as to the i n h i b i t i o n o f t h i s r e a c t i o n . We have been able t o prepare a s t a b l e , c r y s t a l l i n e f e r r i c t h i o l a t e complex, 1, as i l l u s t r a t e d i n e q . 2. The


30


CONCEPTS

complex Fe(TPP)(SCgHc)(HSCgHs) i s v e r y u n u s u a l i n t h a t i t i s the only i s o l a t e d low-spin t h i o l a t e f e r r i c p o r p h y r i n and t h e o n l y i s o l a t e d f e r r i c p o r p h y r i n h a v i n g two d i f f e r e n t a x i a l l i g a n d s t o our knowledge. In s o l u t i o n t h i s t h i o l a t e complex i s u n s t a b l e and undergoes a redox d i s p r o p o r t i o n a t i o n r e a c t i o n s i m i l a r t o t h a t shown i n eq. 1. However i n t h e c r y s t a l l i n e s t a t e , the f e r r i c t h i o l a t e complex 1 i s q u i t e s t a b l e . The p h y s i c a l p r o p e r t i e s o f 1 a r e v e r y u n u s u a l and o f f e r a p o s s i b l e analogy to~the f a c i l e s p i n e q u i l i b r i u m e x h i b i t e d by cytochrome P-450 systems which pass from low t o h i g h s p i n f e r r i c (stage A t o B) upon substrate binding. Single c r y s t a l s of 1 are pred o m i n a t e l y h i g h s p i n (]i =5.4 BM) a t 25°; however, as the temperature i s l o w e r e d , a low s p i n form i s o b s e r v e d which e v e n t u a l l y becomes dominant. This u n u s u a l temperature-dependent e q u i l i b r i u m i s q u i t e r e v e r s i b l e and does n o t a f f e c t t h e m o s a i c i t y o f s i n g l e crystals. We have been m o n i t o r i n g t h i s change w i t h a b a t t e r y o f p h y s i c a l t e c h n i q u e s w h i c h i n c l u d e X-ray d i f f r a c t i o n , Mossbauer s p e c t r o s c o p y , e s r , and magnetic measurements (14). These s t u d i e s a r e s t i l l i n p r o g r e s s but some p o i n t s a r e becoming c l e a r . The e s r o f t h e dominant h i g h s p i n form has g v a l u e s (-8.6, -3.4) comparable w i t h t h o s e o f t h e s u b s t r a t e - b o n d e d P-450 (stage B ) . A t -196° t h e e s r shows a dominant low s p i n form whose g v a l u e s (2.40, 2.25, 1.97) a r e s i m i l a r t o t h o s e o f the r e s t i n g form o f P-450 (stage A ) . X-ray d i f f r a c t i o n a t -160° shows t h e low s p i n form t o r e p r e s e n t -*70% o f t h e m o l e c u l e s . This r e s u l t i s c o n s i s t e n t w i t h p r e l i m i n a r y Mossbauer s t u d i e s w h i c h i n d i c a t e t h a t a s p i n e q u i l i b r i u m i s o c c u r r i n g between two s p i n s t a t e s o f a s i n g l e s p e c i e s . Two v e r y d i f f e r e n t i r o n - s u l f u r d i s t a n c e s a r e a p p a r e n t from t h e X - r a y d a t a a t 25°, c o n s i s t e n t w i t h t h e assumption t h a t one s u l f u r i s n o t c o o r d i n a t e d i n the h i g h s p i n form. Under vacuum J. l o s e s b e n z e n e t h i o l , p r o d u c i n g the h i g h s p i n complex 2, w h i c h has a s i n g l e a x i a l t h i o l a t e l i g a n d (eq. 3 ) • S m a l l gaseous l i g a n d s s u c h as ammonia


2.


Reaction


P-450

31

and methylamine w i l l p e n e t r a t e s o l i d samples o f 2 a f f o r d i n g l o w - s p i n complexes w i t h e s r g v a l u e s s i m i l a r t o t h o s e o f s t a g e A o f t h e P-450 c y c l e ( F i g u r e 3 ) . S o l u t i o n s o f 2 i n t h e p r e s e n c e o f a x i a l bases a r e u n s t a b l e w i t h r e s p e c t t o t h e redox r e a c t i o n (eq. 1 ) . However, when c o l d t o l u e n e s o l u t i o n s o f 2 a r e mixed w i t h v a r i o u s a x i a l bases and t h e n f r o z e n , t h e r e s u l t i n g g l a s s e s e x h i b i t e s r g v a l u e s t y p i c a l o f t h e low s p i n s t a g e A o f P-450. S i m i l a r s t u d i e s have been c a r r i e d o u t by Holm (13) who employed p r o t o p o r p h y r i n I X d i m e t h y l e s t e r , PPIXDME, r a t h e r t h a n t e t r a p h e n y l p o r p h y r i n , TPP. T a b l e I I e x h i b i t s r e p r e s e n t a t i v e g values f o r low-spin f e r r i c porphyrins having d i f f e r e n t combinations o f a x i a l l i g a t i o n along w i t h r e p r e s e n t a t i v e v a l u e s f o r cytochrome P-450 and cytochrome c . Comparison o f t h e s e v a l u e s c l e a r l y i n d i c a t e s t h a t one a x i a l t h i o l a t e l i g a n d i n combination w i t h v i r t u a l l y a l l o t h e r p o s s i b l e modes o f l i g a t i o n w i l l a f f o r d g v a l u e s s i m i l a r t o t h o s e found f o r t h e r e s t i n g s t a g e A o f P-450. However t h e g v a l u e s f o r o t h e r c o m b i n a t i o n s o f l i g a t i o n w i t h o u t an a x i a l m e r c a p t i d e do n o t c o r respond as c l o s e l y t o t h o s e o f P-450. On t h e b a s i s o f such i n s i t u s t u d i e s we c o n c l u d e t h a t one a x i a l l i g a n d i n s t a g e A o f P-450 i s v e r y p r o b a b l y t h e c y s t e i n e t h i o l a t e b u t t h e o t h e r a x i a l base cannot be d i s t i n g u i s h e d from t h e f o l l o w i n g p o s s i b i l i t i e s : oxygen (water, amide c a r b o n y l , s e r i n e h y d r o x y l ) , n i t r o g e n ( i m i d a z o l e o r l y s i n e amino), o r n e u t r a l s u l f u r (methionine t h i o e t h e r o r c y s t e i n e t h i o l ) . The r o l e o f s u l f u r as one l i g a n d i n s t a g e A i s a l s o c o n s i s t e n t w i t h P e i s a c h ' s r e c e n t s t u d i e s u s i n g e s r i n an e l e c t r i c f i e l d (15) and w i t h Holm's l i m i t e d s t u d y o f low temperature U V - v i s i b l e s p e c t r a o f model compounds generated i n s i t u (13). One experiment p r o v i d e s a p o s s i b l e e x p l a n a t i o n f o r t h e s t a b i l i z a t i o n o f t h e low s p i n f e r r i c - t h i o l a t e p o r p h y r i n by t h e p r o t e i n o r t h e l i p i d i n cytochrome P-450, s t a g e A. R e a c t i o n between c o l d t o l u e n e s o l u t i o n s o f t h e h i g h - s p i n complex 2 and p o l y s t y r e n e bonded i m i d a z o l e a f f o r d s a s o l i d l o w - s p i n p o l y m e r i c complex, F e T P P ( S C H ) ( N ^ N - p o l y s t y r e n e ) / 3, which i s s t a b l e f o r months a t 25°C i n t h e absence o f s o l v e n t (12). I t i s a p p a r e n t t h a t i m m o b i l i z a t i o n k i n e t i c a l l y s t a b i l i z e s such low s p i n t h i o l a t e complexes. In t h e same v e i n , a m e r c a p t o p r o p y l group c o v a l e n t l y a t t a c h e d t o s i l i c a g e l r e a c t s w i t h [FePjÔ i n t h e p r e s e n c e o f a base B ( B = p y r i d i n e o r N-Melm) t o give s t a b l e species with epr g values i d e n t i c a l t o t h o s e o f t h e c o r r e s p o n d i n g complexes g e n e r a t e d i n s i t u 6

5


32


® F e L O W SPIN

CONCEPTS

-(|)Fe HIGH SPIN

3 +

3+

ESR: g=2.45, 2.26,191

ESR:g=8,4,1.8

X max: 417,534,568 nm

Xmax:391,520,540,646r

©

Fe «(0 ) -

•©Fe

2+

2

MOSSBAUER A E INDEPENDENT OFT

2 +

A E INDEPENDENT OFT Q

Q

©Fe »(CO) 2+

Figure 2. Distinctive properties for the reaction stages of P-45Q wm

X max:=370,450,555 nm CHARACTERISTIC MCD SPECTRUM

i CH

3

Figure 3. Scheme for the preparation of models for the resting stage (A) of P-450

B = NH

3

CH NH 3

2

3

4

5

5

5

5

5

SC H

SC H

SC H

SC H

2.40

S, N

cytochrome C

3.06

2.40

2.25

2.25

2.26

1.24

1.93

1.91

1.95

( r e f . 13).

P=TPP; o t h e r p o r p h y r i n s g i v e s i m i l a r r e s u l t s

s-, ?

P-450 (TBM)

2.45

2.27

1.97

1.94

1.96

1.96

1.57

1.92

1.85

1.95

1.93

(b)

s", ?

p-450 (cam)

2.36

2.25

2.27

2.25

2.22

2.29

2.15

2.21

2.25

2.26

Most s p e c t r a were o b t a i n e d i n f r o z e n t o l u e n e g l a s s e s a t -196°

S~, S

THT

5

S~, S

6

HSC H

2.37

2.38 2.34

S~, 0

S~, N

camphor

2

2.90

S~, 0

N H

N, N

0~, N

2.43

2.61

N

0~,

3 THF

C H

N-Melm

N-Melm

N-Melm

2.37

2.39

^1

S~, N

S~, N

Donor Set

(a)

6

6

6

6

6

SC H

N-Melm

OCH

6

OC H N0

3

2

?

SC H N-Melm

N-Melm

5

SC H

6

B

X

Table I I EPR S p e c t r a f o r Low S p i n F e r r i c P o r p h y r i n Complexes, F e ( P ) ( X ) ( B ) * , b


34


a t low t e m p e r a t u r e ( F i g u r e 4 ) ( 1 6 ) . A t t e m p t s t o remove the base B under vacuum have been m a r g i n a l l y s u c c e s s f u l , l e a d i n g t o some d e c o m p o s i t i o n o f t h e p o r p h y r i n . F u r t h e r work aimed a t p r e p a r i n g t h e f i v e - c o o r d i n a t e a l k y l t h i o l a t e complexes i m m o b i l i z e d on s i l i c a i s s t i l l i n p r o g r e s s , as w e l l as p a r a l l e l s t u d i e s on t h i o methylpolystyrene, (p)— CH2SH (16). H i g h S p i n F e r r i c S t a g e B. Upon s u b s t r a t e b i n d i n g , the r e s t i n g l o w - s p i n form A o f P - 4 5 0 m i s c o n v e r t e d i n t o a h i g h - s p i n f e r r i c stage B (Figure 1). The u n u s u a l epr and e l e c t r o n i c s p e c t r a l c h a r a c t e r i s t i c s o f B have been r e p r o d u c e d i n model f e r r i c p o r p h y r i n s h a v i n g a s i n g l e a x i a l t h i o l a t e l i g a n d (12^,13,r7) . Examples o f such i s o l a t e d f e r r i c complexes a r e presented i n Table I I I . I t i s i n t e r e s t i n g to note t h a t Fe(OEP) and Fe(PPIXDME) form f i v e - c o o r d i n a t e t h i o l a t e complexes d i r e c t l y whereas w i t h F e ( T P P ) , t h e s i x c o o r d i n a t e s p e c i e s 1 i s o b t a i n e d which must be f u r t h e r t r e a t e d t o give"the d e s i r e d h i g h - s p i n adduct. The d i s s i m i l a r p h y s i c a l p r o p e r t i e s ( T a b l e I I I ) e x h i b i t e d by t h e s e complexes may r e f l e c t some c r y s t a l p a c k i n g f o r c e s which a r e a f u n c t i o n o f the mode o f preparation. Holm, e t a l . have c h a r a c t e r i z e d by X-ray d i f f r a c t i o n one such model complex, Fe(PPIXDME)(SC6H4NO2), 4 , and s t u d i e d t h i s compound w i t h a wide range o f s p e c t r o s c o p i c t e c h n i q u e s (13). He found t h a t d i s t i n c t i v e f e a t u r e s i n t h e u v - v i s i b l e , MCD, epr, and Mossbauer s p e c t r a o f 4 c l o s e l y p a r a l l e l t h e a n a l o g o u s s p e c t r a l f e a t u r e s o f cytochrome P - 4 5 0 s t a g e B whereas o t h e r a x i a l l i g a n d s gave l e s s s a t i s f a c t o r y correspondence. Our own more l i m i t e d s t u d i e s (12) show t h a t F e ( T P P ) ( S C H ) , 2 , has e p r £-values s i m i l a r t o t h o s e o f s t a g e B, a l t h o u g h our complex i s l e s s w e l l d e f i n e d due t o i t s r e l a t i v e instability. In f a c t , even a t low t e m p e r a t u r e , F e ( T P P ) ( S C H ) undergoes a slow d e c o m p o s i t i o n t o g i v e a s p e c i e s w i t h e p r £-values o f 6 . 0 and 2 . 0 . Thus, the a b s o r b a n c e s o f £ = 8 . 6 and £ = 3 . 4 appear as s h o u l d e r s on t h e £ = 6 peak and can o n l y be approximated; i n r e a l i t y , they a r e p r o b a b l y c l o s e r t o 8 and 4 . Another consequence o f t h e i n s t a b i l i t y o f 2 i s t h e appearance of a broad a b s o r p t i o n a t £ = 2 which obscures the h i g h f i e l d r e g i o n and t h e e x p e c t e d t h i r d r e s o n a n c e a t £ = 1 . 8 . T a b l e IV shows t h a t no o t h e r t y p e o f e x p e r i m e n t a l l y a c c e s s i b l e a x i a l l i g a t i o n w i l l produce s i m i l a r epr spectral features. O g o s h i has p r e p a r e d a model f o r s t a g e B i n which t h e a x i a l l i g a n d i s an a l k y l t h i o l a t e (1J7) (Table I I I )


c a

c a m

6

6

5

5

2.


Reaction


P-450

35

Table I I I Models f o r S u b s t r a t e Bonded H i g h S p i n F e r r i c P-450 Ref.

9 Values

y (B.M.)

Complex

12

5.8

~8.6 , -3.4,

FePPIXDME ( S C ^ C l )

5.9

7.2, 4.8, 1.8

13

Fe(OEP)(SC H ) 5

5.9

7.2, 4.7, 1.9

13

Fe(OEP)(t-BuS)

5.9

6.4, 4.4, 2.0

17

P-450

5.2

8.0, 4.0, 1.8

3

6

5

g

•substrate

—

a

FeTPP(SC H )


cam (a)

See t e x t f o r e x p l a n a t i o n .

T a b l e IV EPR Data f o r H i g h S p i n F e r r i c Complexes

Porphyrin

Ref.

Donor

Complex

—

12

4.8

1.8

13

6.4

4.4

2.0

17

o"

6.6

5.3

1.9

16

FePPIXDME(OC H N0 )

o"

5.9

5.9

2.0

13

FePPIXDME(0 CCH )

0"

5.9

5.9

2.0

13

Met-Hb

N

6.0

6.0

2.0

18

P-450 (TBM)

s"

8.3

3.3

—

19

P-450 (cam)

s"

8.0

4.0

1.8

s"

-8.6

-3.4

FePPIXDME(SC H C1)

s"

7.2

FeOEP(S-t-Bu)

s"

FeTPP(OCH )

FeTPP(SC.H ) c

D

0

6

4

3

6

2

4

2

3

3

36


i n c o n t r a s t w i t h t h e a r y l t h i o l a t e s employed by Holm, e t a l * and our own group. Such a l k y l t h i o l a t e f e r r i c porphyrins are f a r l e s s s t a b l e than the a r y l analogues with regard to the s e l f - d e g r a d a t i v e redox r e a c t i o n s u c h as t h a t shown i n eq. 1 , which l e d us t o our a t t e m p t s t o i m m o b i l i z e such complexes on s i l i c a . The P 2 p a r a m e t e r s r e p o r t e d by O g o s h i a r e n o t as s i m i l a r t o t h o s e o f s t a g e B as t h o s e o f t h e a r y l t h i o l a t e complexes; however, O g o s h i s £ v a l u e s a r e somewhat s u s p e c t due t o t h e u n u s u a l s p l i t t i n g o f t h e low f i e l d resonance. I n v i e w o f t h i s d i s c r e p a n c y i t seems d e s i r a b l e t o examine a d d i t i o n a l models f o r s t a g e B using a l k y l t h i o l a t e s . e

r


v

Stage C. To d a t e , w e l l d e f i n e d f i v e - c o o r d i n a t e h i g h - s p i n f e r r o u s p o r p h y r i n s h a v i n g an a x i a l t h i o l a t e o r an a x i a l t h i o l l i g a n d have n o t been i s o l a t e d o r even c h a r a c t e r i z e d i n s i t u so t h a t comparison cannot be made w i t h t h e u n i q u e p h y s i c a l p r o p e r t i e s o f t h e i r o n c e n t e r i n cytochrome P - 4 5 0 stage C (Figure 2 ) . Recent Mossbauer s t u d i e s o f s t a g e C i n t h e p r e s e n c e o f s t r o n g a p p l i e d m a g n e t i c f i e l d s have shown d i s t i n c t i v e parameters w h i c h have so f a r n o t been r e p r o d u c e d by model s t u d i e s and a r e d i f f e r e n t from t h o s e p r o p e r t i e s o f deoxymyoglobin i n w h i c h i m i d a z o l e i s t h e a x i a l l i g a n d (2J)) . c a m

S t a g e D. The f e r r o u s c a r b o n y l form ( s t a g e D) o f cytochrome P - 4 5 0 i s c h a r a c t e r i z e d by an u n u s u a l S o r e t a t about 450 nm w h i c h i s r e d s h i f t e d from t h e S o r e t o f "normal" f e r r o u s c a r b o n y l hemes w h i c h a p p e a r s around 420 nm. The u n u s u a l 450 nm a b s o r p t i o n has been i n v a l u a b l e as an a n a l y t i c a l a i d f o r d e t e r m i n i n g t h e p r e s e n c e o f t h i s cytochrome. Small d i f f e r e n c e s i n the p o s i t i o n o f t h i s band (from 446 t o 453 nm) have been o b s e r v e d i n t h e s p e c t r a o f d i f f e r e n t members o f t h e cytochrome P - 4 5 0 f a m i l y . Hanson, e t a l . (21) have proposed a t h e o r e t i c a l e x p l a n a t i o n f o r t h i s unusual S o r e t on t h e b a s i s o f t h e o r e t i c a l c a l c u l a t i o n s and the experimental o b s e r v a t i o n of another o p t i c a l t r a n s i t i o n a t 363 nm h a v i n g t h e same p o l a r i z a t i o n as t h e 446 peak i n t h e b a c t e r i a l cytochrome P " 4 5 0 . These "hyperbands" can be e x p l a i n e d by an i n t e r a c t i o n between a p — e (TT*) ( p o r p h y r i n ) and t h e normal porphyrin a ( i r ) , a2 (ir) — e (ir*) S o r e t t r a n s i t i o n r e s u l t i n g i n t h e o b s e r v e d p a i r o f bands. Such t r a n s i t i o n s c o u l d be c a u s e d by b i n d i n g o f an a x i a l thiolate ligand. S t e r n and P e i s a c h f i r s t r e p o r t e d t h a t t h e unu s u a l 450 nm S o r e t c o u l d be g e n e r a t e d i n s i t u i n an c a m

g

l u

U

g

2.


Reaction


37

P-450

Fe(PPIX) t h i o l / C O DMSO-EtOH s o l u t i o n under v e r y s t r o n g l y b a s i c c o n d i t i o n s (22) . I n t e r p r e t a t i o n o f t h e i r d a t a i s c o m p l i c a t e d by t h e l a r g e e x c e s s o f t h i o l and base r e q u i r e d , t h e p o t e n t i a l f o r c o o r d i n a t i o n o f the DMSO and e t h a n o l s o l v e n t , and t h e p r e s e n c e o f a n o t h e r "normal" S o r e t , as w e l l as t e m p o r a l c h a r a c t e r i s t i c s o f t h e spectrum. Under c o n d i t i o n s o f c o n t r o l l e d s t o i c h i o m e t r y and s o l v e n t environment we have been a b l e t o q u a n t i t a t i v e l y r e p r o d u c e t h e f u l l e l e c t r o n i c and magnetic c i r c u l a r d i c h r o i s m s p e c t r a o f s t a g e D i n t h e P-450 sequence. T h i s was a c c o m p l i s h e d by combining e q u i v a l e n t amounts o f t h e sodium crown-ether m e t h y l m e r c a p t i d e s a l t w i t h i r o n ( I I ) p o r p h y r i n s and CO i n anhydrous benzene as shown i n F i g u r e 5 (23). This f e r r o u s c a r b o n y l complex i s e x t r e m e l y s e n s i t i v e towards oxygen. The i n f r a r e d vCO band a t 1945 cm" is c l o s e t o t h e 1938 cm"l v a l u e r e p o r t e d by Caughey, e t a l . f o r t h e s u b s t r a t e bonded s t a g e D, P-450 m (24). However t h i s s i m i l a r i t y may n o t be m e a n i n g f u l i n t h a t s e v e r a l f a c t o r s such as l o c a l p o l a r i t y and t i l t i n g o f t h e CO group from t h e a x i s normal t o the p o r p h y r i n can influence vco values (25). The e l e c t r o n i c and MCD s p e c t r a (26) o f our mercaptide f e r r o u s carbonyl porphyrins generated i n s i t u are d i s p l a y e d along with those of h i g h l y p u r i f i e d P - 4 5 0 i i n F i g u r e s 6 and 7. The q u a n t i t a t i v e s i m i l a r i t y between the PPIXDEE d e r i v a t i v e and t h e n a t u r a l system i s s t r i k i n g . The s p e c t r a l band a t 370 nm, f i r s t d e s c r i b e d and e x p l a i n e d by Hanson, e t a l . (21) i s c l e a r l y e v i d e n t i n each. These s p e c t r a l comparisons p r o v i d e s t r o n g e v i d e n c e t h a t an a x i a l m e r c a p t i d e l i g a n d i s p r e s e n t i n s t a g e D o f cytochrome P-450. I t i s noteworthy t h a t no o t h e r l i g a n d (RO", PhO~, RC05, RSH, i m i d a z o l e , RCONH2, ROH, o r RNH2) i s c a p a b l e o f p r o d u c i n g t h i s u n u s u a l chromophore. We a l s o d i s c o v e r e d t h a t t h e 449 nm S o r e t c o u l d be s h i f t e d t o l o n g e r wavelengths by i n c r e a s i n g t h e p o l a r i t y o f the s o l v e n t . T h i s o b s e r v a t i o n provides a p o s s i b l e e x p l a n a t i o n f o r the v a r i a t i o n i n the Soret of stage D o b s e r v e d f o r d i f f e r e n t members o f t h e P-450 f a m i l y . Subsequently, Dolphin, e t a l reported s i m i l a r o p t i c a l s p e c t r a f o r models o f s t a g e D g e n e r a t e d i n s i t u (27). We a r e c u r r e n t l y a t t e m p t i n g t o p r e p a r e a c r y s t a l l i n e model f o r s t a g e D. V a r i o u s r e a g e n t s d e n a t u r e cytochrome P-450 y i e l d i n g a p r o t e i n whose f e r r o u s c a r b o n y l e x h i b i t s a S o r e t band a t 420 nm. We have r e p r o d u c e d t h e MCD spectrum o f t h i s s o - c a l l e d P-420 by combining F e ( I I ) P P I X D E E , CO, and e i t h e r N-methyl i m i d a z o l e o r an a l k y l mercaptan (26). These s p e c t r a a r e shown~Tn c a m


1

ca

m

M

38


CONCEPTS

-3 Et OH (Si0 ) + (Et 0) - Si CH CH CH SH 2

x

3

2

2

2

(Fe PPIXDME) 0 2

(Si0 ) -SiCH CH CH SH 2

x

2

2

2

(Si0 ) - SiCH CH CH S-0e - B Figure 4.

x

2

2

2

:

B= PYRIDINE, g=2.34, 2.25,1.96 N-Melm, g=2.38, 2.26,1.95

Scheme for immobilization

of low spin ferric porphyrin thiolate complexes on silica gel [(SiO»)z]


2

E

P

R

B

=

V CH S-SCH 2

3

I) Na/NH

3

(Na )SCH +

3

2) CROWN ETHER IN BENZENE

Fe (PPIXDEE) CO INC H 31

6

A max = 375,449,555nm Figure 5. Scheme for the prepa- »co = 1945 cm" ration of a model for the carbonyl (P i/Co i938cm ) stage (D) of P-450

6

1

H

450

s

(g)

e :

\H

3

3

2. C O L L M A N A N D S O R R E L L

Reaction


P-450

39

F i g u r e 8. T h i s r e s u l t i n d i c a t e s that the nature o f the a x i a l l i g a n d i n P-420 cannot be a s s i g n e d a t p r e s e n t and r a i s e s t h e p o s s i b i l i t y t h a t P-420 c o u l d r e p r e s e n t a v a r i e t y o f d i f f e r e n t modes o f a x i a l l i g a t i o n . However t h e s e r e s u l t s demonstrate t h a t s t a g e D o f P-450 does n o t have an a x i a l t h i o l l i g a n d . Oxygenated Stage E. The n a t u r e o f t h e o t h e r a x i a l l i g a n d i n t h e cytochrome P - 4 5 0 oxygen complex (stage E F i g u r e 1) i s u n c e r t a i n . On t h e b a s i s o f t h e above mentioned m o d e l l i n g s t u d i e s o f s t a g e s A, B, and D i t would be r e a s o n a b l e t o assume t h a t t h e a x i a l l i g a n d t r a n s t o d i o x y g e n i s m e r c a p t i d e . However, D o l p h i n has r e c e n t l y d e s c r i b e d low temperature s p e c t r a o f t h e c o m b i n a t i o n o f r e d u c e d i r o n p o r p h y r i n s , oxygen, and e x c e s s m e r c a p t i d e i n d i m e t h y l a c e t a m i d e c o n t a i n i n g 5% water (enough t o a f f o r d e q u i v a l e n t c o n c e n t r a t i o n s o f t h i o l ) (23). These s p e c t r a e x h i b i t a S o r e t a t 476 nm, whereas oxygenated P " 4 5 0 ( s t a g e E) i s r e p o r t e d t o have a normal S o r e t a t 418 nm. Dolphin r e p o r t s a 1:1 O2 t o Fe s t o i c h i o m e t r y from gas a b s o r p t i o n measurements on t h e d i l u t e ( 1 0 ~ * M ) , c o l d (-45°) s o l u t i o n s . The assignment o f t h i s spectrum t o t h a t o f an oxygen complex i s c r i t i c a l l y dependent on t h i s s t o i c h i o m e t r y i n view o f t h e f a c i l e o x i d a t i o n o f i r o n ( I I ) and m e r c a p t i d e and t h e redox r e a c t i o n o f t h e f e r r i c t h i o l a t e c o m b i n a t i o n shown i n eq. 1. D o l p h i n ' s r e s u l t s i n d i c a t e t h a t an a x i a l base d i f f e r e n t from m e r c a p t i d e must be p r e s e n t i n t h e oxygenated s t a g e E o f t h e P-450 c y c l e . The oxygenated s t a g e E o f P-450 e x h i b i t s a MSssbauer spectrum d i f f e r e n t from t h a t o f oxyhemoglobin (3). An e s p e c i a l l y d i s t i n c t i v e f e a t u r e i n t h e Mossbauer spectrum o f s t a g e E i s t h e l a c k o f tempera t u r e dependence o f t h e q u a d r u p o l e s p l i t t i n g p a r a meter which i s q u i t e temperature dependent i n oxyhemoglobin. We have i s o l a t e d c r y s t a l l i n e d i o x y g e n i r o n p o r p h y r i n complexes h a v i n g t h r e e d i f f e r e n t modes o f axial ligation: i m i d a z o l e n i t r o g e n (29), t e t r a h y d r o f u r a n oxygen (30), and t e t r a h y d r o t h i o p h e n e s u l f u r (31). The Mossbauer spectrum o f each o f t h e s e oxygenated complexes d i f f e r s from t h e spectrum o f P-450 s t a g e E — e s p e c i a l l y w i t h r e s p e c t t o the temperature dependence o f t h e q u a d r u p o l e s p l i t t i n g . However, t h e a x i a l l i g a n d may have l i t t l e e f f e c t on t h i s s p l i t t i n g parameter (32) i n which c a s e no good probe w i l l be a v a i l a b l e t o d e t e r m i n e t h e n a t u r e o f t h e a x i a l base i n s t a g e E. The t h i o e t h e r d i o x y g e n " p i c k e t f e n c e " p o r p h y r i n c a m


f

c a m



40

*7

Figure 8. MCD spectra for P'420 carbonyl (—), FePPlXDEE + N-Melm + CO in benzene ( ), and FePPlXDEE + C H SH + CO in benzene (—)

CONCEPTS

15H

LM

S

7

350

400

450

500

550

WAVELENGTH (nm)

600

650

700


2.

C O L L M A N A N D SOBRELL

Reaction


P-450

41

i s r e l e v a n t t o t h e P-450 models because t h i o e t h e r i s e x p e c t e d t o have a l i g a n d f i e l d c h a r a c t e r s i m i l a r t o t h a t o f t h i o l (33). The r e s u l t s o f an X-ray d i f f r a c t i o n study o f t h i s complex a r e i l l u s t r a t e d i n F i g u r e 9 (31). The s t r u c t u r a l f e a t u r e s o f t h i s t e t r a h y d r o t h i o p h e n e complex a r e s i m i l a r t o t h o s e o f our e a r l i e r N-methylimidazole " p i c k e t fence" dioxygen complex e x c e p t t h a t t h e t e r m i n a l oxygen atom does n o t appear t o be d i s o r d e r e d i n t h e former and i s f o u r way d i s o r d e r e d i n t h e l a t t e r . The poor q u a l i t y o f o u r c r y s t a l s and t h e r e l a t i v e l y s m a l l number o f r e f l e c t i o n s do n o t p e r m i t a s u f f i c i e n t l y a c c u r a t e d e t e r m i n a t i o n o f i n t e r e s t i n g bond d i s t a n c e s t o make r e a l i s t i c comparisons. We have a l s o p r e p a r e d an oxygen complex from a new t y p e o f p o r p h y r i n h a v i n g t h r e e " p i c k e t s " and a b u i l t - i n a x i a l t h i o e t h e r base. The s y n t h e t i c scheme i s shown i n F i g u r e 10 (3_1). O x y g e n a t i o n o f c o l d t o l u e n e s o l u t i o n s o f t h i s complex l e a d s t o slow d e c o m p o s i t i o n b u t t h e p r e s e n c e o f t h e oxygen complex has been demonstrated by Mossbauer s t u d i e s on f r o z e n solutions. However, we have n o t y e t i s o l a t e d and c h a r a c t e r i z e d a s t a b l e oxygen complex from t h i s system, a l t h o u g h a c r y s t a l l i n e c a r b o n y l adduct has been o b t a i n e d . T h i s system s h o u l d l e n d i t s e l f t o t h e s y n t h e s i s and f u l l c h a r a c t e r i z a t i o n o f model f e r r o u s complexes h a v i n g a x i a l t h i o l and t h i o l a t e l i g a n d s (3£) . E x p e r i m e n t s d i r e c t e d towards t h i s end a r e i n p r o g r e s s . An i m i d a z o l e d e r i v a t i v e o f t h i s " t h r e e p i c k e t " system undergoes r e v e r s i b l e o x y g e n a t i o n and we have r e c e n t l y i s o l a t e d an oxygen complex which i s i n t h e process of being f u l l y c h a r a c t e r i z e d (35). Summary Model complexes have been g e n e r a t e d which a c c u r a t e l y r e f l e c t s p e c t r o s c o p i c features of stages A, B, and D o f cytochrome P - 4 5 0 , a l t h o u g h a c r y s t a l l i n e model f o r D has n o t y e t been i s o l a t e d . The a x i a l l i g a n d common t o s t a g e s A, B. and D i s mercaptide. However t h e p r e s e n c e o f an a x i a l merc a p t i d e l i g a n d i n s t a g e s C and E i s u n c e r t a i n a t present. The r e a s o n b e h i n d N a t u r e ' s c h o i c e o f t h i s u n u s u a l l i g a t i o n i s u n c l e a r . One p o s s i b i l i t y has t o do w i t h s t a b i l i z i n g t h e a c t i v e P-450 o x i d a n t w h i c h must be two o x i d a t i o n l e v e l s above t h e f e r r i c s t a g e . In t h i s s t a g e perhaps one e l e c t r o n i s removed from i r o n a f f o r d i n g FeIV=0 and t h e o t h e r from m e r c a p t i d e g e n e r a t i n g a t h i o l r a d i c a l as suggested by Marchon (36). I t i s c l e a r t h a t more work must be done i n c a m


42


Figure 9. Crystal structure of Fe(TpivPP) (THT)O g

(CH3)3CC0CI

I

R

V"}

heat NH, CH 3 S(CH 2 ) 5 -COCI

Figure 10. Scheme for preparing "picket fence" porphyrins having an appended axial thioether ligand. X=NH and R=(CH ),CCONH-. t

t

» 1

I

I

NN
cytochrome P-450c. TCDD, B-naphthaf l a v o n e , and 3-methylcholanthrene are r e l a t e d inducers o f c y t o chrome P-450. These t h r e e cytochrome P-450 p r e p a r a t i o n s react s t r o n g l y w i t h antibody prepared a g a i n s t cytochrome P-450 LM^ o r P-450c. They a l s o e x h i b i t i d e n t i c a l m o b i l i t i e s i n 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 experiments. These experiments were performed i n Dr. M.J. Coon's and our l a b o r a t o r i e s . The work we have d e s c r i b e d i n d i c a t e s that benzphetamine N-demethylase, benzo(a)pyrene 3-hydroxylase, and a c e t a n i l i d e hydroxylase are each a s s o c i a t e d w i t h d i s t i n c t forms o f cytochrome P-450. The demonstration o f s u b s t r a t e s p e c i f i c i t i e s f o r m u l t i p l e forms o f cytochrome P-450 i s an important aspect o f t h i s i n v e s t i g a t i o n and i n d i c a t e s each form may f u n c t i o n i n d i f f e r e n t metab o l i c pathways. In a d d i t i o n , i t may be p o s s i b l e t o use these o r o t h e r assays t o detect i n d i v i d u a l cytochromes i n t i s s u e preparations. a

n

d

o

u

r

80

DRUG M E T A B O L I S M C O N C E P T S

This research was supported by National Institutes of Health Grants HD-04445 and CA-17735 (Dr. U. Muller-Eberhard) and by a California Division, American Cancer Society Junior Fellowship Number J-301 (Dr. E . F . Johnson). We would like to thank Dr. Betty Sue Siler Masters for supplying a preprint of her work, and Dr. Russell Prough for providing samples of 7-ethoxyresorufIn and resoruftn. We would also like to express our appreciation to K. Cox, G. Schwab, and M. Zounes for their help in this investi gation. Literature Cited: 1.


2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

G i l l e t t e , J . R . , Davis, D . C . , and Sasame, H . A . , Ann. Rev. Pharm. (1972) 12, 57-84. Nebert, D.W., Robinson, J . R . , Niwa, Α . , Kumaki, Κ., and Poland, A . P . , J. C e l l . Physiol. (1975) 85, 393-414. Alvares, A . P . , S c h i l l i n g , G . , and Levin, W., J. Pharm. Exp. Therap. (1970) 175, 4-11. Nebert, D.W., Robinson, J . R . , and Kon, H., J. B i o l . Chem. (1973) 248, 7637-7647. Kawalek, J . C . and Lu, A . Y . H . , Mol. Pharmacol. (1975) 11, 201-210. Atlas, S . A . , Thorgeirsson, S . S . , Boobis, A . R . , Kumaki, Κ., and Nebert, D.W., Blochem. Pharmacol. (1975) 24, 2111-2116. Prough, R., personal communication. van der Hoeven, T.A. and Coon, M . J . , J. B i o l . Chem. (1974) 249, 6302-6310. Johnson, E . F . and Muller-Eberhard, U., in preparation. Weir, D.M., "Handbook of Experimental Immunology", F.A. Davis C o . , Philadelphia (1967). van der Hoeven, T . A . , Haugen, D . A . , and Coon, M . J . , Blochem. Biophys. Res. Commun. (1974) 60, 569-575. Yasukochi, Y. and Masters, B . S . S . , J. B i o l . Chem. (1976) 251, 5337-5344. Ryan, D . , Lu, A . Y . H . , Kawalek, J., West, S . B . , and Levin, W., Biochem. Biophys. Res. Commun. (1975) 64, 1134-1141. Huang, M . I . , West, S . B . , and Lu, A . Y . H . , J. B i o l . Chem. (1976) 251, 4659-4665. Philpot, R.M. and Arinc, E., Mol. Pharmacol.(1976) 12, 483-493. Haugen, D . A . , van der Hoeven, T . A . , and Coon, M . J . , J. B i o l . Chem. (1975) 250, 3567-3570. Kawalek, J.C., Levin, W., Ryan, D . , Thomas, P . E . , and Lu, A . Y . H . , Mol. Pharmacol. (1975) 11, 874-878.

5 Enantiomeric Selectivity and Perturbation of Product Ratios as Methods for Studying the Multiplicity of Microsomal Enzymes WILLIAM F. TRAGER


Department of Pharmaceutical Sciences, School of Pharmacy, University of Washington, Seattle, WA 98195 I t i s n o w well established that t h e process o f t h e biotransformation o f ingested exogenous substances m a y form reactive electrophilic intermediates which in turn may be responsible for various kinds of potentially lethal toxicities including carcinogenesis. Covalent binding of such intermediates to nucleophilic sites in critical catalytic and structural proteins or nucleic acids is being recognized as one of the major molecular mechanisms for the manifestation of such toxicities. As a consequence, intensified investigations have been initiated t o delineate a n d categorize the multiple forms of the enzymes present in the endoplasmic reticulum of mammalian tissues since these enzymes are largely responsible for such reactions. Intimate knowledge of the specific, catalytic and structural spectrum of the system would at the very least alert society to the real scope of the problem and would undoubtedly suggest methods by which potential toxic hazards could be recognized and therefore be either circumvented or corrected. The suggestion that more than one enzyme system was responsible f o r t h e oxidations o f drugs w a s m a d e almost t w o decades ago (1,2) and was based on the differential induction of drug metabolism by phenobarbital and polycyclic aromatic hydrocarbons. Subsequent studies directed towards the elucidation of t h e multiplicity o f t h e microsomal cytochrome P - 4 5 0 e n z y m e s have segregated along two main lines of investigation, direct and indirect. Direct studies a r e t h e m o s t recent a n d a r e largely biochemical in nature. Investigators employing such studies have focused on the isolation, purification, characterization and reconstitution of both the normal enzymes and various inducible forms from several different species. Levin and Lu et al.(3-11) utilizing chromatographic e l e c t r o p h o r e t i c and immunologic techniques have succeeded i n s e p a r a t i n g and p u r i f y i n g t h e m i c r o somal cytochrome P-450 s from both r a t and r a b b i t l i v e r a f t e r i n d u c t i o n w i t h e i t h e r phénobarbital (PB) o r 3-methylcholanthrene (3-MC). S i m i l a r l y Coon e t a l . (12-15) have i s o l a t e d and charact e r i z e d m u l t i p l e forms o f cytochrome P-450 as haveAust e£aj.. (16,17). 1

81


82


I n d i r e c t s t u d i e s are much more numerous and have a r r i v e d a t the c o n c l u s i o n of the m u l t i p l e nature of the cytochrome P-450's present i n crude microsomal p r e p a r a t i o n s by i n f e r e n c e . G e n e r a l l y d i f f e r e n c e s produced i n the c a t a l y t i c p r o f i l e s of the system produced by some p e r t u r b a t i o n are i n t e r p r e t e d as evidence f o r m u l t i p l i c i t y . T y p i c a l l y changes produced by e i t h e r i n d u c i n g agents (1,2,19-27) o r i n h i b i t o r s (28,29) are s t u d i e d and o f t e n c o n c l u s i o n s regarding m u l t i p l i c i t y are b u r i e d w i t h i n the manus c r i p t s i n c e the o r i g i n a l i n t e n t of the study was focused on some other q u e s t i o n (27,30,31). Two recent examples from the l i t e r a t u r e i l l u s t r a t e the b a s i s of the i n d i r e c t approach. Selander, J e r i n a and Daly (32) s t u d i e d changes i n the r a t i o s of the £, m and jg-chlorophenol m e t a b o l i t e s o f chlorobenzene produced by the hemoprotein-monoxygenase system a t v a r i o u s stages o f r e s o l u t i o n . I n a d d i t i o n they s t u d i e d the d i f f e r e n t i a l e f f e c t s produced by p e r t u r b a t i o n s such as the i n d u c i n g agents PB and 3-MC, the i n h i b i t o r s , carbon monoxide, metyrapone, g l u t a t h i o n e , SKF-525a and 7,8-benzoflavone and changes i n the dependence of product r a t e on s u b s t r a t e c o n c e n t r a t i o n . These d i f f e r e n t i a l e f f e c t s allowed the authors to conclude t h a t a t l e a s t three cytochrome P-450 s d i f f e r i n g i n r e g i o s e l e c t i v i t y and o p e r a t i n g by two d i s t i n c t mechanisms were i n v o l v e d i n the h y d r o x y l a t i o n o f chlorobenzene. Burke and Bridges (33) i n a c o n c e p t u a l l y analogous approach s t u d i e d changes i n the r a t i o s of the 2- and 4- hydroxybiphenyl m e t a b o l i t e s of b i p h e n y l produced by a range of p e r t u r b a t i o n s . These authors concluded t h a t a t l e a s t two independent enzyme systems must be r e s p o n s i b l e i n order to account f o r the data. I t i s somewhat s u r p r i s i n g t h a t i n the wealth of d i f f e r e n t p e r t u r b a t i o n s such as i n d u c e r s , i n h i b i t o r s , age, sex, s p e c i e s , aging o f microsomes, d i f f e r e n t p r e p a r a t i o n s , e t c . , t h a t have been used as t o o l s to probe the m u l t i p l i c i t y of the microsomal system, stereochemical f a c t o r s have been e s s e n t i a l l y ignored. The r e mainder o f t h i s chapter w i l l focus on work done i n our l a b o r a t o r i e s (Sprague-Dawley r a t s ) and the New York S t a t e Department of H e a l t h (Wistar r a t s ) on the use o f stereochemical f a c t o r s f o r such s t u d i e s and the i n t r o d u c t i o n and i n i t i a l development o f a systematic framework f o r the i n t e r p r e t a t i o n of such data. The o r a l a n t i c o a g u l a n t w a r f a r i n , jL, e x i s t s i n two e n a n t i o meric forms. In both man (34,35) and the r a t (36-38) the 5- enantiomer i s f i v e to s i x times as potent as the R-enantiomer. Moreover, i t i s known t h a t the drug i s s t e r e o s e l e c t i v e l y metabolized both q u a n t i t a t i v e l y and q u a l i t a t i v e l y i n man (39) and t h a t the metabolic p a t t e r n s are q u a n t i t a t i v e l y a f f e c t e d by p r i o r a d m i n i s t r a t i o n of other drugs (40,41). I n the r a t the metabolic p a t t e r n s both i n v i v o (42) and i n v i t r o (43) had been r e p o r t e d but the metabolic p a t t e r n s o f the i n d i v i d u a l enantiomers were r e p o r t e d only r e c e n t l y (44,45). Since the drug i s metabolized to f i v e i s o m e r i c hydroxylated products i t i s w e l l s u i t e d f o r probing the microsomal system from the viewpoint o f 1

5.

TRAGER

Enantiomeric

Selectivity and Perturbation

of Product Ratios

83

stereochemical f a c t o r s i n order t o e x p l o r e both t h e phenomenon of drug i n t e r a c t i o n s and m u l t i p l i c i t y . The Michaelis-Menten k i n e t i c parameters f o r the formation o f the i n d i v i d u a l hydroxylated m e t a b o l i t e s from each o f the enantiomers a r e g i v e n i n Table I and F i g u r e 1. I n the a n a l y s i s t o f o l l o w the r e s u l t s obtained from each o f the enantiomers w i l l be d i s c u s s e d s e p a r a t e l y and then i n combination. Table I . Comparative K i n e t i c s o f the O x i d a t i o n o f R and j5 W a r f a r i n by Normal Microsomes From Rat L i v e r (Sprague Dawley) Apparent V (nmolesAng p r o t e i n , 10 min i n c u bations*) d*f (R) 22 0.754±0.049 22 1.65310.113 22 0.44810.052 0.50010.042 19 19 1.69310.527 m a x


Warfarin Metabolite

Apparent Km(mM)* (R)

6-hydroxy 7-hydroxy 8-hydroxy 4'-hydroxy b e n z y l i c hydroxy

0.096±0.028 0.046±0.024 0.093±0.051 0.109±0.038 0.803±0.400

(S)

(S) 6-hydroxy 7-hydroxy 8-hydroxy 4'-hydroxy b e n z y l i c hydroxy

0.032±0.011 0.050±0.010 0.19810.048 0.067±0.021 0.22110.157

0.64410.023 0.45510.013 0.20010.015 0.65210.036 0.23610.060

23 23 19 23 19

*Data were d e r i v e d from weighted l e a s t - s q u a r e s l i n e a r r e g r e s s i o n o f [ S ] / v s . [ S ] . The data i s expressed as the means 1 standard e r r o r s w i t h degrees o f freedom ( d * f ) as shown. v

H y d r o x y l a t i o n o f R-Warfarin The apparent Km f o r the 6-, 7- and 8-hydroxylation o f R w a r f a r i n a r e s t a t i s t i c a l l y i n d i s t i n g u i s h a b l e a t t h e 95% confidence l e v e l . Since a s i n g l e s u b s t r a t e i s being transformed i n a chemic a l l y and s p a t i a l l y d i s c r e t e p a r t o f t h e molecule (coumarin r i n g ) i n t o three s t r u c t u r a l l y d i s t i n c t products, i t i s reasonable t o assume based on t h i s evidence alone t h a t the products a r e formed a t a s i n g l e enzymatic s i t e and t h a t product formation i s r a t e l i m i t i n g . S i m i l a r l y the Km f o r 4 - h y d r o x y l a t i o n i s i n d i s t i n g u i s h a b l e from those o f coumarin r i n g h y d r o x y l a t i o n . Hence t h i s l i n e o f evidence i s c o n s i s t e n t w i t h a s i n g l e hemoprotein being r e s p o n s i b l e f o r a l l the aromatic hydroxylated products obtained from R-warfarin and the observed d i f f e r e n c e s i n reflect d i f f e r e n c e s i n t h e v a r i o u s a c t i v a t i o n energies f o r product format i o n i r r e s p e c t i v e o f mechanism. Although t h e Km f o r b e n z y l i c h y d r o x y l a t i o n , Table I , appears to be l a r g e r than t h e o t h e r s , the i m p r e c i s i o n o f i t s determination 1



S-(->-ISOMER

CONCEPTS

R-(+)-ISOMER 7 OH?

Figure 1. The theoretical velocity for the formation of each of the hydroxylated products was calculated from the experimentally determined Km and V values and plotted as a function of log [S], Such plots approach V asymptotically and have inflection point /"SJ — Km. W<M?

maa

5.

TRAGER

Enantiomeric

Selectivity and

Perturbation

of Product Ratios

85

(due to the l a c k of a s y n t h e t i c standard) does not a l l o w i t to be d i s t i n g u i s h e d s t a t i s t i c a l l y . However, other evidence suggests t h a t the Km f o r b e n z y l i c h y d r o x y l a t i o n i s l a r g e r than the Km f o r any o f the aromatic h y d r o x y l a t i o n s . For example, a s m a l l e r q u a n t i t y of the b e n z y l i c hydroxy m e t a b o l i t e was obtained i n a s i n g l e c o n c e n t r a t i o n study r e l a t i v e to the q u a n t i t i e s of the aromatic h y d r o x y l a t i o n producte (44) but the V for benzylic h y d r o x y l a t i o n i s g r e a t e r than the Vmax f o r e i t h e r 4'- or 8-hydroxylation. This can o n l y be t r u e i f the Km f o r b e n z y l i c h y d r o x y l a t i o n i s indeed l a r g e r than the Km f o r e i t h e r 4 - o r 8-hydroxylation. The f a c t t h a t the Km f o r 6- or 7-hydroxylations are no l a r g e r than the Km f o r 4 - or 8 - h y d r o x y l a t i o n i m p l i e s t h a t the Km f o r b e n z y l i c h y d r o x y l a t i o n must be l a r g e r than the Km f o r any of the aromatic h y d r o x y l a t i o n processes. This r e s u l t i s perhaps not too s u r p r i s i n g s i n c e b e n z y l i c h y d r o x y l a t i o n r e s u l t s from a fundamentally d i f f e r e n t chemical r e a c t i o n which i n v o l v e s o x i d a t i o n a t a s t e r i c a l l y hindered a l i p h a t i c s i t e . m a x

f


1

H y d r o x y l a t i o n of j[ W a r f a r i n U n l i k e the r e s u l t s obtained f o r R-warfarin the Km f o r 8-hydroxylation from ^ - w a r f a r i n i s s i g n i f i c a n t l y l a r g e r than the Km f o r e i t h e r 6- or 7-hydroxylation. Thus, i t i s c l e a r t h a t two k i n e t i c a l l y d i s t i n c t enzymatic processes must be i n v o l v e d d e s p i t e the p r o x i m i t y of the s i t e s o f metabolic a t t a c k . I f product formation i s r a t e l i m i t i n g the c o n t r i b u t i o n of the r a t e constant f o r product formation to Km should be n e g l i g i b l e ; hence, a l l the Km should be the same w h i l e the Vmax n&y he d i f f e r e n t . I f product formation i s not r a t e l i m i t i n g but a common intermediate i s i n v o l v e d , two cases are p o s s i b l e ; the r a t e of formation i s comparable to the r a t e of d i s s o c i a t i o n of the common intermediate back to s u b s t r a t e or the r a t e of formation of the common i n t e r mediate i t s e l f may be r a t e l i m i t i n g . In the f i r s t case the product which i s formed w i t h the g r e a t e s t must a l s o have the g r e a t e s t Km w h i l e i n the second case the Km f o r a l l the products must be the same. Since 8-hydroxylation occurs w i t h the l a r g e s t Km coupled to the s m a l l e s t V ax> c o n d i t i o n s are f u l f i l l e d . Hence, there can.be no common intermediate i n the formation of 8-hydroxywarfarin and e i t h e r 6- or 7-hydroxy m e t a b o l i t e s . As was the case f o r R-warfarin, the Km f o r 4 - and b e n z y l i c h y d r o x y l a t i o n alone y i e l d l i t t l e new i n f o r m a t i o n . Although the evidence i s l e s s convincing i n t h i s case, the data suggests t h a t Km f o r b e n z y l i c h y d r o x y l a t i o n i s l a r g e r than the r e s t . n

o

n

e

o f

t l i e

a

t

o

v

e

m

f

Comparative H y d r o x y l a t i o n of the Enantiomers A h i g h degree of s t e r e o s e l e c t i v i t y as evidenced by the d i f f e r i n g V x values f o r each of the enantiomeric p a i r of products i s d i s p l a y e d . The i n d i s t i n g u i s h a b i l i t y of the Km m a


86


f o r 6- and 7 - h y d r o x y l a t i o n o f a given enantiomer suggests on f i r s t a n a l y s i s that a common enzymatic s i t e i s r e s p o n s i b l e f o r t h e generation o f these two products. Since arene oxides are w e l l e s t a b l i s h e d intermediates i n aromatic h y d r o x y l a t i o n processes by microsomal mixed f u n c t i o n oxidases (45), the p o s s i b l e intermediacy o f a 6,7-epoxide i n t h e i r formation i s an a t t r a c t i v e i n i t i a l P o s t u l a t e . However s e v e r a l l i n e s o f evidence argue a g a i n s t t h i s p o s s i b i l i t y . When the Km f o r the two 6-hydroxylation products are compared they are found t o j u s t d i f f e r a t the 95% confidence l e v e l . This i s n o t t r u e f o r 7-hydroxylation. I f 6- and 7-hydroxylation occur v i a the intermediacy o f an epoxide, a s t e r e o s e l e c t i v e d i f f e r e n c e i n Km f o r t h e 6-hydroxy m e t a b o l i t e s should be r e f l e c t e d i n a corresponding i d e n t i c a l d i f f e r e n c e i n the Km f o r the 7-hydroxy m e t a b o l i t e s . This does n o t appear t o be t h e case i f the Km d i f f e r e n c e s a r e r e a l . Studies o f the a r o m a t i z a t i o n s o f arene oxides have shown t h a t the r a t e - l i m i t i n g step o f t h i s process i n v o l v e s the spontaneous opening o f t h e arene oxide. The d i r e c t i o n i n which the opening occurs depends on the s t a b i l i t y o f the c a r b o c a t i o n o i d t r a n s i t i o n s t a t e (47-54). In the case o f the p o s t u l a t e d arene oxide t h i s would imply t h a t r i n g opening should occur t o y i e l d p r i m a r i l y t h e 6-hydroxylated product because o f the ortho and para d i r e c t i n g e f f e c t o f t h e coumarin l a c t o n e oxygen. However, s i n c e twice as much 7- as 6-hydroxy product i s obtained from R-warfarin i t i s reasonable to conclude t h a t an epoxide i s probably not i n v o l v e d i n t h e formation o f the 6- and 7-hydroxy m e t a b o l i t e s from t h i s isomer. S i m i l a r e l e c t r o n i c arguments permit one t o conclude t h a t a 7,8-epoxide cannot be an i n t e r m e d i a t e i n the 7-hydroxylation of R-warfarin w h i l e the non involvement o f a common i n t e r m e d i a t e i n the formation o f t h e 7- and 8-hydroxy m e t a b o l i t e s o f ^-warf a r i n i s based on the k i n e t i c arguments presented e a r l i e r . Therefore, i f 7,8-epoxidation occurs i n the metabolism o f e i t h e r isomer i t must y i e l d 8-hydroxywarfarin almost e x c l u s i v e l y . U n l i k e coumarin h y d r o x y l a t i o n , 4 ' - h y d r o x y l a t i o n i s s t e r e o s p e c i f i c f o r the j>-enantiomer but these r e s u l t s a r e c o n s i s t e n t w i t h e i t h e r a s i n g l e o r multi-enzyme system. The apparently l a r g e r Km f o r b e n z y l i c h y d r o x y l a t i o n o f both isomers suggests t h a t i f product formation i s r a t e l i m i t i n g , a l i p h a t i c h y d r o x y l a t i o n i s probably the r e s u l t o f a d i s t i n c t microsomal enzyme. Thus, the data i s c o n s i s t e n t w i t h involvement o f a t l e a s t three k i n e t i c a l l y d i s t i n c t enzymes o r enzymatic s i t e s . One enzyme would be r e s p o n s i b l e p r i m a r i l y f o r the o x i d a t i o n o f jS-warfarin to 8-hydroxywarfarin. A second could be r e s p o n s i b l e f o r a l l t h e remaining p h e n o l i c products and a t h i r d f o r the formation o f b e n z y l i c hydroxywarfarin. To f u r t h e r probe t h e system the s t u d i e s were repeated a t a s i n g l e c o n c e n t r a t i o n a f t e r pretreatment o f the animals w i t h e i t h e r PB o r 3-MC (55). The r e s u l t s o f t h i s study are shown i n Table I I (Sprague-Dawley), Table I I I (Wistar) and F i g u r e 2. The data can most r e a d i l y be analyzed i n terms o f the f o l l o w i n g

5.

TRAGER

Enantiomeric

Table I I .

Selectivity

of Product Ratios

87

I n v i v o Comparative O x i d a t i o n o f R- and Warfarin by Normal, PB and 3-MC Induced Hepatic Microsomes from Sprague-Dawley Rats.

Warfarin Metabolites

Product Normal (R)

6-hydroxy 7-hydroxy 8-hydroxy 4 -hydroxy b e n z y l i c hydroxy Total f


and Perturbation

6-hydroxy 7-hydroxy 8-hydroxy 4'-hydroxy h e n z y l i c hydroxy Total

(nmoles/mg protein/10 min*) PB

3-MC (R)

0.69±0.05 1.92±0.13 0.4310.02 0.3810.02 0.2610.03 3.68

1.3510.04 4.3610.26 0.9210.04 0.8910.03 0.4010.01 7.92

2.7810.17 0.9410.13 4.9110.09 0.1610.02 0.0910.01 8.88

(1)

(1)

(S)

0.6410.02 0.4310.01 0.1310.01 0.6410.04 0.1110.01 1.95

1.2310.07 1.7010.05 0.4210.02 1.1210.12 0.9410.04 5.41

1.7010.06 0.4010.02 0.5410.03 0.2710.04 0.0910.01 3.00

*The data i s expressed as the means 1 standard d e v i a t i o n s and represents three analyses. A w a r f a r i n c o n c e n t r a t i o n o f 0.26 mM was employed i n these s t u d i e s .

6-OH R

S

7-OH R S

8-OH R S

4'and Benzylic OH R S

Normal microsomes

o c

4

3

6

•AjOc^-H^) ( k _ + k ) 2

k

and Km

k

+ k

< _l"*4> < - 2 5

)

( k

-3

+ k

6

)

5

5.

TRAGER

Enantiomeric

Selectivity

and Perturbation

of Product Ratios

93

The Km again c h a r a c t e r i z e s the e n t i r e system. Since the v e l o c i t i e s f o r product formation are dPi/dt« k^(E*S),dP2/dt « k5(E*S ) and d P / d t » k (E*S") and s i n c e the r a t i o o f (dPx/dt)/dP2dt) f

3

(

k

6

+

k


Vl -2 5 k k ^ — t h e

r a t i o o f any two products i s a constant which i s

independent o f e i t h e r enzyme o r s u b s t r a t e c o n c e n t r a t i o n . I t should be noted t h a t i n d e r i v i n g the r a t e laws f o r Case 1 and Case 2 the o n l y assumptions t h a t were made were t h a t steady s t a t e k i n e t i c s were a p p l i c a b l e and t h a t product formation was i r r e v e r s i b l e . I t was not necessary t o assume t h a t product formation i s rate l i m i t i n g . Consider a microsomal p r e p a r a t i o n which a c t s on a s i n g l e s u b s t r a t e t o y i e l d m u l t i p l e products. Information r e g a r d i n g t h e enzymatic m u l t i p l i c i t y o f the p r e p a r a t i o n can now be gained by p e r t u r b i n g the system. Fundamentally two types o f p e r t u r b a t i o n s are p o s s i b l e ; 1) those t h a t a f f e c t enzyme o r s u b s t r a t e concent r a t i o n without a l t e r i n g the a c t i v e s i t e o r i n d i v i d u a l r a t e constants and 2) those t h a t can a l t e r the a c t i v e s i t e o r i n d i v i d u a l r a t e constants. Type-1 p e r t u r b e r s i n c l u d e such f a c t o r s as i n h i b i t o r s , i n ducers and s u b s t r a t e c o n c e n t r a t i o n s t u d i e s . F o r such p e r t u r b a t i o n s any s t a t i s t i c a l l y v a l i d d i f f e r e n c e i n the measured Km s o r any change i n product r a t i o s f o r e i t h e r Case 1 o r Case 2 systems i n d i c a t e s the e x i s t e n c e o f a t l e a s t two independent enzymatic s i t e s . Conversely l a c k o f changes i n Km o r product r a t i o s a r e not c o n c l u s i v e evidence f o r the presence o f a s i n g l e enzymatic s i t e but become i n c r e a s i n g l y c o n v i n c i n g as the number o f p e r t u r bations studied i s increased. Type-2 p e r t u r b e r s i n c l u d e such f a c t o r s as pH (by a f f e c t i n g i o n i z a b l e groups a t the a c t i v e s i t e ) i o n i c s t r e n g t h (change s o l v a t i o n o f the a c t i v e s i t e ) temperature ( s h i f t the steady s t a t e constants) and a l l o s t e r i c i n t e r a c t i o n s (by i n d u c i n g conformational changes a t the a c t i v e s i t e ) . For such p e r t u r b a t i o n s a l l combin a t i o n s o f changes i n Km and product r a t i o s are p o s s i b l e . That i s , three r e s u l t s are p o s s i b l e : 1) Km and product r a t i o s do n o t change. This r e s u l t i n d i c a t e s that the p e r t u r b a t i o n d i d n o t a f f e c t e i t h e r the r a t e constants o r the a c t i v e s i t e ; 2) Km changes but product r a t i o s do not (e.g., a temperature e f f e c t ) o r product r a t i o s change and Km does n o t (e.g., an a l l o s t e r i c i n t e r a c t i o n ) . These r e s u l t s i n d i c a t e a s i n g l e enzyme but are n o t c o n c l u s i v e (to have the s i t u a t i o n where two independent enzymes gave e x a c t l y the same product r a t i o s but had d i f f e r e n t Km* s o r the converse would be h i g h l y f o r t u i t o u s ) ; 3) Both Km and product r a t i o s change. This r e s u l t y i e l d s no i n f o r m a t i o n regarding multiplicity. Based on the above a n a l y s i s and assuming the model i s a p p l i c a b l e , i t would appear t h a t i n f o r m a t i o n r e g a r d i n g t h e m u l t i p l i c i t y o f microsomal cytochrome P-450's can most r e a d i l y 1


94

CONCEPTS

be obtained by p e r t u r b i n g the system by f a c t o r s which o n l y a f f e c t enzyme o r s u b s t r a t e c o n c e n t r a t i o n s . To t e s t the method c o n s i d e r the w a r f a r i n data presented e a r l i e r i n Table's I and I I . R-Warfarin


The s t a t i s t i c a l i n d i s t i n g u i s h a b i l i t y o f t h e Km data f o r Itw a r f a r i n suggests t h a t a l l the h y d r o x y l a t e d products c o u l d be formed by a s i n g l e enzyme. I f one accepts the arguments advanced f o r b e n z y l i c hydroxywarfarin having a d i f f e r e n t Km then a t l e a s t two independent c a t a l y t i c s i t e s o r enzymes a r e r e s p o n s i b l e . The product r a t i o s f o r each o f the h y d r o x y l a t e d products from PB induced versus normal animals as determined from Table I I are shown below: 6-0H|^ - = 1.96 ± .15 Normal

. = 2.27 ± .20 Normal

benzylic-OHg^-1.5*

.18

The aromatic h y d r o x y l a t i o n processes a r e s t a t i s t i c a l l y i n d i s t i n g u i s h a b l e w h i l e a t t h e 90-95% confidence l e v e l b e n z y l i c h y d r o x y l a t i o n i s d i f f e r e n t i a l l y induced. ^-Warfarin The PB/Normal product r a t i o s f o r ^ - w a r f a r i n a r e shown below:

6

"

0 H

Srmal

β

X

8

"

0 H

^rmal " '

3

9 2

2 3

* °±

'

1 2

7

2 9

benzylic-OH ^

4

0 H

"

^

0

s m

a

l

Η

3

Srmal " ' fifrmal

m

Χ

·

7

9 5

5

* "

1 5

* °'

2 2

8.54 ± 0.86

In c o n t r a s t t o R - w a r f a r i n t h r e e product groupings a r e immediately apparent. These a r e 6- and 4 ' - h y d r o x y l a t i o n , 7- and 8 - h y d r o x y l a t i o n and b e n z y l i c h y d r o x y l a t i o n . Based on the d i f f e r e n c e i n Km a f u r t h e r d i s t i n c t i o n between 7- and 8- h y d r o x y l a t i o n can be made. Thus, ^ - w a r f a r i n i s h y d r o x y l a t e d by a t l e a s t f o u r d i s t i n c t enzymatic processes.

5.

TRAGER

Enantiomeric

Selectivity

and Perturbation

of Product Ratios

95


R + S-Warfarin Thus f a r the a n a l y s i s has considered a s i n g l e s u b s t r a t e o n l y . To compare two d i f f e r e n t s u b s t r a t e s one must e s t a b l i s h t h a t they are i n t e r a c t i n g w i t h the same enzymatic systems. Since b i o l o g i c a l systems are c h i r a l , enantiomers must be considered as d i f f e r e n t s u b s t r a t e s . I n the a n a l y s i s t o f o l l o w we assume t h a t the R and j> enantiomers are i n t e r a c t i n g w i t h the same group o f enzymes. Experiments are i n progress t o c l a r i f y t h i s assumption by u t i l i z i n g one enantiomer as a c o m p e t i t i v e i n h i b i t o r f o r the other. Assume f o r example, t h a t a s i n g l e enzyme generates-both 6-(R) and 6-(£)-hydroxywarfarin then the r a t i o o f β-ΟΗ^ should be constant upon p e r t u r b a t i o n o f enzyme o r s u b s t r a t e concentra t i o n . Therefore R R 6-0H-4- Normal « 6-0tt-=- PB

which i m p l i e s t h a t R-6-0H ^

r

a

a

l

- S-6-0H

T h i s r e l a t i o n s h i p holds f o r the comparison o f any two products and t h e r e f o r e a l l the necessary c a l c u l a t i o n s have a l r e a d y been done. Groups o f s t a t i s t i c a l l y n o n - d i s t i n g u i s h a b l e products obtained from the product r a t i o s c a l c u l a t e d above are shown below. Group 1 R-6-0H = 1.961.15 R-7-0H » 2.271.20 R-8-0H = 2.131.14 R-4'-0H * 2.341.15 S-6-0H » 1.921.12 S-4'-0H = 1.7510.22

Group 2 R-benzylic-OH 1.51.18 Group 3 ^-benzylic-OH 8.541.86

Group 4 S-7-0H = 3.951.15 Group 5 (Based on Kn) S-8-0H = 3.231.29

Thus the a n a l y s i s indicates that the combination of normal and PB induced microsomes c o n t a i n a minimum o f f i v e d i s t i n c t enzymatic processes. F u r t h e r d i f f e r e n t i a t i o n and determination o f the i n d i v i d u a l c a t a l y t i c r e g i o and s t e r e o s p e c i f i c i t i e s r e q u i r e s the study o f a g r e a t e r number o f p e r t u r b a t i o n s and u l t i m a t e l y the study o f the i s o l a t e d hemoproteins themselves. Such s t u d i e s are c u r r e n t l y i n progress. For example a s i m i l a r a n a l y s i s o f the 3-MC data i n d i c a t e s t h a t t h i s agent induces the formation o f a p p a r e n t l y abnormal isozymes which c a t a l y z e 6- and 8-hydroxyla t i o n . The isozymes r e s p o n s i b l e f o r the remaining h y d r o x y l a t i o n r e a c t i o n s are not induced. The a p p l i c a t i o n o f the product r a t i o technique t o the i n t e r p r e t a t i o n o f microsomal data appears t o be reasonably s u c c e s s f u l and i f v a l i d , g r e a t l y s i m p l i f i e s such i n t e r p r e t a t i o n s . The a p p l i c a b i l i t y o f t h i s method t o cases where M i c h a e l i s Menton

96


k i n e t i c s a r e n o t f o l l o w e d such as: product i n h i b i t i o n , s u b s t r a t e i n h i b i t i o n , non h y p e r b o l i c k i n e t i c s i n g e n e r a l ; t o d i f f e r e n t mechanisms f o r P-450 o x i d a t i o n ; t o the i n f l u e n c e o f epoxide hydrase e t c . , a r e being s t u d i e d . Acknowledgements The author wishes t o express h i s g r a t i t u d e t o h i s c o l l a b o r a t o r s and c o l l e a g u e s a t the New York S t a t e Department o f H e a l t h ; Dr.'s M i c h a e l Fasco, John Fenton, I I I and Mr. F r e d e r i c k Baker, h i s former students, D r . s Lance P o h l , W i l l i a m P o r t e r and Sidney Nelson and h i s present student, Mr. R i c h a r d Branchflower f o r t h e i r c o n t r i b u t i o n s t o the work d e s c r i b e d i n t h i s chapter. We a l s o a p p r e c i a t e the p e r m i s s i o n granted by Biochemical Pharmacology, t o reproduce the F i g u r e s and Tables. Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch005

1

Literature Cited (1) Conney, Α. Η., Gillette, J. R., Inscoe, J. Κ., Trams, E. R. and Posner, H. S., Science (1959), 130, 1478. (2) Conney, Α. Η., Pharmac. Rev. (1967), 19, 317. (3) L u , Α. Υ. Η., and L e v i n , W., Biochem. Biophys. Res. Commun. (1972), 46, 1334. (4) L e v i n , W., L u , Α. Υ. Η., Ryan, D., West, S., Kuntzman, R. and Conney, A. H., Arch. Biochem. Biophys. (1972), 153, 533. (5) L u , Α. Y. H., West, S. Β., Ryan, D. and L e v i n , W., Drug Metab. Dispos. (1973), 1, 29. (6) L e v i n , W., Ryan, D., West, S., and Lu, Α. Υ. H., J. Biol. Chem. (1974), 249, 1747. (7) Kawalek, J. C., L e v i n , W., Ryan, D., Thomas, P. E. and L u , A. Y. H., Mol. Pharmacol. (1975), 153, 533. (8) Ryan, D., Lu, Α. Υ. Η., West, S., and Levin, W., J. Biol. Chem. (1975), 250, 2157. (9) Ryan, D., Lu, Α. Y. H., Kawalek, J., West, S. B., and L e v i n , W., Biochem. Biophys. Res. Commun. (1975), 64, 1134. (10) Kawalek, J. C., L e v i n , W., Ryan, D. and L u , Α. Υ. Η., Drug Metab. Dispos. (1976), 4, 190. (11) Thomas, P. E., Lu, Α. Υ. Η., Ryan, D., West, S. B., Kawalek, J. and Levin, W., J. Biol. Chem. (1976), 251, 1385. (12) Van der Hoeven, T. A. and Coon, M. J., J. Biol. Chem. (1974), 249, 6302. (13) Van der Hoeven, Τ. Α., Haugen, D. A. and Coon, M. J., Biochem. Biophys. Res. Commun. (1974), 60, 569. (14) Haugen, D. Α., Van d e r Hoeven, T. A. and Coon, M. J., J . Biol. Chem. (1975), 250, 3567. (15) Haugen, D. A. and Coon, M. J., Pharmacol. (1975), 17, 186. (16) Haugen, D. Α., Coon, M. J. and Nebert, D. W., J. Biol. Chem. (1976), 251, 1817. (17) Welton, A. F. and Aust, S. D., Biochem. Biophys. Res. Commun. (1974), 56, 898.


5. TRAGER

Enantiomeric

Selectivity

and Perturbation

of Product Ratios

97

(18) Welton, A. F., O'Neal, F. O., Chaney, L. C. and Aust, S. D., J. Biol. Chem. (1975), 250, 5631. (19) L e v i n , W., A l v a r e s , Α., Jacobson, M. and Kuntzman, R., Biochem. Pharmacol. (1969), 18, 883. (20) Pederson, T. C. and Aust, S. D., Biochem. Pharmacol. (1969), 18, 883. (21) Gloumann, Η., Chem. Biol. Interactions (1970), 2, 369. (22) Aust, S. D., and Stevens, J. B., Biochem. Pharmacol. (1971), 20, 1061. (23) Conney, A. H., L u , Α. Υ. Η., L e v i n , W., Somogyi, Α., West, S., Jacobson, Μ., Ryan, D. and Kuntzman, R., Drug Metab. Dispos. (1973), 1, 199. (24) Stonard, M. D., Biochem. Pharmacol. (1975), 24, 1959. (25) Hook, G. E. R., Orton, T. C., Moore, J. A. and Lucifer, G. W., Biochem. Pharmacol. (1975), 24, 335. (26) U l b r i c h , V., Wever, P., and Woolenberg, P., Biochem. Biophys. Res. Commun. (1975), 64, 808. (27) May, H. E., Bose, R. and Reed, D. J., Biochem, (1975), 14, 4723. (28) W e r r i n g l o e r , J. and Estabrook, R. W., Arch. Biochem. Biophys. (1975), 167, 270. (29) Wiebel, F. J. and G e l b o i n , H. V., Biochem. Pharmacol. (1975) 24, 1511. (30) Townsend, M. G., Odam, Ε. M. and Page, J. M. J., Biochem. Pharmacol. (1975), 24, 729. (31) Juchau, M. R., Namkung, M. J., B e r r y , D. L. and Zachariah, P. K., Drug Metab. Dispos. (1975), 3, 494. (32) Selander, H. G., Jerina, D. M. and Daly, J. W., A r c h i v . Biochem. Biophys. (1975), 168, 309. (33) Burke, M. D. and B r i d g e s , J. W., X e n o b i o t i c a (1975),5.,357. (34) Hewick, D. and McEwen, J., J. Pharm. Pharmacol. (1973), 25, 458. (35) O ' R e i l l y , R. Α., Clin. Pharmacol. Therap. (1974), 16, 348. (36) E b l e , Ν., West, Β. and L i n k , Κ., Biochem. Pharmacol. (1966), 15, 1003. (37) Breckenridge, A. and L'E Orme, M., Life Sciences (1974), 11 (Part II), 337. (38) Hewick, D., J. Pharm. Pharmacol. (1972), 24, 661. (39) Chan, Κ. Κ., Lewis, R. J. and Trager, W. F., J. Med. Chem. (1972), 15, 1265. (40) Lewis, R. J., Trager, W. F., Chan, Κ. Κ., Breckenridge, Α., L'E Orme, M., Rowland, R. and Scharry, W., J. Clin. Invest. (1974), 53, 1607. (41) O ' R e i l l y , R. Α., New E n g l . J. Med. (1976), 295, 354. (42) Barker, W. M., Hermodson, M. A. and L i n k , K. P., J . Pharmacol. Exp. Therap. (1970), 171, 307. (43) Ikeda, Μ., Ullrich, V. and Staudinger, Η., Biochem. Pharmacol. (1968), 17, 1663. (44) P o h l , L. R., Nelson, S. D., P o r t e r , W. R., Trager, W. F., Fasco, M. J., Baker, F. D. and Fenton, J. W., Biochem.



98

Pharmacol. (1976), 25, 2153. (45) P o h l , L. R., B a l e s , R. and Trager, W. F., Res. Commun. Chem. Path. Pharmacol., in press. (46) Jerina, D. M. and Daly, J. W., Science (1974), 185, 573. (47) Daly, J. W., Jerina, D. H. and Witkop, Β., E x p e r i e n t i a (1972), 28, 1129. (48) Kasperek, G. J., and Bruce, T. C., J. Amer. Chem. Soc. (1972), 94, 198. (49) Kasperek, G. J., Bruice, T. C., Y a g i , H. and Jerina, D. Μ., J . Chem. Soc. D., Chem. Commun. (1972), 784. (50) Y a g i , Η., Jerina, D. Μ., Kasperek, G. J. and B r u i c e , T. C., Proc. N a t ' l . Acad. Sci., U.S.A. (1972), 69, 1985. (51) Kasperek, G. J., B r u i c e , T. C., Y a g i , H., Kaubisch, N. and Jerina, D. M., J. Amer. Chem. Soc. (1972), 94, 7876. (52) J e r i n a , D. Η., Kaubisch, N. and Daly, J. W., Proc. N a t ' l . Acad. Sci., U.S.A. (1971), 68, 2545. (53) Kaubisch, Ν., Daly, J. and Jerina, D. Μ., Biochem. (1972), 11, 3080. (54) Richardson, J. D., B r u i c e , T. C., Warasziewiez, S. M. and B e r c h t o l d , G. Α., J. Org. Chem. (1974), 39, 2088. (55) P o h l , L. R., P o r t e r , W. R., Trager, W. F., Fasco, M. J. and Fenton, J. W., Biochem. Pharmacol., in press. (56) Comai, K. and Gaylor, J. L., J. Biol. Chem. (1973), 248, 2947. (57) A l v a r e z , A. P. and Siekevitz, P., Biochem. Biophys. Res. Commun. (1973), 54, 923. (58) Muna, W. J., "Cytochrome P-450 and Cytochrome b L e v e l s in Rat L i v e r Microsomes During Prolonged A d m i n i s t r a t i o n o f Pheno barbital: Changes in the Topographical R e l a t i o n s h i p s o f Cytochrome b ," Ph.D. T h e s i s , U n i v e r s i t y o f Washington (1974). (59) P o h l , L. R., Nelson, S. D., Garland, W. A. and Trager, W. F., Biomed. Mass Spect. (1975), 2, 23. (60) P o r t e r , W. R., Branchflower, R. V. and Trager, W. F., Biochem. Pharmacol., in p r e s s . (61) B r i g g s , G. E. and Haldane, J. B. S., Biochem. J. (1925), 19, 388. (62) K i n g , E. L. and Altman, C., J. Phys. Chem. (1956), 60, 1375. (63) Segel, I. Η., "Enzyme Kinetics," John Wiley and Sons, I n c . , New York (1975), 506-515. 5

5

6 Role of Purified Cytochrome P-448 and Epoxide Hydrase in the Activation and Detoxification of

Benzo[α]pyrene


W. LEVIN, A. W. WOOD, A. Y. H. LU, D. RYAN, S. WEST, and A. H. CONNEY Department of Biochemistry and Drug Metabolism, Hoffman-La Roche Inc., Nutley, NJ 07110 D. R. THAKKER, H. YAGI, and D. M. JERINA National Institute of Arthritis, Metabolism and Digestive Disesases, National Institutes of Health, Bethesda, MD 20014

The liver microsomal monoxygenase system is a membranebound, multicomponent electron transport system which is responsible for the oxidative metabolism of a variety of endogenous and exogenous substrates such as steroids, fatty acids, drugs, insecticides and chemical carcinogens (1). Of the three components involved in microsomal drug metabolism (cytochrome P-450, NADPH-cytochrome c reductase and phosphatidylcholine), cytochrome P-450 is undoubtedly the most important because of its vital role in oxygen activation, substrate binding and in determining the overall substrate specificity of the enzyme system (2,3). The rate at which various compounds are metabolized by this enzyme system varies widely and depends on the species, strain, age, tissue and pretreatment of the animal (1). Over the last decade, numerous studies have suggested that different forms of cytochrome P-450 exist in liver microsomes, and, more recently, the purification and reconstitution of the monoxygenase system have established the existance of multiple forms of cytochrome P-450 having different substrate specificities. The various purified forms of cytochrome P-450 differ from one another not only in their substrate specificity but also in their spectral and immunological properties as well as in their minimum molecular weights as determined by SDS-gel electrophoresis (4-9). The importance of this rather versatile enzyme system has become increasingly apparent during the last 10 years. Today we live in a society that has become increasingly aware of the potential dangers of environmental pollutants which include chemical carcinogens. It has been estimated that 60-80% of a l l human cancers are caused by environmental factors (10-12), and many chemicals in our environment are metabolically activated to ultimate carcinogens by the microsomal monoxygenase system (13). One 99

100


of these environmental p o l l u t a n t s , the p o l y c y c l i c aromatic hydro carbon benzolajpyrene (BP), may b e o n e o f t h e m o s t p r e v a l e n t c h e m i c a l c a r c i n o g e n s t o w h i c h man i s e x p o s e d (14).


1

S i n c e p o l y c y c l i c h y d r o c a r b o n s s u c h a s BP a r e c h e m i c a l l y i n e r t , t h e i r c a r c i n o g e n i c i t y i s thought to r e s u l t from metabolic a c t i v a t i o n by t h e m i c r o s o m a l monoxygenase system t o a c h e m i c a l l y reactive iηtermediate(s)(13,15-17). The o x i d a t i v e m e t a b o l i s m o f BP p r o c e e d s i n i t i a l l y t h r o u g h t h e f o r m a t i o n o f r e a c t i v e a r e n e oxides which spontaneously isomerize to phenols, are hydrated t o d i h y d r o d i o l s by microsomal epoxide hydrase or are c o n j u g a t e d with glutathione v i a the soluble glutathione S-transferases (ljj>« 16,18). In o r d e r t o e l u c i d a t e t h e r o l e o f m e t a b o l i s m i n t h e m u t a g e n i c i t y and c a r c i n o g e n i c i t y o f BP, a b a s i c understanding o f t h e p r o p e r t i e s and mechanism o f a c t i o n o f t h e enzymes involved i n t h e a c t i v a t i o n a n d i n a c t i v a t i o n o f BP i s e s s e n t i a l . Thus, t h e p u r i f i c a t i o n and r e c o n s t i t u t i o n o f t h e monoxygenase s y s t e m (19) i n t h e p r e s e n c e o r absence o f p u r i f i e d e p o x i d e hydrase (20) has e n a b l e d us t o s t u d y t h e r o l e o f t h e s e enzymes i n t h e m e t a b o l i s m o f BP a n d B P d e r i v a t i v e s a n d t o m a n i p u l a t e t h e s o u r c e o f t h e p u r i f i e d c y t o c h r o m e P-450 as w e l l as t h e l e v e l o f e p o x i d e hydrase to generate mutagenic metabolites of t h i s p o l y c y c l i c aromatic hydrocarbon. F i n a l l y , the synthesis of approximately t h i r t y BP d e r i v a t i v e s a n d m e t a b o l i t e s ( 2 1 ) h a s p e r m i t t e d u s t o u t i l i z e t h e s e compounds as s u b s t r a t e s f o r t h e p u r i f i e d monoxy g e n a s e s y s t e m i n an e f f o r t t o i d e n t i f y t h e b i o a c t i v a t e d m e t a b o l i t e s o f BP. M e t a b o l i s m o f B e n z o l a j p y r e n e and B e n z o l a j p y r e n e A r e n e O x i d e s b £ t h e P u r i f i e ? Wônoxygenase System and E p o x i d e H y d r a s e . Tïïë p u r i f i e d , r e c o n s t i t u t e d monoxygenase system w i t h and w i t h o u t a d d i t i o n o f p u r i f i e d e p o x i d e hydrase has been u t i l i z e d t o s t u d y the metabolism o f L CJ-benzolajpyrene (Table I). Although high pressure l i q u i d chromatography i s h i g h l y e f f i c i e n t f o r the separ a t i o n and q u a n t i t a t i o n o f d i h y d r o d i o l s , p h e n o l s and q u i n o n e s formed from BP, a l l p o t e n t i a l m e t a b o l i t e s w i t h i n each group have not been i d e n t i f i e d i n t h e s e s t u d i e s ( 2 2 J . Thus, d i o l fractions 1 , 2 a n d 3 c o r r e s p o n d t o BP 9 , 1 0 - , 4 , 5 - a n d 7,8-dihydrodiols, q u i n o n e f r a c t i o n 1 c o r r e s p o n d s t o BP 1 , 6 - , 3 , 6 - and 4,5-quinones, q u i n o n e f r a c t i o n 2 c o r r e s p o n d s t o BP 1 1 , 1 2 - a n d 6 , 1 2 - q u i n o n e s a n d BP 4 , 5 - o x i d e , p h e n o l f r a c t i o n 1 c o r r e s p o n d s t o 2 - , 6 - , 8and 9-HOBP and p h e n o l f r a c t i o n 2 c o r r e s p o n d s t o t h e o t h e r 8 i s o m e r i c p h e n o l s o f BP ( 1 - , 3 - , 4 - , 5 - , 7 - , 1 0 - , 1 1 - a n d 1 2 - H O B P ) . In t h e a b s e n c e o f e p o x i d e h y d r a s e , t h e r e c o n s t i t u t e d c y t o c h r o m e P - 4 4 8 c o n t a i n i n g m o n o x y g e n a s e s y s t e m m e t a b o l i z e s BP t o p h e n o l s and q u i n o n e s ( T a b l e I ) . Upon a d d i t i o n o f p u r i f i e d e p o x i d e h y d r a s e , t h e r a t e o f t o t a l BP m e t a b o l i s m i s u n c h a n g e d , b u t s i g n i f i c a n t amounts o f d i h y d r o d i o l s a r e p r o d u c e d a t t h e expense o f phenols. F o r m a t i o n o f d i o l f r a c t i o n s 2 and 3 (BP 4 , 5 - and 7 , 8 d i h y d r o d i o l s , r e s p e c t i v e l y ) r e a c h e d a maximum l e v e l w i t h t h e a d d i t i o n of 5 u n i t s of epoxide hydrase whereas f u r t h e r a d d i t i o n

6.

LEVIN E T A L .

Activation

and Detoxification

of Benzolajpyrene

101

o f t h e enzyme r e s u l t e d i n a c o n t i n u e d i n c r e a s e i n d i o l f r a c t i o n 1 (BP 9 , 1 0 - d i h y d r o d i o l ) , p r o b a b l y as a r e s u l t o f t h e marked i n s t a b i l i t y o f BP 9 , 1 0 - o x i d e ( t , < 2 m i n u t e s a t 3 7 ° i n 1 0 0 mM p o t a s s i u m p h o s p h a t e b u f f e r ) c o m p a r e d t o BP 4 , 5 - a n d 7 , 8 - o x i d e s . T h u s , h i g h e r amounts o f e p o x i d e h y d r a s e w o u l d be r e q u i r e d t o


/

2

Table

I

Metabolism o f B e n z o l a j p y r e n e b y a P u r i f i e d Cytochrome P-448 Dependent Monoxygenase System and Epoxide Hydrase

Epoxide Hydrase

Diol 1

(units)

None

2

3

(nmol

product

1

Quinone 2

formed/nmol

0.06

0.05

0.07

1.54

5

0.59

0.41

0.57

1.66

15

0.79

0.47

0.61

50

1.19

0.48

0.56

0.52

1

Phenol 2

Total

hemeprotein/min)

0.87

1.31

4.42

-

0.63

1.00

4.86

1.62

-

0.35

0.90

4.74

1.39

-

0.13

0.74

4.49

I n c u b a t i o n m i x t u r e s c o n t a i n e d 0 . 2 nmol c y t o c h r o m e P - 4 4 8 , 120 u n i t s o f NADPH c y t o c h r o m e c r e d u c t a s e , 0 . 1 mg o f l i p i d , 0 . 5 p m o l o f NADPH, 3 y m o l o f M g C l , 100 y m o l o f p o t a s s i u m p h o s p h a t e b u f f e r (pH 6.a) a n d 95 nmol o f ( ^ C J - B P i n a f i n a l v o l u m e o f 1 m l . One u n i t o f e p o x i d e h y d r a s e p r o d u c e s 1 nmol o f s t y r e n e g l y c o l p e r m i n from styrene oxide. M e t a b o l i t e s o f BP w e r e a n a l y z e d b y h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y as d e s c r i b e d b y H o l d e r e t jrt. ( 2 2 ) . 2

102



c o m p l e t e l y c o n v e r t BP 9 , 1 0 - o x i d e t o t h e c o r r e s p o n d i n g dihydrodiol. A g o o d s t o i c h i o m e t r i c r e l a t i o n s h i p was f o u n d b e t w e e n t h e i n c r e a s e i n t h e d i o l 2 f r a c t i o n and t h e d e c r e a s e i n q u i n o n e f r a c t i o n 2 w h i c h c o n t a i n s m a i n l y BP 4 , 5 - o x i d e . Direct confirm a t i o n t h a t a r e n e o x i d e s o f BP a r e s u b s t r a t e s f o r t h e p u r i f i e d e p o x i d e h y d r a s e i s shown i n T a b l e I I . BP 4 , 5 - , 7,8and 9 , 1 0 oxides are a l l metabolized to the corresponding dihydrodiols a t c o m p a r a b l e r a t e s u s i n g t h e p u r i f i e d e n z y m e w h i l e BP 1 1 , 1 2 o x i d e i s a r e l a t i v e l y poor s u b s t r a t e f o r t h e enzyme. No d e t e c t a b l e BP 1 1 , 1 2 - d i h y d r o d i o l i s formed by t h e p u r i f i e d cytochrome P-448 c o n t a i n i n g monoxygenase system i n the presence of epoxide hydrase. T h e r e s u l t s o f o u r s t u d i e s o n t h e m e t a b o l i s m o f BP i n the presence or absence of epoxide hydrase demonstrate t h a t t h e d i h y d r o d i o l s a n d p h e n o l i c m e t a b o l i t e s o f BP s h a r e a r e n e o x i d e s a s common precursors. Requirements f o r t h e M e t a b o l i c A c t i v a t i o n o f Benzol a Ipyrene t o M u t a g e n i c P r o d u c t s by a P u r i f i e d Monoxygenase System. Mutag e n i c i t y t e s t s u t i l i z i n g m i c r o o r g a n i s m s o r c u l t u r e d mammalian c e l l s have been used w i t h i n c r e a s i n g f r e q u e n c y t o i d e n t i f y b i o activated metabolites of carcinogens. Metabolic activation of c h e m i c a l s t o m u t a g e n i c m e t a b o l i t e s has been most commonly p e r f o r m e d b y a p r o c e d u r e d e v e l o p e d b y Ames a n d h i s a s s o c i a t e s (23, 24). G e n e r a l l y , t h e c h e m i c a l , b a c t e r i a and a p p r o p r i a t e c o f a c E o r s are i n c u b a t e d w i t h microsomes or a 9000 xg s u p e r n a t a n t f r a c t i o n o f l i v e r i n a s e m i s o l i d agar g e l f o r 48 h o u r s . When l i t t l e is known a b o u t t h e m e t a b o l i s m o f a p a r t i c u l a r compound, o r a l a r g e number o f d i v e r s e c h e m i c a l s a r e b e i n g e v a l u a t e d f o r m u t a g e n i c a c t i v i t y , r e l a t i v e l y c r u d e t i s s u e homogenates s h o u l d be used as t h e s o u r c e o f enzymes t o e n s u r e t h a t a l l p o s s i b l e m e t a b o l i c pathways are being e v a l u a t e d . W h i l e t h i s p r o c e d u r e has been u s e d s u c c e s s f u l l y t o a c t i v a t e a number o f c a r c i n o g e n s t o b a c t e r i a l mutagens, i t has l i m i t a t i o n s f o r i d e n t i f y i n g t h e m u t a g e n i c m e t a b o l i t e s f o r m e d f r o m a compound w h i c h u n d e r g o e s oxidation v i a m u l t i p l e pathways. The 9000 xg s u p e r n a t a n t f r a c t i o n i s r e l a t i v e l y c r u d e and c o n t a i n s many e n z y m a t i c and s t r u c t u r a l p r o t e i n s , n u c l e i c a c i d s and numerous n u c l e o p h i l i c and e l e c t r o p h i l i c groups which could i n t e r a c t with the b i o a c t i v a t e d metabolites before they reach the b a c t e r i a . Moreover, regardless of the source of the monoxygenase a c t i v i t y , long i n c u b a t i o n times can r e s u l t i n spontaneous or enzymatic breakdown o f p r i m a r y m e t a b o l i t e s t o as y e t i l l - d e f i n e d p r o d u c t s ( 2 5 , 2 6 ) . An e x a m i n a t i o n o f t h e m e t a b o l i t e p r o f i l e o b t a i n e d f r o m BP w h e n l i v e r m i c r o s o m e s a r e i n c u b a t e d f o r 30 m i n u t e s w i t h l i m i t i n g s u b s t r a t e c o n c e n t r a t i o n s r e v e a l e d t h a t a l l p r i m a r y o x i d a t i v e p r o d u c t s o f BP ( d i h y d r o d i o l s , q u i n o n e s and p h e n o l s ) undergo e x t e n s i v e s e c o n d a r y m e t a b o l i s m by t h e monoxygenase system (26). Thus, use of prolonged i n c u b a t i o n times f o r m e t a b o l i c a c t i v a t i o n s t u d i e s i n agar gel would a l l but e l i m i n a t e the p o s s i b i l i t y of o b t a i n i n g a p r o f i l e of the m e t a b o l i t e s formed under t h e c o n d i t i o n s which induce mu-

6.

LEVIN ET A L .

Activation

and Detoxification

Table


Oxides

445

BP

321

BP 9 , 1 0 - o x i d e

BP

11,12-oxide

by P u r i f i e d

Epoxide

D i h y d r o d i o l Formed (nmol/mg p r o t e i n / m i n )

BP 4 , 5 - o x i d e

7,8-oxide

103

II

Metabolism o f Benzolajpyrene Arene "Hydrase

Substrate

of Benzolajpyrene

390

31

Incubation m i x t u r e s c o n t a i n e d 2-6 y g o f p u r i f i e d epoxide hydrase, 30 y g o f p h o s p h a t i d y l c h o l i n e , 12.5 ymol o f T r i s b u f f e r and 10-25 nmol o f t r i t i u m l a b e l l e d s u b s t r a t e i n a f i n a l v o l u m e o f 8 0 y l .


104

tations. F i n a l l y , t h e a b i l i t y t o m a n i p u l a t e t h e amount o f v a r i o u s BP m e t a b o l i t e s f o r m e d t h r o u g h a l t e r a t i o n o f t h e r a t i o o f e p o x i d e hydrase t o t h e monoxygenase system ( c f T a b l e I) should be o f v a l u e i n d e t e r m i n i n g t h e n a t u r e o f t h e b i o a c t i v a t e d m e t a b o l i t e s formed from BP. We, t h e r e f o r e , s o u g h t t o d e v e l o p an e n z y m a t i c a l l y w e l l - d e f i n e d monoxygenase system which would m e t a b o l i z e BP a n d BP d e r i v a t i v e s t o m u t a g e n i c p r o d u c t s u n d e r c o n d i t i o n s w h i c h w o u l d p e r m i t t h e a n a l y s i s and i d e n t i f i c a t i o n o f the metabolites (27). Except f o r tRê presence of b a c t e r i a (Salmonella typhimurium s t r a i n T A 9 8 ) i n t h e r e a c t i o n m i x t u r e , t h e m e t a b o l i s m o f BP t o m u t a g e n i c p r o d u c t s by t h e r e c o n s t i t u t e d s y s t e m was p e r f o r m e d e s s e n t i a l l y as d e s c r i b e d f o r m e t a b o l i t e i d e n t i f i c a t i o n . Bacteria (2 χ 1 0 c e l l s ) were suspended i n a t o t a l i n c u b a t i o n volume o f 0 . 5 ml c o n t a i n i n g 2 . 5 y m o l o f s o d i u m p h o s p h a t e , 75 y m o l o f sodium c h l o r i d e , 0 . 0 8 y m o l (50 y g ) o f p h o s p h a t i d y l c h o l i n e , 150 u n i t s o f N A D P H - c y t o c h r o m e c r e d u c t a s e , 0 . 0 2 - 0 . 2 nmol o f c y t o c h r o m e P - 4 5 0 o r P - 4 4 8 , 25 nmol o f BP ( i n 1 2 . 5 y 1 a c e t o n e ) a n d 0 . 1 y m o l o f NADPH. T h e f i n a l pH o f t h e i n c u b a t i o n m i x t u r e was 6 . 8 . After i n c u b a t i o n a t 37 f o r 5 m i n u t e s , 9 nmol o f m e n a d i o n e was a d d e d t o s t o p t h e r e a c t i o n f o l l o w e d i m m e d i a t e l y by 2.0 ml o f m o l t e n t o p a g a r , and t h e s t a n d a r d Ames p o u r p l a t e p r o c e d u r e ( 2 4 ) was performed. S t u d i e s on t h e r e q u i r e m e n t s f o r t h e enzymaTTc acti v a t i o n o f BP b y t h e r e c o n s t i t u t e d c y t o c h r o m e P - 4 4 8 s y s t e m i n dicated that optimal metabolic a c t i v a t i o n to mutagenic metabolites r e q u i r e d t h e p r e s e n c e o f NADPH, N A D P H - c y t o c h r o m e c r e d u c t a s e , c y t o c h r o m e P-448 and p h o s p h a t i d y l c h o l i n e ( T a b l e I I I ) . A 5 minute i n c u b a t i o n w i t h 0.1 nmol o f c y t o c h r o m e P - 4 4 8 and s a t u r a t i n g a m o u n t s o f N A D P H - c y t o c h r o m e c r e d u c t a s e , p h o s p h o l i p i d , NADPH a n d BP i n d u c e d a p p r o x i m a t e l y a 1 5 - f o l d i n c r e a s e i n h i s t i d i n e i n d e p e n d e n t c o l o n i e s i n s t r a i n TA 9 8 . An a b s o l u t e r e q u i r e m e n t was o b s e r v e d f o r a l l c o m p o n e n t s o f t h e s y s t e m e x c e p t p h o s p h a t i d y l c h o l i n e which i s i n agreement w i t h t h e r e q u i r e d components for t h e m e t a b o l i s m o f BP t o p h e n o l i c m e t a b o l i t e s ( 1 9 ) . In o t h e r e x p e r i m e n t s ( 2 7 ) , i t was e s t a b l i s h e d t h a t t h e number o f m u t a t i o n s i n d u c e d i n S a l m o n e l l a t y p h i m u r i u m s t r a i n TA 9 8 was p r o p o r t i o n a l t o t h e amount o f added c y t o c h r o m e P - 4 4 8 and t o t h e t i m e o f i n cubation (Figure 1).


B

M e t a b o l i s m o f Benzol a j p y r e n e t o M u t a g e n i c M e t a b o l i t e s by Various Pur i f i e d T o r m s of Cytochrome P-450. Γη t h e l a s t d e c a d e , numerous l a b o r a t o r i e s have i n v e s t i g a t e d t h e p o s s i b i l i t y t h a t m u l t i p l e hydroxylase systems e x i s t i n l i v e r microsomes. The p u r i f i c a t i o n and r e c o n s t i t u t i o n o f t h i s enzyme s y s t e m has p r o v i d e d t h e means t o s t u d y i n d e t a i l t h e p h y s i c a l p r o p e r t i e s o f e a c h o f t h e components and t h e c a t a l y t i c a c t i v i t y o f c y t o c h r o m e P-450. These s t u d i e s have p r o v i d e d d i r e c t e v i d e n c e f o r t h e e x i s t ance of m u l t i p l e forms of cytochrome P-450, each of which have d i f f e r e n t , but o v e r l a p p i n g , s u b s t r a t e s p e c i f i c i t i e s ( 4 - 7 ) . T a b l e I V s h o w s a c o m p a r i s o n o f t h e m e t a b o l i s m o f BP t o m u t a g e n i c

LEVIN E T A L .

6.

Activation

and Detoxification

Table

of Benzo[a]pyrene

105

III

Requirements f o r t h e M e t a b o l i c A c t i v a t i o n o f BenzoI a)pyrene t o mutagenic"ProducTs i n Salmonella Typïïimuriûm"Strain TA""98


Addition

His

Revertants/Plate

None

36

BP

37

Benzolajpyrene Metabolism (% A c t i v i t y )

-

440

100

30

0

32

1

32

2

-Phosphatidylcholine

74

18

-NADPH

34

0

Complete Monoxygenase

System

-BP -NADPH-cytochrome -Cytochrome

c

P-448

reductase

The c o m p l e t e monoxygenase system c o n s i s t e d o f 50 y g o f p h o s p h a t i d y c h o l i n e , 150 u n i t s o f NADPH-cytochrome c r e d u c t a s e , 0 . 1 nmol o f c y t o c h r o m e P - 4 4 8 , 0 . 1 y m o l o f NADPH a n d 2 5 n m o l o f B P i n a f i n a l volume o f 0 . 5 ml c o n t a i n i n g 2 χ 1 0 b a c t e r i a . Incubations were a t 3 7 ° f o r 5 m i n . 8


106

Table

Metabolism

of

Benzolajpyrene Purified

Forms

to Mutagenic of


Source of Cytochrome P-450 Species Pretreatment of Animals

Rat

Rabbit

Products

by

Various

P-450

BenzolaJpyrene Metabolism (pmol f o r m e d / pmol h e m e p r o t e i n )

Mutations in S t r a i n TA 9 8 (His Revertants/pmol hemeprotein) +

38.0

1254

13.0

26.5

Phénobarbital

0.7

Aroclor

Rat

Cytochrome

15.0

3-Methylcholanthrene

Rat

IV

0.55

3-Methylcholanthrene

0.7

0.25

Phénobarbital,

0.5

0.05

0.1

0.05

Mouse

Fraction Mouse

A

2

Phénobarbital, Fraction C 2

A l l i n c u b a t i o n m i x t u r e s c o n t a i n e d t h e complete monoxygenase s y s t e m a n d 2 χ IQr b a c t e r i a i n a f i n a l v o l u m e o f 0 . 5 m l a s d e s c r i b e d in Table III. C y t o c h r o m e P - 4 5 0 was p u r i f i e d f r o m t h e l i v e r s o f r a t s ( 5 , 2 9 ) , r a b b i t s (6) o r m i c e (7) as p r e v i o u s l y d e s c r i b e d . The f i n a l s u b s t r a t e c o n c e n t r a t i o n w a s 75 y M . After incubation for 5 min at 37°C, t h e r e a c t i o n s were t e r m i n a t e d by t h e a d d i t i o n of 9 nmol o f m e n a d i o n e . B e n z o l a j p y r e n e h y d r o x y l a t i o n was m e a s u r e d as fluorescent phenols (28). f t

6.

LEVIN E T A L .

Activation

and Detoxification

of Benzo[a]pyrene

107

m e t a b o l i t e s by t h e p u r i f i e d and r e c o n s t i t u t e d monoxygenase system using cytochrome P-450 p u r i f i e d from animals t r e a t e d w i t h d i f f e r e n t i n d u c e r s as w e l l as from s e v e r a l d i f f e r e n t animal s p e c i e s . These r e s u l t s c l e a r l y demonstrate t h e marked d i f f e r e n c e s i n t h e m e t a b o l i s m o f BP t o f l u o r e s c e n t p h e n o l s b y v a r i o u s p u r i f i e d f o r m s of cytochrome P-450. T h e m e t a b o l i c a c t i v a t i o n o f BP t o p r o d u c t s m u t a g e n i c t o s t r a i n TA 9 8 o f S. t y p h i m u r i u m b y v a r i o u s p u r i f i e d c y t o c h r o m e P - 4 5 0 s was s i m i l a r t o t h e r a t e o f m e t a b o l i s m o f BP to fluorescent phenols (Table IV). Cytochrome P-450 i s o l a t e d from rats pretreated with e i t h e r 3-methylcholanthrene or A r o c l o r 1254 were t h e most e f f i c i e n t h e m e p r o t e i n s f o r t h e m e t a b o l i s m o f BP t o f l u o r e s c e n t p h e n o l s a n d m u t a g e n i c p r o d u c t s w h i l e t h e o t h e r p u r i f i e d c y t o c h r o m e P - 4 5 0 s w e r e much l e s s e f f e c t i v e . 1


1

E f f e c t o f Epoxide Hydrase on t h e M e t a b o l i c A c t i v a t i o n o f Benzol a lpyrene t o Mutagenic MetâEoTTtes. Epoxide hydrase i s an i m p o r t a n t enzyme i n t h e m e t a b o l i s m o f BP s i n c e i t c o n v e r t s t h e i n t e r m e d i a t e arene o x i d e s formed by t h e monoxygenase system to the corresponding trans dihydrodiols (15,16). In t h e absence of f u r t h e r metabolic a c t i v a t i o n , t h e d i h y d r o d i o l s produced from these arene oxides are e s s e n t i a l l y nontoxic (21,30-32). Addi t i o n o f h i g h l y p u r i f i e d e p o x i d e hydrase t o t h e monoxygenase s y s t e m d u r i n g t h e m e t a b o l i s m o f BP d e c r e a s e d t h e m u t a t i o n f r e q u e n c y b y a maximum o f 3 0 % i n S. t y p h i m u r i u m s t r a i n TA 9 8 ( F i g u r e 2 ) , i n d i c a t i n g t h a t a t l e a s t some o f t h e m u t a g e n i c m e t a b o l i t e s o f BP a r e a r e n e o x i d e s . In c o n t r a s t t o these r e s u l t s , mutations i n d u c e d b y BP 4 , 5 - o x i d e w e r e c o m p l e t e l y a b o l i s h e d b y t h e a d d i t i o n of 5 units o f epoxide hydrase (Figure 2 ) . Thus, t h e i n a b i l i t y of epoxide hydrase t o completely abolish the metabolic a c t i v a t i o n o f BP t o m u t a g e n i c p r o d u c t s s u g g e s t e d t h a t n o n - a r e n e o x i d e m e t a b o l i t e s o f BP m a y a l s o h a v e m u t a g e n i c a c t i v i t y o r t h a t some m u t a genic epoxide m e t a b o l i t e s a r e poor substrates f o r epoxide hydrase. Metabolic Activation of Benzolajpyrene Phenols. Phenols, formed e i t h e r by spontaneous i s o m e r i z a t i o n o f arene oxides (15) or d i r e c t oxygen i n s e r t i o n r e a c t i o n s ( 3 3 ) , a r e t h e p r i m a r y o x i d a t i v e p r o d u c t s when BP i s m e t a b o l i z e c T E y t h e p u r i f i e d m o n o x y genase system i n t h e absence o f epoxide hydrase ( c f Table I ) . Of t h e t w e l v e p o s s i b l e i s o m e r i c p h e n o l s o f B P , o n l y 6 - and 1 2 HOBP h a v e s i g n i f i c a n t i n t r i n s i c m u t a g e n i c a c t i v i t y i n s e v e r a l s t r a i n s o f S. t y p h i m u r i u m ( 3 2 ) . H o w e v e r , b a s e d on t h e amount o f phenols produced from B l H ï y t h e p u r i f i e d monoxygenase system (cf Table I ) , i t i s u n l i k e l y that these phenols contribute s i g n i f i c a n t l y t o t h e m u t a g e n i c i t y o b s e r v e d f r o m BP m e t a b o l i s m . S i n c e s e v e r a l s t u d i e s h a v e shown t h a t p r i m a r y o x i d a t i v e m e t a b o l i t e s o f BP, i n c l u d i n g phenols, can be f u r t h e r m e t a b o l i z e d b y t h e m o n o x y g e n a s e s y s t e m ( 2 6 , 3 4 ) , we u t i l i z e d a l l t w e l v e o f t h e phenols as s u b s t r a t e s f o r t h e p u r i f i e d monoxygenase system to determine i f f u r t h e r o x i d a t i v e metabolism would r e s u l t i n the formation o f mutagenic products. T a b l e V shows t h e m u t a g e n i c


108


CONCEPTS

0.1 nmol CYTOCHROME

Figure I. Effect of cytochrome P-448 concentration and time of incubation on the metabolism of BP to products mutagenic to strain TA 98 of Salmonella typhimurium. Reaction mixtures were similar to those described in Table III. Each value represents the mean ± S.D. from three replicate incubation mixtures.

Figure 2. Effect of epoxide hydrase on mutations induced bu the metabolism of BP by the purified monoxygenase system (left) and on the mutagenic activity of BP 4,5-oxide (right). Incubation mixtures for the metabolic activation of BP were similar to those described in Table 111. The effect of epoxide hydrase on the mutagenic activity of BP 4,5-oxide was assayed using 0.4 nmole of BP 4,5-oxide in 0.5 ml containing 2 X 10* bacteria. All samples were incubated for 5 min at 37° before addition of the top agar.

10

Î 2 " 2 0

UNITS OF EPOXIOE HYDRASE

•i-^—k

6.

LEVIN ET A L .

Activation

and Detoxification

Table

of Benzo[a]pyrene

109

V

Metabolism o f Benzo|aIpyrene Phenols t o Mutagenic Products by a Pur i f i e T t y t o c h r o m e P-448""Dependen£ Monoxygenase System

Substrate

Cytochrome 0


Hi s

None BP 1-H0BP 2-H0BP 3-H0BP 4-HOBP 5-H0BP 6-HOBP 7-H0BP 8-HOBP 9-HOBP 10-H0BP 11-H0BP 12-H0BP

17 18 43 17 32 15 19 94 35 15 33 14 11 219

P-448 (pmol) 25 50 Revertants/Plate

-

280 109 67 117 22 19 116 43 35 93 22 21 302

17 670 140 73 158 29 24 261 49 45 180 21 21 372

I n c u b a t i o n m i x t u r e s c o n t a i n e d t h e complete monoxygenase system and t y p h i m u r i u m s t r a i n TA 98 a s d e s c r i b e d i n T a b l e I I I u s i n g f i x e d c o n c e n t r a t i o n s o f a l l components e x c e p t cytochrome P-448. The f i n a l c o n c e n t r a t i o n o f BP o r BP p h e n o l s was 25 μ Μ .


110


CONCEPTS

a c t i v i t y o f t h e t w e l v e i s o m e r i c p h e n o l s o f BP i n s t r a i n T A 9 8 b e f o r e and a f t e r m e t a b o l i c a c t i v a t i o n by v a r i o u s amounts o f c y t o chrome P-448. The number o f r e v e r t a n t c o l o n i e s i n t h e a b s e n c e of the cytochrome i s a measure o f the i n t r i n s i c mutagenic a c t i v i t y of the phenols s i n c e the r e c o n s t i t u t e d system i s i n a c t i v e in the absence of the cytochrome. Of t h e t w e l v e p o s s i b l e p h e n o l s o f B P , o n l y 1 - , 2 - , 3 - , 6 - , 9 - and 12-HOBP w e r e m e t a b o l i c a l l y a c t i v a t e d to mutagenic products. However, s i n c e none o f t h e p h e n o l s was as e f f e c t i v e l y c o n v e r t e d t o m u t a g e n i c p r o d u c t s as B P , i t a p p e a r s t h a t f u r t h e r m e t a b o l i s m o f BP p h e n o l s t o m u t a g e n i c products is not responsible f o r the mutagenic a c t i v i t y of metab o l i c a l l y a c t i v a t e d BP. This data, together with the lack of i n h e r e n t m u t a g e n i c a c t i v i t y o f s i x p o s s i b l e BP q u i n o n e s (32), s t r o n g l y s u g g e s t s t h a t n o n e p o x i d e d e r i v a t i v e s o f BP a r e n o t r e s p o n s i b l e f o r t h e m u t a t i o n s o b s e r v e d w h e n BP i s m e t a b o l i z e d t o mutagenic p r o d u c t s i n t h e p r e s e n c e of t h e p u r i f i e d monoxygenase s y s t e m and e p o x i d e h y d r a s e . Metabolic A c t i v a t i o n of Benzolajpyrene Dihydrodiols. Since a d d i t i o n of e p o x i d e hydrase t o t h e p u r i f i e d monoxygenase system was u n a b l e t o b l o c k t h e f o r m a t i o n o f m u t a g e n i c m e t a b o l i t e s o f B P , i t w a s p o s s i b l e t h a t a n a r e n e o x i d e o f BP w a s c o n v e r t e d t o a d i h y d r o d i o l b y a d d i t i o n o f e p o x i d e h y d r a s e w h i c h was t h e n f u r t h e r m e t a b o l i c a l l y a c t i v a t e d t o a potent mutagen. In t h i s r e g a r d , B o r g e n e t a l . (35) have shown t h a t m e t a b o l i c a c t i v a t i o n o f BP 7 , 8 - d i h y d r o d i o l " T 5 y l i v e r m i c r o s o m e s r e s u l t s i n a m u c h g r e a t e r b i n d i n g t o DNA t h a n d o e s s u c h m e t a b o l i c a c t i v a t i o n o f B P , BP 4 , 5 - d i h y d r o d i o l o r BP 9 , 1 0 - d i h y d r o d i o l . Sims e t a l . ( 3 6 ) have p r e s e n t e d e v i d e n c e t h a t a BP 7 , 8 - d i o l - 9 , 1 0 - e p o x " u i e ~ T s Tiïe b i o a c t i v a t e d m e t a b o l i t e o f BP 7 , 8 - d i h y d r o d i o l w h i c h b i n d s t o DNA. I t h a s s i n c e b e e n e s t a b l i s h e d t h a t t h e s t e r e o i s o m e r i c BP 7,8-diol-9,10-epoxides a r e among t h e m o s t p o t e n t m u t a g e n s y e t described (21,31,37-39). We h a v e u t i l i z e d t h e p u r i f i e d m o n o x y g e n a s e s y s t e m i n an a t t e m p t t o m e t a b o l i c a l l y a c t i v a t e t h e f o u r BP d i h y d r o d i o l s w h i c h h a v e b e e n s y n t h e s i z e d i n o u r l a b o r a t o r i e s (21). A s s h o w n i n F i g u r e 3 , BP 7 , 8 - d i h y d r o d i o l w a s a c t i v a t e d t o a t l e a s t a 4 - f o l d g r e a t e r e x t e n t t h a n was B P . In c o n t r a s t t o t h e s e r e s u l t s , t h e BP 4 , 5 - , 9 , 1 0 - a n d 1 1 , 1 2 - d i h y d r o d i o l s were not m e t a b o l i c a l l y a c t i v a t e d to mutagenic m e t a b o l i t e s . The h i g h m u t a g e n i c a c t i v i t y o f a m e t a b o l i t e ( s ) o f BP 7 , 8 - d i h y d r o d i o l t o w a r d s S. t y p h i m u r i u m s t r a i n TA 98 p r o m p t e d f u r t h e r s t u d i e s o n t h e m e t a b o l i c a c t i v a t i o n o f BP 7 , 8 - d i h y d r o d i o l b y t h e p u r i f i e d monoxygenase system. Microsomal epoxide hydrase converts the a r e n e o x i d e s f o r m e d by t h e c y t o c h r o m e P-450 monoxygenase system into trans, v i c i n a l dihydrodiols (15). T a b l e VI shows t h a t ç i s BP 7 , 8 - d i h y d r o d i o l c a n a l s o b e m e t a i ï o l i c a l l y a c t i v a t e d t o a p o t e n t mutagen(s) by t h e monoxygenase s y s t e m , a l t h o u g h i t i s s l i g h t l y less a c t i v e than the trans isomer. These r e s u l t s i n d i c a t e that the r e l a t i v e p o s i t i o n of the hydroxyl groups i s not a c r i t i c a l f a c t o r in determining the mutagenic a c t i v i t y of the

BP 7 , 8 -

2±1

( ± ) - t r a n s BP 7 , 8 dihydroxy-7,8,9, 10-H BP

76+10

130±8

9±2

2

34±3

6±4

320+18

10±3

60±5

20

9±2

120±10

(pmol)

ΉΓ

Revertants7P1ate

522±45

His

+

Cytochrome P-448 5 80

Products

29±4

800±45

t o Mutagenic

I n c u b a t i o n m i x t u r e s c o n t a i n e d t h e c o m p l e t e monoxygenase system as d e s c r i b e d i n T a b l e I I I . The f i n a l s u b s t r a t e c o n c e n t r a t i o n was 25 y M . Background mutation f r e q u e n c i e s have been s u b t r a c t e d . V a l u e s r e p r e s e n t t h e mean + S . E . f o r t h r e e determinations.

4

1±0.5

6±1

1±0.6

( t ) - c i s BP 7 , 8 dihydrodiol

dihydrodiol

(±)-trans

BP

VI

A c t i v a t i o n o f Benzol aipyrene 7 , 8 - D i h y d r o d i o l

Substrate

Metabolic

Table


112



bioactivated metabolite(s). However, 7,8-dihydroxy-7,8,9,10t e t r a h y d r o B P , a c o m p o u n d r e l a t e d t o BP 7 , 8 - d i h y d r o d i o l b u t w i t h t h e d o u b l e bond removed f r o m t h e 9 , 1 0 - p o s i t i o n o f t h e m o l e c u l e , c a n n o t be m e t a b o l i c a l l y a c t i v a t e d t o mutagenic m e t a b o l i t e s by t h e monoxygenase system. These r e s u l t s suggest t h a t the mutag e n i c p r o d u c t f o r m e d o n m e t a b o l i c c o n v e r s i o n o f BP 7 , 8 - d i h y d r o d i o l i s a BP 7 , 8 - d i o l - 9 , 1 0 - e p o x i d e s i n c e t h e d o u b l e b o n d i n t h e 9,10-position of the molecule is a requirement f o r the formation o f an e p o x i d e a t t h a t p o s i t i o n . Metabolic A c t i v a t i o n of Benzolajpyrene 7,8-Oxide. Since BP 7 , 8 - d i h y d r o d i o l w a s r e a d i l y m e t a b o l i z e d t o a m u t a g e n i c p r o d u e t t s ) b y t h e c y t o c h r o m e P - 4 4 8 m o n o x y g e n a s e s y s t e m , we e x a m i n e d t h e e f f e c t o f t h e monoxygenase s y s t e m on t h e m u t a g e n i c a c t i v i t y o f i t s a r e n e o x i d e p r e c u r s o r , BP 7 , 8 - o x i d e ( T a b l e V I I ) . In t h e a b s e n c e o f a d d e d e p o x i d e h y d r a s e , t h e weak i n t r i n s i c m u t a g e n i c a c t i v i t y o f BP 7 , 8 - o x i d e ( 4 0 ) w a s u n a f f e c t e d b y a d d i t i o n o f t h e p u r i f i e d monoxygenase system. Addition of epoxide hydrase to t h e monoxygenase system r e s u l t e d i n a marked i n c r e a s e i n the m u t a t i o n f r e q u e n c y i n S. t y p h i m u r i u m s t r a i n TA 9 8 . These r e s u l t s d e m o n s t r a t e t h a t BP 7 , ï ï - o x i d e i s h y d r a t e d t o t h e c o r r e s p o n d i n g d i h y d r o d i o l w h i c h i n t u r n i s o x i d a t i v e l y m e t a b o l i z e d t o an a c t i v e m u t a g e n ( s ) , p r e s u m a b l y t h e s t e r e o i s o m e r i c BP 7,8-diol-9,10-epoxides. Metabolism of Benzolajpyrene 7,8-Dihydrodiol b^ the P u r i f i e d Monoxygenase System. Based on t h e s t u d i e s o f Borgen e t a l . ( 3 5 ) , Sims e t a l . ( 3 6 ) p r o p o s e d a BP 7 , 8 - d i o l - 9 , 1 0 - e p o x i d e a s t h e DTOa c t i v a t e ï ï " m e T â b o l i t e o f B P 7 , 8 - d i h y d r o d i o l w h i c h b i n d s t o DNA. T h e o x i d a t i v e m e t a b o l i s m o f BP 7 , 8 - d i h y d r o d i o l a t t h e 9 , 1 0 - p o s i t i o n o f t h e m o l e c u l e c o u l d r e s u l t i n t h e f o r m a t i o n o f two p o s s i b l e stereoisomers of the d i o l epoxide (Figure 4). Recently o u r l a b o r a t o r i e s (41) s y n t h e s i z e d and u n e q u i v o c a l l y assigned t h e r e l a t i v e s t e r e o c h e m i s t r y o f t h e t w o s t e r e o i s o m e r s o f BP 7 , 8 d i o l - 9 , 1 0 - e p o x i d e ( d i o l e p o x i d e s 1 and 2 ) , b o t h o f w h i c h a r e p o t e n t mutagens ( 3 1 , 3 7 - 3 9 ) . A d e t a i l e d study of the metabolism of BP-7,8-dihydrodiol is thus necessary in order to determine w h e t h e r one o r b o t h o f t h e s t e r e o i s o m e r s a r e r e s p o n s i b l e f o r t h e m u t a g e n i c a c t i v i t y o f m e t a b o l i c a l l y a c t i v a t e d BP 7 , 8 - d i h y d r o diol. R e c e n t l y , h i g h p r e s s u r e l i q u i d chromatography has been u s e d t o s e p a r a t e m e t a b o l i t e s o f BP 7 , 8 - d i h y d r o d i o l (42«43). Although a d i r e c t demonstration of the formation of the d i o l e p o x i d e s has n o t been p o s s i b l e due t o t h e i r e x t r e m e i n s t a b i l i t y i n aqueous s o l u t i o n s , t e t r a o l s have been i d e n t i f i e d as m e t a b o l i t e s f r o m BP 7 , 8 - d i h y d r o d i o l ( F i g u r e 4 ) . D i o l e p o x i d e s 1 and 2 u n d e r go c i s and t r a n s a d d i t i o n o f w a t e r a t t h e 1 0 - p o s i t i o n o f t h e oxirane (42,43) to form stereoisomeric p a i r s of (±)-7,8,9,10tetrahydroxy-/,8,9,10-tetrahydrο BP d e r i v a t i v e s i n w h i c h t h e r e l a t i v e s t e r e o c h e m i s t r y o f t h e 7 - and 8 - h y d r o x y l group i s a l w a y s t r a n s and t h e r e l a t i v e s t e r e o c h e m i s t r y b e t w e e n t h e 8 - and 9 -

6.

Activation

LEVIN E T A L .

and Detoxification

Table

Metabolic


BP

7,8-oxide

(25 μΜ)

Benzo[z]pyrene

113

VII

A c t i v a t i o n o f BP 7 , 8 - o x i d e

Substrate

of

t o Mutagenic

Metabolites

Monoxygenase System

Epoxide Hydrase

-

-

91±4

+

-

132±10

+

+

514±22

+

His

Revertants/Plate

The c o m p l e t e monoxygenase s y s t e m c o n s i s t e d o f 0 . 0 5 nmol o f c y t o chrome P-448, 150 u n i t s o f NADPH-cytochrome c r e d u c t a s e , 50 ug o f p h o s p h a t i d y l c h o l i n e , 0 . 1 p m o l o f NADPH a n d 2 χ 1 0 b a c t e r i a i n a f i n a l volume o f 0.5 m l . Four u n i t s o f p u r i f i e d epoxide h y d r a s e was added t o t h e a p p r o p r i a t e r e a c t i o n m i x t u r e s ( s e e Table I f o r d e f i n i t i o n of u n i t s of epoxide hydrase activity). The m u t a t i o n s o b s e r v e d i n t h e absence o f t h e monoxygenase system and e p o x i d e h y d r a s e a r e a r e s u l t o f t h e i n h e r e n t m u t a g e n i c i t y o f BP 7 , 8 - o x i d e . Background m u t a t i o n f r e q u e n c i e s have been s u b tracted. V a l u e s r e p e s e n t t h e mean + S . E . f o r t h r e e d e t e r m i n a t i o n s . 8


114 80

CONCEPTS

r

φ

! » Ul g

60

t υ

50

1

40

I

30

χ ο

α

0 - a l k y l ) are r e s p o n s i b l e f o r t h i s e f f e c t (43). Extensive m e t h y l a t i o n o f DNA at N o f guanine by tf-methy1-tfn i t r o s o u r e a i s t y p i c a l o f m e t h y l a t i n g agents. R e a c t i v e aromatic compounds o f h i g h e r molecular weight show d i f f e r e n t s e l e c t i v i t y . For example, tf-acetoxy-2-acetylaminofluorene 3 reacts selectively at C o f guanosine 6 b (44) and t o a l e s s e r degree at the 2-amino group o f guanosine (45). 7-Bromomethylbenζ[a]anthracene 4 (46) and 7,12-dimethylbenz[a]anthracene 5,6-oxide 5 (47) a l k y l a t e pre dominantly at the e x o c y c l i c 2-amino group o f guanosine. P r i o r o r i e n t a t i o n o f the p l a n a r r e a c t a n t by i n t e r c a l a t i o n between the base p a i r s may account f o r s e l e c t i v e a l k y l a t i o n o f the 2-amino group (48). I n t e r c a l a t i o n i n t o n u c l e i c a c i d s a l s o tends t o dra m a t i c a l l y i n c r e a s e the s o l u b i l i t y o f PAH s i n aqueous s o l u t i o n s (49,50). Thus, i n t e r c a l a t i o n o f PAH d e r i v a t i v e s may serve the dual f u n c t i o n o f i n c r e a s i n g the e f f e c t i v e c o n c e n t r a t i o n o f the r e a c t i v e form o f the PAH near the n u c l e i c a c i d and o f o r i e n t a t i n g t h i s r e a c t i v e form f o r s p e c i f i c s i t e s o f the polymer. D i o l epoxides 1 and 2 have been shown t o a t t a c k the e x o c y c l i c 2-amino group o f guanosine i n n u c l e i c a c i d s . D i o l epoxide 2, on r e a c t i o n w i t h p o l y g u a n y l i c a c i d (poly (G)) i n 75% acetone/water, has been shown t o form a p a i r o f d i a s t e r e o m e r i c adducts by t r a n s opening o f the epoxide at C-10 (22). One o f these diastereomers and other u n i d e n t i f i e d products were d e t e c t e d i n UNA i s o l a t e d from bovine b r o n c h i a l e x p i a n t s which had been exposed t o [ H]-BP (23). D i o l epoxide 1 has been shown t o form both c i s and t r a n s adducts at C-10 w i t h the 2-amino group o f guanosine i n p o l y (G) (50% ace tone/water) as w e l l as a l k y l a t e the phosphate backbone t o form p h o s p h o t r i e s t e r s (26). The products formed from the in vitro r e a c t i o n s o f the d i o l epoxides w i t h DNA have been s t u d i e d (9,18,23, 25). We d e s c r i b e here d e t a i l s o f the r e a c t i o n s o f d i o l epoxides 1 and 2 w i t h p o l y (G) and p r o v i d e evidence f o r the s t r u c t u r e o f the RNA adducts which form when [ H]-BP i s p a i n t e d on mouse s k i n . Adducts from both d i o l epoxides 1 and 2 are formed i n t h i s in vivo 7

3

6


7

6

7

8

1

3

3

MOORE ET A L .

Diastereomeric


g,

9,10-Epoxides

from

c/s-2

Benzo[a]pyrene

trans-Z

Figure 1. Hydrolysis of diol epoxides to tetraoh. Unless specifically designated, diol epoxide 1 and diol epoxide 2 refer to racemic compounds. The structures 1 and 2 are intended to show the rehtive stereochemistry between the two sets of diastereomers and to show the absolute stereochemistry of the diol epoxide products derived from the (—) enantiomer of BP 7,8-aihydrodiol.


130

experiment w i t h a t i s s u e toward which BP i s c a r c i n o g e n i c . These adducts are o p t i c a l l y a c t i v e and t h e i r absolute stereochemistry has been assigned through the use o f the o p t i c a l l y pure enantio mers o f d i o l epoxides 1 and 2. M a t e r i a l s and Methods D i o l Epoxides. D i o l epoxide 1 was synthesized from BP 7,8d i h y d r o d i o l v i a a b r o m o - t r i o l , w h i l e d i o l epoxide 2 was obtained by m-chloroperbenzoic a c i d o x i d a t i o n o f the same BP 7,8-dihydro d i o l (15,16). T r i t i a t e d d i o l epoxides were s y n t h e s i z e d from [9, 10- H]-BP 7,8-dihydrodiol as p r e v i o u s l y d e s c r i b e d (27) and were used at a s p e c i f i c a c t i v i t y o f 2.3 pCi/ymol. O p t i c a l l y a c t i v e d i o l epoxides 1 and 2 were s y n t h e s i z e d from (+) and (-)-BP 7,8d i h y d r o d i o l which had been r e s o l v e d v i a the d i a s t e r e o m e r i c b i s (-)-a-methoxy-a-trifluoromethylphenylacetates (51-53). B i n d i n g o f D i o l Epoxides t o Poly (G). The potassium s a l t o f p o l y g u a n y l i c a c i d ( 5 ) (mol. wt. > 150,000) was obtained from Sigma. [ 8 - H ] - P o l y g u a n y l i c a c i d was purchased from M i l e s Research Products and used at a s p e c i f i c a c t i v i t y o f 0.31 yCi/ymol o f phos phorous. For a t y p i c a l b i n d i n g experiment, 1.7 mg o f p o l y (G) was d i s s o l v e d i n 0.5 ml o f d i s t i l l e d water and the pH was adjusted by a d d i t i o n o f d i l u t e HC1 o r KOH. A pH o f 7.0 (monitored w i t h a pH e l e c t r o d e ) was used i n a l l cases except those s p e c i f i c a l l y d e s i g nated. An equal volume o f s p e c t r o s c o p i c grade acetone was added and the s o l u t i o n was t r e a t e d w i t h 0.4 mg o f e i t h e r d i o l epoxide i n 100 μΐ o f dry t e t r a h y d r o f u r a n (THF). The s o l u t i o n s were incubated at 37 C f o r 18 hours. T e t r a o l s which r e s u l t from h y d r o l y s i s were removed by e x t r a c t i o n w i t h t h r e e equal volumes o f e t h y l a c e t a t e . The modified polymer was i s o l a t e d by a c i d i f i c a t i o n o f the s o l u t i o n w i t h 0.1 volume o f 2.5 M sodium acetate (pH 5.0) and p r e c i p i t a t i o n o f the polymer w i t h 3 volumes o f e t h a n o l . The mixture was s t o r e d at 0°C overnight and then c e n t r i f u g e d f o r 15 min. at 3,000 rpm. The supernatant was removed and the p r e c i p i t a t e d polymer was wash ed w i t h 2.0 ml o f ethanol. The polymer could be r e d i s s o l v e d i n water and the p r e c i p i t a t i o n procedure was repeated without s i g n i f i c a n t change i n the amount o f bound hydrocarbon absorbing at 350 nm (54). H y d r o l y s i s o f P o l y (G) t o Nucleosides. M o d i f i e d p o l y (G) was d i s s o l v e d i n 1.0 Ν KOH and incubated f o r 18 hours at 37°C. The s o l u t i o n was n e u t r a l i z e d w i t h 2 N HCIO4 which r e s u l t e d i n p r e c i p i t a t i o n o f KCIO4. A f t e r c e n t r i f u g a t i o n the s a l t was separated by decanting the aqueous s o l u t i o n . Tris-(hydroxymethyl)-aminomethane was added t o the aqueous supernatant t o produce a f i n a l concentra t i o n o f 1.0 mg/ml and the pH was adjusted t o 8.4. Bacterial alka l i n e phosphatase (1 unit/mg o f p o l y (G)) was added and the s o l u t i o n was incubated f o r 24 hours at 37°C, which a f f e c t e d complete h y d r o l y s i s t o n u c l e o s i d e s as monitored by paper chromatography. Removal o f Ribose by N - M e t h y l a t i o n w i t h Dimethyl S u l f a t e . A f t e r base h y d r o l y s i s , the modified n u c l e o t i d e mixture from 50 mg o f p o l y (G) was d i s s o l v e d i n 10 ml o f 2 M KH2PO4 at pH 7.0 and


3

f

3

e

7

7.

MOORE E T A L .

Diastereomeric

9,10-Epoxides

from Benzofajpyrene

131

t r e a t e d w i t h three 100 y l a l i q u o t s o f dimethyl s u l f a t e a t 2 hour i n t e r v a l s . The pH was maintained > 6.0 by o c c a s i o n a l a d d i t i o n o f d i l u t e KOH. When the uv spectrum o f the mixture showed the spec t r a l s h i f t s w i t h pH which are c h a r a c t e r i s t i c o f 7-methylguanosine (55), the pH was adjusted t o 6.0 and the mixture was r e f l u x e d f o r 1 hour t o remove the r i b o s e from the methylated base. The d i o l epoxide-7-methylguanine base was s e l e c t i v e l y e x t r a c t e d w i t h t h r e e equal volumes o f e t h y l acetate. Treatment o f Mice w i t h [ H]-BP. The backs o f eleven C57BL/6J female mice were shaved and, two days l a t e r each mouse was p a i n t e d w i t h 100 ]xg o f [ H]-BP (Amersham S e a r l e ) (1.4 mCi/mouse) i n 100 y l o f acetone. Twenty f o u r hours l a t e r the mice were s a c r i f i c e d , and the epidermal l a y e r was i s o l a t e d and homogenized as d e s c r i b e d (56) The RNA was, i s o l a t e d by a p h e n o l - c r e s o l method (57). The 1.3 mg o f RNA thus obtained contained % 2.8 χ 1 0 dpm/mg. High-pressure L i q u i d Chromatography. The n u c l e o s i d e adducts from 1 and 2 were c o n v e n i e n t l y separated from unmodified guanosine w i t h a Poragel PN column (3/8" χ 3 ) e l u t e d w i t h 85% methanol i n water a t a f l o w r a t e o f 5.0 ml/min. For high r e s o l u t i o n a n a l y s i s o f the n u c l e o s i d e adducts, a Waters yC^g-Bondapak column (1/4" χ l ) was e l u t e d a t a constant r a t e o f 1.2 ml/min w i t h 39% methanol/ water f o r 50 minutes f o l l o w e d by a 60 minute l i n e a r gradient t o 50% methanol/water. 3

3


5

f

f

Results At pH 7.0 d i o l epoxides 1 and 2 were found t o b i n d t o p o l y (G) t o about the same extent. Binding was c h a r a c t e r i z e d by the uv a b s o r p t i o n p a t t e r n o f the 7,8,9,10-tetrahydro-BP moiety i n the 320 - 355 nm r e g i o n (Figure 3). The a b s o r p t i o n maximum at 352 nm r e presents a bathochromic s h i f t o f 9 nm and a s u b s t a n t i a l decrease i n the e x t i n c t i o n c o e f f i c i e n t which would be expected f o r the 7, 8,9,10-tetrahydro-BP chromophore i n t h i s r e g i o n . These changes i n the chromophore l a r g e l y disappear upon a l k a l i n e h y d r o l y s i s o f the modified p o l y (G) ( i n s e t F i g u r e 3 ) . This c l e a r l y i n d i c a t e s t h a t the hydrocarbon i s c l o s e l y a s s o c i a t e d w i t h the polymer (50). Evidence p r o v i n g t h a t t h i s a s s o c i a t i o n i n v o l v e s covalent bond formation i s presented l a t e r . E f f e c t o f pH on Rate and Extent o f Binding. The r a t e and extent o f b i n d i n g were explored w i t h [9,10-^H]-diol epoxides s i n c e t o t a l b i n d i n g could be a c c u r a t e l y determined r a d i o c h e m i c a l l y . B u f f e r s o l u t i o n s were avoided s i n c e the d i o l epoxides r e a c t w i t h phosphate i o n s . For example, from 5 - 10% o f the hydrocarbon becomes non-extractable due t o a l k y l a t i o n o f 0.05 M phosphate a t pH 7.0 (26). When these phosphate-diol epoxide products were heated f o r 1 hour on a steam bath, a l l o f the hydrocarbon was l i b erated from i t s phosphate e s t e r s . When d i o l epoxide was added t o pure water o n l y a t r a c e ( « 1%) o f hydrocarbon remained a f t e r e x t r a c t i o n , which i n d i c a t e s t h a t very l i t t l e non-extractable s o l v o l y s i s products are formed.



132

Figure 3. Ultraviolet spectra of poly (G), diol epoxide 1, and poly (G) modified by diol epoxide 1. The following c values were observed, poly (G) in water € o = 9,000 and diol epoxide 1 in dry THF € s = 46,500. The inset shows repetitive scans of poly (G)-diol epoxide 1 in 0.5N KOH at 25°C. 26

Si

7. MOORE E T A L .

Diastereomeric

9,10-Epoxides

from Benzo[a]pyrene

133

The e f f e c t o f pH on t o t a l b i n d i n g i s shown i n F i g u r e 4. Maximum b i n d i n g takes p l a c e i n s l i g h t l y a c i d i c media. Both d i o l epoxides showed s i m i l a r pH-dependent r a t e s o f b i n d i n g t o p o l y (G). Binding o f d i o l epoxide 1 was s l i g h t l y f a s t e r but q u a l i t a t i v e l y the same as t h a t o f d i o l epoxide 2. Figure 5 shows t h e r a t e o f b i n d i n g f o r d i o l epoxide 2 a t v a r i o u s pH v a l u e s . The r a t e o f b i n d i n g a t pH 4.0 i s a t l e a s t twenty times g r e a t e r than t h a t a t pH 7.0. Even though b i n d i n g a t pH 4.0 i s much f a s t e r than t h a t a t pH 7.0, t h e t o t a l amount bound a f t e r 2 hours o f i n c u b a t i o n i s s l i g h t l y l e s s . These observations a r e c o n s i s t e n t w i t h a c i d c a t a l y z e d a l k y l a t i o n o f the polymer competing w i t h a c i d c a t a l y z e d hyd r o l y s i s o f the d i o l epoxide t o t e t r a o l s .


Reaction on the Phosphate Backbone o f P o l y (G) When p o l y (G) was modified by e i t h e r d i o l epoxide and p u r i f i e d by s o l v e n t e x t r a c t i o n and m u l t i p l e p r e c i p i t a t i o n , non-bound t e t r a o l s were completely removed. The uv spectrum o f t h i s p u r i f i e d modified p o l y (G) showed a d i s t i n c t v a l l e y a t 343 nm. When the modified polymer was heated t o 100°C a t n e u t r a l pH a small amount o f f r e e t e t r a o l s was l i b e r a t e d and could be detected by t h e i r uv spectrum. The l i b e r a t i o n o f t e t r a o l s was confirmed by co-chromatography w i t h standards and by the mass spectrum o f t h e t e t r a a c e t y l d e r i v a t i v e (M a t m/e 488). A f t e r e x t r a c t i o n o f these t e t r a o l s prolonged h e a t i n g (4 hours) o f the modified p o l y (G) f a i l e d t o l i b e r a t e f u r t h e r q u a n t i t i e s o f t e t r a o l s . Base hydrolys i s o f modified p o l y (G) which had not been heated i n water a l s o l e d t o the formation o f t e t r a o l s . Q u a n t i t a t i v e measurements i n d i cated t h a t 10 - 15% o f the t o t a l bound hydrocarbon could be r e leased as t e t r a o l s . Since p o l y c y c l i c aromatic hydrocarbons, such as t h e t e t r a o l s or the d i o l epoxides themselves, can p h y s i c a l l y i n t e r c a l a t e i n t o n u c l e i c a c i d s t h e p o s s i b l e presence o f r e s i d u a l non-covalently1inked hydrocarbon d e r i v a t i v e s cannot, a priori, be e l i m i n a t e d . A d d i t i o n o f p o l y (G) modified by 1 t o 0 enriched water (18%) f o l l o w e d by h e a t i n g f o r 15 minutes a t 100°C generated t e t r a o l s which i n c o r p o r a t e d 0.96 atom % o f s o l v e n t oxygen. When c i s - 1 and trans-l t e t r a o l s were added t o t h i s same p o l y (G) s o l u t i o n and s i m i l a r l y t r e a t e d , no i n c o r p o r a t i o n o f 0 i n the r e i s o l a t e d t e t r a o l s was observed. This r e s u l t s t r o n g l y argues against the presence o f p h y s i c a l l y bound t e t r a o l s . Since the h a l f - l i f e f o r b i n d i n g i s l e s s than 10 minutes a t pH 7.0, i t seems u n l i k e l y t h a t i n t a c t d i o l epoxides could have s u r v i v e d d u r i n g the i n c u b a t i o n w i t h out e i t h e r b i n d i n g t o the p o l y (G) o r undergoing h y d r o l y s i s t o ext r a c t a b l e t e t r a o l s . I n a d d i t i o n , i t i s u n l i k e l y t h a t t h e d i o l epoxides could s e l e c t i v e l y r e s i s t e x t r a c t i o n compared t o t e t r a o l s . R e l a t i v e amounts o f c i s and t r a n s t e t r a o l s which form i n v a r i o u s c o n d i t i o n s are shown i n Table I I . Base h y d r o l y s i s o f d i o l epoxide 1 leads t o s u b s t a n t i a l trans-l t e t r a o l w h i l e the t e t r a o l s gener+

i 8

1 8



Ο·

4.0

,

1

eo

ao

1—

ιαο

PH

Figure 4. Extent of binding as a function of pH. Poh (G) solutions of 3.4 mg/ml were adjusted to pH 4.0, 6.0, 7.0, 8.0, ana 10.0, then diluted with an equal volume of acetone. 2.95 ftCi [9,10H] diol epoxide I or 2 was added to 1.0 ml of the stock poly (G) solu tions at 37°C, and 50-μΙ aliquots of the reaction mixture were removed at convenient time intervals. Each aliquot was added to a tube containing 1.0 ml of H 0 and 5.0 ml of ethyl acetate. This mixture was shaken vig orously, and the ethyl acetate was removed. The aqueous solution of poly (G) was then extracted twice with 5.0 ml of ethyl acetate. Since tetraols are readily extracted from aqueous poly(G) solutions the [ H] counts present were assumed to be bound to poly (G). Results obtained when uv absorbance at 350 nm was used to quantitate binding indicated that the above extraction procedure was correct to ±10%. Diol epoxide 1 was examined after 60 min of reaction and diol epoxide 2 after 50 min of reaction. Points represent the average of two separate determinations. 3

2

3

7.

M O O R E ET A L .

Diastereomeric

9,10-Epoxides

from Benzolajpyrene

135

Table I . M e t h y l a t i o n o f Salmon Sperm DNA by tf-Methyl-tfn i t r o s o u r e a . Data o f Lawley and Shah (38).

Site

Base

Guanine

% Total i d e n t i f i e d m e t h y l a t i o n o f bases

7

Ν 0 N3

75.7 7.3 1.1

6

3 Adenine


Thymidine

i N N

Source

1

7

N 0

3

N

3

Cytidine

Table I I .

11.2 1.4 2.5

N

0.3 0.1

4

0.6

Percent c i s and t r a n s T e t r a o l s from D i o l Epoxides 1 and 2 and M o d i f i e d Poly (G).

Conditions

50% acetone/water, pH 7.0 ( c o n d i t i o n s of binding)

c i s - 1 trans-l

85

trans-2

15

50% acetone/water, pH 7.0 ( c o n d i t i o n s of binding) 75

cis-2

15

85

39

61

2

98

25

P o l y (G)

100°C, pH 7.0, 15 minutes

Poly (G)

100°C, pH 7.0, 15 minutes

1

1.0 Ν KOH, 20% THF/H 0, 37°C, 24 h r s .

18

81

Poly (G)

1.0 Ν KOH, 37°C, 24 h r s .

95

5

P o l y (G)

1.0 Ν KOH, 37°C, 24 h r s .

2


136


ated from base h y d r o l y s i s o f p o l y (G) modified by 1 are almost ex c l u s i v e l y c i s - 1 t e t r a o l s . T h i s provides another argument t h a t the l a b i l e hydrocarbon products are not due t o r e s i d u a l d i o l epoxides. These r e s u l t s are c o n s i s t e n t w i t h the presence o f a heat and a l k a l i n e l a b i l e covalent d i o l epoxide adduct. The formation o f phosphate-diol epoxide monoesters which could be cleaved by simple h e a t i n g at pH 7.0 together w i t h the chemical and thermal s t a b i l i t y o f the guanosine adducts d i s c u s s e d below s t r o n g l y suggest t h a t the l i b e r a t e d t e t r a o l s are d e r i v e d from a l k y l phosphates o f p o l y (G) (37,40-42,58). Reaction on the Guanine Base o f P o l y (G). When modified p o l y (G) was hydrolyzed i n base, the t e t r a o l s r e l e a s e d from the phosphate adducts were r e a d i l y removed by e x t r a c t i o n o f the neu t r a l i z e d h y d r o l y s a t e w i t h e t h y l a c e t a t e . A f t e r a l k a l i n e phospha tase treatment, the n u c l e o s i d e adducts became s u f f i c i e n t l y nonp o l a r t o a l l o w e x t r a c t i o n i n t o organic s o l v e n t s . However, the n u c l e o s i d e adducts were more c o n v e n i e n t l y i s o l a t e d by reverse phase chromatography o f the h y d r o l y s a t e on a Poragel PN high-pres sure l i q u i d chromatography column (Figure 6 ) . Guanosine, nucleo s i d e adducts, and t e t r a o l s are a l l separated. The h i g h c a p a c i t y o f the column a l l o w s s e p a r a t i o n o f 10 - 100 mg samples w i t h a s i n g l e i n j e c t i o n . The n u c l e o s i d e adduct f r a c t i o n contains > 95% o f a l l non-tetraol-hydrocarbon products. High r e s o l u t i o n chromatography on yC -Bondapak separated the n u c l e o s i d e adduct f r a c t i o n i n t o a number o f peaks. Each d i o l ep oxide formed f o u r products which, on the b a s i s o f peak a r e a , ap peared t o c o n s i s t o f two groups. N u c l e o s i d e - d i o l epoxide 1 prod u c t s were e l u t e d i n the approximate r a t i o o f 1:2:1:2. Products from d i o l epoxide 2 were o v e r a l l s l i g h t l y more p o l a r and e l u t e d i n r a t i o o f 3:1:1:3 (Figure 7 ) . The CD s p e c t r a (Figure 8) o f the i n d i v i d u a l peaks e s t a b l i s h e d t h a t the products o f equal peak area were a c t u a l l y p a i r s o f diastereomers which r e s u l t e d from the r e a c t i o n o f racemic d i o l epoxides w i t h o p t i c a l l y pure p o l y (G). F u r t h e r p r o o f t h a t the p a i r s o f products are diastereomers was ob t a i n e d through the use o f o p t i c a l l y pure d i o l epoxides 1 and 2, s i n c e o n l y one component o f each p a i r was obtained from each o f the d i o l epoxide enantiomers (Table I I I ) . Thus, when ( + ) - d i o l ep oxide 1 d e r i v e d from (+)-BP 7,8-dihydrodiol was reacted w i t h p o l y (G), o n l y n u c l e o s i d e adducts F and D were formed. S i m i l a r l y , (+)d i o l epoxide 2 from (-)-BP 7,8-dihydrodiol produced o n l y nucleo s i d e adducts Β and G. S t r u c t u r e o f the Guanosine Adducts. Normally the p o s i t i o n at which a p u r i n e base i s a l k y l a t e d can r e a d i l y be e s t a b l i s h e d by uv spectroscopy. C h a r a c t e r i s t i c changes i n the uv s p e c t r a o f the a l k y l a t e d bases as a f u n c t i o n o f pH g e n e r a l l y p r o v i d e s s u f f i c i e n t evidence f o r assignment o f the s i t e o f a l k y l a t i o n (37). This ap proach proved t o be o f l i m i t e d value i n the present study s i n c e the uv s p e c t r a o f the n u c l e o s i d e adducts as a f u n c t i o n o f pH showed l i t t l e change. The a b s o r p t i o n spectrum o f the 7,8,9,1018

Diastereomenc

MOORE ET AL,


7.

40

9,10-Epoxides

from Benzo[a]pyrene

137

Figure 5. Rate of binding of diol epoxide 2 to poly (G) as a function of pH. Conditions were the same as in Figure 4 with the exception that the binding was examined at the indicated time intervals.

60

Time (min)

guanosine

nucleoside adducts

tetraols 5

10

15

20

25

Figure 6. Purification of guanosinediol epoxide 1 products via Poragel PN chromatography (see Materiats and Methods" for details) u

Tîme(min)


138


CONCEPTS

E; Diol Epcoode 2

10

20

30

40

50 60 Time (min)

70

80

90

100

110

Figure 7. High resolution chromatography of the guanosine adducts from diol epoxides 1 ana 2. Conditions are as described in "Materials and Methods." The peak with a retention time of 39 min in the lower trace is caused by trans-2 tetraol from diol epoxide 2 which was a contaminate in this sample.

MOORE ET

AL.

Diastereomeric

9,10-Epoxides

from

Benzo[a]pyrene


•125-1

-I25H

Figure 8. CD spectra of the major pairs of guanosine adducts from diol epoxide 1(D and H) and diol epoxide 2 (A and G). Spectra were determined in methanol, and Δε is based on e = 37,000. Both sets of products were isolated as in Figure 7 and will be shown later to arise from trans addition of the 2-amino group of guanosine at C-10 of the respective diol epoxide. The two pairs of diastereomeric cis addition products show almost identi cal mirror image CD spectra simUar to Ό and H. sil


140

Table I I I .

R e l a t i o n between t h e Nucleoside Adducts and t h e O p t i c a l l y A c t i v e BP 7,8-Dihydrodiol and D i o l Epoxide Products from Which They Are Derived.

BP 7 , 8 - D i h y d r o d i o l

a

Diol Epoxide

a

Nucleoside Adduct" (+)- c i s '

C+) - d i o l epoxide 1

S y m b o l

m n

F

(-)-trans

79

D

(-)- c i s

70

C

108

H

(+)-cis

89

Ε

(-)-trans

56

A

64

Β

93

G

d

0

(-)- d i h y d r o d i o l

(+)- d i h y d r o d i o l

(-)- d i o l epoxide 1

(-)- d i o l epoxide 2

(+)-trans

(-)-cis° (-)- d i h y d r o d i o l

(+)- d i o l epoxide 2

(•)-trans

e

r i \e 92

c

(+)- d i h y d r o d i o l


CONCEPTS

cd

* Sign o f [o] . Sign o f CD Cotton e f f e c t a t 250 nm. Nucleoside adducts i d e n t i f i e d i n the RNA i s o l a t e d from t h e s k i n of mice which had been t r e a t e d w i t h BP. Due t o co-chromatography, n u c l e o s i d e products F and G can not be d i s t i n g u i s h e d . Chromatographic p r o f i l e o f n u c l e o s i d e adducts (see F i g u r e 7 ) . D

b

c

e


7.

Diastereomeric

MOORE ET AL.

9,10-Epoxides

from Benzolajpyrene

141

tetrahydro-BP chromophore, whose e x t i n c t i o n c o e f f i c i e n t a t 270 nm i s almost 10 times as l a r g e as t h a t o f guanosine (Figure 9 ) , masked changes t h a t presumably occur i n the p u r i n e chromophore on changing pH. I n a d d i t i o n , the a b s o r p t i o n spectrum near 340 nm, which i s due e n t i r e l y t o the hydrocarbon p o r t i o n o f the adduct, was observed t o change w i t h pH. D i f f e r e n c e uv s p e c t r a between the n u c l e o s i d e adducts and the t e t r a o l s p r o v i d e d l i t t l e h e l p s i n c e the a b s o r p t i o n p a t t e r n i n the n u c l e o s i d e adducts i s s l i g h t l y s h i f t e d compared t o t h a t o f t e t r a o l s . Without the conventional s p e c t r a l method f o r assignment o f the p o s i t i o n o f a l k y l a t i o n , chemical methods were used t o l o c a t e the s i t e o f s u b s t i t u t i o n on the guanine base. A c i d H y d r o l y s i s o f the Nucleoside Adducts. When the d i a s t e r eomeric mixture o f p u r i f i e d n u c l e o s i d e adducts from 1 was heated i n 0.1 N HC1, two t e t r a o l s (cis-1 and trans-1) and guanosine were r a p i d l y generated. At 85°C the h a l f - l i f e f o r r e l e a s e o f t e t r a o l s i s approximately 15 minutes. When these n u c l e o s i d e adducts were t r e a t e d w i t h 0.1 N HC1 i n 1 0 enriched water (18%) a t 85°C, mass s p e c t r a l a n a l y s i s o f the t e t r a o l mixture a f t e r a c e t y l a t i o n showed i n c o r p o r a t i o n o f 0.96 atom % 0 i n t o the i s o l a t e d t e t r a o l s . O v e r a l l recovery o f the t e t r a o l s exceeded 80%. Under these cond i t i o n s the t e t r a o l s , c i s - 1 and trans-1, i n c o r p o r a t e d 0,86 atom % o f one s o l v e n t oxygen (26). Since a carbon-carbon bond would not be expected t o be a c i d - l a b i l e , the experiment suggest an oxygen o r n i t r o g e n t o carbon bond i n the s i t e o f attachment. Reaction o f D i o l Epoxides 1 and 2 w i t h [ 8 - H ] - P o l y (G)• At pH 7.0 the most r e a c t i v e s i t e on the guanosine base towards a l k y l a t i n g reagents i s g e n e r a l l y the N p o s i t i o n (37). A f t e r methyla t i o n a t N the hydrogen a t C becomes r e a d i l y exchangeable w i t h water (59). Thus, treatment o f [ 8 - H ] - p o l y (G) w i t h d i o l epoxides 1 o r 2 would p r o v i d e a s e n s i t i v e t e s t f o r C o r N a l k y l a t i o n . Binding o f the d i o l epoxides 1 and 2 w i t h [ 8 - H ] - p o l y (G) was performed i n 50% acetone/water a t pH 7.0. A f t e r i n c u b a t i o n overn i g h t , the s o l u t i o n s were d i l u t e d w i t h 5 volumes o f pH 7.0 phosphate b u f f e r , and the water was recovered by d i s t i l l a t i o n a t 25°C under h i g h vacuum. Release o f t r i t i u m above the blank o f 0.5% was not observed f o r e i t h e r d i o l epoxide d e s p i t e m o d i f i c a t i o n o f 10% o f the guanosine s i t e s . The r e s i d u a l m o d i f i e d p o l y (G) was hydrol y z e d t o n u c l e o s i d e s which were p u r i f i e d by Poragel PN chromatography. These n u c l e o s i d e adducts had a s p e c i f i c a c t i v i t y which was > 80% o f t h a t o f the s t a r t i n g guanosine i n [ 8 - H ] - p o l y (G). T h i s r e s u l t i s c o n s i s t e n t w i t h a l k y l a t i o n a t a s i t e other than a t C o r N o f guanosine. However, exchange o f the C hydrogen o f 7-methylguanosine r e l i e s on the s t a b i l i t y o f the 7-methylimmonium i o n . The s t a b i l i t y o f the h i g h l y hindered immonium i o n which would form i f the d i o l epoxides r e a c t e d a t N i s p r e s e n t l y unknown. The h y p o t h e t i c a l pathway i n F i g u r e 10 f o l l o w s the p r o posed r o u t e o f a l k a l i n e (pH 9 - 10) decomposition o f 7-methylguanosine and would r e s u l t i n r e t e n t i o n o f t r i t i u m a t C . Strong 8

1 8

3

7

7

8

3

8

7

3

3

8

7

8

7

8



CONCEPTS

nm

Figure 9. Ultraviolet spectra of nucleoside adducts from diol epox ide 1 in methanol as a function of pH. Neutral ( ); alkaline (— ); acidic ( ). The c values of the adduct mixture are based on the specific activity of the [9J.0~ H] diol epoxides. S

no exchange of

Ή

Figure 10. A possible decomposition pathway of"N -substitutedguano sine leading to retention of tritium in the product. DEBP represents diol epoxide lor 2 after opening of the 9,10-epoxide at C-10. 7

7.

MOORE

Diastereomeric

ET AL.

9,10-Epoxides

143

from Benzofajpyrene

base treatment o f the mixture o f formamides would produce the non v o l a t i l e s a l t o f [ l - H ] - f o r m i c a c i d (60). To t e s t f o r t h i s pos s i b i l i t y , the p u r i f i e d n u c l e o s i d e adducts from [8- H]-poly (G) were heated a t 100°C i n 2 Ν KOH, c o n d i t i o n s where [8- H]-guanosine exchanges. Most o f the t r i t i u m d i s t i l l e d from t h i s b a s i c s o l u t i o n . A f t e r a c i d i f i c a t i o n and r e d i s t i l l a t i o n v o l a t i l e [ 1 - H ] formic a c i d was not found i n the d i s t i l l a t e . The f a i l u r e t o r e lease v o l a t i l e [1- H]-formic a c i d upon a c i d i f i c a t i o n e l i m i n a t e d a l k y l a t i o n a t N and the mechanism i n Figure 10. Taken t o g e t h e r , these r e s u l t s completely exclude N and C as the s i t e o f a t t a c h ment o f the hydrocarbon. Base S t a b i l i t y o f Nucleoside Adducts. I n 1.0 Ν KOH a t 100°C c e r t a i n (Table IV) s u b s t i t u t e d p u r i n e r i b o s i d e s r a p i d l y degrade. This degradation can proceed by a t t a c k o f hydroxide on the i m i d azole r i n g a t C o r a t v a r i o u s s i t e s o f the p y r i m i d i n e r i n g . However, p u r i n e r i b o s i d e s w i t h a hydrogen on the N p o s i t i o n , except N d e r i v a t i v e s , are deprotonated t o y i e l d a monoanion and become s t a b i l i z e d towards f u r t h e r r e a c t i o n w i t h base (60). A c c o r d i n g l y , Ν , Ν , N , and 0 s u b s t i t u t e d guanosines are known t o be degraded (Table IV) w h i l e N s u b s t i t u t e d guanosine i s s t a b l e towards base treatment (61,62). Treatment o f n u c l e o s i d e adducts w i t h 1 Ν KOH a t 100°C f o r 2 hours r e s u l t e d i n o n l y minor degra d a t i o n and allowed h i g h recovery o f unchanged adducts. This s t r o n g l y suggests the presence o f an amidic proton i n the nucleo s i d e adducts. Determination o f the p K Values o f the Nucleoside Adducts. Measurement o f the p K s o f the n u c l e o s i d e adducts could e s t a b l i s h whether o r not a f r e e amidic proton i s present a t N . Spectrophotomeric t i t r a t i o n was p o s s i b l e but subject t o u n c e r t a i n t y due t o the s m a l l change i n the uv spectrum t h a t occurred upon i o n i z a t i o n . Since the sample s i z e precluded d i r e c t t i t r a t i o n , a more s e n s i t i v e and r e l i a b l e method was sought. The n u c l e o s i d e adducts are s o l u b l e i n water but can be e x t r a c t e d i n t o p o l a r o r g a n i c s o l vants. Since the i o n i z e d form o f guanosine i s much more s o l u b l e i n water than the n e u t r a l form, a p K should be d e t e c t a b l e by a change i n p a r t i t i o n c o e f f i c i e n t w i t h pH (26,63) (Figure 11). The a c i d i c pKa a t 9.8 i n d i c a t e s deprotonation from n e u t r a l n i t r o g e n and the b a s i c pKa a t 1.5 i n d i c a t e s p r o t o n a t i o n o f n e u t r a l n i t r o gen. Although the pKa values obtained are approximate due t o the s e l e c t i v e removal o f one component from the acid-base e q u i l i b r i u m , the presence o f a f r e e N proton i s c l e a r l y demonstrated. This r e s u l t , i n combination w i t h the s t a b l e nature o f the n u c l e o s i d e adducts towards base treatment, e l i m i n a t e s N i , N , and 0 as pos s i b l e s i t e s o f attachment. Since N and C s u b s t i t u t i o n had pre v i o u s l y been e l i m i n a t e d , an N s u b s t i t u t e d guanosine becomes the s o l e p o s s i b l e s t r u c t u r e f o r the n u c l e o s i d e adducts. 3

3

3

3

3

7

7

8

8


1

7

1

3

7

6

2

a

f

a

1

a

1

3

7

2

8

6

144



Table IV. E f f e c t o f 1.0 Ν KOH a t 100°C on S u b s t i t u t e d Guanosines.

7.

MOORE

ET AL.

Diastereomeric

9,10-Epoxides

from Benzo[a]pyrene

145

Nmr and Mass S p e c t r a o f the Adducts. F i n a l d e t e r m i n a t i o n o f the s t r u c t u r e s o f t h e n u c l e o s i d e adducts from 1 by mass s p e c t r o metry and p r o t o n nmr was g r e a t l y handicapped by the added mass and the i n t e r f e r i n g resonances o f the r i b o s e moiety. S i n c e a c i d treatment cleaved the hydrocarbon-guanosine bond much more r e a d i l y than the g l y c o s i d i c l i n k a g e , the normal method o f d i r e c t d e p u r i n a t i o n w i t h a c i d was not u s e f u l . An a l t e r n a t i v e m i l d method f o r cleavage o f the g l y c o s i d i c l i n k a g e was sought. S i n c e the N p o s i t i o n was known not t o be s u b s t i t u t e d and s i n c e dimethyl s u l f a t e shows a marked p r e f e r e n c e f o r a l k y l a t i o n o f N , the mixture o f f o u r n u c l e o t i d e adducts o b t a i n e d from d i o l epoxide 1 was t r e a t e d w i t h dimethyl s u l f a t e t o l a b i l i z e the r i b o s e l i n k a g e (55) (Figure 12). Mass spectrometry o f the d e r i v e d t r i a c e t a t e o f the methylated adducts by chemical i o n i z a t i o n w i t h methane showed t h a t 7-methylguanine-tetrahydro-BP t r i a c e t a t e s had been formed. Chromatography on ODS allowed the i s o l a t i o n o f two products (Figure 13) 8 and 9 w i t h i d e n t i c a l mass s p e c t r a (26). Only two adducts were expected s i n c e l o s s o f the o p t i c a l l y pure r i b o s e moiety reduces each p a i r o f o p t i c a l l y a c t i v e diastereomers w i t h m i r r o r image CD s p e c t r a i n t o s i n g l e racemic compounds. F o u r i e r t r a n s f o r m 220 MHz p r o t o n nmr o f the major product showed t h r e e a c e t y l s i n g l e t s a t 1.98, 2.04, and 2.25, one broad N-methyl s i n g l e t a t 3.38, the f o u r one proton s i g n a l s from p o s i t i o n s 7,8,9, and 10 o f the tetrahydro-BP r i n g a t 6.71, 5.52, 5.58, and 6.14, r e s p e c t i v e l y , and the remain i n g C and pyrene s i g n a l s from 7.95 t o 8.28 ppm. Comparison o f the chemical s h i f t s and c o u p l i n g constants o f t h i s product ( ^7 « = 5.2, J q = 5.4, J q i o = 2.6 Hz) w i t h those eq, eq eq, eq ' eq, eq 7


7

8

3

3

7

3

8f t

8

9

y

l u

o f the 9 , 1 0 - t r a n s - a n i l i n e adduct (--NHC H a t C-10 o f 8) from d i o l epoxide 1 (28) c o n v i n c i n g l y e s t a b l i s h e d t h a t the major prod u c t was d e r i v e d from t r a n s a d d i t i o n o f the 2-amino group o f gua n i n e t o the C-10 p o s i t i o n o f 1 . S i m i l a r l y , the p r o t o n nmr o f the minor product showed t h r e e a c e t y l s i n g l e t s a t 1.95, 2.00, and 2.13, one N-methyl s i n g l e t a t 3.93, the f o u r one p r o t o n s i g n a l s from p o s i t i o n s 7,8,9, and 10 o f the tetrahydro-BP r i n g a t 6.93, 6.18, 5.61, and 6.29, r e s p e c t i v e l y , and the remaining C and pyrene s i g n a l s from 7.95 t o 8.40 ppm. Comparison o f the c o u p l i n g con s t a n t s o f t h i s product ( J =8.0, J 9 =12.0, 6

5

8

3

3

7

3 j

o

y

8

ax,

8

ax

ax,

ax

ι λ = 4.0 Hz) w i t h those o f the 9,10-cis-phenol adduct ax, eq i U

(--0C H a t C-10 o f 9) from d i o l epoxide 1 (28) showed t h a t 9 r e s u l t e d from c i s opening o f the o x i r a n e r i n g o f 1 by the 2-amino group o f guanine. A r e c e n t p u b l i c a t i o n (22) has assigned the major product from d i o l epoxide 2 and p o l y (G) as a t r a n s N s u b s t i t u t e d guanosine based s o l e l y on the nmr and mass s p e c t r a o f the m o d i f i e d nucleo s i d e . S t r u c t u r a l i n t e r p r e t a t i o n o f the mass s p e c t r a a t t h e 6

5

2

146


0

I—«


10

,

•

,

,

•

,

.

•

20

30

4.0

50

60

70

80

ao

CONCEPTS

ι IQO

IIJO

rI2J0

PH

Figure 11. Estimation of pK by change in partition coefficient with pH. Solutions (pH 2.0-9.0) were prepared by mixing 0.05M citric acid pH 1.8, 0.05M KJ/POj 7.0, and 0.05M NaHCO pH 9.0. Solutions outside the range of pH 2.0-9.0 were obtained adding HCl or KOH to 0.05M citric acid or NaHC0 . Nucleoside adducts from 1 or 2 wer partitioned with 25% n-butanol in ethyl acetate. Guanosine triacetate was partitioned wi n-butanol in ethyl ether. Distribution between the two phases was determined spectro metrically as described (26,63). a

s

3

Figure 12. Labilization of the glycosidic linkage by N methylation. Diol epoxide 1 (DEBP) guanosine products were treated with dimethyl sulfate as described in materials and methods. 7

7.

MOORE ET AL.

Diastereomeric

9,10-Epoxides

from Benzo[a]pyrene

147

n u c l e o s i d e l e v e l i s somewhat d i f f i c u l t s i n c e s t r u c t u r e 7 (Figure 14) which has a mass 18 u n i t s h i g h e r than the N s u b s t i t u t e d anal o g might be expected t o g i v e a s i m i l a r cleavage p a t t e r n a f t e r e l i m i n a t i o n o f water. A major argument presented f o r N s u b s t i t u t i o n was an observed s p i n - s p i n c o u p l i n g between the 2-amino hydrogen and the hydrogen a t C-10. A s i m i l a r c o u p l i n g between the hydrogen a t C-10 and the amino hydrogen a t N i n 7 would be expected. The formamide s i g n a l i n 7 would be obscured by the pyrene resonances (64). Although the data presented by these authors (22) does not exclude s t r u c t u r e 7 , the present t r i t i u m r e l e a s e s t u d i e s w i t h [ 8 - H ] - p o l y (G) and d i o l epoxide 2 does. Thus, both d i o l epoxides 1 and 2 a t t a c k the e x o c y c l i c 2-amino group o f guanosine (22,26). Assignment o f the A b s o l u t e C o n f i g u r a t i o n o f the Guanosine Adducts from D i o l Epoxides 1 and 2 . For d i o l epoxide 1 , the minor p a i r o f diastereomers were shown t o r e s u l t from c i s opening and the major p a i r o f diastereomers by t r a n s opening o f the o x i r a n e r i n g a t C-10. Each o f the enantiomers o f d i o l epoxide 1 leads t o the formation o f a c i s and t r a n s adduct. The CD s p e c t r a o f t h i s c i s / t r a n s p a i r are almost m i r r o r images w i t h o p p o s i t e signs a t t h e i r s t r o n g e s t t r a n s i t i o n s (Figure 8 ) . The c i s and t r a n s adducts from a given enantiomer o f d i o l epoxide 1 d i f f e r o n l y i n t h e i r c o n f i g u r a t i o n a t C-10. Since each enantiomer o f d i o l epoxide 2 a l s o produces a p a i r o f adducts w i t h o p p o s i t e CD s p e c t r a and s i n c e the major adduct from each enantiomer o f d i o l epoxide 2 c o n s t i t u t e a p a i r o f t r a n s diastereomers (22), i t i s reasonable t o conclude on the b a s i s o f the CD s p e c t r a t h a t the minor p a i r o f adducts from d i o l epoxide 2 c o n s t i t u t e a p a i r o f c i s diastereomers. S i n c e the a b s o l u t e c o n f i g u r a t i o n o f the enantiomeric BP 7,8d i h y d r o d i o l s and the f o u r p o s s i b l e corresponding d i o l epoxides has been assigned (53) and s i n c e these d i o l epoxides have been s e p a r a t e l y r e a c t e d w i t h p o l y (G), the assignments i n Table I I I are p o s s i b l e . A l l o f these assignments are based on the e x c i t o n c h i r a l i t y CD spectrum o f the Jbis-(p-N,i\r-dimethylaminobenzoate) ester o f o p t i c a l l y active trans-7,8-dihydroxy-4,5,7,8,9,10,11,12octahydro-BP. 2

2

7


3

3

Formation o f RNA-Nucleoside Adducts from [ H]-BP on Mouse S k i n Once the s t r u c t u r e s and the chromatographic p r o p e r t i e s o f the adducts which form when d i o l epoxides 1 and 2 a l k y l a t e p o l y (G) had been e s t a b l i s h e d , i t became p o s s i b l e t o determine whether these products form from BP in vivo. Since we have determined the c a r c i n o g e n i c i t y o f BP 7,8-oxide and BP 7 , 8 - d i h y d r o d i o l on the s k i n o f C57BL/6J mice, we examined t h e . s k i n epidermis o f t h i s mouse s t r a i n f o r the formation o f d i o l epoxide-nucleoside adducts a f t e r t o p i c a l a p p l i c a t i o n o f [ H]-BP t o the mouse. Mice were p a i n t e d w i t h [ H]-BP and the i s o l a t e d RNA was hyd r o l y z e d t o a mixture o f n u c l e o s i d e s . A f t e r a d d i t i o n o f c a r r i e r amounts o f the e i g h t p o s s i b l e guanosine adducts, the r a d i o a c t i v e 3

3

American Chemical Society Library 1155 16th St. n. w. Washington, D. C. 20036


8, 9, \0-trans


9

9,10-c/s

Figure 13. Structure of the guanosine adducts of diol epoxide 1 after methylation at N 7

Figure 14. Possible alternative structure of guanosine adducts from trans opening at C-10 of diol epoxide 2 (DEBP)

Ribose

7

CONCEPTS


7.

MOORE

ET AL.

Diastereomeric

9,10-Epoxides

from Benzo[a]pyrene

149

n u c l e o s i d e s were p u r i f i e d by Poragel PN chromatography t o remove both t e t r a o l s and unmodified n u c l e o s i d e s . The r a d i o c h e m i c a l y i e l d of the products e l u t e d i n the guanosine adduct f r a c t i o n was > 30%. Most o f the remaining r a d i o a c t i v i t y was e l u t e d i n the f i r s t peak (Figure 6) and i s o f unknown s t r u c t u r e . On t h i s Poragel PN column n u c l e o s i d e adducts from d i o l epoxides 1 and 2 and p o l y (A) a r e e l u t e d i n the t e t r a o l f r a c t i o n which contained < 5% o f the t o t a l radioactivity. When the g u a n o s i n e - d i o l epoxide f r a c t i o n which had been pur i f i e d by Poragel PN chromatography was examined by HPLC on μΟχβ" Bondapak (Figure 15), r a d i o a c t i v e guanosine adducts w i t h chromato graphic r e t e n t i o n times i d e n t i c a l t o adduct peaks B , D, F, and 6 were found. F u r t h e r c o n f i r m a t i o n o f the nature o f the i n d i v i d u a l r a d i o a c t i v e peaks was obtained by d e t e r m i n a t i o n o f t h e i r e x t r a c t i o n versus pH p r o f i l e (as i l l u s t r a t e d i n F i g u r e 11) (26,63). A l l t h r e e r a d i o a c t i v e peaks showed a change i n p a r t i t i o n co e f f i c i e n t i n the range o f pH 9.0 - 11.0 i n d i c a t i n g t h a t each i s a N o r C s u b s t i t u t e d guanosine adduct. Adducts a t C are pre sumed not t o be present based on the model experiments w i t h p o l y (G). The p a i r o f adducts Β and D a r i s e from d i o l epoxide 2 and 1 , r e s p e c t i v e l y , and p r o v i d e the f i r s t evidence t h a t these d i o l ep oxides are formed and b i n d t o RNA i n mouse s k i n . The formation of both o f these d i o l epoxides was f u r t h e r confirmed by a c i d hy d r o l y s i s o f the t r i t i a t e d n u c l e o s i d e adducts t o r a d i o a c t i v e t e t r a o l s r e l a t e d t o d i o l epoxides 1 and 2. The formation o f at l e a s t t h r e e o f the e i g h t d i a s t e r e o m e r i c guanosine d e r i v a t i v e s ( B and D along w i t h e i t h e r o r both o f F and G) p r o v i d e s i n s i g h t i n t o the metabolism o f BP i n mouse s k i n . Peak Β i s d e r i v e d from ( + ) - d i o l epoxide 2 and guanosine by c i s a d d i t i o n a t C-10 w h i l e peak D i s d e r i v e d from ( + ) - d i o l epoxide 1 by t r a n s a d d i t i o n a t C-10. (+)-Diol epoxide 2 i s formed from (-)BP 7 , 8 - d i h y d r o d i o l , w h i l e ( + ) - d i o l epoxide 1 i s formed from the (+)-enantiomer. Furthermore, the c i s / t r a n s counterpart o f both peaks Β and D (peaks F and G ) are known t o co-chromâtograph w i t h the t h i r d r a d i o a c t i v e peak. These r e s u l t s imply t h a t the monooxygenases i n mouse s k i n which produce the 9,10-epoxides are h i g h l y s t e r e o s e l e c t i v e i n t h a t predominantly ( + ) - d i o l epoxide 1 from (+)-BP 7 , 8 - d i h y d r o d i o l and ( + ) - d i o l epoxide 2 from (-)-BP 7,8-dihydrodiol form d e t e c t a b l e RNA adducts. 2

8

8

Conclusions We have observed t h a t both d i o l epoxides 1 and 2 r e a c t s i m i l a r l y w i t h p o l y (G) i n 50% acetone/water. A l k y l a t i o n occurs a t phosphate s i t e s (10 - 15%) and a t the e x o c y c l i c 2-amino group o f guanosine. The formation o f both c i s and t r a n s a d d i t i o n products to the o x i r a n e r i n g s o f d i o l epoxides 1 and 2 suggests t h a t these r e a c t i o n s may proceed a t l e a s t i n p a r t by Sjjl mechanism. T h i s r e a c t i v i t y under n e u t r a l c o n d i t i o n s i s a l s o demonstrated by a l k y l -


CONCEPTS


150

10

20

30

40

50

60

70

80

90

HO

100

Fraction Number

Figure 15. Coinjection of Poragel PN-purified nucleoside adducts from [ H] BP on mouse skin and 2-amino guanosine-diol epoxide adducts from both diol epoxides 1 and 2 on high resolution μϋ Bondapak. Refer to Figure 7 and Table III for definition of the nucleoside symbols. Cochromatography of the longest retained radioactive peak with uv markers F and G suggests that very little radioactive Ε was present. 3

18


7.

MOORE ET AL.

Diastereomeric 9,10-Epoxides from Benzo[a]pyrene

151

ation of inorganic phosphate. The relative biological importance of 2-amino substitution and phosphate alkylation can not as yet be estimated. Little is known about the effect of 2-amino substitu tion by large aromatic groups on factors such as the conformation of the polymer or the fidelity of transcription. Alkylation at phosphate, however, has been suggested to have high biological significance (41,42). This report describes the structural assignment of the covalently bound guanosine adducts derived from RNA, which are form ed when mouse skin is exposed to BP. Both (+)-diol epoxide 1 and (+)-diol epoxide 2 from the (+)- and (-)-enantiomers of BP 7,8dihydrodiol, respectively, are produced in vivo and react with cellular RNA on the 2-amino group of guanosine by cis and trans addition. When bovine bronchial expiants were exposed to BP (23) only diol epoxide 2 was observed to form an adduct at the 2-amino group of guanosine. The differences in these two results may be explained by a difference in metabolism of BP between mouse skin and bovine bronchial expiants or by the fact that only about half of the radioactivity in the chromatographic region for diol ep oxide adducts of guanosine was characterized in the latter study. Whether or not the enantiomers of diol epoxides 1 and 2 are impor tant in the binding of BP to the DNA of mouse skin is presently under study. Acknowledgment This research has been supported in part by Grant CA 18580 from the National Cancer Institute of the US Public Health Service (to M. K.). Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Brookes, P. and Lawley, P. D., Nature (London) (1964) 202, 781. Miller, J., Cancer Res. (1970) 30, 559. Sims, P. and Grover, P. L . , Adv. Cancer Res. (1974) 20, 165. Berwald, Y. and Sachs, L . , J. Natl. Cancer Inst. (1965) 35, 641. Ames, Β. N . , Durston, W. E . , Yamasaki, E . , and Lee, F. D., Proc. Natl. Acad. Sci. U.S.A. (1973) 70, 2281. Grover, P. L. and Sims, P., Biochem. J. (1968) 110, 159. Gelboin, H. V., Cancer Res. (1969) 29, 1272. Jerina, D. M. and Daly, J . W., Science (1974) 185, 573. Sims, P., Grover, P. L . , Swaisland, Α., Pal, K., and Hewer, Α., Nature (London) (1974) 252, 326. Ts'o, P. O. P., Caspary, W. J., Cohen, Β. I., Leavitt, J. C., Lesko, S. Α., Lorentzen, R. J., and Schechtman, L. M. in "Chemical Carcinogenesis, Part A", Ts o, P. O. P. and DiPaolo, J . Α., eds., p.113, Marcel Dekker, New York, Ν. Υ., 1974. '


152

DRUG METABOLISM CONCEPTS

11. Nagata, C., Tagashira, Y . , and Kodama, Μ., ibid., p. 87. 12. Fried, J., ibid., p. 197. 13. Cavalieri, E., Roth, R., and Rogan, E. G. in "Carcinogenesis", Vol. 1; Polynuclear Aromatic Hydrocarbons: Chemistry, Meta bolism, and Carcinogenesis, Freudenthal, R. and Jones, P. W., eds., p. 181, Ravern Press, New York, Ν. Υ., 1976. 14. Borgen, Α., Darvey, Η., Castagnoli, Ν., Crocker, T. T., Rasmussen, R. E., and Wang, I. Y . , J. Med. Chem. (1973) 16, 502. 15. Yagi, H., Hernandez, O., and Jerina, D. M., J. Am. Chem. Soc. (1975) 97, 6881. 16. McCaustland, D. J., Fischer, D. L . , Kolwyck, K. C., Duncan, W. P., Wiley, J . C., Jr., Menon, C. S., Engel, J. F., Selkirk, J . K., and Roller, P. P. in ref. 13, p. 349. 17. Daudel, P., Duquesne, M., Vigny, P., Grover, P. L . , and Sims, P., FEBS Lett. (1975) 57, 250. 18. Nebert, D. W., Kouri, R. E., Yagi, H., Jerina, D. M., and Boobis, A. R. in "Reactive Intermediates: Formation, Toxicity, and Inactivation", Jollow, D., Kocsis, J., Snyder, R., Vainio, Η., eds., Plenum Press, New York, Ν. Y . , in press. 19. Meehan, T., Warshawsky, D., and Calvin, Μ., Proc. Natl. Acad. Sci. U.S.A. (1976) 73, 1117. 20. Osborne, M. R., Thompson, M. H., Tarmy, E. M., Beland, F. Α., Harvey, R. G., and Brookes, P., Chem.-Biol. Interact. (1976) 13, 343. 21. Osborne, M. R., Beland, F. Α., Harvey, R. G., and Brookes, P., Int. J . Cancer (1976) 18, 362. 22. Jeffrey, A. M., Jennette, K. W., Blobstein, S. Η., Weinstein, I. Β., Beland, F. Α., Harvey, R. G., Kasai, H., Miura, I., and Nakanishi, K., J. Am. Chem. Soc. (1976) 98, 5714. 23. Weinstein, I. Β., Jeffrey, Α. Μ., Jennette, K. W., Blobstein, S. H., Harvey, Ri G., Harris, C., Autrup, Η., Kasai, H., and Nakanishi, Κ., Science (1976) 193, 592. 24. King, H. W. S., Thompson, Μ. Η., Tarmy, Ε. Μ., Brookes, P., and Harvey, R. G., Chem.-Biol. Interact. (1976) 13, 349. 25. King, H. W. S., Osborne, M. R., Beland, F. Α., Harvey, R. G., Brookes, P., Proc. Natl. Acad. Sci. U.S.A. (1976) 73, 2679. 26. Koreeda, M., Moore, P. D., Yagi, H., Yeh, H. J. C., and Jerina, D. M., J. Am. Chem. Soc. (1976) 98, 6720. 27. Thakker, D. R., Yagi, H., Lu, Α. Y. H., Levin, W., Conney, A. H., and Jerina, D. Μ., Proc. Natl. Acad. Sci. U.S.A. (1976) 73, 3381. 28. Yagi, H., Thakker, D. R., Hernandez, O., Koreeda, Μ., and Jerina, D. M., J. Am. Chem. Soc. (1977) 99, in press. 29. Yang, S. K., McCourt, D. W., Roller, P. P., and Gelboin, H. V., Proc. Natl. Acad. Sci. U.S.A. (1976) 73, 2594. 30. Wood, A. W., Wislocki, P. G., Chang, R. L . , Levin, W., Lu, Α. Y. H., Yagi, Η., Hernandez, O., Jerina, D. Μ., and Conney, A. H., Cancer Res. (1976) 36, 3358.

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Diastereomeric 9,10-Epoxides from Benzo[a]pyrene

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31. Levin, W., Wood, A. W., Yagi, H., Dansette, P., Jerina, D. M., and Conney, A. H., Proc. Natl. Acad. Sci. U.S.A. (1976) 73,


243.

32. Levin, W., Wood, A. W., Yagi, Η., Jerina, D. M., and Conney, A. H., ibid. (1976) 73, 3867. 33. Conney, A. H., Wood, A. W., Lu, A. Y. H., Chang, R. L . , Wislocki, P. G., Holder, G. M., Dansette, P., Yagi, H., and Jerina, D. M. in ref. 18, in press. 34. Wislocki, P. G., Wood, A. W., Chang, R. L . , Levin, W., Yagi, H., Hernandez, O., Jerina, D. M., and Conney, A. H., Biochem. Biophys. Res. Commun. (1976) 68, 1006. 35. Newbold, R. F. and Brookes, P., Nature (London) (1976) 261, 52. 36. Huberman, E., Sachs, L . , Yang, S. K., and Gelboin, H. V., Proc. Natl. Acad. Sci. U.S.A. (1976) 73, 607. 37. Singer, B., Prog. Nucleic Acid Res. Mol. Biol. (1975) 15, 219 and references cited therein. 38. Lawley, P. D. and Shah, S. Α., Chem.-Biol. Interact. (1973) 7, 115. 39. 40. 41.

Loveless, Α., Nature (London) (1969) 223, 206. Lawley, P. D. and Thatcher, C. J . , Biochem. J . (1970) 116, 693. Singer, B. and Fraenkel-Conrat, H., Biochemistry (1975) 14, 772.

Singer, B., Nature (London) (1976) 264, 333. 43. Goth, R. and Rajewsky, M. F . , Proc. Natl. Acad. Sci. U.S.A.

42.

(1974) 71, 639.

44. Kriek, E., Miller, J . Α., Juhl, U., and Miller, E. C., Biochemistry (1967) 6, 177. 45. Westra, J . G., Kriek, E., and Hittenhausen, H., Chem.-Biol. Interact. (1976) 15, 149. 46. Dipple, Α., Brookes, P., Mackintosh, D. S., and Rayman, M. P., Biochemistry (1971) 10, 4323. 47. Jeffrey, A. M., Blobstein, S. H., Weinstein, I. B., Beland, F. Α., Harvey, R. G., Kasai, Η., and Nakanishi, K., Proc. Natl. Acad. Sci. U.S.A. (1976) 73, 2311. 48. Brown, D. M. in "Basic Principles of Nucleic Acid Chemistry", Ts'o, P. O. P., ed., Vol. II, p. 66, Academic Press, New York, Ν. Y . , 1974. 49. Boyland, E. and Green, B., Brit. J . Cancer (1962) 16, 507. 50. Lesko, S. Α., Smith, Α., Ts'o, P. O. P., and Umans, R. S., Biochemistry (1968) 7, 434. 51. Dale, J . Α., Dull, D. L . , and Mosher, H. S., J . Org. Chem. (1969) 34, 2543.

52. Thakker, D. R., Yagi, H., Akagi, H., Koreeda, M., Lu, Α. Υ. Η., Levin, W., Conney, Α. Η., and Jerina, D. M., Chem.-Biol. Interact. (1977) in press. 53. Yagi, H., Akagi, H., Mah, H. D., Koreeda, Μ., and Jerina, D. Μ., submitted. 54. Blobstein, S. Η., Weinstein, I. Β., Dansette, P., Yagi, H., and Jerina, D. Μ., Cancer Res. (1976) 36, 1293.


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55. Haines, J. Α., Reese, C. Β., and Todd, L . , J. Chem. Soc. (1962) 5281. 56. Bowden, G. T., Shapas, B. G., and Boutwell, R. Κ., Chem.-Biol. Interact. (1974) 8, 379. 57. Irving, C. C. and Veazey, R. Α., Biochem. Biophys. Acta (1968) 166, 246. 58. Holy, A. and Scheit, Κ. H., ibid. (1967) 138, 230. 59. Tomasz, Μ., ibid. (1970) 199, 18. 60. Kochetkov, Ν. K. and Budovskii, Ε. I., "Organic Chemistry of Nuclei Acids", Part B, p. 381, Plenum Press, New York, Ν. Y . , 1972. 61. Holmes, R. E. and Robins, R. K., J. Org. Chem. (1963) 28, 3483. 62. Chambers, R. W., Moffatt, J . G., and Khorana, H. G., J . Am. Chem. Soc. (1957) 79, 3747. 63. Moore, P. D. and Koreeda, Μ., Biochem. Biophys. Res. Commun. (1976) 73, 459. 64. Hecht, S. Μ., Adams, B. L . , and Kozarich, J. W., J. Org. Chem. (1976) 41, 2303.

8 Role of Metabolic Activation in Chemical-Induced Tissue Injury SIDNEY D. NELSON, MICHAEL R. BOYD, and JERRY R. M I T C H E L L


Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, Bethesda, MD 20014

An important result of metabolism studies in recent years has been the realization that many chemical compounds are metabolized by the liver and various other tissues to potent alkylating and arylating intermediates (1-12). Such studies demonstrate how chemically stable compounds can produce serious tissue lesions in man and experimental animals, including hepatic, renal, and pulmonary necrosis, bone marrow aplasia, neoplasia and other injuries. Although these lesions are rare, such toxic effects are of great c l i n i c a l concern because they often lead to irreversible failure of the liver, lungs, kidneys or other organs, and subsequent death of the patient. Many of the initial concepts of metabolic activation were developed during studies of chemical carcinogenesis; the work of the Millers in the United States (1,2) and of Magee and co-workers in England (3) has been especially illuminating. The realization that the enzyme pathways responsible for the conversion of certain chemicals to proximate carcinogens are the same microsomal mixed-function oxygenases that metabolize most drugs and other xenobiotics led to the concept that drug-induced tissue lesions might also be mediated through the covalent binding of reactive metabolites (6-11). The lack of reactivity of most chemically stable compounds and the frequent localization of tissue damage only in those organs or to those animal species having the necessary drug-metabolizing enzymes supported this view. Additionally, these studies frequently demonstrated a role for sulfhydryl-containing compounds, particularly glutathione, in protecting tissues from such toxic reactions. Most drugs and foreign compounds that enter the body are converted to chemically stable metabolites that are readily excreted into urine and b i l e , or are expired. Thus, it has become important to distinguish those toxicities that are mediated by chemically reactive metabolites and those reactions due to an exaggerated therapeutic effect or unwanted secondary effect caused by the drug or one of i t s stable metabolites. The toxicologic activity produced by the latter class of reactions usually can be monitored by 155

156



measuring the c o n c e n t r a t i o n of the compound and i t s m e t a b o l i t e s i n body f l u i d s * However, when the response i s t i s s u e damage caused by the covalent b i n d i n g of c h e m i c a l l y r e a c t i v e m e t a b o l i t e s t o t i s s u e macromolecules, r a r e l y can a r e l a t i o n s h i p between t i s s u e l e v e l s of the m e t a b o l i t e and the s e v e r i t y of the l e s i o n be d e t e r mined* Indeed, h i g h l y r e a c t i v e m e t a b o l i t e s may e x i s t f o r o n l y a few seconds o r l e s s and w i l l t h e r e f o r e never accummulate i n body fluids. Parameters f o r s t u d y i n g r e a c t i v e m e t a b o l i t e s . How then can the formation of such c h e m i c a l l y u n s t a b l e and r e a c t i v e m e t a b o l i t e s be studied? Based on s t u d i e s where an animal model has been developed f o r a p a r t i c u l a r chemical-induced t i s s u e l e s i o n , a r e l a t i o n s h i p can o f t e n be made between the s e v e r i t y of the t i s s u e l e s i o n and the amount of m e t a b o l i t e t h a t i s c o v a l e n t l y bound to the damaged t i s s u e . That i s , covalent b i n d i n g of the r e a c t i v e m e t a b o l i t e can be used as an index of formation of the m e t a b o l i t e . Furthermore, t h i s parameter might w e l l be the most r e l i a b l e e s t i mate of the a v a i l a b i l i t y of the m e t a b o l i t e i n s i t u f o r causing t i s s u e damage, s i n c e much of the m e t a b o l i t e o f t e n decomposes or i s f u r t h e r metabolized before i t can be i s o l a t e d i n body f l u i d s . Thus, one approach t o the problem i s t o determine whether r a d i o l a b e l e d drugs administered t o animals over a wide dose range are c o v a l e n t l y bound t o macromolecules i n t a r g e t t i s s u e s t h a t subsequently become n e c r o t i c . Pretreatment of animals w i t h inducers o f drug metabolism, such as phénobarbital, or w i t h i n h i b i t o r s of drug metabolism, such as p i p e r o n y l butoxide, c o b a l t c h l o r i d e , o r ©(-naphthylisothiocyanate, s i m i l a r l y should a l t e r the r a t e of metabolism of t o x i n , the extent of covalent b i n d i n g of r e a c t i v e m e t a b o l i t e , and the s e v e r i t y of t i s s u e i n j u r y . I n c o n j u n c t i o n w i t h these s t u d i e s i n animals, experiments can be performed i n v i t r o w i t h microsomal enzymes i s o l a t e d from the t a r g e t organ t i s s u e . Covalent b i n d i n g of r e a c t i v e m e t a b o l i t e s may be one u s e f u l index of product formation when v a r i o u s a d d i t i o n s or d e l e t i o n s from the system are made, or when animals are p r e t r e a t e d w i t h v a r i o u s enzyme inducers and i n h i b i t o r s . Another u s e f u l index of r e a c t i v e product formation i n t h i s system i s the t r a p p i n g of e l e c t r o p h i l i c intermediates w i t h a l t e r n a t e n u c l e o p h i l e s such as c y s t e i n e or g l u t a t h i o n e . S t r u c t u r a l e l u c i d a t i o n of such i n t e r m e d i a t e s may o f t e n p r o v i d e i n s i g h t i n t o the s t r u c t u r e o f the i n i t i a l r e a c t i v e m e t a b o l i t e . U l t i m a t e l y , i s o l a t i o n and s t r u c t u r e e l u c i d a t i o n of the r a d i o l a b e l e d m a t e r i a l bound to the t i s s u e macromolecules (RNA, DNA, p r o t e i n ) can be c a r r i e d out. The approach d e s c r i b e d has been used to i m p l i c a t e t o x i c m e t a b o l i t e s as mediators of the t o x i c i t i e s caused by s e v e r a l drugs. Hepatic n e c r o s i s has been a s s o c i a t e d w i t h the use of hydrazides i s o n i a z i d ( I ) , a t u b e r c u l o s t a t i c agent, and i p r o n i a z i d ( I I ) , an a n t i d e p r e s s a n t . Both h e p a t i c and r e n a l i n j u r y are a s s o c i a t e d w i t h the use of h i g h doses of two s u b s t i t u t e d aminophenol a n a l g e s i c s , acetaminophen ( I I I ) and phenacetln ( I V ) . The f u r a n - c o n t a i n i n g d i u r e t i c agent, furosemide (V), and the thiophene-containing

8.

NELSON E T A L .

Chemical-Induced

Tissue

Injury

157

a n t i b i o t i c , c e p h a l o r i d i n e ( V I ) , are a s s o c i a t e d w i t h r e n a l i n j u r y i n man. Ipomeanol ( V I I ) , a f u r a n - c o n t a i n i n g d e r i v a t i v e produced by moldy sweet potatoes, i s an example o f a chemical t o x i n which produces pulmonary l e s i o n s v i a r e a c t i v e m e t a b o l i t e formation. These and other experimental s t u d i e s w i t h model compounds w i l l be presented t o i l l u s t r a t e the concepts which u n d e r l i e the r o l e o f metabolic a c t i v a t i o n i n chemical-induced t i s s u e i n j u r y and the parameters used t o e s t a b l i s h these concepts. Hydrazines and Hydrazides


I s o n i a z i d . A good example of t o x i c drug r e a c t i o n s caused by metabolic a c t i v a t i o n i s i s o n i a z i d - i n d u c e d l i v e r i n j u r y . This drug provides a unique o p p o r t u n i t y to show how a study can be pursued from a c l i n i c a l l y manifest t i s s u e l e s i o n t o the proposal o f a r a t i o n a l chemical mechanism f o r the t o x i c i t y . C l i n i c a l f i n d i n g s . Three c l i n i c a l s t u d i e s (13-15) provided evidence t h a t metabolic a c t i v a t i o n was i n v o l v e d i n the s e r i o u s h e p a t i t i s caused by i s o n i a z i d when t h i s drug was administered i n t h e r a p e u t i c doses. F i r s t was a p r o s p e c t i v e study c a r r i e d out i n 1972 (13). SGOT and serum b i l i r u b i n concentrations were examined monthly i n 250 p a t i e n t s r e c e i v i n g i s o n i a z i d f o r one year. These b i o c h e m i c a l i n d i c e s i n d i c a t e d that i s o n i a z i d was hepatotoxic i n a l a r g e p r o p o r t i o n o f i n d i v i d u a l s but most adapted t o the i n s u l t and recovered r a t h e r than developing severe h e p a t i t i s . Measurement of plasma concentrations o f i s o n i a z i d i n these p a t i e n t s , f a i l e d t o show a c o r r e l a t i o n between plasma l e v e l s o f i s o n i a z i d and l i v e r i n j u r y . I n t h i s study, no a n t i - i s o n i a z i d a n t i b o d i e s were found and no c o r r e l a t i o n was seen between h e p a t i c i n j u r y and a n t i n u c l e a r a n t i b o d i e s measured a t the end o f the study. The seoncd study was a r e t r o s p e c t i v e a n a l y s i s o f 114 p a t i e n t s w i t h i s o n i a z i d - r e l a t e d h e p a t i t i s (14). Some o f the important f i n d i n g s were t h a t : 1) i s o n i a z i d - r e l a t e d l i v e r i n j u r y was c l i n i c a l l y i n d i s t i n g u i s h a b l e b i o c h e m i c a l l y and m o r p h o l o g i c a l l y from i p r o n i a z i d - i n d u c e d l i v e r damage or from other causes o f acute h e p a t o c e l l u l a r i n j u r y such as v i r a l h e p a t i t i s ; 2) no c l i n i c a l evidence such as r a s h , f e v e r , a r t h r a l g i a s o r e o s i n o p h i l i a was found f o r h y p e r s e n s i t i v i t y mechanism; 3) about 30% o f the p a t i e n t s w i t h h e p a t i c r e a c t i o n s were r e s i d e n t s o f Honolulu and o f O r i e n t a l a n c e s t r y ; on g e n e t i c b a s i s , 90% or more o f these p a t i e n t s would be expected t o be r a p i d a c e t y l a t o r s o f i s o n i a z i d i n c o n t r a s t t o b l a c k and w h i t e populations i n whom 45% are r a p i d a c e t y l a t o r s (16). I n the t h i r d study (15), 21 n o n - O r i e n t a l p a t i e n t s who had recovered from i s o n i a z i d h e p a t i t i s were g e n e t i c a l l y phenotyped as r a p i d o r slow a c e t y l a t o r s o f i s o n i a z i d u s i n g the sulfamethazine method. E i g h t y - s i x percent o f them d i s p l a y e d the r a p i d a c e t y l a t o r phenotype f o r i s o n i a z i d metabolism. Metabolism s t u d i e s i n man. Based on these c l i n i c a l f i n d i n g s , we re-examined the metabolism of i s o n i a z i d and i d e n t i f i e d the


158


m e t a b o l i t e s by co-chromatography, reverse i s o t o p e d i l u t i o n w i t h s y n t h e s i z e d standards and by mass s p e c t r a l a n a l y s i s (15). T r i t i u m r i n g - l a b e l e d i s o n i a z i d and a c e t y l i s o n i a z i d , the major primary m e t a b o l i t e of i s o n i a z i d , were administered to human v o l u n t e e r s i n s i n g l e 300 mg doses and u r i n a r y m e t a b o l i t e s were c o l l e c t e d f o r 24 h r s . As shown i n F i g u r e 2, about 55% of a dose of a c e t y l i s o n i a z i d was metabolized by h y d r o l y s i s t o i s o n i c o t i n i c a c i d and f r e e a c e t y l h y d r a z i n e r e g a r d l e s s of g e n e t i c phenotype of the p a t i e n t s f o r a c e t y l a t i n g i s o n i a z i d * I n c o n t r a s t , p a t t e r n of m e t a b o l i t e s a f t e r a d m i n i s t r a t i o n of i s o n i a z i d was very dependent upon the r a t e a t which i s o n i a z i d was a c e t y l a t e d * On the b a s i s of the r e l a t i v e amounts of a c e t y l i s o n i a z i d and i s o n i c o t i n i c a c i d excreted i n t o the u r i n e , we c a l c u l a t e d t h a t almost a l l of the i s o n i c o t i n i c a c i d was formed by way of a c e t y l i s o n i a z i d * We a l s o c a l c u l a t e d that p a t i e n t s who were f a s t m e t a b o l i z e r s of i s o n i a z i d converted about 94% of an i s o n i a z i d dose to a c e t y l i s o n i a z i d ; o n l y 2.8% of the drug was exc r e t e d unchanged i n the u r i n e and 3.6% as hydrazone conjugates. Slow a c e t y l a t o r s , on the other hand, excreted almost 37% of the drug i n the u r i n e e i t h e r f r e e or as a hydrazone. Thus, o n l y 63% was converted t o a c e t y l i s o n i a z i d and subsequently to i s o n i c o t i n i c a c i d and a c e t y l h y d r a z i n e . We concluded, t h e r e f o r e , that f a s t a c e t y l a t o r s are exposed t o much more a c e t y l i s o n i a z i d and a c e t y l hydrazine than are slow a c e t y l a t o r s . Hepatic n e c r o s i s i n animals. A c e t y l i s o n i a z i d and i s o n i a z i d were given t o r a t s , mice and hamsters to see i f they could produce h e p a t i c n e c r o s i s (17,18). These hydrazines were given i n a doseresponse manner to s e v e r a l hundred animals. I s o n i a z i d d i d not cause n e c r o s i s i n any of the animals. However, a c e t y l i s o n i a z i d produced o c c a s i o n a l s i n g l e c e l l n e c r o s i s i n r a t s and mice. Moreover, as shown i n Table I , pretreatment of r a t s w i t h phénobarbital, which i s known to i n c r e a s e drug m e t a b o l i z i n g enzymes, g r e a t l y p o t e n t i a t e d the n e c r o s i s . The l i v e r damage was prevented by p r e treatment of r a t s w i t h c o b a l t c h l o r i d e , which i n h i b i t s s y n t h e s i s of cytochrome P-450 m e t a b o l i z i n g enzymes. S i m i l a r l y when hydrol y s i s of a c e t y l i s o n i a z i d was i n h i b i t e d by pretreatment of r a t s w i t h b i s - p a r a - n i t r o p h e n y l phosphate (BNPP), the n e c r o s i s was prevented. The e f f e c t on the l i v e r of the h y d r o l y s i s product, a c e t y l h y d r a z i n e , was t h e r e f o r e examined. This hydrazine i s a very potent hepatotoxin which produces h e p a t i c n e c r o s i s i n phenobarbital-pret r e a t e d r a t s a f t e r s i n g l e doses of 10 mg/kg. The n e c r o s i s was p o t e n t i a t e d by pretreatment w i t h phénobarbital and prevented by pretreatment w i t h c o b a l t c h l o r i d e (Table I ) . However, BNPP, which i n h i b i t e d the h y d r o l y s i s of a c e t y l i s o n i a z i d and prevented the n e c r o s i s , had no e f f e c t on n e c r o s i s produced by a c e t y l h y d r a z i n e . Thus, the metabolic a c t i v a t i o n of the l i b e r a t e d a c e t y l h y d r a z i n e moiety of a c e t y l i s o n i a z i d to a t o x i c m e t a b o l i t e s a t i s f a c t o r i l y accounts f o r the h e p a t i c n e c r o s i s produced by i s o n i a z i d . Subsequently, i s o n i a z i d i t s e l f was shown to produce acute h e p a t i c n e c r o s i s i n p h e n o b a r b i t a l - t r e a t e d r a t s . The p r o p o r t i o n or the i s o n i a z i d that i s a c e t y l a t e d i n r a t s decreases markedly

8.

NELSON

C-N-NH

Chemical-Induced

ETAL.

S H H

Tissue

Injury

159

/C 3 H

C-N-N-CH

2

Ô

ô

un

(I)

9

HN-C-CH

HN—C—CH

5

3

0-CH -CH

OH (III)

2

3

(IV)


HOOC-"

HOOC

OAc

(VI)

(V)

CH,^CH,

HO'' ΓΗ

Figure 1. Structures of the compounds discussed in the text

(VII)

X Of DOSE DRUG

PATIENTS ACETYLATIOtl RATE 00

AcIMH

FAST (2)

AcINH

INH HTDRAZONES

AcINH

INA DERIVATIVES

ESTIMATED ACETYL HYDRAZINE

...

54.912.2

45.112.7

45.112.7

...

SLOW (3)

—

53.811.2

46.211.1

46.211.1

...

INH

FAST (3)

2.810.4

3.610.4

49.211.9

44.413.9

41.013.8

3.410.1

INH

SLOW (4)

10.910.8 26.5±4.8

32.111.2

30.513.5

26.813.3

3.7*0.2

INH

ESTIMATED HYDRAZINE

JLS-CCH CH C-

o -

3

ACETYLISONIAZID

ISONIAZID

ISONIAZID HÏDRAZONES

ACETYLHYDRAZINB

ISONICOTINIC ACID

Figure 2. Twenty-four hour urinary excretion of metabolites after administration of 300 mg of acetylisoniazid- H-ring-labeled (AcINH) or isoniazid- H-ring-labeled (INH) to male volunteers (See Ref. 15) s

3

160


Table I


ACUTE HEPATIC NECROSIS IN RATS PRODUCED BY ISONIAZID (INH), ACETYLISONIAZID (AcINH), ACETYLHYDRAZINE (AcHz), IPRONIAZID (IpINH), AND ISOPROPYLHYDRAZINE (IpHz)

Treatments

INH 100 mg/kg*

AcINH 200 mg/kg

AcHZ 20 mg/kg

0 or +

IpINH 200 mg/kg

IpHz 20 mg/kg

+

+

None

0

0 or +

Phénobarbital

+

++

-H+

Phénobarbital + CoCl

0

0

0

0 or+

0 or +

Phénobarbital + BNPP

0

0

-H-+

0 or+

+4+4-

2

*Admlnisterd every hour for 6 hours* +CoCl • cobalt chloride. 2

ÎBNPP • bls-para-nitrophenyl phosphate.

+++

8.

NELSON

ET AL.

Chemical-Induced

Tissue

Injury

161


above 100 mg/kg i n d i c a t i n g a s a t u r a b l e mechanism. Thus, a s i n g l e l a r g e dose does not cause l i v e r n e c r o s i s , but the a d m i n i s t r a t i o n o f i s o n i a z i d i n s i x s i n g l e doses o f 100 mg/kg per hour caused acute h e p a t i c n e c r o s i s (Table I ) . Covalent b i n d i n g s t u d i e s i n v i v o . As f u r t h e r support f o r the hypothesis t h a t a c e t y l i s o n i a z i d i s converted i n the b o d y j j o a c h e m i c a l l y r e a c t i v e form v i a a c t i v a t i o n o f a c e t y l h y d r a i n e , Ca c e t y l i s o n i a z i d r a d i o l a b e l e d i n the a c e t y l moiety and C - a c e t y l hydrazine were given t o r a t s and evidence f o r covalent b i t i d i n g t o t i s s u e macromolecules was sought (18,19). A l a r g e amount o f cov a l e n t b i n d i n g was found upon d i g e s t i o n o f the p r o t e i n s i n the l i v e r , the t a r g e t organ f o r t o x i c i t y , but l i t t l e was found i n other t i s s u e s . This b i n d i n g was p r o p o r t i o n a l t o dose, was i n creased by pretreatment w i t h phénobarbital and was markedly decreased by pretreatment w i t h c o b a l t c h l o r i d e (Table I I ) . However, no c o v a l e n t l y bound r a d i o l a b e l e d m a t e r i a l was found when a c e t y l i s o n i a z i d r a d i o l a b e l e d i n the p y r i d i n e r i n g was administered. Thus, the r e a c t i v e m e t a b o l i t e came only from the a c e t y l h y d r a z i n e moiety. BNPP, which b l o c k s the h y d r o l y s i s o f a c e t y l i s o n i a z i d , decreased the covalent b i n d i n g o f ^ C - a c e t y l i s o n i a z i d |> t h a t o f C - a c e t y l h y d r a z i n e , p a r a l l e l i n g the e f f e c t o f BNPP on the hepatic necrosis. ut n

o

t

14

Covalent b i n d i n g s t u d i e s i n v i t r o . Based on the e f f e c t s o f mixed f u n c t i o n oxygenase inducers and i n h i b i t o r s on the h e p a t i c n e c r o s i s and covalent b i n d i n g found i n animals, experiments were c a r r i e d out u s i n g l i v e r microsomes i n v i t r o t o determine t h e enzyme requirements f o r the b i n d i n g r e a c t i o n . The r e s u l t s o f experiments w i t h a c e t y l h y d r a z i n e and r a t l i v e r microsomes under v a r i o u s c o n d i t i o n s (Table I I I ) showed t h a t a s u b s t a n t i a l amount o f covalent b i n d i n g occurred a t 37 C i n the presence o f l i v e r microsomes, a i r and NADPH. The b i n d i n g was almost a b o l i s h e d by l a c k o f NADPH, heat dénaturât i o n o f the enzymes, o r l a c k o f oxygen. A carbon monoxide: oxygen atmosphere, SKF-525A, p i p e r o n y l butoxide pretreatment, o r an antibody a g a i n s t NADPH cytochrome £ reductase i n h i b i t e d covalent b i n d i n g , thereby i n d i c a t i n g i n v o l v e ment o f a cytochrome P-450 mixed f u n c t i o n oxygenase. Furthermore, experiments w i t h h e p a t i c microsomes prepared immediately f o l l o w i n g the traumatic death o f a h e a l t h y young a d u l t male demonstrate t h a t the a c t i v a t i o n system i s present i n human t i s s u e s (19, Table III). — G l u t a t h i o n e and c y s t e i n e , n a t u r a l l y o c c u r r i n g s u l f h y d r y l compounds, s u b s t a n t i a l l y decreased covalent b i n d i n g i n v i t r o by formation o f the adducts, S - a c e t y l g l u t a t h i o n e and N - a c e t y l c y s t e i n e . The work o f Smith and G o r i n (21), which showed t h a t S - a c e t y l c y s t e i n e rearranges r a p i d l y a t n e u t r a l pH values t o the thermodynamically more s t a b l e N - a c e t y l c y s t e i n e , suggests t h a t the i n i t i a l product might have been S - a c e t y l c y s t e i n e which subsequently r e arranged t o the observed product, N - a c e t l y c y s t e i n e . Both N - a c e t y l c y s t e i n e and S - a c e t y l g l u t a t h i o n e were i s o l a t e d from e

162


CONCEPTS

Table I I EFFECT OF TREATMENTS ON IN VIVO HEPATIC COVALENT BINDING OF 3

3

ISONIAZID- H-RING-LABELED (INH),* ACETYLISONIAZID- H-RING LABELED 14

(AcINH)*, ACETYLISONIAZID- C-ACETYL-LABELED (AçINH), ACETYL-HYDRA14

3

ZINE- C-ACETYL-LABELED (AcHz), IPRONIAZID- H-RING-LABELED

(IpINH),

3

IPR0NIAZID-2- H-IS0PR0PYL-LABELED (ΙηΙΝΗ), and ISOPROPYLHYDRAZINE3

2- H-IS0PR0PYL-LABELED (IpHz) IN RATS.


R e s u l t s a r e expressed as means + standard e r r o r s o f 3 separate experiments u s i n g 3 animals i n each experiment.

Treatment

AcINH IpINH IDINH AcHz IpHz 200 200 20 200 20 mg/kg mg/kg mg/kg mg/kg mg/kg Covalent B i n d i n g Covalent B i n d i n g nmole/mg p r o t e i n nmole/mg p r o t e i n (6 h r a f t e r dose) (6 h r a f t e r dose)

None

0.20 + .021

0.15 + .012

0.09 + .015

0.28 + .029

0.35 + .023

Pb**

0.31 + .021

0.19 + .012

0.10 + .015

0.53 + .038

0.44 + .038

0.15 + .039

0.09 + .008

0.18 + .017

0.22 + .029

0.11 + .033

0.23 + .035

0.17 + .019

0.32 + .025

Pb + C o C l

2

Pb + ΒΝΡΡΦ

Covalent b i n d i n g f o r these two compounds was p r o t e i n f o r a l l treatments. + CoCl

β 2

< 0.05 nmole/mg

phénobarbital + c o b a l t c h l o r i d e

tpb + BNPP » phénobarbital + b i s - p a r a - n i t r o p h e n y l phosphate

8.

NELSON

ET

AL.

Chemical-Induced

Tissue

Injury

163

Table I I I 14

14

COVALENT BINDINGQIN VITRO OF ACETYL-( C)-HYDRAZINE ( C-AcHz) AND ISOPROPYL-(2-nQ-HYDRAZINE CH-IpHz) TO RAT LIVER MICROSOMES A

Conditions

X-AcHz (1 mM)

~Ή-ΙρΗζ (0.1 mM)

** % of C o n t r o l


A.

C o n t r o l * ( a i r atmosphere) B o i l e d microsomes - C o f a c t o r (NADPH generating system) +NADH (-NADPH generating system) 100% N atmosphere 2

6%

10%

7% 15%

7% 11%

atmosphere

92%

97%

C0:0

(9:1) atmosphere

37%

48%

64% 25% 35% 49%

70% 45% 58% 65%

2

: 0

2

+SKF 525-A (0.2 mM) P i p e r o n y l butoxide+ +GSH (1 mM) +Cysteine (1 mM)

C.

100% 12%

2

N

B.

100% 13%

$

Preimmune - g l o b u l i n

0.099

nmoles/mg/15 min 0.328

Immune Ï - g l o b u l i n (NADPH-cytochrome c reductase antibody)

0.048

0.159

C o n t r o l ( a i r atmosphere) - c o f a c t o r (NADPH generating system)

0.16

Human Microsomes 0.37

0.02

0.03

Microsomes were prepared from r a t l i v e r and human l i v e r , incubated as d e s c r i b e d i n Table IV and covalent b i n d i n g was determined ( 1 9 ) . The c o n t r o l b i n d i n g o f AcHz w i t h r a t l i v e r microsomes was 0.55 nmoles/mg/15 min and f o r IpHz was 0.58 nmoles/mg/15 min. Âdministered

(0.3 ml) i . p . 30 min p r i o r t o s a c r i f i c i n g

the animal.

"Êach i n c u b a t i o n contained 7 mg of p a r t i a l l y p u r i f i e d preimmune o r immune jf - g l o b u l i n per mg microsomal p r o t e i n , as p r e v i o u s l y described (20).

164



i n c u b a t i o n mixtures by g e l f i l t r a t i o n on Sephadex f o l l o w e d by anion exchange chromatography* The products were then c h a r a c t e r i z e d by chemical i o n i z a t i o n mass spectrometry (22,23)* I p r o n i a z i d * Studies i n animals o f the metabolism of i p r o n i a z i d ( I I ) , an antidepressant drug removed from c l i n i c a l use because o f a h i g h i n c i d e n c e of h e p a t i t i s s i m i l a r to t h a t o f i s o n i a z i d r e v e a l e d t h a t i p r o n i a z i d a l s o r e q u i r e d enzymatic h y d r o l y s i s to produce the h e p a t i c l e s i o n (Table I ) . S p e c i f i c r a d i o l a b e l l n g and covalent b i n d i n g s t u d i e s showed that i s o p r o p y l h y d r a z i n e was r e leased by h y d r o l y s i s and then o x i d a t i v e l y a c t i v a t e d i n v i t r o to a potent hepatotoxin (19, Table I I ) . M e t a b o l i c a c t i v a t i o n of i s o p r o p y l h y d r a z i n e , the hepatotoxic m e t a b o l i t e of i p r o n a z i d , t o a r e a c t i v e intermediate showed enzyme requirements v i r t u a l l y i d e n t i c a l t o those f o r the a c t i v a t i o n of a c e t y l h y d r a z i n e (Table I I I ) . Thus, a cytochrome P-450 oxygenase mediated the covalent b i n d i n g o f i s o p r o p y l h y d r a z i n e to t i s s u e p r o t e i n . Trapping experiments w i t h c y s t e i n e and g l u t a t h i o n e showed t h a t S - i s o p r o p y l c y s t e i n e and S - i s o p r o p y l g l u t a t h i o n e were formed (23)· The a c t i v a t i n g enzyme system could be assessed k i n e t i c a l l y u s i n g covalent b i n d i n g o f r a d i o l a b e l e d m e t a b o l i t e as an index o f r e a c t i v e product formation* A d o u b l e - r e c i p r o c a l p l o t of the enzyme-dependent b i n d i n g o f a c e t y l h y d r a z i n e to microsomal p r o t e i n s ( F i g u r e 3A) shows t h a t the r e a c t i o n r a t e i s markedly increased by phénobarbital pretreatment, which p o t e n t i a t e d the h e p a t i c n e c r o s i s and b i n d i n g i n v i v o , whereas i t i s decreased by pretreatment o f the animals w i t h c o b a l t c h l o r i d e , which blocked the h e p a t i c n e c r o s i s and b i n d i n g i n v i v o . The same e f f e c t s were found f o r the b i n d i n g r e a c t i o n o f i s o p r o p y l h y d r a z i n e to r a t l i v e r microsomes, except that the Κ f o r b i n d i n g was 1/10 that f o r a c e t y l h y d r a z i n e (Figure 3B). This may account f o r the greater h e p a t o t o x i c i t y observed w i t h i s o p r o p y l hydrazine when compared to t h a t of a c e t y l h y d r a z i n e . I n a d d i t o n , the e v o l u t i o n o f propane from microsomal r e a c t i o n s was determined by gas chromatography and gas chromatography-mass spectrometry .As shown i n Table IV, phénobarbital i n c r e a s e d both covalent b i n d i n g and propane formation, whereas c o b a l t c h l o r i d e decreased both. Double-isotope experiments w i t h a c e t y l i s o n i a z i d - a c e t y l h y d r a z i n e and i p r o n i a z i d - i s o p r o p y l h y d r a z i n e . In order to study the metabolic a c t i v a t i o n process f o r the covalent b i n d i n g of a c e t y l i s o n i a z i d and a c e t y l h y d r a z i n e i n more d e t a i l , we administered to r a t s a mixture o f a c e t y l i s o n i a z i d and a mixture of a c e t y l h y d r a z i n e l a b e l e d w i t h t r i t i u m and carbon-14 i n the a c e t y l moiety. The H/ r a t i o o f the c o v a l e n t l y bound m e t a b o l i t e from e i t h e r a c e t y l i s o n i a z i d or a c e t y l h y d r a z i n e was almost i d e n t i c a l to the ^H/^C r a t i o o f the administered mixture (19, Table V ) . This i n d i c a t e d 3r ij y ! group was bound. Moreover, Incubation of the ^H- and ^ C - a c e t y l h y d r a z i n e w i t h r a t l i v e r microsomes i n v i t r o gave the same r e s u l t s (19, Table V ) . 3

t h a t

t h e

e n t

e

a c e t

8.

Chemical-Induced

NELSON E T A L .

Tissue

Injury

165

200

PHENOBARBITAL + COBALTOUS CHLORIDE PRETREATMENT K^-0.98 aM V^-0.03 NMOLES/MG/MIN

150 1

(NMOLES/MG/MIN)"

NORMAL 1^-0.95^ V ax-0.06 NMOLES/MG/MIN B


PHENOBARBITAL 1^-1.13 «M V - _ - 0 . l l NMOLES/MG/MIN

1

1/S («Μ" ) 100

r

PHENOBARBITAL + CoCl, Κ - 0.07 m V ? " 0.04 naolM/ag/aln

NORMAL K^- 0.09 WÊL V_ • 0.07 naol*s/ag/aln

PHENOBARBITAL K_« 0·10 «M - 0.13 naoUs/ae/aln

-15

-10

-5

Figure S. Lineweaver-Burk plots of mixed function oxidase-dependent covalent binding of acetylhydrazine (A) ana isopropylhydrazine (B) to rat microsomal protein in vitro. For each incubation, rat microsomes were prepared, incubated under air with either Cacetylhydrazine (A) or isopropyl-2 ( H)-hydrazine (B) and a NADPH-generating system, and covalent binding was determined. 14

s

166


CONCEPTS


TABLE IV CORRELATION OF PROPANE EVOLUTION WITH IN VITRO COVALENT BINDING OF ISOPROPYL-(2- H)-HYDRAZINE TO HEPATIC MICROSOMES Ice-cold incubation mixtures (3 ml) contained rat l i v e r microsomal protein (2 mg/ml) isolated from rats pretreaÇed as i n dicated; phosphate buffer, pH 7.4, 83 mM; isopropyl-(2- H)hydrazine, 0.1 mM; and a NADPH-generating system (NADP, 0.64 mM; glucose-6-phosphate, 15.5 mM; glucose-6-phosphate dehydrogenase, 2U/ml; MgC^, 10 mM). Reactions were incubated under a i r i n septum-sealed incubation vessels for 15 min with shaking (Dubnoff shaker incubator) at 37 C and covalent binding determined (19). The head-space gases were analyzed by GLC as described i n Ref. 19} the propane effluent was trapped in Aquasol s c i n t i l l a n t cooled i n dry ice-acetone, and radioa c t i v i t y was counted by s c i n t i l l a t i o n spectrometry. Results are expressed as means + standard deviations. Numbers i n parentheses are number of determinations.

Treatment

None Phénobarbital (75 mg/kg i.p.

I n v i t r o Covalent B i n d i n g (nmoles/mg p r o t e i n / 1 5 min)

0.58 + 0.051 (9)

χ 4 days)1.06 + 0.059 (6)

Phénobarbital (75 mg/kg i.p. χ 4 days) + cobalt chloride (30 mg/kg s.c. 12 hourly χ 4 doses) 0.33 + 0.006 (6)

Propane evolved (% o f t o t a l radioactivity i n 15 min)

13.0% + 1.00 (9)

19.5% + 0.75 (6)*

9.4% + 0.81 (6)*

*P jC 0.05 when compared to respective control values as determined by Student's t test.

8.

Chemical-Induced

NELSON E T A L .

Tissue

Injury

167

Table V 3

14

RATIOS ( H / C ) RADIOLABEL BOUND TO HEPATIC PROTEIN VERSUS THAT IN INITIAL SUBSTRATE MIXTURES Mixtures o f

14 3 C-carbonyl- and H-methyl-labeled t/

i s o n i a z i d (AcINH; 200 mg/kg; sp. a c t .

acetyl*x

C, 0.15 mCi/mmole;

H,

0.53 mCi/mmole) and s i m i l a r mixtures o f a c e t y l h y d r a z i n e (AcHz, 1 4

3

20 mg/kg; sp. a c t . C , 0.36 mCi/mmole; H , 1.01 mCi/mmole) were administered t o male F i s c h e r r a t s . I n a d d i t i o n , mixtures o f 14 3 s p e c i f i c a l l y l a b e l e d i s o p r o p y l - ( 2 - C ) - and i s o p r o p y l - ( 2 - H ) l a b e l e d i p r o n i a z i d (IpINH. 200 mg/kg; sp. a c t C , 0.50 mCi/ 1 4


3

mmole; sp. a c t .

H, 1.43 mCi/mmole) and i s o p r o p y l h y d r a z i n e 14 3 (IpHz, 20 mg/kg; sp. a c t . C, 0.30 mCi/mmole; sp. a c t . H,

0.87 mCi/mmole) were administered t o F i s c h e r r a t s . I n other experiments, mixtures o f H- and C-acetylhydrazine (1 mM) 3 14 and i s o p r o p y l - ( 2 - H)- and i s o p r o p y l - ( 2 - C ) - hydrazine (0.1 mM) were incubated i n a i r w i t h an NADPH-generating system and w i t h microsomes i s o l a t e d from F i s c h e r r a t l i v e r .

Covalent b i n d i n g o f

r a d i o l a b e l t o h e p a t i c t i s s u e p r o t e i n was determined by methods p r e v i o u s l y d e s c r i b e d (19) and found t o be 0.20 nmoles/mg ( i n v i v o , AcHz), 0.28 nmoles/mg ( i n v i t r o , IpHz). 3

Values a r e r e p o r t -

4

ed as H/^* C r a t i o s o f the c o v a l e n t l y bound r a d i o l a b e l , as determined by the c h a n n e l s - r a t i o method u s i n g i n t e g r a l counting, 3

14

d i v i d e d by the H / C r a t i o o f the i n i t i a l s u b s t r a t e mixture as determined by the same method. R e s u l t s a r e expressed as means + standard e r r o r s o f 4 such determinations.

Conditions

Substrate

AcINH

AcHz

IpINH

IpHz

In vivo (6 h r a f t e r dosing) 0.90 + .055 0.92+0.021 0.92+. 032 0.96+0.060 In v i t r o (15 min i n c u b a t i o n s )

-

0.94+0,012

-

0.98+0.022


168


CONCEPTS

L i v e r microsomes and NADPH were a l s o incubated w i t h c y s t e i n e and approximately equimolar amounts o f a c e t y l - and t r i d e u t e r o a c e t y l h y d r a z i n e (22). N - a c e t y l c y s t e i n e was i s o l a t e d from i n cubation mixtures c o n t a i n i n g NADPH, oxygen, c y s t e i n e and a c e t y l hydrazine. Chemical i o n i z a t i o n mass spectrometry showed q u a s i molecular i o n s (QM ) a t m/e 164 and 167 f o r the non- and t r i d e u t e r a t e d N - a c e t y l c y s t e i n e . These i o n s were monitored and found t o have t h e same H/D r a t i o as t h e quasimolecular ions o f the a c e t y l hydrazine s u b s t r a t e mixture (m/e 75 and 78, F i g u r e 4 ) . This study e l i m i n a t e d ketene as the r e a c t i v e i n t e r m e d i a t e . S i m i l a r experiments w i t h i p r o n i a z i d and i s o p r o p y l h y d r a z i n e , l a b e l e d w i t h t r i t i u m and carbon-14 i n the methine carbon o f the i s o p r o p y l group, showed t h a t e q u i v a l e n t amounts o f t r i t i u m and carbon-14 were bound both i n v i v o and i n v i t r o , demonstrating t h a t methine hydrogen was r e t a i n e d and t h e r e f o r e e l i m i n a t i n g acetone as t h e intermediate i n the b i n d i n g r e a c t i o n (19). T h i s was confirmed by a t w i n - i o n study u s i n g s p e c i f i c a l l y C-2 deuterated i s o p r o p y l h y d r a z i n e . The r e a c t i v e m e t a b o l i t e was trapped from microsomal i n c u b a t i o n s i n v i t r o w i t h c y s t e i n e . Mass s p e c t r a l a n a l y s i s o f t h e i s o l a t e d c y s t e i n e d e r i v a t i v e showed t h a t S - i s o p r o p y l c y s t e i n e was formed and t h a t no deuterium was l o s t from the i s o p r o p y l group (23). M e c h a n i s t i c i m p l i c a t i o n s f o r the metabolic a c t i v a t i o n o f t o x i c m e t a b o l i t e s o f i s o n i a z i d and i p r o n i a z i d . From the r e s u l t s i n v i v o showing c o r r e l a t i o n s between t i s s u e n e c r o s i s and covalent b i n d i n g , s t u d i e s i n v i t r o showing a microsomal P-450 oxygenase requirement, and t r a p p i n g experiments w i t h c y s t e i n e and g l u t a t h i o n e , we propose the f o l l o w i n g r e a c t i o n scheme (Figure 5) f o r formation o f t o x i c m e t a b o l i t e s from i s o n i a z i d and i p r o n i a z i d . I s o n i a z i d i s a c e t y l a t e d t o i t s major m e t a b o l i t e a c e t y l i s o n i a z i d . I n man, r a p i d a c e t y l a t o r s convert a t l e a s t 35% more i s o n i a z i d t o a c e t y l i s o n i a z i d than slow a c e t y l a t o r s . A c e t y l i s o n i a z i d i s then e f f i c i e n t l y hydrolyzed t o i s o n i c o t i n i c a c i d and a c e t y l hydrazine. A c e t y l h y d r a z i n e i s f u r t h e r metabolized by a P-450 oxygenase, p o s s i b l y t o a N-hydroxy hydrazine. T h i s intermediate would probably dehydrate t o a c e t y l d i a z e n e which could be t h e e l e c t r o p h i l i c a c y l a t i n g s p e c i e s . However, mono-substituted d i a zenes are known t o fragment i n the presence o f oxygen most l i k e l y t o r a d i c a l s (24) and t h i s could be t h e t o x i c i n t e r m e d i a t e . Ketene has been e l i m i n a t e d as the r e a c t i v e a c y l a t i n g s p e c i e s o f a c e t y l hydrazine by chemical i o n i z a t i o n mass s p e c t r a l t w i n - i o n study w i t h t r i d e u t e r o - a c e t y l h y d r a z i n e , which i n d i c a t e d t h a t t h e e n t i r e a c e t y l group was bound (22). By mechanisms t h a t are not understood t h e r e a c t i v e m e t a b o l i t e i n i t i a t e s processes t h a t l e a d t o h e p a t i c necrosis. I p r o n i a z i d i s hydrolyzed t o i s o n i c o t i n i c a c i d and i s o p r o p y l hydrazine. Isopropylhydrazine i s then f u r t h e r o x i d i z e d t o a r e a c t i v e a l k y l a t i n g agent. Since the e v o l u t i o n o f propane p a r a l l e l s covalent b i n d i n g , we suspect t h e two r e a c t i o n s d e r i v e from common

NELSON E T A L .

Chemical-Induced

Injury

0AC75

100

I

Tissue

S

ΐθΜ,+78

80

Η R-C-N-NH

2

R-CH *CD3 3

60


•Ι

40 20

100

90

80 M/E

70

100 Ο H Il H I R-C-N-C-CH2-SH

δ

1 C

80

QM+167

COOH R=CH +CD 3

60

3

40 '22 UO 121 1 2 3 |

20

1

80

100

120 M/E

3

3

140

160

180

Figure 4. Chemical ionization mass spectra (isobutane reactant gas) of a sample of the substrate mixture of acetyl- and trideuteroacetylhydrazine (A) and of the cysteine adduct iso lated from a microsomal incubation containing the substrate mixture, NADPH and cysteine (B) (See Ref. 22)

170


CONCEPTS


i n t e r m e d i a t e . Furthermore, a t w i n - i o n study w i t h s p e c i f i c a l l y C-2 deuterated i s o p r o p y l h y d r a z i n e , s i m i l a r t o the study c a r r i e d out with trideuteroacetylhydrazine, indicated that the e n t i r e isopro p y l group was r e t a i n e d i n t h e bound m e t a b o l i t e . A r e a c t i o n scheme ( F i g u r e 5 ) compatible w i t h these r e s u l t s i s the formation o f the i s o p r o p y l r a d i c a l o r c a t i o n from i s o p r o p y l d i a z e n e . These r e a c t i v e a l k y l a t i n g agents then c o v a l e n t l y b i n d t o t i s s u e macromolecules. Whatever t h e intermediate may be, i t i s c l e a r t h a t o x i d a t i v e a c t i v a t i o n o f these hydrazines by microsomal enzymes mediates l i v e r n e c r o s i s i n animals. Since these enzymes a r e present i n human l i v e r t i s s u e , these intermediates probably cause the s e r i o u s and o c c a s i o n a l l y l e t h a l h e p a t i t i s seen w i t h i s o n i a z i d and i p r o n i a z i d therapy i n man.

t Covalent Binding to Macromolecules Λ

Propane

* •

Expired Propane

Hepatic Necrosis

Figure 5.

Proposed metabolic activation pathways for isoniazid, acetyliso niazid, and isopropylisoniazid (iproniazid)

8.

NELSON

ET AL.

Chemical-Induced

Tissue Injury

171


Acetaminophen S t u d i e s i n v i v o i n man and l a b o r a t o r y animale» Acetaminophen ( p - h y d r o x y a c e t a n i l i d e , I I I ) i s a commonly used m i l d a n a l g e s i c which i s a p p a r e n t l y q u i t e s a f e when taken i n normal t h e r a p e u t i c doses. However, l a r g e overdoses cause l i f e - t h r e a t e n i n g l i v e r l e s i o n s i n man (2J5,26), r a t s (27,28), mice (28), and hamsters (29). P r i o r treatment o f animals w i t h inducers o f drug metabolism, such as phénobarbital o r 3-methylcholanthrene, g r e a t l y p o t e n t i a t e s t h e s e v e r i t y o f t h e n e c r o s i s (28,30). I n c o n t r a s t , pretreatments w i t h i n h i b i t o r s o f drug metabolism, such as p i p e r o n y l b u t o x i d e , c o b a l t c h l o r i d e , o r «-naphthylisothiocyanate, prevent t h e n e c r o s i s (28,30). A l a c k o f c o r r e l a t i o n between acetaminophen t i s s u e l e v e l s and acetaminophen-induced h e p a t i c n e c r o s i s i n d i c a t e s t h a t a t o x i c m e t a b o l i t e r a t h e r than acetaminophen i t s e l f causes the h e p a t i c t i s s u e i n j u r y . Acetaminophen r a d i o l a b e l e d w i t h t r i t i u m o r w i t h carbon-14 was g i v e n t o normal mice and mice p r e t r e a t e d w i t h compounds t h a t a l t e r e d acetaminophen-induced h e p a t i c n e c r o s i s . The animals were k i l l e d a t v a r i o u s times and t h e l i v e r s examined f o r c o v a l e n t l y bound m e t a b o l i t e s o f acetaminophen. Autoradiograms showed covalent b i n d i n g o f acetaminophen p r e f e r e n t i a l l y i n t h e n e c r o t i c c e n t r i l o b u l a r area o f t h e l i v e r , i . e . , there was a d i r e c t c o r r e l a t i o n between the two measurable parameters t i s s u e n e c r o s i s and c o v a l e n t b i n d i n g (31). Pretreatment w i t h an inducer o f microsomal metabolism, phénobarbital, i n c r e a s e d b i n d i n g , whereas p r e t r e a t ments w i t h d i f f e r e n t i n h i b i t o r s o f metabolism decreased b i n d i n g . Thus, t h e e f f e c t o f treatment on covalent b i n d i n g c o r r e l a t e d d i r e c t l y w i t h treatment e f f e c t s on h e p a t i c n e c r o s i s . Evidence f o r the c o v a l e n t nature o f t h e b i n d i n g was obtained by d i g e s t i o n o f s o l v e n t - e x t r a c t e d l i v e r p r o t e i n w i t h protease and i s o l a t i o n o f the r a d i o l a b e l bound t o amino a c i d and peptide fragments. These s t u d i e s i n d i c a t e d t h a t acetaminophen was converted by microsomal enzymes, t o a r e a c t i v e a r y l a t i n g agent which c o v a l e n t l y bound t o macromolecules i n the t a r g e t t i s s u e f o r damage, t h e l i v e r . Concept o f a dose-threshold f o r t o x i c i t y . Because o f t h e s t r i k i n g c o r r e l a t i o n between t h e s e v e r i t y o f h e p a t o t o x i c i t y and the extent o f covalent b i n d i n g by t h e a r y l a t i n g m e t a b o l i t e o f acetaminophen, i t was s u r p r i s i n g t h a t s i g n i f i c a n t b i n d i n g d i d not occur u n t i l over 60% o f t h e drug had been e l i m i n a t e d from t h e l i v e r . G l u t a t h i o n e i s depleted from t h e l i v e r o f animals r e c e i v i n g acetaminophen because i t combines w i t h a minor m e t a b o l i t e o f the drug and forms a r e a d i l y excreted mercapturic a c i d (4,30,32, 33). Thus t h e p o s s i b i l i t y a r i s e s t h a t t h e a r y l a t i n g m e t a b o l i t e o f acetaminophen i n t i a l l y i s d e t o x i f i e d by r e a c t i n g p r e f e r e n t i a l l y w i t h g l u t a t h i o n e (Figure 6 ) . A f t e r t h e major r o u t e s o f acetaminophen metabolism ( s u l f a t i o n and g l u c u r o n i d a t i o n pathways) become s a t u r a t e d , and a f t e r t h e l i v e r i s depleted o f g l u t a t h i o n e , t h e r e a c t i v e m e t a b o l i t e can combine w i t h l i v e r macromolecules and by undefined mechanisms cause c e l l death.


172

CONCEPTS

ACETAMINOPHEN HNCOCH

HNCOCH,

HHCOCH-

3

0

OH P-450 MIXEDJFUNCTION OXIDASE HO-NCOCH "


3

•

NCOCH,

0 -

POSTULATED TOXIC INTERMEDIATES

NUCLEOPHILIC CELL MACROMOLECULES HNCOCH,

HNCOCH,

OLUTATHIONE

0 =

CELL MACROMOLECULES

•

ERCAPTURIC ACID

• CELL DEATH

Figure 6. Pathways of acetaminophen metabolism


8.

NELSON E T A L .

Chemical-Induced

Tissue

Injury

173

I n support o f t h i s view, covalent b i n d i n g and l i v e r n e c r o s i s occurred o n l y a f t e r doses o f acetaminophen s u f f i c i e n t l y l a r g e t o exceed t h e a v a i l a b i l i t y o f g l u t a t h i o n e f o r d e t o x i f i c a t i o n (Figure 7). S i m i l a r l y , when g l u t a t h i o n e concentrations i n the l i v e r were compared w i t h t h e extent o f covalent b i n d i n g a t v a r i o u s times a f t e r t h e a d m i n i s t r a t i o n o f acetaminophen, s i g n i f i c a n t b i n d i n g had occurred o n l y a f t e r g l u t a t h i o n e was s e v e r e l y depleted (30,32). I n accord w i t h t h i s view, p r i o r a d m i n i s t r a t i o n o f d i e t h y l maleate, which decreases t h e g l u t a t h i o n e c o n c e n t r a t i o n i n l i v e r without causing l i v e r n e c r o s i s , markedly p o t e n t i a t e s t h e l i v e r damage caused by acetaminophen (30,32), and d i e t s t h a t lower the conc e n t r a t i o n o f g l u t a t h i o n e enhance the t o x i c i t y as w e l l (34). On the other hand, the a d m i n i s t r a t i o n o f the a l t e r n a t e s u l f h y d r y l compounds, c y s t e i n e o r cysteamine, prevented t h e l i v e r n e c r o s i s (6,32). Recent s t u d i e s w i t h acetaminophen have supported t h e view t h a t a g l u t a t h i o n e t h r e s h o l d i s o p e r a t i v e i n man as w e l l as l a b o r a t o r y animals (33.35,36). Therefore, s u l f h y d r y l reagents such as c y s t e i n e , cysteamine, d i m e r c a p r o l , and g l u t a t h i o n e i t s e l f a r e being s u c c e s s f u l l y used i n the therapy o f acetaminophen-overdosed p a t i e n t s (37). T h i s emphasizes t h e importance o f understanding b i o c h e m i c a l mechanisms o f t o x i c i t y before r a t i o n a l approaches t o treatment can be made. Phenacetin Phenacetin ( p - e t h o x y a c e t a n i l i d e , IV) has been i m p l i c a t e d i n r e n a l i n j u r y i n man (38). Therefore, we considered t h e p o s s i b i l i t y t h a t t h i s s p e c i a l type o f n e p h r i t i s , c a l l e d a n a l g e s i c nephropathy, was r e l a t e d t o metabolic a c t i v a t i o n . Although no c o n s i s t e n t l y r e p r o d u c i b l e l e s i o n could be obtained i n l a b o r a t o r y animals t r e a t e d w i t h l a r g e doses o f phenacetin, l i v e r n e c r o s i s was observed, e s p e c i a l l y i n hamsters (39)» a species u n u s u a l l y s u s c e p t i b l e t o acetaminophen-induced h e p a t i c n e c r o s i s (29,30). As w i t h acetaminophen, phenacetin-induced l i v e r n e c r o s i s i n hamsters i s p o t e n t i a t e d by pretreatment w i t h 3-methylcholanthrene but not by phénobarbital. For example, phenacetin doses o f 400 mg/kg produce massive c e n t r i l o b u l a r n e c r o s i s i n 3-methylcholanthrene-treated animals. Moreover, t h e s e v e r i t y o f n e c r o s i s p a r a l l e l s t h e magnitude o f t h e covalent b i n d i n g o f r a d i o l a b e l e d phenacetin t o h e p a t i c p r o t e i n s and t h e d e p l e t i o n o f h e p a t i c g l u t a t h i o n e (39). L i t t l e b i n d i n g o r h e p a t i c n e c r o s i s occurs a t doses that deplete h e p a t i c g l u t a t h i o n e l e s s than 80%. However, c o n s i d e r a b l e b i n d i n g and n e c r o s i s occur at doses t h a t deplete g l u t a t h i o n e more than 80%. Pretreatment o f hamsters w i t h 3-methylcholanthrene i n c r e a s e s d e p l e t i o n o f h e p a t i c g l u t a t h i o n e , t h e covalent b i n d i n g , and the s e v e r i t y o f n e c r o s i s a f t e r phenacetin, whereas pretreatment w i t h cobaltous c h l o r i d e o r p i p e r o n y l butoxide decreases them. These f i n d i n g s i n d i c a t e t h a t g l u t a t h i o n e i n t h e l i v e r prevents covalent b i n d i n g and n e c r o s i s by combining w i t h a r e a c t i v e a r y l a t i n g m e t a b o l i t e o f phenacetin.



IN v m

2* SO OOSC OF^-ACCTAMINOPMCNfmc/kt,

·?

CONCEPTS

*»·

I.p.)

S 5

*

as

so

OOSt OF ^N-ACtTAMIMOPNtN f • I S / M l !·»·>

Figure 7. Relationship in vivo between hepatic glutathione concentration, the formation of an acetaminophen-glutathione conjugate (measured in unne as acetaminophen mercapturic acid), and covalent binding of an acetaminophen metabolite to liver proteins (See Ref. 33)


8.

NELSON E T A L .

Chemical-Induced

Tissue

Injury

175

M e c h a n i s i t i c I m p l i c a t i o n s f o r the metabolic a c t i v a t i o n o f acetaminophen and phenacetin based on s t u d i e s u s i n g hamster microsomal enzymes* Although the i d e n t i t y of the a r y l a t i n g m e t a b o l i t e s o f acetaminophen and phenacetin are u n c e r t a i n , the involvement o f N-hydroxy d e r i v a t i v e s o r arene oxides as r e a c t i v e m e t a b o l i t e s has been p o s t u l a t e d (30, 40). Experiments i n v i t r o w i t h hamster l i v e r microsomes suggests t h a t the a r y l a t i n g m e t a b o l i t e s o f a c e t aminophen and phenacetin are d i f f e r e n t , a t l e a s t i n t h i s microsoma l system (41)* The evidence i s as f o l l o w s : l ) T h e maximum v e l o c i t y of covalent b i n d i n g f o r phenacetin exceeds t h a t f o r acetaminophen, showing t h a t phenacetin i s not f i r s t deethylated to acetaminophen which i s then a c t i v a t e d * 2)Pretreatment o f hamsters w i t h 3-methylcholanthrene i n c r e a s e s the r a t e o f covalent b i n d i n g f o r acetaminophen but decreases the r a t e of b i n d i n g f o r phenacetin* 3) Phénob a r b i t a l pretreatment i n c r e a s e s the r a t e o f covalent b i n d i n g f o r phenacetin without a f f e c t i n g the r a t e of b i n d i n g f o r acetaminophen* 4) When covalent b i n d i n g was prevented by t r a p p i n g o f the r e a c t i v e m e t a b o l i t e s w i t h g l u t a t h i o n e d u r i n g i n c u b a t i o n s c a r r i e d out under atmospheres o f oxygen-18, r e d u c t i o n by Raney-nickel o f the g l u t a t h i o n e conjugates formed from e i t h e r acetaminophen or phenacetin y i e l d e d acetaminophen; however, the acetaminophen conjugate formed d u r i n g i n c u b a t i o n w i t h phenacetin i n c o r p o r a t e d 50% oxygen-18 i n t o the 4 - p o s i t i o n , whereas the acetaminophen-glutathione conjugate d u r i n g i n c u b a t i o n w i t h acetaminophen i n c o r p o r a t e d v i r t u a l l y no oxygen-18. Mechanisms based on these r e s u l t s are presented i n F i g u r e 8. The l a c k of i n c o r p o r a t i o n of oxygen-18 i n t o the g l u t a t h i o n e conjugate d e r i v e d from acetaminophen i s c o n s i s t e n t w i t h e i t h e r an N-hydroxylation o r 2,3-epoxidation mechanism. I n d i r e c t evidence i n v i v o supports an N-hydroxy l a t i o n mechanism f o r the metabolic a c t i v a t i o n of acetaminophen. Masking of the amide n i t r o g e n , as i n N-methyl-4-hydroxy a c e t a n i l i d e , b l o c k s h e p a t o t o x i c i t y (39). Pretreatment o f hamsters w i t h 3-methylcholanthrene, which i n creases the h e p a t o t o x i c i t y of acetaminophen, correspondingly i n creases the N - h y d r o x y l a t i o n of 4 - c h l o r o a c e t a n i l i d e and 2 - a c e t y l a minofluorene and the covalent b i n d i n g o f acetaminophen (20, 42, 43) By c o n t r a s t , phénobarbital pretreatment n e i t h e r a l t e r s the r a t e o f N-hydroxylation o f 4 - c h l o r o a c e t a n i l i d e nor the covalent b i n d i n g of acetaminophen (43). I t seems l i k e l y t h a t i f an N-hydroxylated m e t a b o l i t e were formed i t would subsequently undergo dehydration t o a c h e m i c a l l y r e a c t i v e imidoquinone before a r y l a t i n g t i s s u e macromolecules (Figure 8 ) . The i n c o r p o r a t i o n of 50% oxygen-18 i n t o the 4 - p o s i t i o n of the a r y l a t i n g m e t a b o l i t e of phenacetin i s p e r p l e x i n g . The s t r o n g imp l i c a t i o n i s t h a t the carbon atom i n t h i s p o s i t i o n (C-4 o f the a r o matic r i n g ) becomes t e t r a h e d r a l , b i n d i n g e q u i v a l e n t oxygen-18, der i v e d from molecular oxygen, and oxygen-16. Mechanisms c o n s i s t e n t w i t h t h i s i n t e r p r e t a t i o n are presented i n F i g u r e 8. Although these mechanisms are c o n s i s t e n t w i t h the r e s u l t s obt a i n e d i n v i t r o w i t h phenacetin, they may have l i t t l e b e a r i n g on the s i t u a t i o n i n v i v o . The major m e t a b o l i t e of phenacetin i n v i v p


176


OH

CONCEPTS

OH PHENACETIN

R R R.R-H R-R-

Figure 8.

^M^HJ'

Possible reaction mechanisms for reactive metabolite formation from acetaminophen and phenacetin using hamster liver microsomes

8.

NELSON E T A L .

Chemical-Induced

Tissue

Injury

177


i s acetaminophen and those pretreatments i n hamsters which i n crease h e p a t i c n e c r o s i s and c o v a l e n t b i n d i n g f o r acetaminophen i n c r e a s e the h e p a t i c n e c r o s i s and c o v a l e n t b i n d i n g f o r phenacetin (39). A d d i t i o n a l s t u d i e s i n v i v o w i t h phenacetin, s p e c i f i c a l l y deuterated i n theot-methylene carbon atom o f the 4-ethoxy group, a l s o i n d i c a t e d t h a t d e e t h y l a t i o n o f phenacetin t o acetaminophen i s a t least p a r t i a l l y rate-determining f o r hepatic tissue i n j u r y 45). Renal i n j u r y w i t h a c e t a n i l i d e s . R e a c t i v e i n t e r m e d i a t e s o f acetaminophen, phenacetin, and other a c e t a n i l i d e s may a l s o mediate the r e n a l i n j u r y caused by these compounds. Nery (46) has suggested t h a t the N-hydroxylated d e r i v a t i v e o f phenacetin i s an i n t e r m e d i a t e f o r minor u r i n a r y m e t a b o l i t e s o f phenacetin. Calder e t a l . (47) subsequently examined the n e p h r o t o x i c i t y o f N-hydroxyphenacetin, 4-aminophenol, hydroquinone, and p-benzoquinone and found acute r e n a l t u b u l a r n e c r o s i s i n r a t s . I t t h e r e f o r e seemed p o s s i b l e t h a t acetaminophen and phenacetin c o u l d be n e p h r o t o x i c through t h e i r N-hydroxy m e t a b o l i t e s and u l t i m a t e l y through the common r e a c t i v e d e r i v a t i v e , N-acetyl-4-benzoimidoquinone. Both acetaminophen and phenacetin c o v a l e n t l y b i n d t o a s m a l l extent t o F i s c h e r r a t kidney and acetaminophen causes r e n a l tubul a r n e c r o s i s (39). Both compounds d e p l e t e r e n a l g l u t a t h i o n e , w i t h acetaminophen much more potent than phenacetin. Experiments i n v i t r o w i t h r a t kidney microsomes show t h a t these compounds can be a c t i v a t e d t o r e a c t i v e m e t a b o l i t e s . A l t e r n a t i v e l y , acetaminophen and phenacetin may be N-hydroxylated i n the l i v e r and t r a n s ported t o the k i d n e y , p o s s i b l y as N-O-glucuronides. The g l u c u r o n i d e s might then be h y d r o l y z e d under the a c i d i c c o n d i t i o n s i n the u r i n e o r by glucuronidases i n the kidney o r u r i n e t o cause the observed r e n a l i n j u r y . However, 3-methylcholanthrene pretreatment i n c r e a s e s the h e p a t i c n e c r o s i s , but s l i g h t l y decreases the r e n a l n e c r o s i s produced by acetaminophen, suggesting t h a t the acetaminophen a c t i v a t i o n r e s p o n s i b l e f o r r e n a l i n j u r y occurs i n the kidney i t s e l f . Furans and Thiophenes Furosemide T o x i c i t y i n v i v o o f furosemide. Furosemide (V), a f r e q u e n t l y used d i u r e t i c drug, i s c o n t r a i n d i c a t e d i n pregnancy because o f i t s recognized t e r a t o g e n i c p o t e n t i a l (48). The drug a l s o has been reported t o p o t e n t i a t e r e n a l i n j u r y when used i n combination w i t h c e p h a l o r i d i n e (49, 50). Furosemide produces massive h e p a t i c n e c r o s i s i n mice and the n e c r o s i s i s prevented when metabolism i s i n h i b i t e d by pretreatment o f mice w i t h p i p e r o n y l b u t o x i d e , c o b a l t c h l o r i d e and «c-naphthylisothiocyanate (51). Covalent b i n d i n g o f the drug t o h e p a t i c t i s s u e i n mice i s a l s o blocked by these p r e treatments and occurs a few hours b e f o r e h i s t o l o g i c a l l y r e c o g n i s a b l e n e c r o s i s . Thus, the formation o f a r e a c t i v e furosemide metab o l i t e i s c a u s a l l y r e l a t e d t o the development o f furosemideinduced h e p a t i c n e c r o s i s .


178


As w i t h acetaminophen and phenacetin, the h e p a t i c n e c r o s i s produced by furosemide a l s o e x h i b i t s a dose-threshold f o r t o x i c i t y . No covalent b i n d i n g o r n e c r o s i s occurs u n t i l a dose of 100 mg/kg i s exceeded. U n l i k e the dose t h r e s h o l d f o r acetaminophen and phen e a c e t i n h e p a t o t o x i c i t y , the furosemide t h r e s h o l d i s not due t o a p r o t e c t i v e r o l e of g l u t a t h i o n e , s i n c e furosemide does not deplete h e p a t i c g l u t a t h i o n e . Studies of metabolism, d i s t r i b u t i o n , and r e v e r s i b l e plasma p r o t e i n b i n d i n g of furosemide a f t e r t o x i c and nont o x i c doses i n d i c a t e t h a t the dose-threshold f o r t o x i c i t y r e s u l t s from e i t h e r s a t u r a t i o n o f the organic anion b i n d i n g s i t e s on plasma p r o t e i n s a f t e r t o x i c doses, o r p o s s i b l y s a t u r a t i o n of b i l i a r y or r e n a l e x c r e t i o n of the drug (52). P o s s i b l e mechanism of metabolic a c t i v a t i o n . The h e p a t i c i n j u r y produced by furosemide apparently r e s u l t s from the metabolic a c t i v a t i o n of the f u r a n r i n g , p o s s i b l y by an e p o x i d a t i o n s i m i l a r t o t h a t proposed i n F i g u r e 9. Furosemide, r a d i o l a b e l e d w i t h t r i tium i n i t s f u r a n moiety, i s bound c o v a l e n t l y t o h e p a t i c microsomes i n the presence of oxygen and NADPH t o the same extent as furosemide r a d i o l a b e l e d s p e c i f i c a l l y w i t h s u l f u r - 3 5 i n i t s sulfonamide moiety, demonstrating t h a t the bound m e t a b o l i t e cont a i n s both p a r t s of the furosemide molecule. To determine where the b i n d i n g occurred on the molecule, the m e t a b o l i t e - p r o t e i n conj u g a t e s i s o l a t e d from the l i v e r were hydrolyzed under m i l d a c i d c o n d i t i o n s t h a t s p l i t furosemide i n t o i t s methylfuran and s u l f a m y o l a n t h r a n i l i c a c i d p o r t i o n s . The b i n d i n g of f u r a n - r a d i o l a b e l e d ^ r o s e m i d e t o p r o t e i n was unchanged, whereas the b i n d i n g of S-labeled furosemide was l o s t . The r e s u l t s suggested t h a t the f u r a n r i n g was being m e t a b o l i c a l l y a c t i v a t e d (51, 52). A d d i t i o n a l s t u d i e s i n v i t r o reported by W i r t h e t a l . (53) u s i n g ^ r f - H] furosemide, [35S] furosemide], k ~ H] furosemide, and H ] furosemide i n d i c a t e d t h a t formation of a p o s s i b l e e l e c t r o p h i l i c imine i n t e r m e d i a t e was u n l i k e l y and t h a t theoC-carbon was not a s i t e of metabolic a c t i v a t i o n . This f u r t h e r i m p l i c a t e d the f u r a n r i n g . Since covalent b i n d i n g was enhanced by an epoxide hydrase i n h i b i t o r and d i d not occur when tetrahydro [35S] furosemide was used as s u b s t r a t e , the authors speculated t h a t an arene oxide i n t e r m e d i a t e of the f u r a n moiety was i n v o l v e d i n the b i n d i n g . 4-Ipomeanol T o x i c i t y i n v i v o of 4-ipomeanol. Chemicals which r e p r o d u c i b l y produce an acute, s p e c i f i c pulmonary t o x i c i t y by r o u t e s of admini s t r a t i o n other than i n h a l a t i o n are r a r e and t h e i r mechanisms of a c t i o n are p o o r l y understood. 4-Ipomeanol ( V I I ) , a 3 - s u b s t i t u t e d f u r a n and the major component of "lung edema f a c t o r " produced i n sweet potatoes (Ipomoea b a t a t a s ) i n f e c t e d w i t h a common mold, has provided a v a l u a b l e t o o l w i t h which t o probe chemical-induced lung disease(54).The i n g e s t i o n of mold-damaged sweet potatoes has been i m p l i c a t e d f o r many years i n outbreaks of p o i s o n i n g i n c a t t l e . A f f e c t e d animals s u f f e r severe and o f t e n f a t a l r e s p i r a t o r y d i s t r e s s .


8.

NELSON

ET AL.

Chemical-Induced

Tissue

Injury

179

The same k i n d o f lung damage observed i n c a t t l e can be produced i n l a b o r a t o r y animals by a d m i n i s t r a t i o n o f s y n t h e t i c 4-ipomeanol (55» 56). The lung i s t h e primary t a r g e t organ i n most species* P a t h o l o g i c a l changes, such as p l e u r a l e f f u s i o n s , i n t r a a l v e o l a r and p e r i v a s c u l a r edema a r e apparent w i t h i n 6-24 hours a f t e r administration of the t o x i n . Studies were undertaken t o determine t h e p o s s i b l e formation of a c h e m i c a l l y r e a c t i v e m e t a b o l i t e i n t h i s pulmonary t o x i c i t y . Rats have been used as t h e experimental species i n a l l experiments d e s c r i b e d here. Pretreatments o f animals w i t h metabolic i n h i b i t o r s such as p y r a z o l e , p i p e r o n y l butoxide, and c o b a l t c h l o r i d e a l l markedly reduced the t o x i c i t y o f 4-ipomeanol i n the lung (57, 58). Phénobarbital, an inducer o f mixed f u n c t i o n oxygenase a c t i v i t y d i d not a l t e r t h e nature o f the lung t o x i c i t y but s i g n i f i c a n t l y increased the LD50 v a l u e , p o s s i b l y by i n c r e a s i n g d e t o x i f i c a t i o n pathways more than t o x i c pathways (59). Another type o f inducer, 3-methylcholanthrene showed a s t r i k i n g phenomenon when i t was used t o p r e t r e a t r a t s . M o r t a l i t y was decreased because o f marked decrease i n lung damage. I n c o n t r a s t , t i s s u e i n j u r y increased d r a m a t i c a l l y i n t h e l i v e r w i t h appearance o f widespread c e n t r i l o b u l a r n e c r o s i s (60). Thus, the t a r g e t organ f o r t o x i c i t y had switched from t h e lung t o the l i v e r . Covalent b i n d i n g i n v i v o and i n v i t r o . I n non-pretreated r a t s , r a d i o a c t i v i t y from 14C-4-ipomeanol becomes c o v a l e n t l y bound, p r e f e r e n t i a l l y t o t h e lungs, a f t e r a l l routes o f a d m i n i s t r a t i o n . Pretreatments w i t h mixed-function oxygenase i n h i b i t o r s , which decreased lung t o x i c i t y , caused a p a r a l l e l decrease i n covalent b i n d i n g t o lung t i s s u e i n v i v o and t o lung microsomes i n v i t r o (57, 58, 61). Phénobarbital pretreatment a l s o decreased the covalent b i n d i n g o f t o x i n t o both lung and l i v e r t i s s u e i n v i v o . However, t h e maximal r a t e o f b i n d i n g t o l i v e r microsomes i n v i t r o was increased whereas no change occurred i n b i n d i n g t o lung microsomes. The a l t e r a t i o n o f t a r g e t organ s p e c i f i c i t y f o r t i s s u e damage observed w i t h 3-methylcholanthrene pretreatment was p a r a l l e l e d by s i m i l a r a l t e r a t i o n o f covalent b i n d i n g o f r a d i o l a b e l e d 4-ipomeanol i n v i v o (60). The l e v e l o f c o v a l e n t l y bound t o x i n was markedly e l e v a t e d i n l i v e r s o f 3-methylcholanthrene-induced r a t s , whereas b i n d i n g t o lung was s i g n i f i c a n t l y reduced. The r a t e o f b i n d i n g t o l i v e r microsomes i n v i t r o was a l s o markedly i n c r e a s e d , whereas the r a t e o f b i n d i n g t o lung microsomes was unchanged (60). An important c o n c l u s i o n can be drawn from the experiments i n 3-methylcholanthrene p r e t r e a t e d animals. The t o x i c m e t a b o l i t e o f 4- ipomeanol i s so r e a c t i v e t h a t l i t t l e , i f any, o f i t escapes t h e organ i n which i t i s formed. I t t h e r e f o r e appears that i n normal animals the s p e c i f i c lung t o x i c i t y produced by 4-ipomeanol r e s u l t s p r i m a r i l y from pulmonary metabolism o f t h e agent; t h e l i v e r i s not a s i g n i f i c a n t source o f t h e r e a c t i v e m e t a b o l i t e t h a t binds t o and damages t h e lungs. The i n c r e a s e i n h e p a t i c t o x i c i t y i n 3-methylcholanthrene p r e t r e a t e d animals i s due t o an increased h e p a t i c metabolism o f 4-ipomeanol; t h e r e d u c t i o n i n pulmonary t o x i c i t y


180

CONCEPTS


probably i s due t o an e l e v a t e d r a t e o f h e p a t i c clearance o f t o x i n . Nature o f the c h e m i c a l l y r e a c t i v e m e t a b o l i t e o f 4-ipomeanol. Studies i n v i v o and i n v i t r o i n d i c a t e t h a t the r e a c t i v e m e t a b o l i t e formed by mixed f u n c t i o n oxygenase-catalyzed metabolism o f 4-ipomeanol i s a h i g h l y e l e c t r o p h i l i c species (57, 58). A d d i t i o n o f the n u c l e o p h i l i c t r i p e p t i d e , g l u t a t h i o n e , markedly i n h i b i t e d covalent b i n d i n g o f 4-ipomeanol i n v i t r o , presumably by a c t i n g as an a l t e r n a t e n u c l e o p h i l e . D e p l e t i o n of endogenous g l u t a t h i o n e i n v i v o by d i e t h y l m a l e a t e pretreatment s i g n i f i c a n t l y increased t o x i c i t y and covalent b i n d i n g o f 4-ipomeanol i n v i v o . Analogs o f 4-ipomeanol i n which the f u r a n moiety was replaced by phenyl o r methyl s u b s t i t u e n t s were not metabolized t o t o x i c e l e c t r o p h i l e s i n v i v o o r i n v i t r o (58). Thus, the furan r i n g appears t o be e s s e n t i a l f o r the observed t o x i c i t y and covalent b i n d i n g . Based on these r e s u l t s and those found f o r the metabolic a c t i v a t i o n o f furosemide, F i g u r e 10 r e v e a l s a p o s s i b l e scheme f o r f u r a n a c t i v a t i o n . Since f u r a n has l e s s a r o m a t i c i t y than benzene, i t i s not u n l i k e l y t h a t t h i s heteroaromatic nucleus could form an epoxide. T h i s epoxide would probably be q u i t e r e a c t i v e , and could y i e l d other e l e c t r o p h i l e s by spontaneous r e arrangement o r r i n g s c i s s i o n r e a c t i o n s . This arene oxide could a l s o be d e a c t i v a t e d by an epoxide hydrase, g l u t a t h i o n e , o r a g l u t a t h i o n e t r a n s f e r a s e . R e l a t i v e a c t i v i t i e s o f the v a r i o u s pathways i n t h e v a r i o u s t i s s u e s would modulate s u s c e p t i b i l i t y t o t h e toxin. H e p a t o t o x i c i t y , r e n a l t o x i c i t y , and pulmonary t o x i c i t y a r e a l s o caused by other f u r a n compounds, some of which show a g l u t a t h i o n e t h r e s h o l d , and others which show no such t h r e s h o l d (51). Furan, 2-hydroxymethylfuran, and 2 - a c e t y l f u r a n a r e hepatot o x i c . Furosemide, f u r a n , 2,3-benzofuran and c e r t a i n other furans produce acute r e n a l t u b u l a r n e c r o s i s . Other simple furans such as 2-methylfuran, 3-methylfuran, and furan i t s e l f produce lung damage, and pulmonary edema (58). Thus, a v a r i e t y of t i s s u e l e s i o n s seen a f t e r the use o f f u r a n - c o n t a i n i n g compounds probably r e s u l t s from metabolic a c t i v a t i o n s i m i l a r t o t h a t proposed f o r 4-ipomeanol and furosemide. Cephaloridine Extension o f the f u r a n s t u d i e s t o thiophene, another h e t e r o aromatic d e r i v a t i v e , has shown t h a t s e v e r a l thiophene-containing compounds produce h e p a t i c and r e n a l n e c r o s i s (39). Cephaloridine ( V I ) , a w i d e l y p r e s c r i b e d cephalosporin a n t i b i o t i c , has i t s use l i m i t e d p r i m a r i l y because o f r e n a l n e c r o s i s a s s o c i a t e d w i t h such therapy (49). Pretreatments o f mice w i t h p i p e r o n y l butoxide and c o b a l t c h l o r i d e decrease r e n a l n e c r o s i s caused by c e p h a l o r i d i n e . F u r t h e r s t u d i e s a r e i n progress t o determine i f t h i s s p e c i f i c r e n a l n e c r o s i s i s mediated by a r e a c t i v e m e t a b o l i t e .

8.

NELSON E T A L .

Chemical-Induced

F«rf«r«M«liyd«.

Tissue

Injury

181

Frmm Wmre—mié* A

Antkranilic Acid A

«lMCMr«nM«

6lucvr«ni

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