Xenobiotic Metabolism: In Vitro Methods Gaylord D . Paulson, D . Stuart Frear, and E d w i n P. Marks, EDITORS U. S. Dep...
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Xenobiotic Metabolism: In Vitro Methods Gaylord D . Paulson, D . Stuart Frear, and E d w i n P. Marks, EDITORS U. S. Department of Agriculture
A symposium sponsored by the A C S Division of Pesticide Chemistry at the 176th Meeting of the American Chemical Society, Miami, Florida, September 10-15, 1978.
ACS SYMPOSIUM SERIES AMERICAN CHEMICAL SOCIETY 1979 WASHINGTON, D. C. In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
97
Library of Congress CIP Data
Xenobiotic metabolism, in vitro methods. (ACS symposium series; 97 ISSN 0097-6156) Includes bibliographies and index. 1. Xenobiotic metabolism—Congresses. I. Paulson, Gaylord D. II. Frear, D. S., 1929III. Marks, Edwin P., 1925- . IV. American Chemical Society. Division of Pesticide Chemistry. V. Series: American Chemical Society. ACS symposium series; 97. QH521.X46 615.9'02 79-789 ISBN 0-8412-0486-1 ASCMC 8 97 1-328 1979
Copyright ©
1979
American Chemical Society A l l Rights Reserved. The appearance of the code at the bottom of the first page of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective works, for resale, or for information storage and retrieval systems. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. P R I N T E D I N THE U N I T E D
STATES
AMERICA
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
ACS Symposium Series Robert F . G o u l d , Editor
Advisory Board Kenneth B. Bischoff
James P. Lodge
Donald G. Crosby
John L. Margrave
Robert E. Feeney
Leon Petrakis
Jeremiah P. Freeman
F. Sherwood Rowland
E. Desmond Goddard
Alan C. Sartorelli
Jack Halpern
Raymond B. Seymour
Robert A. Hofstader
Aaron Wold
James D. Idol, Jr.
Gunter Zweig
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
FOREWORD The A C S S Y M P O S I U M S E R I E S was founded in 1 9 7 4 to provide a medium for publishin format of the Serie I N C H E M I S T R Y SERIES 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. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable since symposia may embrace both types of presentation.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
PREFACE he beneficial effects of a wide variety of pesticides and other xenobiotics i n eliminating or controlling certain insects, plants, and disease processes have been demonstrated conclusively. The standard of living that we now enjoy is attributable, at least in part, to an increased use of xenobiotics. However, there is also a growing awareness and concern that some xenobiotics may have adverse effects on both man and his environment. This concern has resulted i n more extensive testing and evaluation of xenobiotics One type of information an understanding of the metabolic fate of the xenobiotic i n both target and nontarget organisms. In the past, most xenobiotic metabolism studies were conducted with the intact plant or animal. Such i n vivo studies have generated a wealth of useful information and w i l l continue to be the method of choice for many investigations. However, there is a growing realization that i n vitro studies may be superior for generating certain types of information. F o r example, in vitro techniques are often the methods of choice when there is a need to isolate and identify intermediate products of a multistep metabolic sequence. Cofactor requirements and other factors (inhibitors, activators, etc.) affecting the enzymes involved i n xenobiotic biotransformations usually are determined by i n vitro studies. Comparative studies to determine the effect of factors such as species, sex, age, tissue, subcellular fraction, nutritional factors, disease states, etc. on xenobiotic metabolism often are conducted most quickly and easily i n vitro. Usually the mode of action and selectivity of xenobiotics are investigated most effectively by i n vitro studies. Although i n vitro techniques are extremely useful and have broad application in studying the metabolic fate of xenobiotics, they also have definite limitations. F o r example, many i n vitro techniques are not applicable to long-term studies because of the buildup of end products, microbial contamination, and other problems. Some investigators have used i n vitro conditions (temperature, substrate concentrations, etc.) that had little or no relationship to conditions i n the intact organism; therefore, the results obtained were of limited value. Endogenous inhibitors that are retained in subcellular compartments (and therefore have no effect on the metabolism of a xenobiotic i n vivo) may be released K
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In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
during the preparation of an i n vitro system and result i n misleading information. These and other potential problems cited i n the proceedings of this symposium make it clear that i n vitro methods must be used with caution. However, when properly used within the recognized limitations, in vitro techniques are extremely useful i n studying the metabolism of xenobiotics. This symposium was organized because of the growing interest i n the use of i n vitro techniques for xenobiotic metabolism studies. The primary objectives were to critically review, evaluate, and summarize: ( 1 ) how i n vitro techniques are used i n the laboratory with special emphasis on their application to xenobiotic metabolism studies; (2) advantages, disadvantages, and limitations of these techniques; and (3) examples of how i n vitro techniques may be useful i n future studies. It is our hope that the proceeding departure for a more effectiv future research on the metabolism and fate of xenobiotics i n the environment. Metabolism and Radiation Research Laboratory Agriculture Research,
G A Y L O R D D. P A U L S O N D. S T U A R T F R E A R
Science and Education Administration
E D W I N P. M A R K S
U.S. Department of Agriculture Fargo, ND 58102 December 20, 1978
viii
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
1 Xenobiotic Metabolism in Plants: In Vitro Tissue, Organ, and Isolated Cell Techniques R. H . S H I M A B U K U R O and
W.
C.
1
WALSH
Metabolism and Radiation Research Laboratory, Agricultural Research, Science and Education Administration, U . S. Department of Agriculture, Fargo, ND 58105
Interest i n xenobioti p r i m a r i l y on the f a t e o h e r b i c i d e s have been of predominant i n t e r e s t i n p l a n t metabol i s m s t u d i e s , the methods and techniques discussed i n t h i s report are e q u a l l y a p p l i c a b l e to other c l a s s e s of p e s t i c i d e s i n c l u d i n g i n s e c t i c i d e s and f u n g i c i d e s . In t h i s r e p o r t , the term " x e n o b i o t i c s " r e f e r s to s y n t h e t i c p e s t i c i d e s and not to other unnatural compounds. However, the d i s c u s s i o n on i n v i t r o techniques f o r x e n o b i o t i c metabolism i n p l a n t s i s based p r i m a r i l y on research with h e r b i c i d e s . The metabolism of p e s t i c i d e s i n p l a n t s i s discussed e x t e n s i v e l y i n s e v e r a l p u b l i c a t i o n s (1, _2, 3). Much i s known on the metabolism of organic p e s t i c i d e chemicals i n p l a n t s , but the f a t e of most p e s t i c i d e s i n p l a n t s i s s t i l l unknown. I t i s important to know the i d e n t i t y of t r a n s i t o r y intermediate products and the u l t i m a t e f a t e of these chemicals i n p l a n t s s i n c e the intermediate products may be t o x i c . Knowledge of chemical i d e n t i t y and quantity of intermediate products of a h e r b i c i d e i n p l a n t s at d i f f e r e n t times a f t e r treatment i s e s s e n t i a l f o r the e l u c i d a t i o n of the mode of a c t i o n and b a s i s for s e l e c t i v i t y . Whole plants t r e a t e d with p e s t i c i d e s through t h e i r roots or f o l i a g e have been used e x t e n s i v e l y f o r metabolism and " t e r m i n a l " residue s t u d i e s . U s e f u l q u a n t i t i e s of metabolites may be generated from l a r g e - s c a l e treatments of whole p l a n t s ; such metabolites can then be p u r i f i e d f o r chemical c h a r a c t e r i z a t i o n . The development of v a r i o u s chromatographic techniques and improved UV, IR, NMR and mass spectroscopy instrumentation 1
Mention of a trademark or p r o p r i e t a r y product does not c o n s t i t u t e a guarantee or warranty of the product by the U. S. Department of A g r i c u l t u r e and does not imply i t s approval to the e x c l u s i o n of other products that may a l s o be s u i t a b l e . This chapter not subject to U.S. copyright. Published 1979 American C h e m i c a l Society
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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XENOBIOTIC
M E T A B O L I S M
has made i t p o s s i b l e to c h a r a c t e r i z e chemically small amounts of metabolites. Excised p l a n t t i s s u e s and organs and i s o l a t e d c e l l s may be used f o r p e s t i c i d e metabolism s t u d i e s f o r short periods where l i m i t e d q u a n t i t i e s o f metabolites a r e generated. Results from s e l e c t e d r e p o r t s are presented t o i l l u s t r a t e the techniques and methods that may be used. Metabolism of X e n o b i o t i c s The s u c c e s s f u l i s o l a t i o n and chemical c h a r a c t e r i z a t i o n of any x e n o b i o t i c b i o t r a n s f o r m a t i o n product r e q u i r e the generation of s u f f i c i e n t q u a n t i t i e s o f the metabolite. The degradation mechanisms i n p l a n t s may be slower than those i n animals (_1). P l a n t s a l s o l a c k an excretory system comparable to the r e n a l e x c r e t i o n system i n mammals t i o n products o f p e s t i c i d e e x c r e t i o n products as with mammals. P l a n t s metabolize s i g n i f i cant amounts o f p e s t i c i d e s u l t i m a t e l y t o i n s o l u b l e residues ( 3 ) . The chemical nature and q u a n t i t i e s of the metabolites i n a p l a n t are i n f l u e n c e d by the s i t e of absorption of the p e s t i c i d e , t r a n s l o c a t i o n , and the residence time i n the p l a n t . The use of excised plant t i s s u e s , organs, and i s o l a t e d c e l l s f o r s t u d i e s of x e n o b i o t i c metabolism i s an attempt to modify the i n f l u e n c e of the above p h y s i o l o g i c a l f u n c t i o n s i n order to optimize the c o n d i t i o n s f o r maximum metabolite generation. Fundamental f u n c t i o n s of the whole p l a n t , i n c l u d i n g absorption, t r a n s l o c a t i o n , c e l l f u n c t i o n s and senescence should be considered when i n v i t r o techniques with i s o l a t e d p l a n t parts are used. The p h y s i o l o g i c a l s i g n i f i c a n c e of metabolism i n i s o l a t e d plant p a r t s must be evaluated u l t i m a t e l y i n terms of r e s u l t s i n intact plants. Absorption. Regardless o f how a p e s t i c i d e i s a p p l i e d to the p l a n t , the chemical must penetrate the p l a n t and be absorbed s p e c i f i c a l l y i n t o the c e l l s where b i o t r a n s f o r m a t i o n r e a c t i o n s occur. The l e a f surfaces and root t i p s are the primary s i t e s o f p e n e t r a t i o n i n t o the plant (4, 5 ) . The c u t i c l e , a t h i n , l i p o i d a l membrane that covers the e n t i r e s u r f a c e area of the above ground p a r t s o f a p l a n t , i s the primary b a r r i e r to p e n e t r a t i o n by organic p e s t i c i d e s . The p e n e t r a t i o n o f nonpolar organic p e s t i c i d e s i n t o leaves and roots i s b e l i e v e d to be a two-stage process (4, 6 ) . The f i r s t stage i n l e a f a b s o r p t i o n i n v o l v e s passive p e n e t r a t i o n or p a r t i t i o n i n g o f the nonpolar compound i n t o the c u t i c l e and d e s o r p t i o n i n t o the c e l l w a l l s (apoplast) o f the u n d e r l y i n g cells. In r o o t s , the f i r s t stage i n v o l v e s the i n a c t i v e d i f f u s i o n of the compound i n t o the root " f r e e space" ( a p o p l a s t ) . The second stage i n leaves i s the a c t i v e transport of the p e s t i c i d e s across the plasmalemma ( c e l l membrane) i n t o l e a f c e l l s (symplast), and i n some cases i n t o the phloem f o r
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
1.
SHiMABUKURo
A N D W A L S H
Plant Tissues, Organs, and Isolated Cells
symplastic t r a n s p o r t . Symplastic transport i s an a c t i v e , energy-requiring process o c c u r r i n g i n the l i v i n g cytoplasmic continuum of the p l a n t . In r o o t s , the second stage i n v o l v e s an a c t i v e transport o f the p e s t i c i d e s across the endodermis and i n t o the s t e l e where the d i f f e r e n t i a t e d v a s c u l a r s t r u c t u r e i s l o c a t e d . Therefore, symplastic i n t e r c e l l u l a r t r a n s p o r t of p e s t i c i d e s occurs a t one stage during the t r a n s p o r t of p e s t i c i d e s from the e x t e r n a l root s o l u t i o n i n t o the xylem v e s s e l s i n the s t e l e f o r a p o p l a s t i c transport to the shoot. Apoplastic transport from the root t o shoot i s an i n a c t i v e , p h y s i c a l process o c c u r r i n g i n the n o n - l i v i n g extra-protoplasmic component o f the p l a n t under the i n f l u e n c e o f the t r a n s p i r a t i o n stream. The symplast-apoplast concept i s a p p l i e d as defined by C r a f t s and C r i s p (7) Unfortunately l i t t l e i s known about the mechanisms o f absorptio molecules i n r o o t s . Th information regarding the absorption o f i n o r g a n i c ions by r o o t s . T r a n s l o c a t i o n . Once p e n e t r a t i o n i n t o l e a f c e l l s or root t i p s i s accomplished, the p e s t i c i d e must be t r a n s l o c a t e d s y m p l a s t i c a l l y from leaves and a p o p l a s t i c a l l y from roots t o d i f f e r e n t plant organs and d i s t r i b u t e d to s p e c i f i c t i s s u e s and c e l l s where the t a r g e t s i t e s f o r b i o l o g i c a l a c t i v i t y may be l o c a t e d . The t a r g e t s i t e s o f the p e s t i c i d e may o r may not be l o c a t e d i n the same t i s s u e s or c e l l s as the b i o t r a n s f o r m a t i o n s i t e s . D e t a i l e d d i s c u s s i o n s on s t r u c t u r e of the v a s c u l a r system and mechanisms of t r a n s p o r t are discussed i n s e v e r a l p u b l i c a t i o n s (5, 8). I t i s g e n e r a l l y recognized that phloem or symplastic t r a n s p o r t occurs from "sources to s i n k s " or i n broad terms from green leaves t o a c t i v e centers of growth and storage. This r e s u l t s i n d i f f e r e n t i a l or s e l e c t i v e t r a n s l o c a t i o n and accumulation of photosynthates i n young leaves, buds, and meristematic regions of the p l a n t . Most h e r b i c i d e s , and probably other p e s t i c i d e s , do not appear to t r a n s l o c a t e very r e a d i l y i n the symplast (2) although exceptions a r e known ( 5 ) . N o n s p e c i f i c t r a n s l o c a t i o n and uniform d i s t r i b u t i o n to a l l p a r t s of p l a n t s occur by a p o p l a s t i c t r a n s p o r t . This i s g e n e r a l l y observed when h e r b i c i d e s are t r a n s l o c a t e d i n the xylem from roots to shoots under the i n f l u e n c e o f the t r a n s p i r a t i o n stream (9). The rates of p e s t i c i d e s t r a n s l o c a t e d i n the xylem appear to depend on the amount of m a t e r i a l r e l e a s e d by parenchyma c e l l s to the xylem (9). Factors a f f e c t i n g t r a n s p i r a t i o n a l s o i n f l u e n c e a p o p l a s t i c t r a n s p o r t of p e s t i c i d e s from roots t o shoots. The a b s o r p t i o n and t r a n s l o c a t i o n functions i n a whole p l a n t r e f l e c t the f u n c t i o n s o f s p e c i f i c organs, t i s s u e s , and c e l l s organized and i n t e g r a t e d i n t h e i r a c t i v i t i e s t o meet the requirements of growth and maintenance. The i m p l i c a t i o n s o f s e p a r a t i n g the organs, t i s s u e s , and c e l l s f o r use i n i n v i t r o
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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6
XENOBIOTIC
x e n o b i o t i c metabolism s t u d i e s are c l e a r l y
M E T A B O L I S M
evident.
Senescence. Senescence of p l a n t t i s s u e s i s a major f a c t o r that must be considered i n x e n o b i o t i c metabolism s t u d i e s w i t h i s o l a t e d p l a n t organs and c e l l s . In c o n t r a s t to mammals, senescence i n p l a n t s i s not due to i r r e v e r s i b l e changes i n the genome (DNA breakdown), but due to i n t e r n a l p l a n t f a c t o r s that i n h i b i t c e l l metabolism or a l t e r i t s d i r e c t i o n toward a u t o l y t i c pathways (10). Plant senescence i s a hormonally c o n t r o l l e d phenomenon (10, 11) that may be induced, retarded or reversed under d i f f e r e n t circumstances. The process of l e a f senescence begins as soon as leaves are excised or detached from the whole p l a n t . P r o t e i n synthe s i s and c h l o r o p h y l l conten r e s p i r a t i o n , and RNase (12, 13, 14, 15). In whea lipase, esterase, and a c i d phosphatase d e c l i n e d a f t e r detachment. However, the d e c l i n e i n the enzyme l e v e l s was retarded by treatment of leaves w i t h k i n e t i n (15). L i g h t r e t a r d s senes cence i n excised leaves (12). Light-induced r e t a r d a t i o n of senescence was not l i n k e d to phytochrome a c t i o n , but was r e l a t e d d i r e c t l y to photosynthesis (12). However, evidence i n d i c a t e s that l i g h t r e t a r d a t i o n of senescence i s not l i n k e d to CO2 f i x a t i o n or photochemical a c t i v i t y of PS I I . Diuron [ 3 - ( 3 , 4 - d i c h l o r o p h e n y l ) - l , l - d i m e t h y l u r e a ] d i d not e l i m i n a t e or reduce the e f f e c t i v e n e s s of l i g h t i n r e t a r d i n g c h l o r o p h y l l l o s s (16). A d d i t i o n of sucrose a l s o had no e f f e c t on c h l o r o p h y l l l o s s when CO2 f i x a t i o n was i n h i b i t e d by diuron (16). Glucose i n h i b i t e d s i g n i f i c a n t l y the l o s s of c h l o r o p h y l l i n the dark (14). However, the e f f e c t i v e n e s s of glucose at the optimum concentration of 100 μΜ was s t i l l only h a l f the e f f e c t i v e n e s s of k i n e t i n at 10 μΜ (14). I f a high-energy intermediate i s r e q u i r e d to delay senescence, the r e s u l t s i n d i c a t e that l i g h t may be a c t i n g through i t s a c t i o n on c y c l i c photophosphorylat i o n , a system that i s not i n h i b i t e d by diuron. T o t a l p r o t e i n s y n t h e s i s d e c l i n e s i n senescence but a s p e c i f i c proteinase with L - s e r i n e i n i t s a c t i v e center i n creases i n a c t i v i t y as senescence progresses (17). Most of the t o t a l s o l u b l e p r o t e i n l o s t i n e a r l y senescence was account ed f o r by a decrease i n r i b u l o s e - l , 5 - b i p h o s p h a t e carboxylase (13). Therefore, the c h l o r o p l a s t appears to be the o r g a n e l l e i n which the i n i t i a l senescence sequence begins. Excised leaves or l e a f d i s c s have been u t i l i z e d exten s i v e l y to study senescence i n p l a n t s . These systems have two advantages: 1) detached t i s s u e s senesce at a f a s t e r r a t e than when they are attached to the p l a n t , and 2) r e g u l a t o r y compounds can be fed conveniently to the t i s s u e s through the cut s u r f a c e s . However, i t i s u n c e r t a i n whether the biochemical and p h y s i o l o g i c a l changes i n an excised l e a f resemble those i n attached leaves. These c o n s i d e r a t i o n s i n the study of senescence are
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
1.
SHiMABUKURo
A N D W A L S H
Plant Tissues, Organs, and Isolated Cells
r e l e v a n t a l s o to s t u d i e s on metabolism o f x e n o b i o t i c s i n i s o l a t e d plant t i s s u e s . I s o l a t e d Plant Part and C e l l Methods Whole p l a n t s with p e s t i c i d e s a p p l i e d through t h e i r roots or leaves or i n j e c t e d i n t o stems and f r u i t s are the most commonly used experimental m a t e r i a l f o r x e n o b i o t i c metabolism s t u d i e s . However, s t u d i e s with i n t a c t p l a n t s are complicated by v a r i a b l e s r e l a t e d to root and l e a f absorption, t r a n s l o c a t i o n and t r a n s p i r a t i o n . To overcome some of these v a r i a b l e s , researchers have used excised p l a n t p a r t s and enzymatically separated mesophyll c e l l s . Isolated plant protoplasts also may be u s e f u l f o r x e n o b i o t i metabolis s t u d i e s Results of x e n o b i o t i biochemical and p h y s i o l o g i c a response separate plan parts or c e l l s a r e u s u a l l y extrapolated to r e f l e c t r e a c t i o n s o c c u r r i n g i n complex whole p l a n t s . T h i s may o r may not be appropriate and c a u t i o n must be e x e r c i s e d i n e v a l u a t i n g r e s u l t s from separated systems. The d i f f e r e n t methods used f o r xenob i o t i c metabolism s t u d i e s with plant p a r t s and i s o l a t e d c e l l s together with r e s u l t s from s e l e c t e d r e p o r t s are presented here. Excised Leaves and Roots. Whole organs separated from the i n t a c t p l a n t are used i n t h i s method. The leaves of both d i - and monocotyledonous p l a n t s may be used. Method. Dicotyledonous p l a n t s such as cotton (Gossypium hirsutum L.) (18, 19, 20), peanut (Arachis hypogaea L.) (21, 22), c a r r o t (Daucus c a r o t a L.) (20) and soybean [ G l y c i n e max (L.) Merr.] are grown u n t i l t h e i r f i r s t true leaves a r e f u l l y expanded. The p e t i o l e s of the true leaves are excised under water to prevent d i s r u p t i o n o f the water column i n the xylem by the i n t r o d u c t i o n of a i r . This precautionary step minimizes permanent w i l t i n g o f leaves due to the i n t e r r u p t i o n of the t r a n s p i r a t i o n stream. Leaves of monocotyledonous p l a n t s l a c k p e t i o l e s . Theref o r e , s e l e c t e d leaves may be excised near the base of the lamina or blade as described f o r the p e t i o l e s of d i c o t s . Leaves from corn (Zea mays L . ) , sorghum (Sorghum vulgare P e r s . ) , and sugarcane (Saccharum o f f i c i a n a r u m L.) have been excised and used s u c c e s s f u l l y (23, 24). Excised l e a f blades o f b a r l e y (Hordeum vulgare L.) and r i c e (Oryza s a t i v a L.) have been used to study the metabolism of detergents (25). The e n t i r e shoot of young (2- to 3-leaf stage) b a r l e y , wheat ( T r i t i c u m aestivum L.) and w i l d oat (Avena f a t u a L.) seedlings may be excised a t the s o i l l e v e l by the same method and used f o r metabolism s t u d i e s . M o r p h o l o g i c a l l y , the shoots o f young c e r e a l s and other grasses c o n s i s t o f overlapping l e a f sheaths o f emerged and younger leaves. The v a s c u l a r anatomy of the l e a f sheath
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
7
8
XENOBIOTIC
M E T A B O L I S M
would be comparable to that of the p e t i o l e . E x c i s i n g the shoot of young c e r e a l p l a n t s i s n e a r l y equivalent to e x c i s i n g s e v e r a l l e a f blades. The cut edges of e i t h e r p e t i o l e s or l e a f blades are immersed i n a s o l u t i o n of the x e n o b i o t i c compound (18, 25). Excised leaves are normally t r e a t e d i n a controlled-environment chamber with a d e f i n i t e photoperiod. L i g h t not only r e t a r d s senescence but i t s t i m u l a t e s t r a n s p i r a t i o n and increases the uptake of treatment s o l u t i o n . Excised whole r o o t s , separated from the shoots, have not been used e x t e n s i v e l y f o r metabolism s t u d i e s . The r o o t , a h e t e r o t r o p h i c p l a n t organ, i s more conducive f o r use i n t i s s u e c u l t u r e where a carbon source may be provided. Corn roots s u p p l i e d with glucose i n a s e p t i c c u l t u r e metabolized a t r a z i n e (2-chloro~4-ethylamino-6-isopropylamino-^-triazine hr p e r i o d (26). Excise corn, wheat, soybean, oats (Avena s a t i v a L.) and b a r l e y were incubated with simazine [2-chloro-4,6-bis (ethylamino)-s_t r i a z i n e ] i n Hoagland s n u t r i e n t s o l u t i o n f o r 6 hours (27). Excised roots and hypocotyls of soybean were incubated f o r 24 hours i n the dark i n a 0.1% Na2C03 or d i s t i l l e d water s o l u t i o n of amiben (3-amino-2,5-dichlorobenzoic a c i d ) . The amiben metabolite, N-glucosyl amiben [N-(3-carboxy-2,5-dichlorophenyl)glucosylamine], was i s o l a t e d and c h a r a c t e r i z e d from these t i s s u e s (28). Roots of dicotyledonous seedlings with l a r g e endogenous sources of carbon i n t h e i r cotyledons may be c u l t u r e d successf u l l y a f t e r removal of t h e i r e p i c o t y l s (immature shoots). The growth of roots from pea (Pisum sativum L.) seedlings with t h e i r e p i c o t y l s removed was s i m i l a r to growth of roots from i n t a c t seedlings f o r 11 days i n n u t r i e n t s o l u t i o n (29). These r o o t s were t r e a t e d with a t r a z i n e f o r 9 days. The endogenous reserve i n the cotyledons was the only carbon source f o r the roots. f
Discussion. The use of excised leaves f o r x e n o b i o t i c metabolism has s e v e r a l advantages: 1) ease of t r e a t i n g plant m a t e r i a l with the x e n o b i o t i c chemical, 2) r a p i d uptake of the chemical i n t o plant t i s s u e s , and 3) e l i m i n a t i o n of root and l e a f surface absorption as b a r r i e r s . Some l i m i t a t i o n s of t h i s technique i n c l u d e : 1) the treatment period f o r metabolism must be short, 2) senescence of the plant organ begins upon e x c i s i o n from the i n t a c t p l a n t , and 3) r e a c t i o n s i n excised plant organ may not be the same as those o c c u r r i n g i n i n t a c t p l a n t s . A r e l a t i v e l y l a r g e number of excised leaves can be t r e a t e d with a minimum volume of treatment s o l u t i o n to obtain maximum uptake and d i s t r i b u t i o n of a x e n o b i o t i c i n l e a f t i s s u e s . Metabolites of f l u o r o d i f e n ( 2 , 4 - d i n i t r o - 4 - t r i f l u o r o m e t h y l diphenylether) (22) and p e r f l u i d o n e [ l , 1 , 1 - t r i f l u o r o - N - { 2 methyl-4-(phenysulfonyl)phenyl}methanesulfonamide] (21) were 1
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
1.
SHIMABUKURO
A N D W A L S H
Vlant Tissues, Organs, and
Isolated Cells
i s o l a t e d and c h a r a c t e r i z e d from 300 and 3000 excised peanut leaves, r e s p e c t i v e l y . Treatment s o l u t i o n s were prepared at p h y s i o l o g i c a l concentrations of 1 to 100 μΜ and s p a r i n g l y s o l u b l e compounds may be prepared as aqueous s o l u t i o n s c o n t a i n ing up to 1% acetone (22). Acetone at 1% d i d not cause severe i n j u r y to excised leaves. The use of s u r f a c t a n t s and emulsif i e r s common to l e a f s u r f a c e a p p l i c a t i o n s i s not necessary. Absorption and transport of x e n o b i o t i c s to c e l l u l a r b i o transformation s i t e s are f a i r l y r a p i d i n excised leaves. Absorption by d i c o t leaves i s r a p i d , u s u a l l y w i t h i n the f i r s t 3 to 5 hours of treatment. Peanut leaves absorbed approxi mately 3 to 4 ml of f l u o r o d i f e n s o l u t i o n per l e a f w i t h i n 5 hours (22), and cotton leaves absorbed up to 5 ml of c i s a n i l i d e ( c i s - 2 , 5 - d i m e t h y l - l - p y r r o l i d i n e c a r b o x a n i l i d e ) s o l u t i o n per l e a f w i t h i n 3 hours (20). A d d i t i o n a maintain excised leaves hours. The d i s t i l l e d water may be added as a pulse-chase and to replace treatment s o l u t i o n l o s t through t r a n s p i r a t i o n by excised leaves. Generally, the r a t e of x e n o b i o t i c absorption d e c l i n e s s i g n i f i c a n t l y a f t e r the i n i t i a l 3 to 5 hours. Absorption of treatment s o l u t i o n by excised l e a f blades of monocots i s not as r a p i d as with excised leaves of d i c o t s . The r a t e of treatment s o l u t i o n l o s t per u n i t l e a f area was not determined, but excised l e a f blades g e n e r a l l y r e q u i r e d l i t t l e a d d i t i o n a l d i s t i l l e d water. S u f f i c i e n t q u a n t i t i e s of jst r i a z i n e s (23), propachlor (2-chloro-N-isopropylacetamide) (24), and diclofop-methyl [methyl-2-{4-(2 ,4 -dichlorophenoxy) phenoxy}propanoate] (30) were absorbed w i t h i n 24- to 48-hour periods by excised corn and sorghum leaves and wheat shoots, r e s p e c t i v e l y , f o r metabolite i s o l a t i o n and c h a r a c t e r i z a t i o n . Excised leaves may be u s e f u l f o r i n v e s t i g a t i n g p e s t i c i d e i n t e r a c t i o n s . Rapid pulse-chase treatment of excised leaves i s p o s s i b l e with a x e n o b i o t i c preceded o r followed by a second compound. The pulse-chase technique with excised leaves a l s o i s u s e f u l i n s t u d i e s on product-precursor r e l a t i o n s h i p s . I s o l a t e d metabolites may be used i n treatments. Simultaneous treatment with x e n o b i o t i c s may be appropriate i f the compounds are compatible as a mixture. However, before any treatments with two or more compounds are made, the absorption and t r a n s l o c a t i o n c h a r a c t e r i s t i c s of each compound i n excised leaves should be determined to i n s u r e proper e v a l u a t i o n of r e s u l t s . f
f
Absorption and t r a n s l o c a t i o n i n excised leaves. Absorp t i o n of a x e n o b i o t i c i n s o l u t i o n occurs predominantly through the cut edges of p e t i o l e s or l e a f blades or sheaths. There f o r e , any i n f l u e n c e exerted by roots on the absorption and t r a n s l o c a t i o n of a x e n o b i o t i c i s circumvented by the excised l e a f method. This method allows d i r e c t uptake of the x e n o b i o t i c i n t o the xylem (apoplast) and t r a n s p o r t throughout the l e a f under the i n f l u e n c e of the t r a n s p i r a t i o n stream. Maximum
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
9
XENOBIOTIC
10
M E T A B O L I S M
number of degradation s i t e s i n the mesophyll c e l l s o f leaves should be exposed to the x e n o b i o t i c . This i s o f t e n a d i s t i n c t advantage of the excised l e a f method. However, the a p o p l a s t i c t r a n s p o r t of x e n o b i o t i c s , even when introduced d i r e c t l y i n t o the xylem, may not be s o l e l y a f u n c t i o n of t r a n s p i r a t i o n . Several h e r b i c i d e s were s e l e c t e d (Figure 1) to i l l u s t r a t e t r a n s l o c a t i o n d i f f e r e n c e s i n excised leaves. Figures 2 to 5 show the t r a n s l o c a t i o n of [-^C-ring] a m i t r o l e (3-amino-l,2,4-triazole) (A), [ ^ C - r i n g l a t r a z i n e (B), [ C - p h e n y l ] d i c l o f o p - m e t h y l (C), and [ C F ] f l u o r o d i f e n (D) i n r o o t - t r e a t e d i n t a c t p l a n t s and excised leaves o f soybean and oat. A l l h e r b i c i d e s were a p p l i e d a t 10 μΜ (spec. a c t . 0.54 mCi/mmol) concentration. Two excised soybean leaves and oat shoots were t r e a t e d with 10 l d 5 ml r e s p e c t i v e l y f h h e r b i c i d e f o r 24 hours soybean and oat p l a n t wer , r e s p e c t i v e l y , of each h e r b i c i d e f o r 48 hours (Figures 3 and 5). A l l p l a n t m a t e r i a l s were exposed to a 14-10-hour l i g h t - d a r k c y c l e a t 13 klux l i g h t i n t e n s i t y , 26 C day and 20 C night temperatures. The r e l a t i v e absorption date of soybean and oat p l a n t s (Tables I and I I ) cannot be compared d i r e c t l y s i n c e the surface areas and the l ^ C a p p l i e d d i f f e r e d between the two s p e c i e s . However, apparent d i f f e r e n c e s between a d i c o t (soy bean) and monocot (oat), as discussed p r e v i o u s l y , are evident. 14
l 5
3
Table I .
Absorption and t r a n s l o c a t i o n of [14c]herbicides b y excised soybean leaves and excised shoots of oat.u/
Herbicide Amitrole(A) Atrazine(B) Diclofopme thy1(C) Fluorodifen(D)
t
% of applied 14c absorbed^/
D i s t r i b u t i o n o f absorbed oat soybean
soybean
immerseaSJ
oat
1 4
l e a f immersedSJ
C(%)
shoot
99 99
32 28
6 1
94 99
53 22
47 78
92 94
77 56
36 49
64 51
75 81
25 19
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
SHiMABUKURo
Plant Tissues, Organs, and Isolated
A N D W A L S H
Cells
CHEMICALS USED FOR TRANSLOCATION -CI
CH
CI
AMITROLE
_/ C
Figure 1.
N 0
2
_
H
I i / * H C-CH -N^N^N-CH CH, ATRAZINE 3
3
WCLOFOP— METHYL (HOE23408)
CI
3
3
0-è-COOCH
2
2
H
H
V
FLU0R0D4FEN
C-14 herbicides applied to excised soybean leaves, excised oat shoots, and roots of both species for absorption and translocation
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
12
XENOBIOTIC
M E T A B O L I S M
Figure 2. Radioaut ο graphs of excised soybean leaves treated with (A), re labeled amitrole; (B), atrazine; (C), diclofop-methyl; and (D), fluorodifen. The bottom are radioaut ο graphs of the leaves above. The lower 3-cm section of each petiole was immersed in C herbicide solution for 24 hr. 14
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
SHiMABUKURo
A N D W A L S H
Plant Tissues, Organs, and Isolated
Cells
Figure 3. Radioaut ο graphs of soybean plants root-treated with (A), C-labeled amitrole; (B), atrazine; (C), diclof op-methyl; and (D), fluorodifen. The bottom are radioaut ο graphs of the phnts above. Plants were root-treated for 48 hr. 14
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
14
XENOBIOTIC
M E T A B O L I S M
Figure 4. Radioaut ο graphs of excised oat shoots treated with (A), C-fobeled amitrole; (B), atrazine; (C), diclof op-methyl; and (D), fluorodifen. The bottom are radioaut ο graphs of the excised shoots above. The lower 3-cm section of each shoot was immersed in C herbicide solution for 24 hr. 14
14
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
SHiMABUKURo
A N D W A L S H
Plant Tissues, Organs, and Isolated
Cells
Figure 5. Radioautographs of oat plants root-treated with (A), C-labeled amitrole; (B), atrazine; (C), diclof op-methyl; and (D), fluorodifen. The bottom are radioautographs of the plants above. Plants were root-treated for 48 hr. 14
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC
16 Table I I .
M E T A B O L I S M
Absorption and t r a n s l o c a t i o n of [ ^ C ] h e r b i c i d e s r o o t - t r e a t e d i n t a c t soybean and oat p l a n t s
Amitrole(A) Atrazine(B) Diclofopmethyl(C) Fluorodifen(D)
1
% of a p p l i e d C absorbed^
D i s t r i b u t i o n of absorbed ^ C ( % ) soybean oat
soybean
root
shoot
root
shoot
1 4
Herbicide
by
oat
27 29
14 8
52 20
48 80
60 31
40 69
34 77
16 48
98 83
2 17
63 78
37 22
—
Q u a n t i t a t i v e data f o r p l a n t s i n Figures 3 and
5.
bf
14Q remaining i n post-treatment s o l u t i o n : A, B, D - 100% parent h e r b i c i d e ; C - 100% a c i d metabolite ( d i c l o f o p ) .
Rapid t r a n s p i r a t i o n r a t e s increased the uptake of [-^c] h e r b i c i d e s i n excised leaves, e s p e c i a l l y i n soybean (Table I ) . D i s t i l l e d water was added to excised soybean leaves a f t e r 5 hours. Excised oat shoots required no a d d i t i o n a l water over the 24-hour p e r i o d . Root uptake v a r i e d according to the h e r b i c i d e (Table I I ) . No a d d i t i o n a l d i s t i l l e d water was added to r o o t - t r e a t e d p l a n t s over the 48-hour p e r i o d . The l ^ C from a t r a z i n e was uniformly d i s t r i b u t e d i n excised leaves and r o o t - t r e a t e d p l a n t s . This i s t y p i c a l of a p o p l a s t i c transport under the i n f l u e n c e of the t r a n s p i r a t i o n stream. The d i s t r i b u t i o n of -^C from a m i t r o l e was l e s s uniform than that from a t r a z i n e i n both s p e c i e s . The d i f f e r e n t i a l l o c a l i z a t i o n of l ^ C from a m i t r o l e i n younger leaves and apex of soybean p l a n t s (Figure 3) i n d i c a t e s r e t r a n s l o c a t i o n or symplastic t r a n s port of 14c out of older l e a v e s . This c o n t r a s t s to c from a t r a z i n e which l o c a l i z e d i n the two u n i f o l i a t e primary leaves with l i t t l e C i n the younger developing leaves. Symplastic transport of [14c]atrazine and/or i t s metabolites from o l d e r leaves does not occur as with a m i t r o l e (Figure 3). Retransloc a t i o n from leaves f o l l o w i n g i n i t i a l a p o p l a s t i c transport from roots i s not as evident i n a monocot (Figure 5) as i n a d i c o t (Figure 3). The v a s c u l a r t r a n s p o r t of m a t e r i a l i n t o and out of leaves, a normal p h y s i o l o g i c a l process, i s a l t e r e d when leaves are excised (Figures 2 and 4). Therefore, the s i g n i f i c a n c e of r e s u l t s from excised leaves must be evaluated i n terms of what may be o c c u r r i n g i n an i n t a c t p l a n t . Diclofop-methyl and f l u o r o d i f e n were r e a d i l y absorbed by excised leaves and r o o t - t r e a t e d plants (Tables I and I I ) . 1 4
1 4
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
1.
SHIMABUKURO
A N D W A L S H
Plant Tissues, Organs,
and Isolated Cells
17
Uptake was enhanced by t r a n s p i r a t i o n , e s p e c i a l l y i n excised leaves of soybean (Table I ) . However, t r a n s p i r a t i o n appeared to have very l i t t l e i n f l u e n c e on t r a n s p o r t . U n l i k e a m i t r o l e and a t r a z i n e , much of the 14c from d i c l o f o p - m e t h y l and f l u o r o d i f e n was immobilized i n the p e t i o l e o f soybean l e a f and the immersed s e c t i o n of excised oat shoot and the roots of both species (Tables I and I I , Figures 2 to 5). A t r a z i n e i s not metabolized i n s i g n i f i c a n t amounts i n soybean (26, 31) and probably i n oat, an a t r a z i n e - s u s c e p t i b l e s p e c i e s . Amitrole metabolism i n soybean may be as r a p i d as i n bean (Phaseolus v u l g a r i s L.) (32) and oat (33). F l u o r o d i f e n metabolism i s r a p i d i n soybean (20) whereas d i c l o f o p - m e t h y l metabolism i s r a p i d i n both oat and soybean (30). Immobilization of x e n o b i o t i c i th xyle b du t adsorption by xylem v e s s e l chyma c e l l s . The exten y the p h y s i c a l and chemical p r o p e r t i e s o f the molecular e n t i t i e s being t r a n s l o c a t e d with only l i m i t e d i n f l u e n c e by the t r a n s p i r a t i o n stream. Therefore, metabolism of a x e n o b i o t i c and the formation of products with d i f f e r i n g p h y s i c a l and chemical p r o p e r t i e s may be a greater f a c t o r i n f l u e n c i n g t r a n s l o c a t i o n than species d i f f e r e n c e s i n the t r a n s l o c a t i o n mechanisms per se. The greater t r a n s l o c a t i o n of 14c from [14c]dipropetryn X 2 - e t h y l t h i o - 4 , 6 - b i s ( i s o p r o p y l a m i n o ) - s - t r i a z i n e ] to the l e a f t i p s i n oat than i n corn excised leaves was reported t o be a f a c t o r i n the higher s u s c e p t i b i l i t y of oat to d i p r o p e t r y n (34). This d i f f e r e n c e i n t r a n s l o c a t i o n between excised leaves of oat and corn was a t t r i b u t e d to an inherent mechanism r e g u l a t i n g t r a n s l o c a t i o n o f dipropetryne w i t h i n the leaves o f these species (34). However, such a c o n c l u s i o n i s questionable s i n c e the molecular forms o f the 14c being t r a n s l o c a t e d were not determined. The t r a n s l o c a t i o n d i f f e r e n c e s between a r e s i s t a n t and s u s c e p t i b l e species may simply r e f l e c t d i f f e r e n c e s i n metabolism of the x e n o b i o t i c and immobilization of i t s metabol i t e s as a f u n c t i o n of t h e i r p h y s i c a l and chemical p r o p e r t i e s . T r a n s l o c a t i o n of 14c-labeled x e n o b i o t i c s i n excised leaves must be determined and not assumed t o be t y p i c a l of a p o p l a s t i c t r a n s p o r t . I f immobilization occurs i n s p e c i f i c p a r t s of excised leaves, e x t r a c t i o n of metabolites from these parts w i l l maximize the concentration of metabolites and minimize the i m p u r i t i e s and n a t u r a l products that must be removed i n the p u r i f i c a t i o n procedure. Xenobiotic metabolism i n excised leaves. The metabolism o f x e n o b i o t i c s i n excised leaves appears to be s i m i l a r q u a l i t a t i v e l y to metabolism i n shoots o f i n t a c t p l a n t s , but may d i f f e r q u a n t i t a t i v e l y . The same water-soluble metabolites of p e r f l u i d o n e were detected i n excised peanut leaves and leaves o r shoots of i n t a c t peanut p l a n t s t r e a t e d through t h e i r roots (21). However, excised leaves t o l e r a t e d treatment with twice the highest
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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concentration used f o r root-treatment with 62% uptake of a p p l i e d [ C ] p e r f l u i d o n e versus 25% uptake by r o o t s . Only 31% of the root uptake was t r a n s l o c a t e d to the shoots. Two water-soluble conjugates of p e r f l u i d o n e accounted f o r 31% of the 14c i n excised peanut leaves a f t e r 48 hours but the same metabolites accounted f o r only 8% of the i n the shoots of r o o t - t r e a t e d i n t a c t p l a n t s a f t e r 8 days (21). Ethylenethiourea (ETU), a decomposition product of ethylenebis(dithiocarbamate) f u n g i c i d e s , was metabolized s i m i l a r l y by excised leaves and shoots of r o o t - t r e a t e d tomato (Lycopersicon esculentum M i l l . ) (35). However, metabolism i n tomato shoots was slower than i n excised leaves when ETU was stem-injected i n t o i n t a c t p l a n t s . The metabolism of diclofop-methyl, a post-emergence h e r b i c i d e , i n wheat and w i l d oat was q u a l i t a t i v e l s i m i l a r i n excised shoots and r o o t - t r e a t e d i n t a c and w i l d oat absorbed 81 methyl, r e s p e c t i v e l y , and metabolized 77% and 72% of the absorbed l^C to water-soluble conjugates w i t h i n 24 hours. Intact r o o t - t r e a t e d wheat and w i l d oat absorbed 68% and 37% of a p p l i e d [ l ^ c j d i c l o f o p - m e t h y l , r e s p e c t i v e l y , but only 8% and 11% of the root uptake was t r a n s l o c a t e d to the shoots. Theref o r e , the use of excised leaves ( d i c o t s ) or shoots (monocots) to study metabolism of f o l i a r l y - a p p l i e d x e n o b i o t i c s such as diclofop-methyl may have d i s t i n c t advantages over root a p p l i c a t i o n or s t e m - i n j e c t i o n . Metabolites of monuron [3-(4-chlorophenyl)-l,1-dimethyl u r e a ] were i s o l a t e d and c h a r a c t e r i z e d from excised cotton leaves (18, 19). Within 24 hours, two 3-D-glucosides c o n s t i tuted 20 to 25% of the methanol-soluble metabolites (18). These were i d e n t i f i e d as conjugates of the hydroxymethyl i n t e r mediates. Longer treatments of up to 4 days i n d i c a t e d that the r a p i d o x i d a t i v e N-dernethylation of monuron, o c c u r r i n g w i t h i n the f i r s t 24 hours, was followed by slow o x i d a t i v e a r y l hydroxyl a t i o n and subsequent conjugation (19). Both r a p i d and slow b i o t r a n s f o r m a t i o n r e a c t i o n s of monuron were e l u c i d a t e d by the use of excised leaves. Q u a l i t a t i v e d i f f e r e n c e s i n the metabolism of a x e n o b i o t i c between roots and shoots are not common. However, such d i f f e r ences have been observed f o r some h e r b i c i d e s (36, 37). Little or no g l u t a t h i o n e conjugates of a t r a z i n e (GS-atrazine) and i t s d e r i v a t i v e s were detected i n the roots of i n t a c t r o o t - t r e a t e d corn p l a n t s (36). The major metabolite i n corn roots was hydroxyatrazine (2-hydroxy-4-ethylamino-6-isopropylamino-sjtriazine). S i g n i f i c a n t concentrations (10 to 20% of 1*C i n i n t a c t plant) of GS-atrazine were found only i n the shoots. GS-atrazine and i t s d e r i v a t i v e s accounted f o r 60 to 70% of the a t r a z i n e absorbed d i r e c t l y i n t o excised corn l e a f blades w i t h i n 24 hours. Very l i t t l e hydroxyatrazine was detected (36). The q u a l i t a t i v e d i f f e r e n c e s i n a t r a z i n e metabolism between roots and shoots of corn were due to the l o c a l i z a t i o n of g l u t a t h i o n e S14
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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t r a n s f e r a s e i n the shoots and not roots of corn (37). The metabolites of chlorpropham (isopropyl-m-chlorocarbanilate) d i f f e r e d between roots and shoots i n r o o t - t r e a t e d i n t a c t soy bean p l a n t s (38). Conjugates of 2-hydroxy-chlorpropham and 4-hydroxy-chlorpropham were present i n the shoots, but only the conjugate of 2-hydroxy-chlorpropham was found i n root t i s s u e (38). This suggests that l e a f t i s s u e probably formed both hydroxylated d e r i v a t i v e s , but root t i s s u e s formed only 2hydroxy--chlorpropham. Studies on chlorpropham metabolism i n excised soybean leaves should confirm the above c o n c l u s i o n . Leaf Discs and Leaf Sections. Leaf d i s c s of uniform s i z e , l e a f s e c t i o n s of lamina ( l e a f blade) from grasses or t h i n l e a f s t r i p s may be used f o x e n o b i o t i metabolis s t u d i e s Th s t r u c t u r a l and metaboli t a i n e d , but the normal v a s c u l a from the l e a f t i s s u e s i s e l i m i n a t e d . Penetration of x e n o b i o t i c s i n t o the mesophyll and p a l i s a d e c e l l s i s predominantly through the c e l l s along the cut edges of l e a f d i s c s or s e c t i o n s (39). The c u t i c l e , a major b a r r i e r to l e a f p e n e t r a t i o n by s u r f a c e a p p l i e d chemicals, i s circumvented by the use of l e a f d i s c s or s e c t i o n s as i n the use of excised leaves or shoots described p r e v i o u s l y . The uptake and e f f l u x of organic compounds i n plant c e l l s are not w e l l understood (40). The mechanism of uptake i n v o l v i n g physico-chemical i n t e r a c t i o n s at the plasmalemma and tonoplast (vacuole membrane) (41) i s a p p l i c a b l e to l e a f d i s c s and l e a f s e c t i o n s . Method. The s i z e of l e a f d i s c s and l e a f s e c t i o n s used, the i n c u b a t i o n medium f o r the l e a f t i s s u e , and the method of a p p l y i n g the t e s t chemicals vary according to the o b j e c t i v e s of the s p e c i f i c experiments. Leaf d i s c s are u s u a l l y cut with a cork borer (8 to 15 mm diameter) from i n t e r v e i n a l areas of the l e a f blade (41, 42, 43, 44). Leaf s e c t i o n s or s l i c e s from monocots v a r i e d from b a r l e y leaves cut t r a n s v e r s e l y at 0.75 mm wide s t r i p s (45) to sorghum lamina cut i n t o 1 cm χ 1 cm s e c t i o n s (46). Dicotyledonous cotton leaves were cut i n t o 0.4 mm wide s t r i p s (47). The i n c u b a t i o n medium f o r l e a f d i s c s or s e c t i o n s may or may not c o n t a i n an osmoticum. Sucrose (0.25 M, pH 6.2) (41) and mannitol at 0.26 M (pH 4 to 6) (45) and 0.35 M (42, 48) are commonly used osmoticums. Leaf t i s s u e s were a l s o incubated i n water (43, 44, 46, 49, 50). Other than an osmoticum and b u f f e r , i n o r g a n i c s a l t s are not commonly added to the i n c u b a t i o n medium. The s i m p l i c i t y of the i n c u b a t i o n medium f o r l e a f d i s c s or sec t i o n s i s i n marked c o n t r a s t to the medium r e q u i r e d f o r separated c e l l s or a s e p t i c t i s s u e c u l t u r e . The l e a f d i s c s or s e c t i o n s are t r e a t e d with x e n o b i o t i c s or other t e s t compounds by vacuum i n f i l t r a t i o n (41, 42, 43, 48) or by e i t h e r f l o a t a t i o n or submersion of the leaves i n s o l u t i o n s
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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of the compounds (44, 45, 46, 50). I n f i l t r a t i o n of the t e s t chemical i n t o l e a f t i s s u e may be accomplished by evacuating the l e a f t i s s u e i n a s o l u t i o n of the compound to a given pressure (20 cm Hg) (49) before r e l e a s i n g the vacuum or maintaining a given pressure (40 cm Hg) f o r a s p e c i f i c p e r i o d (15 min) (41). The most common p r a c t i c e i s to evacuate to the maximum pressure w i t h a water a s p i r a t o r (42) and e i t h e r maintain the vacuum or r e l e a s e i t immediately. S u c c e s s f u l i n f i l t r a t i o n of the s o l u t i o n i n t o the i n t e r c e l l u l a r a i r spaces of the mesophyll c e l l s can be checked v i s u a l l y by the darker, water-soaked appearance of the l e a f t i s s u e . Discussion. Leaf d i s c s or s e c t i o n s have been used exten s i v e l y f o r b a s i c research on the uptake of i n o r g a n i c s a l t s and organic compounds by p l a n photorespiration. Use o x e n o b i o t i c s has been l i m i t e d . L i t t l e i s known about the pene t r a t i o n of plant c e l l membranes by x e n o b i o t i c s (51). Unlike i n o r g a n i c i o n s , amino a c i d s , and sugars that appear to have s p e c i f i c membrane transport systems, the predominantly l i p o p h i l i c and n e u t r a l x e n o b i o t i c s , such as h e r b i c i d e s , probably cross c e l l membranes by d i f f u s i o n (51). Therefore, vacuum i n f i l t r a t i o n of the x e n o b i o t i c should enhance p e n e t r a t i o n of the compound i n t o mesophyll c e l l s . The x e n o b i o t i c w i l l pene t r a t e more c e l l s than without vacuum i n f i l t r a t i o n , thereby i n c r e a s i n g the concentration of metabolites formed i n l e a f discs. Vacuum i n f i l t r a t i o n of l e a f d i s c s has some d e t r i m e n t a l e f f e c t s . Although r e s p i r a t i o n i n wheat, b a r l e y , and bean l e a f d i s c s was unaffected by vacuum i n f i l t r a t i o n of water, CO2 f i x a t i o n and O2 e v o l u t i o n were t o t a l l y i n h i b i t e d (49). C a r r i e r mediated uptake of amino a c i d s i n t o l e a f s e c t i o n s was dependent on r e s p i r a t i o n and photosynthesis f o r ATP as i t s energy source (45, 52). Amino a c i d uptake was i n h i b i t e d by 2,4-dinitrophenol or anaerobic c o n d i t i o n s i n the dark. L i g h t stimulated amino a c i d uptake under anaerobic c o n d i t i o n s , but t h i s s t i m u l a t i o n was t o t a l l y i n h i b i t e d by 10 μΜ diuron (45). I f uptake of compounds such as 2,4-D (2,4-dichlorophenoxyacetic a c i d ) i s an a c t i v e process i n l e a f d i s c s as i t appears to be i n root t i s s u e (53), then vacuum i n f i l t r a t i o n of l e a f d i s c s may not be a d v i s able f o r s t u d i e s with such compounds. However, f o r compounds such as monuron which i s taken up p a s s i v e l y by d i f f u s i o n i n roots (53), vacuum i n f i l t r a t i o n f o r uptake by l e a f d i s c s may be advantageous. This may be true f o r metabolism s t u d i e s but not f o r experiments on i n h i b i t i o n of photosynthesis by monuron. Studies comparable to those on amino a c i d uptake by p l a n t c e l l s have not been reported f o r h e r b i c i d e s and other p e s t i c i d e s . Therefore, the e f f e c t of vacuum i n f i l t r a t i o n on the uptake of s p e c i f i c h e r b i c i d e s i n l e a f d i s c s i s only s p e c u l a t i v e . L i t t l e i s known of the e f f e c t s of senescence on uptake and
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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metabolism of x e n o b i o t i c s by l e a f d i s c s or s e c t i o n s . The senescence processes described e a r l i e r may have s i g n i f i c a n t e f f e c t s on the processes described above. Metabolism i n l e a f d i s c s or s e c t i o n s . Selected examples are presented to i l l u s t r a t e the v e r s a t i l i t y of the l e a f d i s c or s e c t i o n technique. The use of t h i s method permits: 1) r a p i d assessment of p e s t i c i d e degradation i n plant t i s s u e s , 2) determination of p h y s i o l o g i c a l f a c t o r s that i n f l u e n c e metabolism of p e s t i c i d e s , and 3) e v a l u a t i o n of i n t e r a c t i o n e f f e c t s between p e s t i c i d e s . A thorough study on the metabolism of phenylurea h e r b i c i d e s i n l e a f d i s c s of r e s i s t a n t and s u s c e p t i b l e p l a n t s (54, 55) demonstrates the above p o i n t s . A time-course study on the metabolism of monuron showed r a p i d N-demethylation and metabolism to water-solubl incubation of r e s i s t a n l e a f d i s c s i n a buffered s o l u t i o n of monuron (54). Leaf d i s c s of monuron-sensitive soybean and corn metabolized monuron to a much l e s s e r extent over the same period than cotton or p l a n t a i n . A c t i v e photosynthesis was not r e q u i r e d f o r monuron metabolism s i n c e the r a t e of degradation was s i m i l a r i n the dark and l i g h t under CC>2-free c o n d i t i o n s . A f l u x of a chemical i n t o and out of l e a f d i s c s may be expected (41, 51). The k i n e t i c s of uptake and e f f l u x of none l e c t r o l y t e s have been described (41). However, i n most of these studies i t was assumed that the chemical was not a l t e r e d between i t s uptake and e f f l u x by l e a f d i s c s . Some evidence i n d i c a t e s that t h i s may not be n e c e s s a r i l y t r u e . The e f f l u x from monuron-resistant p l a n t a i n l e a f d i s c s was n e a r l y one-half of the i n i t i a l l y absorbed h e r b i c i d e w i t h i n 2 to 3 hours (54). Subsequent reabsorption and metabolism occurred but 37% of the t o t a l 14c was present i n the e f f l u x a f t e r 7.6 hours. More than 50% of the l ^ c i n the e f f l u x was three metabolites with parent monuron accounting f o r the remainder (54). However, p o l a r water-soluble conjugates, which c o n s t i t u t e the major metabolites of monuron i n l e a f d i s c s , were not detected i n the e f f l u x . S i m i l a r l y , major water-soluble conjugates of a t r a z i n e (56) i n corn l e a f d i s c s and diclofop-methyl (30) i n wheat l e a f s e c t i o n s were not detected i n the e f f l u x from these t i s s u e s . Therefore, a s e l e c t i v e e f f l u x of non-polar parent compounds or t h e i r metabolites appears to occur i n l e a f d i s c s or s e c t i o n s with some x e n o b i o t i c s . Any r e l a t i o n s h i p between the e f f l u x c h a r a c t e r i s t i c s of l e a f d i s c s and r e l e a s e of m a t e r i a l from parenchyma c e l l s i n t o the v a s c u l a r system of i n t a c t p l a n t s i s s t i l l obscure. However, e f f l u x c h a r a c t e r i s t i c s of s p e c i f i c compounds from l e a f d i s c s may be r e l a t e d to t h e i r immobilization or t r a n s l o c a t i o n i n i n t a c t plants. The e v a l u a t i o n of x e n o b i o t i c metabolism as a f a c t o r i n s e l e c t i v i t y , antagonism, or synergism i s p o s s i b l e because of the r a p i d determination of metabolism i n l e a f d i s c s . Metabolism and
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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M E T A B O L I S M
d e t o x i c a t i o n of h e r b i c i d e s i n p l a n t s i s probably the most important s i n g l e f a c t o r i n h e r b i c i d e s e l e c t i v i t y . P h y s i o l o g i c a l responses coupled to time-course metabolism may be r e a d i l y demonstrated i n l e a f d i s c s f o r photosynthetic i n h i b i t o r s such as monuron (55) and a t r a z i n e (44, 48, 56). I n h i b i t i o n of photosynthesis and recovery w i t h i n 8 hours was c o r r e l a t e d w i t h g l u t a t h i o n e conjugation of a t r a z i n e i n sorghum (44), corn (56), and s e v e r a l species of the s u b f a m i l i e s , Festucoideae and Panicoideae (48). Recovery of photosynthesis to n e a r l y the c o n t r o l l e v e l occurred i n monuron-treated cotton l e a f d i s c s w i t h i n 4 to 5 hours with r a p i d N-demethylation of monuron (55). The nature of i n t e r a c t i o n between x e n o b i o t i c s at the molecular l e v e l may be demonstrated r e a d i l y i n l e a f d i s c s . The a p p l i c a t i o n of a mixture of the h e r b i c i d e p r o p a n i l (3,4 dichloropropionanilide) c i d e caused i n j u r y to r e s i s t a n to the i n h i b i t i o n of an a r y l acylamidase i n r i c e by carbamate i n s e c t i c i d e s that prevented d e t o x i c a t i o n of p r o p a n i l i n the r e s i s t a n t species (58, 59). In r e s i s t a n t cotton l e a f d i s c s , c e r t a i n carbamate i n s e c t i c i d e s such as c a r b a r y l (1-napthylmethylcarbamate) s t r o n g l y i n h i b i t e d the degradation of the photosynthetic i n h i b i t o r , monuron. The i n h i b i t i o n of photo s y n t h e s i s was enhanced and normal recovery to c o n t r o l l e v e l s of photosynthetic a c t i v i t y was delayed (55). The i n t e r a c t i o n s between s e v e r a l organophosphate and carbamate i n s e c t i c i d e s and h e r b i c i d e s were t e s t e d i n l e a f d i s c s of s e v e r a l species (60, 61). Organophosphate i n s e c t i c i d e s , dyfonate ( 0-ethyl - S_- ph eny 1 ethylphosphonodithioate) and malat h i o n [£,0-dimethyl-S-(l,2-bis-carbethoxy)-ethyl phosphorod i t h i o a t e j , s t r o n g l y i n h i b i t e d the metabolism of s u b s t i t u t e d phenylurea h e r b i c i d e s . Several carbamates [ c a r b a r y l , carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate), ΡCMC (£-chlorophenyl-N-methylcarbamate)] s t r o n g l y i n h i b i t e d p r o p a n i l metabolism (60). The e f f e c t s of s e v e r a l h e r b i c i d e s on the degradation of c a r b a r y l , dyfonate, and malathion were not as severe as the e f f e c t s of the i n s e c t i c i d e s on h e r b i c i d e metabolism (61). Carbaryl metabolism was stimulated by c h l o r propham while metabolism of dyfonate and malathion was i n h i b i t e d by p r o p a n i l (61). The nature of the i n t e r a c t i o n between carbofuran and the h e r b i c i d e s , alachor (2-ehloro-2 ,6'-diethyl-Nmethoxymethyl a c e t a n i l i d e ) (62), chlorbromuron [3-(4-bromo-3chlorophenyl)-l-methoxy-l-methylurea] (63), and b u t y l a t e (Se t h y l d i i s o b u t y l t h i o c a r b a m a t e ) (64) was explained on the b a s i s of whole-plant experiments. Carbofuran increased the absorption and i n h i b i t e d s l i g h t l y the metabolism of the h e r b i c i d e s . I f the i n h i b i t i o n of h e r b i c i d e metabolism by carbofuran i s s i g n i f i c a n t , i t should be r e a d i l y demonstrated i n l e a f d i s c s over much shorter periods than i s p o s s i b l e with whole p l a n t s . 1
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
1.
S H I M A B U K U R O A N D W A L S H
Phnt
Tissues, Organs, and Isolated Cells
23
I s o l a t i o n and c h a r a c t e r i z a t i o n o f metabolites. The i d e n t i f i c a t i o n of metabolites e x t r a c t e d from l e a f d i s c s i s g e n e r a l l y based on t h i n - l a y e r cochromatography with known standards. L i m i t e d numbers of l e a f d i s c s or s e c t i o n s are t r e a t e d with r a d i o l a b e l e d x e n o b i o t i c s , e x t r a c t e d , and the r a d i o a c t i v e metabolites a r e separated and q u a n t i t a t e d . Leaf d i s c s or s e c t i o n s a l s o may be used to generate s u f f i c i e n t q u a n t i t i e s of metabolites f o r p u r i f i c a t i o n and chemical c h a r a c t e r i z a t i o n . However, use o f l e a f d i s c s or s e c t i o n s f o r such a purpose has been l i m i t e d . The i d e n t i f i c a t i o n o f g l u t a t h i o n e conjugation as one o f the major pathways f o r h e r b i c i d e d e t o x i c a t i o n i n p l a n t s was made through the use of sorghum l e a f d i s c s and s e c t i o n s . A water-soluble metabolite of a t r a z i n e was detected as a major metabolite i n sorghum l e a i n h i b i t e d photosynthesi from a l a r g e - s c a l e treatment o f sorghum l e a f s e c t i o n s bathed i n a s o l u t i o n o f a t r a z i n e f o r 20 hours (46). The metabolites, S-(4-ethylamino-6-isopropylamino-^-triaziny1-2)glutathione ( I I I ) and γ-glutamyl-jv- (4-ethylamino-6-isopropylamino-s_-triazinyl2 ) c y s t e i n e (IV), were s u c c e s s f u l l y i s o l a t e d and c h a r a c t e r i z e d from sorghum l e a f s e c t i o n s (46). Subsequent s t u d i e s with whole p l a n t s e l u c i d a t e d the mercapturic a c i d - l i k e pathway f o r a t r a z i n e metabolism i n p l a n t s (65). The l i m i t e d periods that l e a f d i s c s or s e c t i o n s may be allowed to metabolize x e n o b i o t i c s could be a disadvantage. Metabolites that a r e formed over longer periods cannot be generated i n l e a f d i s c s or s e c t i o n s . The metabolite of a t r a z i n e , N-(4-ethylamino-6-isopropylamino-s-triazinyl-2)lanthionine (VII), was present i n s i g n i f i c a n t q u a n t i t i e s i n leaves o f r o o t t r e a t e d sorghum p l a n t s only a f t e r 5 days (65). M e t a b o l i t e s I I I and IV were major metabolites i n sorghum shoots of whole p l a n t s a f t e r 14.4 hours o f treatment but d e c l i n e d t h e r e a f t e r (65). Therefore, due to temporal requirements i t i s d o u b t f u l i f metabolites such as V I I can be s u c c e s s f u l l y generated and i s o l a t e d from l e a f d i s c s or s e c t i o n s . Separated Leaf C e l l s . In t h i s method the mesophyll and p a l i s a d e c e l l s from leaves a r e separated to give a homo genous l i q u i d suspension of c e l l s that can be manipulated much l i k e u n i c e l l u l a r algae. The separated c e l l system i s u s e f u l f o r studying biochemical, p h y s i o l o g i c a l and cytochemical pro cesses i n p l a n t s . Use of i n d i v i d u a l c e l l s i n suspension permits equal exposure o f a l l c e l l s to t e s t chemicals. This i s u n l i k e the techniques i n v o l v i n g l e a f d i s c s o r s e c t i o n s i n which c e l l s along the cut edges a r e exposed p r e f e r e n t i a l l y to the t e s t chemicals. The presence o f r i g i d c e l l w a l l s permits repeated washing and c e n t r i f u g a t i o n of c e l l s with minimal damage. The metabolic f u n c t i o n o f the l e a f t i s s u e i s maintained i n the c e l l suspension but the s t r u c t u r a l i n t e g r i t y of the l e a f and the
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
24
XENOBIOTIC
M E T A B O L I S M
normal f u n c t i o n of v a s c u l a r transport are no longer r e t a i n e d . Method. M o r p h o l o g i c a l l y i n t a c t mesophyll c e l l s may be separated by: 1) gently g r i n d i n g l e a f t i s s u e and separating i n d i v i d u a l c e l l s from l e a f d e b r i s by s e l e c t i v e f i l t r a t i o n and c e n t r i f u g a t i o n (66, 67); and 2) d i g e s t i o n of l e a f t i s s u e with enzyme preparations c o n t a i n i n g polygalacturonase and c e l l u l a s e (68, 69, 70, 71, 72). The enzyme s e p a r a t i o n methods p r e s e n t l y used are b a s i c a l l y m o d i f i c a t i o n s of those developed by Takebe et a l . (68) and Jensen et a l . (69). Separated l e a f c e l l s are prepared by c u t t i n g leaves i n t o s t r i p s (69) or squares (72) and vacuum i n f i l t r a t i n g these t i s s u e s with a maceration medium c o n t a i n i n g the enzyme (0.5 to 3.0%), i n o r g a n i c s a l t s b u f f e r (pH 5.8) potassium dextran s u l f a t e , a hypertonic s o l u t i o anti-senescent agents (2,4-D The i n f i l t r a t e d t i s s u e i s digested with a d d i t i o n a l maceration medium f o r 10 to 30 min. The c e l l s separated during t h i s f i r s t and a second s i m i l a r maceration periods are discarded. The c e l l s obtained during the t h i r d maceration p e r i o d (30 to 45 min) are most a c t i v e m e t a b o l i c a l l y and are used f o r experimen t a t i o n . The wash and assay media c o n t a i n s i m i l a r s a l t s as the maceration medium, 0.6 to 0.7 M s o r b i t o l and are b u f f e r e d at a higher pH (6.7 to 7.2). Discussion. The d i s c u s s i o n i s l i m i t e d to enzymatically separated c e l l s s i n c e t h i s i s the system that has been used to study the e f f e c t s of x e n o b i o t i c s ( h e r b i c i d e s ) on separated plant c e l l s . Maceration of l e a f t i s s u e i n a hypertonic environment that r e s u l t s i n c e l l p l a s m o l y s i s was necessary to y i e l d photos y n t h e t i c a l l y a c t i v e c e l l s (69). Plasmolyzed c e l l s with i n t a c t plasmalemmas were detected by phase c o n t r a s t microscopy (69). Separated cotton mesophyll c e l l s f i x e d C0£ at l i n e a r r a t e s of 50 to 100 ymoles/mg c h l o r o p h y l l /hour f o r 4 to 8 hours (71). Uptake of l ^ C - l e u c i n e and l ^ C - u r a c i l and i n c o r p o r a t i o n i n t o p r o t e i n and RNA, r e s p e c t i v e l y , occurred i n separated tobacco (Nicotiana tabacum L.) c e l l s (73). Uptake and i n c o r p o r a t i o n of the percursors i n t o macromolecules r e q u i r e d l i g h t and a c t i v e photosynthesis. A d d i t i o n of ATP only p a r t i a l l y s u b s t i t u t e d f o r the l i g h t requirement. A smaller percentage of the absorbed percursors was incorporated i n t o macromolecules when c e l l s were incubated i n the dark than i n l i g h t (73). E f f l u x of photosyn t h e t i c products amounted to 1 to 2% of t o t a l carbon f i x e d per hour (71). E f f l u x was i n h i b i t e d by the a d d i t i o n of Ca + i n the i n c u b a t i o n medium. The metabolic a c t i v i t y i s s i m i l a r between separated c e l l s and c e l l s of i n t a c t leaves and l e a f d i s c s described e a r l i e r . The separated c e l l s were m e t a b o l i c a l l y a c t i v e f o r 20 to 30 hours (69, 73). However, c r i t i c a l experimental periods should 2
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
1.
SHiMABUKURO
A N D W A L S H
Plant Tissues, Organs, and
Isolated Cells
25
not exceed 5 to 12 hours s i n c e the metabolic a c t i v i t y i n the c e l l s may not be sustained beyond t h i s p e r i o d . Metabolism i n separated c e l l s . Separated c e l l s have been used to study p r i m a r i l y the e f f e c t s of h e r b i c i d e s and s u r f a c tants on plant c e l l membranes (74, 75, 76) and s e l e c t e d metab o l i c r e a c t i o n s (70, 72, 77, 78, 79). The biochemical and p h y s i o l o g i c a l changes observed i n the above s t u d i e s were not c o r r e l a t e d with metabolism of the x e n o b i o t i c s i n separated c e l l s . Therefore, the s t u d i e s are not complete. However, the p o t e n t i a l usefulness of t h i s technique has been demonstrated i n the l i m i t e d number of r e p o r t s . C a t i o n i c s u r f a c t a n t s increased s i g n i f i c a n t l y the e f f l u x of 14çQ2 f i x a t i o n products i n separated soybean w i l d onion (Allium canadense L.) an In w i l d onion, CO2 f i x a t i o s u r f a c t a n t s . Nonionic s u r f a c t a n t s had r e l a t i v e l y l e s s e f f e c t on membrane p e r m e a b i l i t y and photosynthesis than the c a t i o n i c s u r f a c t a n t s (74, 75). Wild onion c e l l s were l e s s s e n s i t i v e to the a c t i o n of a c a t i o n i c s u r f a c t a n t than were soybean c e l l s (75). Mixtures of o r y z a l i n O ^ - d i n i t r o - N ^ ^ - d i p r o p y l s u l f a n ilamide) with s e l e c t e d s u r f a c t a n t s enhanced e f f l u x i n soybean c e l l s above that of c e l l s t r e a t e d with o r y z a l i n or s u r f a c t a n t alone (74). O r y z a l i n and s u r f a c t a n t s caused greater e f f l u x of i n t r a c e l l u l a r m a t e r i a l from cotton c e l l s than from soybean c e l l s (74). The s y n e r g i s t i c e f f e c t s between o r y z a l i n and surf a c t a n t s were not observed i n cotton as with soybean c e l l s . The d i f f e r e n c e s observed between c e l l s from d i f f e r e n t species i n response to treatment with a chemical may not be due to inherent d i f f e r e n c e s i n t h e i r membranes. Such d i f f e r e n c e s may be only a r e f l e c t i o n of the d i f f e r e n c e s i n metabolism and d e t o x i c a t i o n of the b i o l o g i c a l l y a c t i v e compound between c e l l s from d i f f e r e n t s p e c i e s . Nonionic s u r f a c t a n t s l i k e T r i t o n X-100 are r a p i d l y metabolized i n p l a n t s when taken up by excised leaves or absorbed from l e a f surfaces (25, 80). The concept that h e r b i c i d e s have m u l t i p l e s i t e s of a c t i o n rather than a s i n g l e s i t e has been supported by r e s u l t s from separated c e l l s (72, 77, 78). A time-course e f f e c t of 13 major h e r b i c i d e s on photosynthesis, r e s p i r a t i o n , p r o t e i n s y n t h e s i s , RNA s y n t h e s i s , and l i p i d synthesis was measured i n separated kidney bean c e l l s a t v a r i o u s concentrations (72). Photosynt h e t i c i n h i b i t o r s such as a t r a z i n e , monuron, bromacil (5-bromo3-sec-butyl-6-methyluracil), and dinoseb (2-sec-butyl-4,6d i n i t r o p h e n o l ) i n h i b i t e d photosynthesis s i g n i f i c a n t l y but stimulated l i p i d synthesis at p h y s i o l o g i c a l concentrations of 0.1 to 1.0 μΜ. RNA synthesis a l s o was i n h i b i t e d at the same concentrations. At 100 μΜ, the same h e r b i c i d e s s t r o n g l y i n hibited l i p i d synthesis. The i n h i b i t i o n of macromolecule s y n t h e s i s by photosynthetic i n h i b i t o r s i s probably a secondary e f f e c t s i n c e the uptake and i n c o r p o r a t i o n of percursors r e q u i r e
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
26
XENOBIOTIC
M E T A B O L I S M
a c t i v e photosynthesis (73). The p h y s i o l o g i c a l s i g n i f i c a n c e of the reported e f f e c t s on metabolic processes may be evaluated b e t t e r i f information on the metabolism and i n t r a c e l l u l a r l o c a l i z a t i o n of the h e r b i c i d e s were known. The metabolism of d i f f e r e n t i a l l y l a b e l e d [ c ] f l u o r o d i f e n i n separated mesophyll c e l l s from r e s i s t a n t peanut i s shown i n Table I I I . The c e l l s were prepared using the method of Jensen et a l . (69) except that P e c t i n o l 41-P (crude pectinase and c e l l u l a s e ) was used i n p l a c e of macerozyme, no 2,4-D was added to any media, K2SO4 was s u b s t i t u t e d f o r potassium dextran s u l f a t e and the assay medium contained 0.6 M s o r b i t o l b u f f e r e d w i t h 0.05 M HEPES (pH 7.0). Assays were conducted i n 2 ml photosynthetic medium (69) that contained 10 μΜ [14c]fluorod i f e n , 1% acetone, 7.5 mM NaHC03 and separated c e l l s (238 μg c h l o r o p h y l l per r e a c t i o bated i n a d i f f e r e n t i a from below with incandescent lamps (5000 l u x ) . The c e l l s were separated from the assay medium and extracted with 80% methanol. The c e l l e x t r a c t and the assay medium were analyzed f o r f l u o r o d i f e n and i t s metabolites as reported p r e v i o u s l y (22, 81, 82). The uptake of f l u o r o d i f e n by peanut mesophyll c e l l s occurred r e a d i l y i n l i g h t or dark (Table I I I ) . The r a t i o of 14 i n the c e l l p e l l e t to that i n assay medium decreased with decreasing c e l l concentrations i n repeated experiments. Maxi mum uptake and metabolism of f l u o r o d i f e n occurred w i t h i n 2 hours. L i g h t appeared to have no i n f l u e n c e on the uptake of f l u o r o d i f e n . However, f l u o r o d i f e n metabolism was enhanced by l i g h t i n the 2-hour treatment p e r i o d . E f f l u x of water-soluble metabolites (glucosides and peptide conjugates) occurred from separated c e l l s . However, e f f l u x of f l u o r o d i f e n could not be determined. The r o l e of l i g h t i n the enhanced metabolism of f l u o r o d i f e n i s not c l e a r . Oxygen e v o l u t i o n was not i n h i b i t e d by f l u o r o d i f e n and ATP was not a r e q u i r e d c o - f a c t o r f o r g l u t a thione S-transferase, the enzyme that c a t a l y z e s the ether cleavage of f l u o r o d i f e n (83). Separated z i n i a ( Z i n i a elegans Jacq.) c e l l s absorbed [14c]fluorodifen and [ 1 4 c ] t r i f l u r a l i n (α,α,α-trifluoro-2,6dinitro-N,N-dipropyl-£-toluidine) w i t h i n 2 hours (70). A f t e r washing c e l l s with s o l u t i o n s of the unlabeled h e r b i c i d e s , [ l ^ c j t r i f l u r a l i n appeared to be bound more s t r o n g l y than [ l ^ C ] f l u o r o d i f e n . However, the s i g n i f i c a n c e of the r e s u l t s i s not c l e a r s i n c e metabolism of the two compounds was not deter mined . The s i n g l e example of x e n o b i o t i c metabolism i n a separated c e l l system i l l u s t r a t e s the importance of metabolism when studying the mechanism of a c t i o n and s e l e c t i v i t y of a compound. Separated c e l l s can be used to measure r a p i d biochemical and p h y s i o l o g i c a l changes i n response to treatment with a xeno b i o t i c . However, the dynamic changes i n uptake, metabolism, 1 4
C
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
4
MeOH-insoluble residue:
3
1 100
58 9
31 1
dark
14
£
2
5 100
30 39
15 11
light
1 100
58 7
29 5
dark
4
2
/
[
27 40
16 10
3
CF ]
7 100
1 4
14
14
2
2
5 100
26 40
13 16
[ C-£-N0 -phenyl]
(light)
4-hour treatment
2.95 mCi/mmol; [ C-£-N0 -phenyl]
[ 4c- -N0 -phenyl]^
1
2-hour treatment
(%)
^/ [ C F 3 ] f l u o r o d i f e n : predominantly S7(2-nitro-4-trifluoromethylphenyl)gluthathione with small amounts of S-(2-nitro-4-trifluoromethylphenyl)-Nmalonylcysteine (82); [l C-£-N0 -phenyl]fluorodifen: mixture of £ nitrophenyl-3-D-glucoside and £-nitrophenyl-6-£-malonyl-$-D-glucoside (81).
1 4
2
D i s t r i b u t i o n of t o t a l l*c
Specific activities: [ CF3 f l u o r o d i f e n ] f l u o r o d i f e n - 2.7 mCi/mmol.
100
29 40
Cell Pellet: fluorodifen. conjugates—
light
CF3
[14 ]£/
18 9
Total
4
Metabolism of [ 1 C F ] - and [14c-£-N0 -phenyl]-labeled f l u o r o d i f e n i n separated peanut mesophyll c e l l s .
Assay medium: fluorodifen conjugates^/
Table I I I .
28
XENOBIOTIC
M E T A B O L I S M
and e f f l u x of the x e n o b i o t i c and i t s metabolites i n p l a n t c e l l s a l s o should be known before conclusions on mechanisms of a c t i o n and s e l e c t i v i t y are made. Plant P r o t o p l a s t s . P r o t o p l a s t s are naked c e l l s obtained by removal of t h e i r c e l l w a l l s . This may be accomp l i s h e d by: 1) m i c r o d i s s e c t i o n , or 2) enzymatic d i g e s t i o n . The l a t t e r i s the more e f f e c t i v e and commonly used procedure. The exposed plasmalemma d i f f e r e n t i a t e s p r o t o p l a s t s from i s o l a t e d mesophyll c e l l s whose r i g i d primary c e l l w a l l s are s t i l l i n t a c t . The same advantages described f o r i s o l a t e d mesophyll c e l l s apply to p r o t o p l a s t s . Because of the exposed plasmalemma, i t i s p o s s i b l e to study membrane absorption and p e r m e a b i l i t y e f f e c t s without i n t e r f e r e n c e by the c e l l w a l l (84) P r o t o p l a s t s have bee s t r u c t u r e and f u n c t i o n s on metabolism of x e n o b i o t i c s or t h e i r mechanisms of a c t i o n . This may be due to the d i f f i c u l t i e s of preparing and maintaining p r o t o p l a s t s ; they are extremely f r a g i l e and must be s t a b i l i z e d by osmotic s t a b i l i z e r s ( s o r b i t o l or mannitol) and C a salts (85, 86). 2 +
Method. Numerous methods f o r the i s o l a t i o n of plant p r o t o p l a s t s have been published, but no standard method e x i s t s s i n c e each s p e c i e s , type of t i s s u e , c e l l - s t r a i n , environmental growth c o n d i t i o n s , e t c . , r e q u i r e new adjustments i n the i s o l a t i o n procedures (85, 86). Generally, the methods are m o d i f i c a t i o n s of that o r i g i n a l l y published by Cocking (87). Each s p e c i a l problem appears to r e q u i r e e m p i r i c a l adjustments to a general scheme o u t l i n e d as follows (85): Leaves are s e l e c t e d from p l a n t s exposed to s p e c i f i c growth regimes and s u r f a c e s t e r i l i z e d . A l l operations are performed a s e p t i c a l l y . The lower epidermis of leaves i s removed to expose mesophyll c e l l s ; the leaves are cut i n t o s e c t i o n s and t r a n s f e r r e d to an enzyme s o l u t i o n . The enzyme s o l u t i o n f o r c e l l w a l l d i g e s t i o n may c o n t a i n pectinase and/or c e l l u l a s e and/or h e m i c e l l u l a s e with an osmotic s t a b i l i z e r and Ca2+. P r o t o p l a s t s , u s u a l l y r e l e a s e d w i t h i n 6 to 24 hours, are washed with a s o l u t i o n of the osmoticum and used f o r experimentation or c u l t u r e d under a s e p t i c c o n d i t i o n s f o r subsequent s t u d i e s . M o d i f i c a t i o n s to t h i s b a s i c procedure are i l l u s t r a t e d i n recent p u b l i c a t i o n s (88, 89, 90). Discussion. I s o l a t e d p l a n t p r o t o p l a s t s are g e n e r a l l y s p h e r i c a l i n appearance with c e l l u l a r components arranged along the c e l l periphery (91). P r o t o p l a s t s may undergo w a l l rejuven a t i o n , c e l l d i v i s i o n , and d i f f e r e n t i a t i o n to produce new p l a n t s under s u i t a b l e c o n d i t i o n s (86, 92). One of the greatest p o t e n t i a l s of p r o t o p l a s t s i s the production of i n t r a - and i n t e r - g e n e r i c hybrids through c e l l f u s i o n . Fusion of naked p r o t o p l a s t s from two tobacco species has been reported (93).
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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A N D W A L S H
Phnt
Tissues, Organs, and
Isolated Cells
29
P r o t o p l a s t s may not be s u i t a b l e f o r l a r g e s c a l e gener a t i o n , p u r i f i c a t i o n , and c h a r a c t e r i z a t i o n of metabolites from x e n o b i o t i c s at present. However, i f the techniques f o r prepara t i o n of p r o t o p l a s t s are f u r t h e r improved and standardized, p r o t o p l a s t s may become another important t o o l f o r the p e s t i c i d e chemist. P r o t o p l a s t s may be u s e f u l f o r studying p h y s i o l o g i c a l and biochemical e f f e c t s of x e n o b i o t i c s i n plant c e l l s . Oat meso p h y l l p r o t o p l a s t s incorporated u r i d i n e and l e u c i n e up to 6 hours and thymidine up to 21 hours. K i n e t i n i n h i b i t e d l e u c i n e i n c o r p o r a t i o n with i n c r e a s i n g concentration of the growth hormone, but 2,4-D, a b s c i s i c a c i d and g i b b e r e l l i c a c i d had no e f f e c t on the same process (94). U r i d i n e i n c o r p o r a t i o n was i n h i b i t e d by 2,4-D at concentration abov 0.1 f the growth hormones had (94). The d i f f e r e n t i a l s u s c e p t i b i l i t y c e p t i b l e oat v a r i e t i e s to the phytotoxin, v i c t o r i n , was demonstrated with i s o l a t e d p r o t o p l a s t s (95). The c y t o k i n i n l i k e a c t i o n of methyl-2-benzimidazole carbamate (MBZ), the f u n g i t o x i c metabolite of benomyl [methyl-l-(butylcarbamoyl)-2benzimidazole carbamate], was confirmed i n oat p r o t o p l a s t s (96). The decrease i n nuclease a c t i v i t y , increase i n l e u c i n e i n c o r p o r a t i o n and decrease i n u r i d i n e and thymidine i n c o r p o r a t i o n were s i m i l a r i n p r o t o p l a s t s t r e a t e d with k i n e t i n or MBZ (96). The e f f e c t of MBZ on the plasmalemma of p r o t o p l a s t s was not resolved. Tomato f r u i t p r o t o p l a s t s absorbed and r e t a i n e d s i g n i f i c a n t amounts of f l u o r o d i f e n and t r i f l u r a l i n (70). The short-term s t r u c t u r a l i n t e g r i t y of the plasmalemma from tomato p r o t o p l a s t s was unaffected by f l u o r o d i f e n , t r i f l u r a l i n , fluometuron [1,1dimethy1-3-(a,α,α-trifluoro-m-tolyl)urea], and chlorbromuron. However, complete membrane breakage and c o l l a p s e of the p r o t o p l a s t s occurred a f t e r 30 minutes of treatment with paraquat ( l , l - d i m e t h y l - 4 , 4 - b y p y r i d i n i u m d i c h l o r i d e ) (97). The p r o t o p l a s t s have not been used to i n v e s t i g a t e metabolism of x e n o b i o t i c s . However, i t appears to be a convenient system to determine the metabolism of x e n o b i o t i c s concurrently with a study on the phytotoxic and s e l e c t i v e a c t i o n of these compounds i n plant c e l l s . ?
f
Conclusion. The survey of the l i t e r a t u r e on i n v i t r o organ, t i s s u e , and i s o l a t e d c e l l techniques f o r x e n o b i o t i c metabolism i n p l a n t s i s not complete. Selected r e p o r t s i n d i c a t e that i s o l a t e d plant systems may be used s u c c e s s f u l l y by p e s t i c i d e chemists to e l u c i d a t e degradation pathways and i n t e r a c t i o n s of x e n o b i o t i c compounds i n p l a n t s . I s o l a t e d p l a n t systems are most u s e f u l f o r short-term i n v e s t i g a t i o n s on metabolism, mechanism of a c t i o n and s e l e c t i v i t y .
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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One of the major advantages of the techniques discussed in this report is the ability to measure rapid biochemical and physiological changes in response to a chemical coupled to its metabolism under carefully controlled conditions. This may not be possible in whole-plant experiments. Reduced growth and other visible manifestations of injury in whole plants are usually much delayed secondary responses to a chemical. How ever, results from isolated plant systems must be carefully evaluated before they are extrapolated to intact whole plants. Also, conclusions based only on whole-plant experiments should be confirmed by in vitro techniques. Abstract. Whole plants have and "terminal" residue using isolated leaves or roots, leaf discs or sections, and separated mesophyll cells have proven to be more useful for short-term investigations on xenobiotic metabolism, mechanism of action and selectivity. The in vitro techniques allow the treatment of large amounts of plant material with a minimum of chemical and facilitate rapid uptake of the chemical into plant tissues. The influence of root and leaf surface absorption is also circumvented by the use of in vitro techniques. Quan titative differences in xenobiotic metabolism between whole plants and isolated plant systems have been observed, but qualitative differences are not common. Results from isolated plant systems must be carefully evaluated before extrapolation to intact plants because of the unknown influence of senescence, absorption, and translocation on in vitro systems. Literature Cited 1. Casida, J. E. and Lykken, L., Annu. Rev. Plant Physiol. (1969) 20, 607. 2. Kearney, P. C. and Kaufman, D. D., eds., "Herbicides Chemistry, Degradation and Mode of Action," Vol. 1 and 2, Marcel Dekker, Inc., New York, NY (1975). 3. Frear, D. S., Hodgson, R. Η., Shimabukuro, R. H. and Still, G. G., Advan. Agron. (1972) 24, 328. 4. Bukovac, M. J., "Herbicides - Physiology, Biochemistry, Ecology," Audus, L. J., ed., Vol. 1, p. 335, Academic Press, New York, NY (1976). 5. Hay, J. R., "Herbicides - Physiology, Biochemistry, Ecology," Audus, L. J., ed., Vol. 1, p. 365, Academic Press, New York, NY (1976). 6. Haynes, R. J. and Goh, Κ. Μ., Scien. Hort. (1977) 7, 291. 7. Crafts, A. S. and Crisp, C. E., "Phloem Transport in Plants," W. H. Freeman and Co., San Francisco, CA (1971).
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1. SHIMABUKURO AND WALSH Plant Tissues, Organs, and Isolated Cells 31 8. Zimmerman, M. H. and Milburn, J. Α., eds., "Transport in Plants I: Phloem Transport," Encyclopedia Plant Physiol. (N.S.), Vol. 1, Springer-Verlag, Heidelberg, West Germany (1975). 9. Crafts, A. S., in "The Physiology and Biochemistry of Herbicides," Audus, L. J., ed., p. 75, Academic Press, New York, NY (1964). 10. Woolhouse, H. W., Sci. Prog. (1974) 61, 123. 11. Thimann, Κ. V., "Hormone Action in the Whole Life of Plants," p. 357, University of Massachusetts Press, Amherst, MA (1977). 12. Goldthwaite, J. J. and Laetsch, W. Μ., Plant Physiol. (1967) 42, 1757. 13. Peterson, L. W. and Huffaker R C., Plant Physiol (1975) 55, 1009. 14. Tetley, R. M. and , , Physiol (1974) 54, 294. 15. Sodek, L. and Wright, S. T. C., Phytochem. (1969) 8, 1629. 16. Haber, Α. Η., Thompson, P. J., Walne, P. L. and Triplett, L. L., Plant Physiol. (1969) 44, 1619. 17. Martin, C. and Thimann, Κ. V., Plant Physiol. (1972) 49, 64. 18. Frear, D. S. and Swanson, H. R., Phytochem. (1972) 11, 1919. 19. Frear, D. S. and Swanson, H. R., Phytochem. (1974) 13, 357. 20. Frear, D. S. and Swanson, H. R., Pest. Biochem. Physiol. (1975) 5, 73. 21. Lamoureux, G. L. and Stafford, L. E., J. Agric. Food Chem. (1977) 25, 512. 22. Shimabukuro, R. Η., Lamoureux, G. L . , Swanson, H. R., Walsh, W. C., Stafford, L. E. and Frear, D. S., Pestic. Biochem. Physiol. (1973) 3, 483. 23. Lamoureux, G. L . , Stafford, L. E. and Shimabukuro, R. Η., J. Agric. Food Chem. (1972) 20, 1004. 24. Lamoureux, G. L., Stafford, L. E. and Tanaka, F. S., J. Agric. Food Chem. (1971) 19, 346. 25. Stolzenberg, G. E. and Olson, P. Α., 173rd Amer. Chem. Soc. Meeting (1977) Abst. Pest. 42. 26. Shimabukuro, R. H., Masteller, V. J. and Walsh, W. C., Weed Sci. (1976) 24, 336. 27. Hamilton, R. H., J. Agric. Food Chem. (1964) 12, 14. 28. Swanson, C. R., Kadunce, R. E., Hodgson, R. H. and Frear, D. S., Weeds (1966) 14, 319. 29. Shimabukuro, R. H., J. Agric. Food Chem. (1967) 15, 557. 30. Shimabukuro, R. Η., Walsh, W. C. and Hoerauf, R. Α., (unpublished data). 31. Shimabukuro, R. Η., Plant Physiol. (1967) 42, 1269. 32. Carter, M. C., "Herbicides - Chemistry, Degradation and Mode of Action," Kearney, P. C. and Kaufmann, D. D., eds., Vol. 1, p. 377, Marcel Dekker, Inc., New York, NY (1975) 33. Lund-Höie, Κ., Weed Res. (1970) 10, 367.
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34. Basler, Ε., Murray, D. S. and Santelmann, P. W., Weed Sci. (1978) 26, 358. 35. Hoagland, R. E. and Frear, D. S., J. Agric. Food Chem. (1976) 24, 129. 36. Shimabukuro, R. Η., Frear, D. S., Swanson, H. R. and Walsh, W. C., Plant Physiol. (1971) 47, 10. 37. Frear, D. S. and Swanson, H. R., Phytochem. (1970) 9, 2123. 38. S t i l l , G. G. and Mansager, E. R., Pestic. Biochem. Physiol. (1973) 3, 87. 39. Shtarkshall, R. Α., Reinhold, L. and Harel, H., J. Exp. Bot. (1970) 21, 915. 40. Nissen, P., Annu. Rev. Plant Physiol. (1974) 25, 53. 41. Morrod, R. S., J. Exp. Bot. (1974) 25, 521. 42. Jensen, Κ. I. Ν. Banden J D. and SouzaMachado V. Can. J. Plant Sci 43. Miflin, B. J., Plant 44. Shimabukuro, R. H. and Swanson, H. R., J. Agric. Food Chem. (1969) 17, 199. 45. Lien, R. and Rognes, S. Ε., Physiol. Plant. (1977) 41, 175. 46. Lamoureux, G. L . , Shimabukuro, R. Η., Swanson, H. R. and Frear, D. S., J. Agric. Food Chem. (1970) 18, 81. 47. Jones, H. G., Aust. J. Biol. Sci. (1973) 26, 25. 48. Jensen, Κ. I. Ν., Stephenson, G. R. and Hunt, L. Α., Weed Sci. (1977) 25, 212. 49. MacDonald, I. R., Plant Physiol. (1975) 56, 109. 50. Chollet, R., Plant Physiol. (1978) 61, 929. 51. Morrod, R. S., "Herbicides - Physiology, Biochemistry, Ecology," Audus, L. J., ed., Vol. 1, p. 281, Academic Press, New York, NY (1976). 52. Cheung, Y. K. S. and Nobel, P. S., Plant Physiol. (1973) 52, 633. 53. Donaldson, T. W., Bayer, D. E. and Leonard, O. Α., Plant Physiol. (1973) 52, 638. 54. Swanson, C. R. and Swanson, H. R., Weed Sci. (1968) 16, 137. 55. Swanson, C. R. and Swanson, H. R., Weed Sci. (1968) 16, 481. 56. Shimabukuro, R. Η., Swanson, H. R. and Walsh, W. C., Plant Physiol. (1970) 46, 103. 57. Bowling, C. C. and Hudgins, H. R., Weeds (1966) 14, 94. 58. Matsunaka, S., Science (1968) 160, 1360. 59. Frear, D. S. and Still, G. G., Phytochem. (1968) 7, 913. 60. Chang, F-Y., Smith, L. W. and Stephenson, G. R., J. Agric. Food Chem. (1971) 19, 1183. 61. Chang, F-Y., Stephenson, G. R. and Smith, L. W., J. Agric. Food Chem. (1971) 19, 1187. 62. Hamill, A. S. and Penner, D., Weed Sci. (1973) 21, 330. 63. Hamill, A. S. and Penner, D., Weed Sci. (1973) 21, 335. 64. Hamill, A. S. and Penner, D., Weed Sci. (1973) 21, 339.
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65. Lamoureux, G. L . , Stafford, L. Ε., Shimabukuro, R. H. and Zaylskie, R. G., J. Agric. Food Chem. (1973) 21, 1020. 66. Gnanam, A. and Kulandaivelu, G., Plant Physiol. (1969) 44, 1451. 67. Edwards, G. E. and Black, C. C. Jr., Plant Physiol. (1971) 47, 149. 68. Takebe, I., Otsuki, Y. and Aoki, S., Plant Cell Physiol. (1968) 9, 115. 69. Jensen, R. G., Francki, R. I. B. and Zaitlin, Μ., Plant Physiol. (1971) 48, 9. 70. Boulware, M. A. and Camper, N. D., Weed Sci. (1973) 21, 145. 71. Rehfeld, D. W. and Jensen, R. G., Plant Physiol. (1973) 52, 17. 72. Ashton, F. Μ., DeVilliers W. B., Pestic. Biochem Physiol (1977) 7, 73. Francki, R. I. B., Zaitlin, M. and Jenson, R. G., Plant Physiol. (1971) 48, 14. 74. Towne, C. Α., Bartels, P. G. and Hilton, J. L . , Weed Sci. (1978) 26, 182. 75. St. John, J. B., Bartels, P. G. and Hilton, J. L . , Weed Sci. (1974) 22, 233. 76. Davis, D. G. and Shimabukuro, R. Η., Weed Sci. Soc. Abst. No. 157 (1973). 77. Porter, Ε. M. and Bartels, P. G., Weed Sci. (1977) 25, 60. 78. Radosevich, S. R. and DeVilliers, O. T., Weed Sci. (1976) 24, 229. 79. DeVilliers, O. T. and Ashton, F. Μ., Agroplantae (1976) 8., 87. 80. Stolzenberg, G. E. and Olson, P. Α., 175th Amer. Chem. Soc. Meeting (1978) Abst. Pest. 67. 81. Shimabukuro, R. Η., Walsh, W. C., Stolzenberg, G. E. and Olson, P. Α., Weed Sci. Soc. Am. (1975) Abst. No. 171. 82. Shimabukuro, R. Η., Walsh, W. C., Stolzenberg, G. E. and Olson, P. Α., Weed Sci. Soc. Am. (1976) Abst. No. 196. 83. Frear, D. S. and Swanson, H. R., Pestic. Biochem. Physiol. (1973) 3, 473. 84. Ruesink, A. W., Plant Physiol. (1971) 47, 192. 85. Constabel, F., in "Plant Tissue Culture Methods," Gamborg, O. L. and Wetter, L. R., eds., p. 11, Prairie Regional Laboratory, Saskatoon, Canada (1975). 86. Cocking, E. C., Annu. Rev. Plant Physiol. (1972) 23, 29. 87. Cocking, E. C., Nature (1960) 187, 962. 88. Farmer, I. and Lee, P. Ε., Plant Sci. Lett. (1977) 10, 141. 89. Cassells, A. C. and Barlass, Μ., Physiol. Plant. (1978) 42, 236. 90. Watts, J. W., Motoyoshi, F. and King, J. Μ., Ann. Bot. (1974) 38, 667. 91. Takebe, I., Otsuki, Υ., Honda, Y., Nishio, T. and Matsui,
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Gamborg, O. L. and Miller, R. Α., Can. J . Bot. (1973) 51, 1795. Carlson, P. S., Smith, H. H. and Dearing, R. D., Proc. N a t l . Acad. S c i . U.S.A. (1972) 69, 2292. Fuchs, Y. and Galston, A. W., Plant C e l l P h y s i o l . (1976) 17, 475. R a n c i l l a c , M., Kaur-Sawhney, R., Staskawicz, B. and Galston, A. W., Plant C e l l P h y s i o l . (1976) 17, 987. Staskawicz, B., Kaur-Sawhney, R., Slaybaugh, R., Adams, W. J r . and Galston, A. W., P e s t i c . Biochem. P h y s i o l . (1978) 8, 106. Boulware, M. A. and Camper, D. Ν., P h y s i o l . P l a n t . (1972) 26, 313. December 20,
1978
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
2 Xenobiotic Metabolism in Higher Plants: In Vitro Tissue and Cell Culture Techniques RALPH O. MUMMA and ROBERT H. HAMILTON Pesticide Research Laboratory and Graduate Study Center and Departments of Entomology and Biology, Pennsylvania State University, University Park, PA 16802 M i l l i o n s of pounds of x e n o b i o t i c s have been a p p l i e d to p l a n t s in our environment f o r th c o n t r o l f pest d p l a n t growth Some of these chemicals b i o l o g i c a l e f f e c t s on animal plants, , such as o i l s , adjuvants, e m u l s i f i e r s and i n e r t m a t e r i a l s , are a p p l i e d i n even greater q u a n t i t y , but have been assumed to cause l i t t l e ecological effect. I t i s important to understand what happens to a l l chemicals a p p l i e d t o our environment and to p r o p e r l y i n t e r p r e t the e c o l o g i c a l s i g n i f i c a n c e of these chemicals and of t h e i r degradation products. I f we cannot know t h i s i n d e t a i l , then a general knowledge of p e r s i s t a n c e and metabolic products i s of importance. Since the t a r g e t organism of these x e n o b i o t i c s i s o f t e n p l a n t s , i t i s of the utmost importance to understand the f a t e of these chemicals i n p l a n t s . U l t i m a t e l y , animals and even humans are exposed to these chemicals and/or t h e i r subsequent metabolites and degradation products. Most i n v e s t i g a t i o n s of x e n o b i o t i c metabolism i n p l a n t s have focused on b i o l o g i c a l l y a c t i v e pest c o n t r o l chemicals. Thus, t h i s review w i l l a l s o focus p r i m a r i l y on p l a n t metabolism of p e s t i c i d e s . There are many ways to study the metabolism of x e n o b i o t i c s by p l a n t s , but whatever technique i s employed, i t should p r e d i c t what would a c t u a l l y happen under f i e l d c o n d i t i o n s . Metabolism s t u d i e s have i n v o l v e d whole p l a n t s , excised p l a n t p a r t s (meristems, shoots, stems, l e a v e s , r o o t s , l e a f d i s k s ) , p l a n t c e l l c u l t u r e s , s u b c e l l u l a r p a r t i c l e s , and i s o l a t e d enzymes. Any metabolism study conducted i n the l a b o r a t o r y i s l e s s than i d e a l because i t i s d i f f i c u l t to d u p l i c a t e many f a c t o r s that a f f e c t the degradation of x e n o b i o t i c s under f i e l d c o n d i t i o n s such as weather, l i g h t , microsymbionts, s o i l or method of a p p l i c a t i o n . Metabolism of xenobiot i c s by p l a n t t i s s u e c u l t u r e o f f e r s the obvious advantages of s t e r i l i t y , space, economical use of l a b e l e d chemicals, l e s s p i g ments, e t c . These advantages w i l l be discussed i n d e t a i l l a t e r . The metabolism of x e n o b i o t i c s by p l a n t t i s s u e c u l t u r e (1) and the metabolism of endogenous and exogenous chemicals (2) have been reviewed r e c e n t l y . W i t h i n the l a s t few y e a r s , many new
0-8412-0486-l/79/47-097-035$10.50/0 © 1979 American Chemical Society
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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XENOBIOTIC
M E T A B O L I S M
i n v e s t i g a t i o n s of x e n o b i o t i c metabolism by p l a n t t i s s u e c u l t u r e s have been reported. This review w i l l focus on these more recent papers with an emphasis on p e s t i c i d e metabolism. We w i l l attempt to evaluate and compare the r e s u l t s obtained w i t h t i s s u e c u l t u r e techniques versus those obtained w i t h whole p l a n t s and to explore the many f a c t o r s ( l i m i t a t i o n s ) that a f f e c t metabolism s t u d i e s with plant tissue culture. H i s t o r y and P r i n c i p l e s of P l a n t T i s s u e Culture Plant t i s s u e c u l t u r e r e f e r s to the growth of r e l a t i v e l y und i f f e r e n t i a t e d p l a n t c e l l s or d i f f e r e n t i a t e d organs on s o l i d or l i q u i d n u t r i e n t medium. The u n d i f f e r e n t i a t e d p l a n t t i s s u e growing on s o l i d i f i e d medium i s u s u a l l y r e f e r r e d to as a c a l l u s c u l t u r e s i n c e they a r e f r e q u e n t l and maintain the appearanc t i s s u e s are placed i n l i q u i d medium w i t h shaking, many small aggregates of c e l l s and even some s i n g l e c e l l s may be obtained. Subcultures of small aggregates or c e l l clumps i n l i q u i d c u l t u r e are u s u a l l y designated as suspension c u l t u r e s . The c u l t u r e of excised d i f f e r e n t i a t e d organs i s , of course, organ c u l t u r e . F r e quently, c a l l u s c u l t u r e s w i l l d i f f e r e n t i a t e w i t h the formation of xylem elements and sometimes buds and/or r o o t s . In many c a l l u s c u l t u r e s with t h i s p o t e n t i a l u s u a l l y a high k i n e t i n / a u x i n concent r a t i o n r a t i o i n the medium favors bud formation while the reverse favors root formation. In a few cases, by proper manipulation of the medium, thousands of pseudoembryos can be induced and grown i n t o normal p l a n t s of the same genotype. Although the c u l t u r e of p l a n t t i s s u e s was attempted i n 1902 (3), success was not achieved u n t i l White c u l t u r e d excised tomato roots i n 1934 and tobacco c a l l u s i n 1939 (4). Gautheret and Nobêcourt a l s o described c u l t u r e of p l a n t c a l l u s t i s s u e at about the same time (5, 6) . White and Gautheret both developed n u t r i e n t media that a r e used widely today (5> 2) · In a d d i t i o n to e s s e n t i a l s a l t s and sucrose, White added thiamine, n i c o t i n i c a c i d , pyridoxine and g l y c i n e while Gautheret added thiamine, pantothenic a c i d , b i o t i n , i n o s i t o l and c y s t e i n e . I t appears that only thiamine i s e s s e n t i a l ; but, i n some cases, b e t t e r growth may be obtained by the a d d i t i o n of other v i t a m i n s . White d i d not add an auxin f o r c u l t u r e of excised root organ c u l t u r e s or tobacco tumor t i s s u e , but Gautheret added naphthaleneacetic a c i d (NAA). Van Overbeek et a l . (8) introduced the use of coconut milk and much l a t e r M i l l e r et a l . (9) found that k i n e t i n was necessary to c u l t u r e tobacco stem p i t h . In g e n e r a l , excised root organ c u l t u r e s , tumor and some c a l l u s c u l t u r e s (so c a l l e d habituated) do not r e q u i r e e i t h e r auxin or k i n e t i n . Some t i s s u e s do not r e q u i r e k i n e t i n e s p e c i a l l y i f 2,4-dichlorophenoxyacetic a c i d (2,4-D) i s used as the auxin (10). The a d d i t i o n of coconut milk i s u s u a l l y not e s s e n t i a l , and a requirement f o r g i b b e r e l l i c a c i d has been reported r a r e l y . I t i s probable that adaptation to the medium
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
2.
M U M M A
A N D
H A M I L T O N
Plant Tissue and
Cell
Cultures
37
and the s e l e c t i o n of t i s s u e that grows w e l l on a p a r t i c u l a r medium accounts f o r the wide v a r i a t i o n i n n u t r i e n t requirements f o r a t i s s u e such as tobacco c a l l u s . Other media used widely f o r plant t i s s u e c u l t u r e i n c l u d e those of Murashige and Skoog (11), N i t s c h and N i t s c h (12) and Gamborg (13). In a d d i t i o n , some media formul a t i o n s are now a v a i l a b l e from a commercial source (Flow Laborat o r i e s , P. 0. Box 2226, 1710 Chapman Ave., R o c k v i l l e , MD 20852). In theory c a l l u s may be derived from any p l a n t t i s s u e cont a i n i n g parenchyma c e l l s . Some species form c a l l u s r e a d i l y and others do not. Tissue s t e r i l i z a t i o n may be accomplished with 70% a l c o h o l (1-2 min dip) and/or a s i m i l a r treatment with 1 to 5 or 1 to 10 d i l u t e d commercial bleach (0.5-1% sodium hypochlor i t e ) containing 0.1% Tween 20. In e i t h e r case, the t i s s u e i s r i n s e d 2-3 times i n s t e r i l e water. Seeds are germinated i n s t e r i l e p e t r i dishes and b i t to s o l i d i f i e d agar mediu (0.5-1.0 mg/1 NAA or 2,4-D). S t e r i l i z e d t i s s u e from bud, leaves or stems may a l s o be used. Formation of enough c a l l u s to s u b c u l ture may vary from 2 weeks to 2 months. Considerable v a r i a t i o n i n appearance and texture between c a l l u s pieces from the same source may be observed. Three to 4 b i t s of c a l l u s (5-7 mm i n dia.) are t r a n s f e r r e d to s o l i d i f i e d agar medium (50 ml i n a 125 ml Erlenmeyer f l a s k ) . D i f f i c u l t t i s s u e s may r e q u i r e the a d d i t i o n of 50 ml of a u t o c l a v e d - f i l t e r e d coconut l i q u i d and/or 2 g of c a s e i n hydrolysate per l i t e r . Growth rates vary, but i t i s convenient to subculture b i t s of c a l l u s onto f r e s h medium every 4^6 weeks. Temperature and l i g h t requirements may not be c r i t i c a l f o r most t i s s u e s . High temperatures (30°C or more) have caused the l o s s of k i n e t i n dependence of tobacco c a l l u s (14) and i n h i b i t e d growth. We have maintained c u l t u r e s under continuous low l e v e l f l u o r e s c e n t l i g h t at 27°C. Simple equipment i s needed f o r p l a n t t i s s u e c u l t u r e s ; a temperature c o n t r o l l e d incubator or c u l t u r e room, an autoclave, and a shaker, i f suspension c u l t u r e s are grown. I t i s a l s o d e s i r a b l e to have a s t e r i l e t r a n s f e r hood. The genetic and p h y s i o l o g i c a l s t a b i l i t y of some plant t i s s u e c u l t u r e s i s of concern. T i s s u e from the same o r i g i n a l c u l t u r e may change i n appearance and growth r a t e over a period of time. It i s commonly observed that the a b i l i t y to regenerate normal p l a n t s by the formation of buds and roots i s l o s t with time. I t may even be necessary to r e i s o l a t e c a l l u s from the same source, i f the c a l l u s t i s s u e appears a t y p i c a l or slow growing. A low growth r a t e i s observed f r e q u e n t l y i n August or September. V a r i ations i n growth r a t e and t i s s u e appearance may be important f a c t o r s i n metabolism studies and should be examined c a r e f u l l y . A m i t o t i c index at two weeks a f t e r t r a n s f e r may give a good i n d i c a t i o n of growth r a t e (15) or f r e s h weights at the end of 4 or 5 weeks may be used. The source of c a l l u s t i s s u e (root, stem, l e a f , cotyledons, etc.) a l s o may i n f l u e n c e metabolism. The appearance of soybean cotyledon, l e a f and root c a l l u s was s i m i l a r and l i m i t e d
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
38
XENOBIOTIC
M E T A B O L I S M
work on 2,4-D metabolism i n d i c a t e d no major q u a l i t a t i v e d i f f e r ences (16). Of much more importance was the age or stage of grow th of the c u l t u r e s (15). Most c a l l u s c u l t u r e s e x h i b i t a l a g i n growth f o r 4-7 days a f t e r t r a n s f e r , a l o g growth phase followed by a slower growth phase a f t e r 4-5 weeks. Cultures 5-9 weeks o l d are more a c t i v e i n metabolism of 2,4-D than 3 week o l d c u l t u r e s (15). I t should be noted that prolonged p e s t i c i d e treatment of o l d e r c a l l u s i n f r e s h medium may put the t i s s u e i n t o l o g phase growth again w i t h probable changes i n metabolism. Metabolism of Xenobiotics
by Plant Tissue
Culture
Most i n v e s t i g a t i o n s w i t h p l a n t t i s s u e c u l t u r e s and xenobiot i c s have been concerned w i t h p e s t i c i d e metabolism. Tables I and I I i l l u s t r a t e the v a r i e t t i s s u e c u l t u r e s that hav metabolism of lindane probably represents one of the more extreme cases where fourteen d i f f e r e n t p l a n t t i s s u e s were used. As i s evident, the source of the p l a n t t i s s u e c u l t u r e , the type of c u l ture (suspension or nonsuspension) and the media used a l s o v a r i e d . This v a r i a t i o n i n p l a n t c u l t u r e s and techniques makes i t extremely d i f f i c u l t to c r i t i c a l l y compare metabolism s t u d i e s . As w i l l be pointed out l a t e r , there are a l s o many other f a c t o r s that a f f e c t metabolism s t u d i e s with p l a n t t i s s u e c u l t u r e s . HERBICIDES I t i s apparent that h e r b i c i d e s exert t o x i c or p h y s i o l o g i c a l e f f e c t s on s e n s i t i v e plant species and that some p h y s i o l o g i c a l e f f e c t s can be expected on t i s s u e c u l t u r e s of these species. An exception may be photosynthetic i n h i b i t o r s . Due to the sugar i n the p l a n t t i s s u e c u l t u r e medium, photosynthetic i n h i b i t o r s should not be s t r o n g l y i n h i b i t o r y unless they have secondary s i t e s of action. I f t o l e r a n c e i s due to metabolism, t h i s might be expected to lead to q u a l i t a t i v e and q u a n t i t a t i v e d i f f e r e n c e s i n metabolism by p l a n t t i s s u e c u l t u r e s from s u s c e p t i b l e and r e s i s t a n t v a r i e t i e s or s p e c i e s . Of course, such t i s s u e c u l t u r e s should a l s o show differences i n actual tolerance. The metabolism of the h e r b i c i d e f l u o r o d i f e n (p-nitrophenyl α,α,a-trifluoro-2-nitro-p-tolyl ether) has been i n v e s t i g a t e d (21) with tobacco c e l l s i n suspension c u l t u r e (42). The c e l l s were incubated f o r 15 days w i t h 1-2 ppm of e i t h e r C i - or CF3-labeled f l u o r o d i f e n . A l l of the a p p l i e d f l u o r o d i f e n was metabolized. Recovery of added r a d i o a c t i v i t y v a r i e d between 52 and 76%. The c e l l s contained 60 to 80% of the recovered r a d i o a c t i v i t y and the remainder was found i n the medium and c e l l wash. With ^Ci-label ed f l u o r o d i f e n , 4-nitrophenol (7%) was i s o l a t e d only from the medium. Aqueous-soluble conjugated forms of 4-nitrophenol (93%), p r i m a r i l y the 3-D-glucoside and other a c i d i c conjugates, were pre sent i n both the c e l l s and the medium as summarized i n Figure 1. l l f
1
lf
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
3
2
ι H
>-N-c-cH cH
Propanil
\=J
CI- • f
0
Diphenimid
CI
^
3
v CH
ÇH3_
Ο
^ N-CCH^ CH ^ ^
Cisanilide
3
ι
0
3
Rice
1
(Oryza s a t i v a L. v a r . Starbonnet)
1
Leaves
Source
Root
Soybean (Glycine max [L.] Merr. Root t i p s Wilkin )
Carrot (Daucus c a r o t a L.) Cotton (Gossypium hirsutum L.)
Species
H, S
B5, S
B5,
Medium and ^ C u l t u r e Type
Plant T i s s u e C u l t u r e
Metabolism of H e r b i c i d e s by P l a n t Tissue C u l t u r e s .
Herbicide
Table I.
20
19
17, 18
Reference.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
2
3
Ν — Ν
Cl
0
NH
2,4-D
/Cl
2
OCH2COOH
Metribuzin
3
^-SCH
Fluorodifen
(CH ) C-/
N0
NO2
Table I - page 2
3
( C u l t i v a r Bragg) ( C u l t i v a r Coker 102)
Cotyledon Cotyledon
Soybean (Glycine max L. v a r Root Mandarin) Soybean (Glycine max L. v a r Cotyledon Acme) Jackbean (Canavalia ensiformis)Pod Endosperm Sweet corn (Zea mays) Tobacco (Nicotiana tobacum) Pith Carrot (Daucus c a r o t a v a r Pith Sativa) Sunflower (Helianthus annus) P i t h Root Rice (Oryza s a t i v a v a r Starbonnet) Wheat ( T r i t i c u m monococcum L. ) Stem F i e l d bindweed (Convolvulus a r v e n i s L.)
Soybean Soybean
Tobacco (Nicotiana tobacum L. var Xanthi)
24, 26, 27 27 27 27 28 29 30
C C C C
M, M, M, M,
M, C M, C B5, S X, C
23
22
21
M, C
B5
X, S X, S
M, S
W Ο Ε
"s
ο
i
w
X m ο
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. Ovular
C
MS,
W,
Bark
C C
W,
Crown-gall W,
Stem
M, S
C
S - Suspension; C - C a l l u s .
f
Geranium (Pelagonium hortorum var N i t t a n y Red) Boston i v y (Parthenocissus tricuspidata) Apple (East M a i l i n g r o o t s t o c k 3430) Shamouti orange ( C i t r u s s i n e n s i s Osb.
Cotyledon
C u l t u r e type:
,CH COOH
L. var
B5 - Gamborg's; H - H e l l e r ' s ; M - M i l l e r s ; MS - Murashige
IAA
2,4,5-T
CI
2
^ - 0 C H COOH
Soybean (Glycine max Acme)
B a s i c media: X - Others.
CI-
Table I - page 3
f
35
1
and Skoog s; W - W h i t e s
36
34,
33
32
31
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
O-C-NHCH3
Potato (Solanum tuberosum)
Purple cockle (Agrostemma githago L.) Soybean (Glycine max) Bedstraw (Galium verum) Carrot (Daucus carota) Clover (Meliotus alba) Tobacco ( N i c o t i a n a tobacum) Tobacco ( N i c o t i a n a g l u t i n o s a ) Tobacco ( N i c o t i a n a s y l v e s t r i s ) Tobacco ( N i c o t i a n a glauca) Lettuce (Lactuca s a t i v a ) (Beta v u l g a r i s ) P a r s l e y (Pstroselinum hortense)
Source
Culture
M,
S
Medium and ^ Culture Type
Plant Tissue
Cultures.
Tobacco ( N i c o t i a n a tobacum L. var Xanthi)
Species
Metabolism of I n s e c t i c i d e s by Plant Tissue
Insecticide
Table I I .
39
37,
38
Reference
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
i
f
3
x x
B a s i c media:
-Cl
-Cl
P a r s l e y (Petroselinum hortense, Hoffm.) Soybean (Glycine max L.)
P a r s l e y (Petroselinum hortense, Hoffm.) Soybean (Glycine max L.)
Bean (Phaseolus v u l g a r i s var Roots & Canadian Wonder) Shoots Potato (Solanum tuberosum var Tuber Majestic)
S
41 41 B5, S
41
40
40
B5, S
B5, S
W, S
MS,
S - Suspension; C - C a l l u s .
M - M i l l e r ' s ; W - White's; MS - Murashige and Skoog's; B5 - Gamborg's; X - Other.
Kelthane (Ascaroside)
CC1
OTO
OH
£,£ -DDT
CCI 3
~ 0 ~ K
^ C u l t u r e type:
a
Cl-
c
H
Table I I - page 2 Ql _ CL Cl'
44
XENOBIOTIC
M E T A B O L I S M
NO
roduct
(79% Journal of Agricultural and Food Chemistry Figure 1.
Metabolism of fluorodifen by tobacco cell in suspension culture
These data from plant t i s s u e c u l t u r e s t u d i e s are c o n s i s t a n t with r e s u l t s obtained w i t h whole p l a n t s where the glucoside of 4n i t r o p h e n o l has been reported as the major product of soybean and maize seedlings (43, 44). Whole p l a n t metabolism s t u d i e s with f l u o r o d i f e n (43, 44) suggested that a small percentage of the a p p l i e d p e s t i c i d e may be reduced to 4-aminophenyl 2-amino-4-(trifluoromethyl) phenyl ether, 4-nitrophenyl 2-amino-4-(trifluoromethyl) phenyl ether or 4-aminophenyl 2 - n i t r o - 4 - ( t r i f l u o r o m e t h y l ) phenyl ether, but none of these compounds, i n c l u d i n g 4-aminophenyl, was detected i n the tobacco t i s s u e c u l t u r e s t u d i e s . P r o p a n i l ( 3 , 4 - d i c h l o r o p r o p i o n a n i l i d e ) i s an h e r b i c i d e used to c o n t r o l weeds i n r i c e . The r e s i s t a n c e of r i c e to p r o p a n i l i s a t t r i b u t e d to the high l e v e l s of an a r y l a c y l amidase ( p r o p a n i l amidase) that hydrolyzes p r o p a n i l to 3 , 4 - d i c h l o r o a n i l i n e and prop i o n i c a c i d . The enzymatic l e v e l of p r o p a n i l amidase i n r i c e p l a n t s and i n r i c e root suspension c u l t u r e s has been i n v e s t i g a t e d (20, 45). The a c t i v i t y of the enzyme was found to be two to four times greater i n o l d e r p l a n t s (four leaves) than i n younger p l a n t s ( l e s s than four l e a v e s ) . P r o p a n i l amidase was a l s o demonstrated i n the r i c e suspension c u l t u r e , but i n t e r e s t i n g l y , the enzymatic a c t i v i t y could only be demonstrated a f t e r the t i s s u e c u l t u r e had developed to s t a t i o n a r y phase (5.5 days). This i n v e s t i g a t i o n i s important because i t documents a change i n the b i o s y n t h e t i c capacity of p l a n t t i s s u e c u l t u r e s w i t h the age of the c u l t u r e . The dependency of s e v e r a l enzymes, i n c l u d i n g phenylalanine ammonia l y a s e , upon i l l u m i n a t i o n of p a r s l e y c e l l c u l t u r e s a l s o has been shown (46). The metabolism of diphenamid (N-N-dimethyl-2,2-diphenylacetamide) by soybean r o o t t i p c e l l suspension c u l t u r e s has been
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
2.
M U M M A
A N D H A M I L T O N
Plant Tissue and Cell
Cultures
45
i n v e s t i g a t e d (19) a t d i f f e r e n t stages o f growth and compared with whole p l a n t s . The metabolites found i n both p l a n t s and suspension c u l t u r e s were N-hydroxymethyl-N-methyl-2,2-diphenylacetamide (MODA), N-methyl-2,2-diphenylacetamide (MMDA), 2,2-diphenylacetamide (DA) and two p o l a r g l y c o s i d e s . One of the g l u c o s i d e s was a c i d i c and was i d e n t i f i e d as an e s t e r of malonic a c i d (Figure 2).
DA
Figure 2.
MMDA
(Acidic Glycoside)
Metabolism of diphenamid by soybean root cells in suspension culture
About 9-22% o f the diphenamid was metabolized by the c e l l c u l t u r e s at e a r l y l o g (3-7 days) and s t a t i o n a r y phases (14-18 days), r e s p e c t i v e l y . However, diphenamid metabolism per gram was about 2 times more r a p i d by e a r l y l o g phase c e l l s than by stationary c e l l s . Cultures o f a l l ages formed the same metabol i t e s with MODA and the d e a l k y l a t e d products predominating. The r e l a t i v e composition o f the metabolites i s presented i n Tables I I I and IV. The hydroxylated m e t a b o l i t e , MODA, was found almost e x c l u s i v e l y i n the medium 92-99%, and the d e a l k y l a t e d products were found predominantly i n the medium (68-94%). Log phase and s t a t i o n a r y phase c e l l s produced l a r g e r amounts of the d e a l k y l a t e d metabolites per mg dry weight per day than d i d e a r l y l o g phase c e l l s . G l y c o s i d e s c o n s i s t e d of only 6-7% of the t o t a l metabolites i n c e l l c u l t u r e s but were the major metabolites (46-48%) of tomato, pepper and soybean p l a n t s . These c e l l suspension c u l t u r e s demonstrate c l e a r l y the same metabolic degradation pathways as i n t a c t p l a n t s , but s i g n i f i c a n t q u a n t i t a t i v e d i f f e r e n c e s do occur e s p e c i a l l y i n the small amount of g l y c o s i d e formation by the soybean root suspension c e l l s . No q u a l i t a t i v e changes occurred i n the metabolism of diphenamid with age of the c u l t u r e . The metabolism of c i s a n i l i d e ( c i s - 2 , 5 - d i m e t h y l - l - p y r r o l i d i n e c a r b o x a n i l i d e ) , a s e l e c t i v e preemergence h e r b i c i d e , has been i n v e s t i g a t e d i n excised leaves and c e l l suspension c u l t u r e s of
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
46
XENOBIOTIC
M E T A B O L I S M
Table I I I . Diphenamid Metabolism by Growth Phase of Soybean C e l l Suspension c u l t u r e s (19)
Growth Phase
Diphenamid or M e t a b o l i t e
nmol per Gram C e l l s Medium C e l l Extract
E a r l y Log (3-7 Days)
Diphenamid MODA Dealkylate Glucosid
12.8 0.1
444.8 31.6
Log (7-14 Days)
Diphenamid MODA Dealkylated Glucoside
41.9 2.2 10.6 1.9
99,4 27.4 27.8 1.1
Stationary (14-18 Days)
Diphenamid MODA Dealkylated Glucoside
42.5 1.2 6.7 0.9
144.3 25.4 14.5 1.9
Table IV. R e l a t i v e Composition of Diphenamid or Metabolites i n C e l l s and Medium i n E a r l y Log Phase (19) Diphenamid or M e t a b o l i t e
% C e l l Extract
Diphenamid MODA Dealkylated Glucoside A c i d i c Glucoside
2,.8 0..3 5.,5 25,,0 100..0
Medium 97.,2 99.,7 94,,5 75.,0 0.,0
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
2.
M U M M A
A N D
Plant Tissue and Cell
H A M I L T O N
Cultures
47
c a r r o t and cotton (17, 18). A f t e r 6 days, excised c a r r o t leaves metabolized ca. 70% o f the a p p l i e d c i s a n i l i d e , but c a r r o t t i s s u e c u l t u r e s metabolized >95% o f the a p p l i e d h e r b i c i d e i n 3 days. Cotton leaves metabolized c i s a n i l i d e t o a greater extent than cotton suspension c u l t u r e s . The metabolism of c i s a n i l i d e by c a r rot and cotton p l a n t s i s shown i n Figure 3 and the r e l a t i v e comp o s i t i o n of metabolites i n p l a n t s and t i s s u e c u l t u r e i s presented i n Table V.
cri
CH
3
Glycoside I
3
Glycoside H Pesticide Biochemistry and Physiology
Figure 3.
Metabolism of cisanilide by carrot and cotton phnts and cells in culture
In the excised p l a n t s , g l y c o s i d e s I and I I were the major methanol s o l u b l e metabolites and only t r a c e q u a n t i t i e s of the aglycons were detected. In c a r r o t and cotton c e l l suspension c u l t u r e s , aglycon I or g l u c o s i d e I was not detected and only t r a c e amounts of g l y c o s i d e I I were detected. Aglycon I I was a major metabolite i n the medium of the c e l l suspension c u l t u r e s . In the excised p l a n t s , l e s s (20-25%) of the * C - l a b e l was found i n the i n s o l u b l e residue f r a c t i o n compared to the c e l l suspension c u l t u r e s (39-40%). ll
American Chemical Society Library
1155 16th St. N. W. In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; Washington, D. C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
V. a
a
Methanol Soluble 25 13 (Unknown) I n s o l u b l e Residue 18 39 G l y c o s i d e I was not detected and g l y c o s i d e I I was t e n t a t i v e l y as a minor component.
identified
Excised Cotton Leaves (2 Days) Cotton Suspension Cultures (7 Days) Fraction % % Cells % Medium T o t a l Metabolized >95 >76 Methanol Soluble 55 — 24 (Glycosides I>II) (Aglycon I I )
40
Carrot Suspension C u l t u r e (3 D a y s ) % Cells % Medium >95 — 40 (Aglycon I I ) 20
R e l a t i v e Composition of C i s a n i l i d e M e t a b o l i t e s (18).
Excised Carrot Leaves (6 Days) Fraction % T o t a l Metabolized 70 Methanol Soluble 50 (Glycoside 1=11) Methanol Soluble 10 (Unknown) I n s o l u b l e Residue 20
Table
2.
M U M M A
A N D H A M I L T O N
Plant Tissue and
Cell
Cultures
49
At f i r s t approximation, i t would be easy to conclude that the metabolic degradation pathways were d i f f e r e n t i n the excised p l a n t s versus the c e l l suspensions s i n c e aglycon I and g l u c o s i d e I were not detected i n the t i s s u e or the medium. T h i s d i f f e r e n c e may i n d i c a t e r a p i d i n c o r p o r a t i o n of aglycon I i n t o a methanoli n s o l u b l e f r a c t i o n , because when aglycon I was administered to the c e l l suspension c u l t u r e , i t was r a p i d l y converted to methanol i n s o l u b l e products. Neither t i s s u e i s able to cleave a p p r e c i a b l y the urea type s t r u c t u r e of c i s a n i l i d e . T h i s i s a l s o the case f o r the metabolism of other urea h e r b i c i d e s by p l a n t s (43). Both t i s s u e s possess the a b i l i t y to form the hydroxy 1 d e r i v a t i v e , aglycon I I , but the c e l l c u l t u r e s e v i d e n t l y have a reduced c a p a c i t y to form the g l y c o s i d e conjugate and consequently aglycon I I accumulate metabolite i s apparentl s o l u b l e p r o d u c t ( s ) . Thus, the metabolic pathways may be somewhat s i m i l a r except f o r l o s s of aglycons i n t o the medium and the increased formation of an i n s o l u b l e product. The p h y t o t o x i c i t y and metabolism of the h e r b i c i d e m e t r i b u z i n (4-amino-6-t-butyl 3-[methylthio]-as-triazin-5-[4H]-one) has been i n v e s t i g a t e d w i t h dark-grown soybean cotyledon suspension c u l t u r e s from s u s c e p t i b l e ("Coker 102") and r e s i s t a n c e ("Bragg") c u l t i v a r s . Bioassays were based on p o p u l a t i o n changes of v i a b l e c e l l s during i n c u b a t i o n with m e t r i b u z i n . V i a b l e c e l l s were c l a s s i f i e d as c e l l s with s t r u c t u r a l i n t e g r i t y and cytoplasmic streaming. Differential r e s i s t a n c e to m e t r i b u z i n was demonstrated by the c e l l suspensions from r e s i s t a n t and s u s c e p t i b l e c u l t i v a r s . M e t r i b u z i n had been reported to i n h i b i t photosynthesis, but the demonstrated phytot o x i c i t y toward both c u l t i v a r s of dark-grown a c h l o r o p h y l l o u s suspension c u l t u r e s i n d i c a t e d that p h y t o t o x i c i t y was not r e s t r i c t e d to photosynthesis. D e t o x i f i c a t i o n of m e t r i b u z i n by soybeans has been a t t r i b u t e d to formation of an N-glucoside. Enzymatic detoxi f i c a t i o n of m e t r i b u z i n d i d not occur i n the s u s c e p t i b l e c u l t i v a r due to the accumulation of a substance which i n h i b i t e d the enzyme. The r e s i s t a n t c u l t i v a r metabolized the i n h i b i t o r to a n o n i n h i b i tory form. Therefore, m e t r i b u z i n r e s i s t a n c e by the Bragg c u l t i v a r was a t t r i b u t e d to the a b i l i t y of t h i s c u l t i v a r to metabolize a common enzymatic i n h i b i t o r . P l a n t t i s s u e c u l t u r e techniques have been used by numerous i n v e s t i g a t o r s (23 - 29) f o r 2,4-D metabolism s t u d i e s . In 1968, the metabolism of 2,4-D-2-i^C by suspension c u l t u r e s of soybean root grown under continuous l i g h t (2000 lux) was examined (23). Most of the C - l a b e l i n the t i s s u e appeared as two spots on paper chromatography. The f a s t e r moving compound has the same Rf as 2,4-D and was assumed to be f r e e 2,4-D. The slower moving spot was a g l y c o s i d e that y i e l d e d glucose and f r e e 2,4-D when t r e a t e d w i t h emulsin. These i n v e s t i g a t o r s assumed that 2,4-D was metabol i z e d only to the 3-D glucose e s t e r of 2,4-D. Presumably, the amino a c i d conjugates, which have been subsequently i d e n t i f i e d as metabolites of 2,4-D, d i d not separate from 2,4-D i n the 1 4
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
50
XENOBIOTIC
M E T A B O L I S M
chromatographic s o l v e n t s used. The a d d i t i o n o f glutamine to the media increased the uptake of 2,4-D, but d i d not change the apparent metabolic products when analyzed by t h e i r chromatographic s o l vent systems. Some 2,4-D was a l s o a s s o c i a t e d with p r o t e i n . Two clones of f i e l d bindweed (Convolvulus a r r e n s i s L.) that d i f f e r e d i n t h e i r s u s c e p t i b i l i t y to 2,4-D under f i e l d and greenhouse c o n d i t i o n s a l s o e x h i b i t e d s i m i l a r d i f f e r e n c e s when stem c e l l s were c u l t u r e d i n l i q u i d and agar media (30). When amino a c i d s were added to the c u l t u r e media, the response to 2,4-D was a l t e r e d . The a b s o r p t i o n o f 2,4-D was increased w i t h glutamine and decreased with glutamic a c i d . Glutamic a c i d increased the t o l e r ance of the s u s c e p t i b l e clone, but reduced the t o l e r a n c e of the r e s i s t a n t clone. Glutamine i n c r e a s e d the s u s c e p t i b i l i t y of the s u s c e p t i b l e clone to a much g r e a t e r degree than i t d i d the r e s i s tant clone. There was and n i t r a t e reductase a c t i v i t y When soybean (Glycine max L.) cotyledon c a l l u s was incubated for 32 days w i t h 2,4-D-l- ^C,metabolites changed q u a l i t a t i v e l y and q u a n t a t i v e l y w i t h time (24). The water s o l u b l e f r a c t i o n from the t i s s u e increased i n r a d i o a c t i v i t y . When i t was t r e a t e d with 3 glucosidase, at l e a s t e i g h t aglycons that changed with time were r e l e a s e d ( F i g . 4 ) . 4-Hydroxy-2,5-dichlorophenoxyacetic a c i d (4OH-2,5-D) was the most abundant aglycon and 4-hydroxy-2,3-dichlorophenoxyacetic a c i d (4-OH-2,3-D) was i d e n t i f i e d as a minor component. Free 2,4-D a l s o was l i b e r a t e d f o l l o w i n g enzymatic treatment. The presumed presence o f 2,4-dichlorophenoxyacetyl-3-0-D glucose, a metabolite reported p r e v i o u s l y (23), was suggested. A
The ether s o l u b l e f r a c t i o n reached a maximum a f t e r 2 days and c o n s i s t e d of seven d i f f e r e n t regions of components on paper chromatography ( E t i - E t 7 ) that v a r i e d with time ( F i g . 5 ) . The major component (Eti*) was i d e n t i f i e d as the glutamic a c i d conjugate of 2,4-D and i t s r e l a t i v e composition was maximal a f t e r one day. The a s p a r t i c a c i d conjugate o f 2,4-D ( E t 2 ) increased g r a d u a l l y i n r e l a t i v e composition. S u r p r i s i n g l y , f r e e 2,4-D ( E t 7 ) d i d not reach i t s maximum u n t i l eight days. These data imply that contrary to some previous s t u d i e s , the metabolism of 2,4-D by plant t i s s u e i s q u i t e complex. Subsequently, f i v e a d d i t i o n a l amino a c i d conjugates of 2,4-D have been i d e n t i f i e d from soybean (27). These i n clude the a l a n i n e , v a l i n e , l e u c i n e , phenylalanine and tryptophan conjugates. A t y p i c a l d i s t r i b u t i o n of 2,4-D metabolites i s o l a t e d from 4-week-old c a l l u s t i s s u e i s presented i n Table VI.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
2.
M U M M A
Plant Tissue and Cell
A N D H A M I L T O N
Cultures
ΙΟΟ r
80
u. 60 I UJ
i
$ 40 I
2CLU
4
8
16
INCUBATION TIME (DAYS) Plant Physiology Figure 4. The relative amounts of the aglycons obtained from the water-soluble fractions of soybean callus tissue incubated with 2,4-D-l- C: (Agi), primarily 4-OH-2,5-D and 4-011-2,3-D; (Ag ), 2,4-D. 14
7
Table V I .
R e l a t i v e Percentage of 2,4-D M e t a b o l i t e s i n Soybean C a l l u s T i s s u e (27). a
Ether-Soluble Metabolite Unk
Metabolites % In T i s s u e 1.2
2,4-D-Asp 2,4-D-Gly Unk 2,4-D-Ala,-Val) 2,4-D 2,4-D-Leu,-Phe -Try) TOTAL a
3.7 12.9 1.4 5.3 33.7 4.0 62.2%
Four-week-old c a l l u s t i s s u e with 2,4-D.
(emulsin) Aglycons % In Tissue Metabolite 26.3 (4-OH-2,5-D, 4-OH-2,3-D) 2.5 Unk 1.1 Unk 1.0 Unk 0.8 Unk 0.9 Unk 0.8 2,4-D 0.4 Unk 33.8% (10g) incubated
f o r 8 days
Journal of Agricultural and Food Chemistry
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
52
XENOBIOTIC
I
2
4
8
INCUBATION TIME
16
24
M E T A B O L I S M
32
(DAYS)
Plant Physiology Figure 5. Relative amounts of ether solubles isolated from soybean callus tissues incubated with 2,4-D-l- C: (Et ), aspartic acid conjugate; (Et ), glutamic acid conjugate; and (Et ), free 2,4-D. 14
2
k
7
A d d i t i o n a l minor aglycons a l s o have been i d e n t i f i e d t e n t a t i v e l y from corn endosperm c a l l u s as 3-hydroxy-2,4-dichlorophenoxyacetic a c i d (3-OH-2,4-D), 4-hydroxy-2-chlorophenoxyacetic a c i d (4-OH-2-C1) (27), and from wheat suspension c u l t u r e s as 6-hydroxy2,4-dichlorophenoxyacetic a c i d (6-OH-2,4-D) and 2-hydroxy-4chlorophenoxyacetic a c i d (2-OH-4-C1) (29). The e t h y l ester o f 2,4-D has been i s o l a t e d from the g l y c o s i d e f r a c t i o n of r i c e c a l l u s t i s s u e c u l t u r e f o l l o w i n g (3-glucosidase treatment (28). The e t h y l ester was presumed to be an a r t i f a c t of the i s o l a t i o n procedure probably being derived from the glucose e s t e r that e x i s t s i n high concentration i n t h i s t i s s u e . The glutamic a c i d conjugate of 2,4-D i s not an end product of 2,4-D metabolism (25) . When C-2,4-D-glutamic a c i d was i n c u bated with soybean c a l l u s t i s s u e , f r e e 2,4-D, the a s p a r t i c a c i d conjugate, and other products were found. These data suggest that amino a c i d conjugates may represent a r e s e r v o i r of bound 2,4-D 1l +
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
2.
M U M M A AND HAMILTON
Plant Tissue and Cell
Cultures
53
that may be i n v o l v e d i n the r e g u l a t i o n of 2,4-D l e v e l s i n the tissue. Comparative metabolism s t u d i e s o f 2,4-D i n c a r r o t , jackbean, sunflower, tobacco, corn and r i c e c a l l u s t i s s u e c u l t u r e s (27, 28) and a wheat suspension c u l t u r e (29) a r e shown i n Tables V I I and V I I I . A l l t i s s u e s examined, except r i c e , formed amino a c i d conjugates and a l l formed g l y c o s i d e s (phenolic g l y c o s i d e s as w e l l as glucose e s t e r s ) . The d i c o t s formed a higher r e l a t i v e percentage of amino a c i d conjugates w h i l e the monocots produced a higher percentage o f g l y c o s i d e s . The metabolism o f 2,4-D i n soybean and corn p l a n t s has been compared w i t h 2,4-D metabolism i n soybean c a l l u s t i s s u e (26). These data are presented i n Tables IX and X. In t h i s experiment, the 2,4-D-l- *C was d i r e c t l injected int th c a l l u tissu growing on agar r a t h e r tha ed c a l l u s . The c a l l u s growth and i t s n u t r i e n t s t a t u s was not changed. The metabolites found i n the p l a n t s a r e a l s o present i n the c a l l u s t i s s u e , but differ quantitatively. Of p a r t i c u l a r note i s the f a c t that f r e e hydroxylated 2,4-D e x i s t s i n both c a l l u s and i n p l a n t s i n c o n t r a s t to e a r l i e r t i s s u e c u l t u r e experiments. Free hydroxylated 2,4-D a l s o has been reported i n bean p l a n t s (48). These experiments a l s o suggest that the unknown compounds, Unki and U n k 2 , a r e amino a c i d conjugates o f hydroxylated 2,4-D metabolites ( p r i m a r i l y the glutamic a c i d conjugate of 4-OH-2,5-D) and are common to a l l t i s sue examined. A summary of the metabolism of 2,4-D i n p l a n t s i s presented i n F i g u r e 6. Recently, the metabolism of 2,4-D has been reported i n s i x i n t a c t p l a n t s : wheat, timothy, green bean, soybean, sunflower and strawberry (47). The r e l a t i v e percentage of amino a c i d conjugates i s low compared to the p r e v i o u s l y c i t e d work w i t h t i s s u e c u l t u r e experiments (Table X I ) . Of p a r t i c u l a r n o t i c e i s the unusually h i g h percentage of the d i c h l o r o p h e n o l g l y c o s i d e i n strawberry. In c o n t r a s t to use of t r u e suspension c u l t u r e s by most other l a b o r a t o r i e s , Feung e t a l . (24-28) u s u a l l y incubated a f a i r l y l a r g e amount (~10 gms) of small p i e c e s of 4-5 week o l d c a l l u s taken from s o l i d medium i n 25-40 ml l i q u i d medium w i t h shaking during treatment w i t h 2,4-D-l- ^C. T h i s technique r e s u l t s i n n e a r l y t o t a l uptake o f the a p p l i e d 2,4-D w i t h i n 48 hours, the accumulation o f s i g n i f i c a n t amounts of g l y c o s i d e metabolites i n the t i s s u e (phenolic g l y c o s i d e s and glucose e s t e r ) , i n s i g n i f i c a n t amounts of primary hydroxylated products and no accumulation of products i n the medium. In c e l l suspension c u l t u r e s , a b s o r p t i o n a l s o i s r a p i d but metabolites o f t e n accumulate i n the medium. In the study (26) where c a l l u s t i s s u e was i n j e c t e d d i r e c t l y w i t h 2,4-D-l- C., a d d i t i o n a l metabolites were found. Thus, the method of p e s t i c i d e a d m i n i s t r a t i o n may be important. Data w i t h i n t a c t p l a n t s (Tables IX, X and XI) a r e c o n s i s t a n t w i t h metabolism data obtained w i t h p l a n t t i s s u e c u l t u r e s , but s i g n i f i c a n t q u a n t i t a t i v e d i f f e r e n c e s do occur. The monocots ll
1
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
ll+
a
4.1 0.8 0.5 0.8 0.6 3.2
2.6 0.4 0.3 0.3 1.1 1.2
12.8
3.2 0.7 0.4 0.1
0.9 1.0
13.2
42.2
2.6 7.7
8.3 2.0 1.9 2.3
17.4
64.6
10.2
1.6
19.4 0.5 1.2
31.7
Corn
A l l were c a l l u s t i s s u e c u l t u r e s (27, 28) except wheat (29).
18.3
8.3
6.9
6.9
(4-OH-2,3-D, 4-OH-2,5-D) Unk Unk Unk Unk Unk Unk 2,4-D Ethyl-2,4-D Others TOTAL
% Total i n Tissue Sunflower Tobacco
Carrot
Jackbean
14.9 12.9 1.5 29.7
0.4
Rice
23.9
Ν/
10. 2
\
Wheat
R e l a t i v e Percentage of Water-Soluble 2,4-D-l- C M e t a b o l i t e s I s o l a t e d from Seven Species of Plant T i s s u e C u l t u r e s as the Aglycons.
Metabolites
Table V I I .
M U M M A AND H A M I L T O N
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In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
56
XENOBIOTIC M E T A B O L I S M
llf
Table IX. R e l a t i v e Percentage of Water-Soluble 2,4-D-l- C M e t a b o l i t e s I s o l a t e d from Soybean C a l l u s , Soybean P l a n t , and Corn Plant as the Aglycons (26).
Soybean Callus
Metabolites Unk Unk Unk (4-OH-2,3-D* 5-OH-2,4-D) 4-OH-2,5-D 2,4-D Unk Ethyl-2,4-D TOTAL
% In T i s s u e Soybean Plant
Corn Plant
5.18 1.67 1.79 8.27
0.39
0.94
0.15 1.47
0.73 2.45
16.28
3.20
1.67
38.15
1.22 17.94
7.70 39.28
*Major m e t a b o l i t e .
Table X.
R e l a t i v e Percentage of the E t h e r - S o l u b l e (at pH 2) 2,4-D-l- *C M e t a b o l i t e s I s o l a t e d from Soybean C a l l u s , Soybean P l a n t , and Corn Plant (26). ll
% In T i s s u e Metabolites Unk (5-OH-2,4-D 4-OH-2,3-D* 4-OH-2,5-D*) Unki Unk Asp-2,4-D (Asp-2,4-D Glu-2,4-D*) Unk Unk Unk (Ala-2,4-D Val-2,4-D*) (Val-2,4-D 2,4-D*) (Phe-2,4-D) Trp-2,4-D Leu-2,4-D*) TOTAL 2
Soybean Callus
Soybean Plant
Corn Plant
0.50 1.48
3.14 3.50
Trace 7.08
9.59 1.49 0.62 11.15
8.87 1.94 Trace 4.02
0.69 0.25 0,16 0.28
2.67 2.00 1.73 4.07
2.22 1.59 1.48 1.37
0.50 0.62 0.36 0.86
11.91
43.55
29.60
0.51
1.63
5.60
73.31
46.00
47.72
*Major metabolite
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
2.
M U M M A AND HAMILTON
Plant Tissue and Cell
57
Cultures
,r y ι, Ethanol » Ethyl Ester-2,4-D OCHgC-R
ι
Ring Hydroxylation?
OH CK^K^
Chain
CI
Ο OCHoC-Ri
0
ι Emulsin ι»
Î A I
Λ
. . .. Ammo Acid Conjugation^
*^^dation 0 C H C 0 0 H 2
ClvJ\
^
I
β
CI OH Amino Acid Conjugation
CI R= Ala, Val, Leu Asp,Glu,Phe,Trp
Amino Acid Conjugation ? 50H-2,4-D 60H-2,4-D 30H-2,4-D 40H-2-CI 20H-4-CI
Conjugation CI 0-Sugar Phenolic Glycosides
Journal of Agricultural and Food Chemistry Figure 6.
Table XI.
Summary of 2,4-D metabolism by plants (26)
R a d i o a c t i v i t y of 2,4-D and i t s M e t a b o l i t e s , Represented as Percentage of T o t a l C Absorbed, i n Plants (47). lk
F r a c t i o n or Substance 2,4-D 2,4-D-Asp and 2,4-D-Glu 4-OH-2,5-D and 4-OH-2,3-D
Wheat 29.9 0 0.8
2,4-Dichlorophenoxy23.9 acetyl/3-D-glucose Glucosides of 4-OH-2,510.2 D and 4-OH-2,3-D Glycoside of 2,4-Di2.2 chlorophenol Unknown 2,4-D M e t a b o l i t e 15.0
Bean
Soy bean
Sun flower
Straw berry
30.9 0
4.0 3.2
10.5 5.6
85.0 1.5
7.0 0
0
3.5
3.0
0
0
21.7
24.8
5.7
3.7
2.0
3.3
50.1
53.0
Timothy
2.4 12.6
—
2.5
—
8.0
0
12.0
—
65.0 0
0
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
58
XENOBIOTIC METABOLISM
c o n s i s t a n t l y produced l a r g e q u a n t i t i e s of the glucose e s t e r . A l though the amino a c i d conjugates were present i n the d i e o t s , t h e i r concentration i n i n t a c t p l a n t s was not as high as i n plant t i s s u e cultures. The l a c k of s u b s t a n t i a l amounts of amino a c i d conjugates i n the p l a n t s may be explained p a r t i a l l y by recent i n v e s t i g a t i o n s (15). In these s t u d i e s , i t was shown that the age of the c a l l u s t i s s u e and the c o n c e n t r a t i o n of the a p p l i e d 2,4-D greatly affected the metabolism of 2,4-D. In young soybean root c a l l u s t i s s u e (3 weeks), 2,4-D was metabolized (2 days) to ether s o l u b l e metabolites (amino a c i d conjugates) and to aqueous s o l u b l e (ether i n s o l u b l e ) metabolites ( g l y c o s i d e s ) . As the concentration of h e r b i c i d e i n creased uptake i n t o the ethanol f r a c t i o n increased and the amount of f r e e 2,4-D increases p r o p o r t i o n a t e l y w h i l e only a s l i g h t i n crease i n metabolites i c a l l u s t i s s u e (5-9 weeks) that the l e v e l of f r e e 2,4-D i n the t i s s u e i s r e g u l a t e d at ca. 4 rim per gram f r e s h weight. As the concentration of 2,4-D administered was increased, a greater amount of 2,4-D was converted to amino a c i d conjugates so the l e v e l of f r e e 2,4-D i n the t i s s u e was regulated ( F i g . 8). This same phenomena, the r e g u l a t i o n of f r e e 2,4-D l e v e l s through the formation of amino a c i d conjugates, was a l s o demons t r a t e d i n soybean l e a f c a l l u s t i s s u e c u l t u r e s . Although the r e g u l a t i o n of the 2,4-D c o n c e n t r a t i o n w i t h i n the t i s s u e i s c o n t r o l l e d by the formation of the ether s o l u b l e metabolites, the aqueous soluble f r a c t i o n i s twice as b i g i n l e a f c a l l u s as i t i s i n root c a l l u s t i s s u e . D i f f e r e n t i a t e d soybean root c u l t u r e behaves b i o chemically l i k e o l d c a l l u s t i s s u e . The 2,4-D l e v e l s are r e g u l a t e d at ca. 2 nm per gram f r e s h weight ( F i g . 9). These experiments with 2,4-D have shown the importance of the types of t i s s u e , the age of the t i s s u e , the concentration of the p e s t i c i d e , and the method of i n c u b a t i o n used i n p l a n t t i s s u e culture studies. Since each l a b o r a t o r y seems to adopt i t s own procedures, which may a f f e c t r e s u l t s , i t i s time to standardize some of the procedures used i n t i s s u e c u l t u r e s t u d i e s . Although the i n c u b a t i o n of the p e s t i c i d e w i t h c a l l u s t i s s u e c u l t u r e s has been used only by a few l a b o r a t o r i e s (24-28, 31-36, 57), t h i s technique should be examined i n more d e t a i l with other p e s t i c i d e s to determine whether i t may be more r e p r e s e n t a t i v e of whole p l a n t studies. The metabolism of 2,4,5-T by soybean root c a l l u s t i s s u e s has been examined r e c e n t l y (31). Studies showed that soybean c a l l u s t i s s u e c u l t u r e s a l s o convert 2,4,5-T i n t o amino a c i d conjugates (glutamic a c i d [90%] and a s p a r t i c a c i d [10%]). No glucose e s t e r formation was demonstrated. Although the metabolism of i n d o l e a c e t i c a c i d (IAA) by p l a n t t i s s u e c u l t u r e s has not been s t u d i e d e x t e n s i v e l y , the metabolic pathways appear s i m i l a r to those of i n t a c t p l a n t s , or excised p l a n t p a r t s . One pathway i s the peroxidase-IAA oxidase
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
M U M M A AND H A M I L T O N
Plant Tissue and Cell
59
Cultures
Ν ANOMOLES OF OLABEL IN ETHANOL FRACTION/g (f.w.) ,4
Plant Physiology Figure 7, Concentration of 2,4-D or 2,4-D metabolites found in various subfrac tions vs. total concentration in the ethanol extract of 3-week-old soybean root callus tissue following incubation for 48 hr with various levels of 2,4-D-l- C (1.8 X 10~ to 1.1 X 10~ M). The free 2,4-D content was subtracted from the total ether-soluble metabolites (mostly amino acid conjugates). 14
6
5
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC
"1
1
1
METABOLISM
Γ
9 Week Old Root Callus
Ether soluble metabolites
Aqueous soluble metabolites
35 NANOMOLES OF
C - L A B E L IN ETHANOL FRACTION /g(f.w.) Plant Physiology
Figure 8. Concentration of or 2,4-D metabolites found in various subfrac tions vs. total concentration in the ethanol extract of 9-week-old soybean root callus tissue following incubation for 48 hr with various levels of 2,4-D-l- C (1.8 X 10 to 1.1 X 10 M). The free 2,4-D content was subtracted from the total ether-soluble metabolites (mostly amino acid conjugates). 14
6
5
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
M U M M A AND H A M I L T O N
Plant Tissue and Cell
π — ι — ι — Γ
!
Cultures
J , J , , , , J , , ,
r
Ether soluble metabolites
Aqueous soluble metabolites
2 ,4-D 5
10
15
20
NANOMOLES OF C-LABEL IN ETHANOL FRACTION/g (f.w. ) ,4
Plant Physiology Figure 9. Concentration of 2,4-D or 2,4-D metabolites found in various subfrac tions vs. total concentration in the ethanol extract of 3-week-old differentiated soy bean roots following incubation for 48 hr with various levels of 2,4-D-l- C (9 X JO" to 2.3 X 10~ M). The free 2,4-D content was subtracted from the total ethersoluble metabolites (mostly amino acid conjugates). 14
7
6
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
62
XENOBIOTIC METABOLISM +
o x i d a t i o n of IAA i n the presence of Mn 2 and p h e n o l i c c o f a c t o r s . The second pathway ( i n common w i t h 2,4-D) i s the conjugation of IAA w i t h amino a c i d s or sugars. I t now appears that peroxidases a r e as widely d i s t r i b u t e d i n p l a n t t i s s u e c u l t u r e s as they a r e i n i n t a c t p l a n t s (49). These s o - c a l l e d isozymes that are observed on g e l e l e c t r o p h o r e s i s o f t e n change w i t h the stage of development o r hormonal treatment (49, 50, 51) of p l a n t s . Since g e n e t i c i n f o r m a t i o n i s not a v a i l a b l e (52) and peroxidase contains carbohydrate (53), the term isozyme i s probably a misnomer. These "isozymes" have v a r y i n g a c t i v i t i e s on peroxidase substances or a c t as an IAA oxidase. An IAA oxidase has been found i n P i c e a glauca c a l l u s c u l t u r e s (54) and the f i r s t e x t r a - c e l l u l a r IAA oxidase was detected i n Parthenocissus t r i c u s p i d a t a crown g a l l c u l t u r e s (55). An e x t r a c e l l u l a r IAA oxidase an oxidase from t h i s t i s s u Although the enzymatic products were 3-hydroxymethyloxindole and u l t i m a t e l y i t s dehydration product 3-methyleneoxindole, n e i t h e r m e t a b o l i t e was found i n the t i s s u e (57). However, s m a l l amounts of 3-methyloxindole were found i n the medium i n c o n f i r m a t i o n of i t s reported occurrence i n pea s e e d l i n g s (58), Extensive decarb o x y l a t i o n of IAA-l- *C s u p p l i e d to t h i s t i s s u e (50% i n 24 hours) suggests the degradation o f IAA by IAA oxidase and conversion of the oxindole to u n i d e n t i f i e d products. In 12 week o l d apple c a l l u s , 90% d e c a r b o x y l a t i o n of I A A - 1 - w a s found i n 4 hours compared to 20% d e c a r b o x y l a t i o n i n 4 hours by 6 week o l d apple c a l l u s (35). S i x week o l d apple c a l l u s accumulated e l e v a t e d IAA l e v e l s i n the t i s s u e , and two m e t a b o l i t e s , thought to be o x i n d o l e s , were detected i n 12 week and 6 week o l d apple c a l l u s (34). li
1
The conjugation of IAA to a s p a r t i c a c i d has been reported i n geranium stem c a l l u s (32) and i n embryogénie habituated o v u l a r c a l l u s o f orange (36) where i t i s suggested to maintain l e v e l s f a v o r a b l e to embryogenesis. The major conjugates found i n Parthenocissus t r i c u s p i d a t a crown g a l l c a l l u s were the g l y c i n e , alanine, and v a l i n e conjugates (33). Minor amounts of the a s p a r t i c and glutamic a c i d conjugates a l s o were found. Chromatographic s e p a r a t i o n of the g l y c i n e conjugate from IAA as w e l l as the aspart i c conjugate from the glutamic conjugate i s d i f f i c u l t (59). Thus, the glutamic and g l y c i n e conjugates may have remained undetected i n i n t a c t p l a n t s or e x c i s e d t i s s u e . The glucose e s t e r of IAA has been detected i n many p l a n t t i s s u e s (60, 61, 62, 63, 64), but does not appear to have been i d e n t i f i e d i n plant tissue cultures. INSECTICIDES The metabolism o f c a r b a r y l (1-naphthyl methylcarbamate) by tobacco c e l l s i n suspension c u l t u r e has been reported (37, 38). The f i r s t r e p o r t (37) i n d i c a t e d that up to 16% of the t o t a l metabo l i t e s of c a r b a r y l might be conjugates of N-hydroxycarbaryl
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
2.
M U M M A AND H A M I L T O N
Plant Tissue and
Cell
Cultures
63
(1-naphthyl N-hydroxy-N-methyl carbamate) a p o t e n t i a l mutagen. In a more thorough i n v e s t i g a t i o n (38) no N-hydroxy c a r b a r y l or d e r i v a t i v e s could be detected. In these l a t e r s t u d i e s , C - l a b e l e d C i - n a p h t h y l , carbonyl and N-methyl c a r b a r y l (9 ppm) were incubated i n the dark with tobacco c e l l suspensions f o r 14 days. The c e l l s metabolized 36% of the c a r b a r y l of which 7.9% represented nine m e t a b o l i t e s . The only m e t a b o l i t e found i n the medium was N-CH2OHcarbaryl. I t represented 18.38% of the m e t a b o l i t e s . Free NCH20H-carbaryl and i t s g l y c o s i d e were found i n the c e l l s , but i n very low c o n c e n t r a t i o n s . The major t i s s u e m e t a b o l i t e was the β-Dg l u c o s i d e of α-naphthol. T h i s m e t a b o l i t e was found present only i n t r a c e amounts i n i n t a c t bean p l a n t s (65). Small amounts of conjugated 4-OH-(0.3%), 5-OH-(0.3%) and 7-OH-(0.82%) c a r b a r y l were a l s o i d e n t i f i e d i n the suspension c u l t u r e s . These conjugates were the major metabolites i i n t e r e s t was the t e n t a t i v of c a r b a r y l , l,4-dihydro-l,4-epiperoxynaphthalene (0.39%) and 0-1n a p h t h y l c h o l e s t e r o l (3.0%), the l a t t e r metabolite being a unique ether. A summary of the metabolism of c a r b a r y l by tobacco suspen s i o n c u l t u r e i s presented i n F i g u r e 10. The r e l a t i v e composition of metabolites i n tobacco c e l l i n suspension c u l t u r e (38) and i n t a c t bean p l a n t s (65) are compared i n Table X I I . 1 k
M e t a b o l i t e s t h a t have been i d e n t i f i e d i n whole p l a n t s are a l s o found i n the tobacco t i s s u e c u l t u r e , but there i s a l a r g e q u a n t i t a t i v e d i f f e r e n c e between bean p l a n t s and tobacco suspension c u l t u r e s . A more d e t a i l e d study of the m e t a b o l i t e s found i n i n t a c t p l a n t s of the same s p e c i e s should be made to see i f any of the newly discovered metabolites found i n tobacco c e l l suspension c u l t u r e s a l s o e x i s t i n i n t a c t tobacco p l a n t s . In bean p l a n t s , the g l y c o s i d e s of 4-OH, 5-OH and N-CR^OH-earbaryl predominate. The primary hydroxylated product N-CH20H-carbaryl i s perhaps excreted i n t o the suspension c u l t u r e media before the g l y c o s i d e i s formed. Strangely, the α-naphthol was found only as the g l y c o s i d e i n t o bacco suspension c u l t u r e c e l l s . In c o n t r a s t , the g l y c o s i d e of NC H 2 0 H - c a r b a r y l was present i n the c e l l s i n only minor amounts. The metabolism of C l - l a b e l e d l i n d a n e (a-hexachlorocyclohexane) by v a r i o u s p l a n t c e l l c u l t u r e s has been compared to i t s me tabolism i n i n t a c t p l a n t s (39), With i n t a c t p l a n t s (28 day i n c u bation) , very l i t t l e C 1 was l i b e r a t e d , but p l a n t suspension c u l t u r e s (12-28 days i n c u b a t i o n ) metabolized from 0% (soybean) to 6.8% (wild c a r r o t ) of the a p p l i e d l i n d a n e . Trace q u a n t i t i e s of 1,2,4-trichlorobenzene (1%) were i d e n t i f i e d i n tobacco c u l t u r e s , but were not detected i n the other c u l t u r e s . The main m e t a b o l i t e of c a r r o t suspension c u l t u r e s had not been reported p r e v i o u s l y i n p l a n t s and was t e n t a t i v e l y i d e n t i f i e d as a g l u c o s i d e of trichlorophenol. T i s s u e c u l t u r e s showed a higher uptake and metabolic r a t e f o r lindane than i n t a c t p l a n t s . No degradation to CO2 was detected, and l e t t u c e p l a n t s were not a b l e to metabolize l i n d a n e to pentachlorophenol as has been reported (66). These s t u d i e s a l s o showed 3 5
35
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
Data taken from 'Data taken from
(65) (38)
0 0 0 0 0 0
+
Carbaryl 1-Naphthol 4-Hydroxy C a r b a r y l 5-Hydroxy C a r b a r y l 7-Hydroxy C a r b a r y l N-Hydroxymethyl C a r b a r y l Dihydrodihydroxy C a r b a r y l 1.0 Trace 33.0 25.0 0 18.1 15.6
Conjugated (Aglycons)
a
Tobacco
64.72 0 0 0 0 1.47
Ether Soluble (Medium)
0. 5.72 0.02 0.02 0.06 0.20
Conjugated (Aglycons) (Cell)
C-Labeled Compound Tobacco Suspension C u l t u r e ^
I s o l a t e d from Bean Plants and
Percentage of 1 k Bean P l a n t s Ether Soluble
1
Percentage of ^ C - C a r b a r y l Metabolites Suspension Cultures.
Carbaryl or Metabolite
Table X I I .
2.
M U M M A
A N D
Figure 10.
H A M I L T O N
Plant Tissue and Cell Cultures
The metabolism of carbaryl by tobacco cell suspension cultures
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
65
66
XENOBIOTIC METABOLISM
that i n t a c t p l a n t s were contaminated with e p i p h y t i c microorganisms that could metabolize l i n d a n e . The metabolism of a l d r i n has been i n v e s t i g a t e d i n suspension c u l t u r e s from bean r o o t s , bean shoots and potato tubers (40). The e f f e c t of media v a r i a t i o n s on metabolism a l s o were s t u d i e d . In i n t a c t p l a n t s , a l d r i n was converted to d i e l d r i n , p h o t o d i e l d r i n , dihydrochlorodene d i c a r b o x y l i c a c i d and a l d r i n t r a n s - d i o l ( F i g . 11). A l d r i n (40%) was metabolized mainly to d i e l d r i n by the p l a n t suspension c u l t u r e s (28 days). The d i e l d r i n was l o c a t e d p r i m a r i l y i n the c e l l s . A l d r i n t r a n s - d i o l was a l s o i s o l a t e d i n small amounts, but only i n the bean shoot and potato tuber c u l t u r e s . Small q u a n t i t i e s of p h o t o d i e l d r i n were detected when l a r g e r q u a n t i t i e s of a l d r i n (10 mg) were added to bean root c u l t u r e s . Conversion of a l d r i n to p h o t o d i e l d r i n was only found i n mixed bean root and shoot c u l t u r e s a l l cultures. These dat d i e l d r i n by p l a n t s i n the dark r a t h e r than by a l i g h t induced photochemical r e a c t i o n or microorganisms. The metabolism of a l d r i n by p l a n t t i s s u e c u l t u r e s i s i n good q u a l i t a t i v e agreement w i t h s i m i l a r i n t a c t p l a n t experiments, except that the d i c a r b o x y l i c a c i d i s o l a t e d i n low l e v e l s from i n t a c t p l a n t s grown under f i e l d c o n d i t i o n s was not detected i n p l a n t suspension c u l t u r e s . V a r i a t i o n i n the c u l t u r e media a f f e c t e d the q u a n t i t a t i v e d i s t r i b u t i o n of m e t a b o l i t e s . D i f f e r e n c e s a l s o exi s t e d between bean root and shoot c u l t u r e s and between potato tuber c u l t u r e s . A l d r i n t r a n s - d i o l was only found i n c u l t u r e s incubated w i t h a l d r i n , never w i t h d i e l d r i n , and suggest that a l d r i n t r a n s - d i o l i s d i r e c t l y formed from a l d r i n . The metabolism of DDT and Kelthane has been examined i n c e l l suspension c u l t u r e s of p a r s l e y and soybean (41). A f t e r 44-48 hr i n c u b a t i o n , only 0.6 to 2.2% of the a p p l i e d DDT was metabolized. DDE ( 1 , l - d i c h l o r o - 2 , 2 - b i s - [ 4 - c h l o r o p h e n y l ] - e t h y l e n e ) was i d e n t i f i e d as the major m e t a b o l i t e of DDT. Other m e t a b o l i t e s were detected but were not i d e n t i f i e d . P a r s l e y suspension c u l t u r e s converted Kelthane to a nonpolar (0.1%) and a p o l a r (0.3%) metabolite. P h y t o t o x i c i t y and
Bioassays
A number of i n v e s t i g a t o r s (22, 67-74) have used p l a n t t i s s u e c u l t u r e s as a means of studying p h y t o t o x i c i t y . These i n v e s t i g a t i o n s u s u a l l y have not focused on the metabolism of the x e n o b i o t i c d i r e c t l y but have concentrated on the p h y s i o l o g i c a l a f f e c t of the x e n o b i o t i c on the p l a n t t i s s u e . I n d i r e c t l y , these i n v e s t i g a t o r s may be measuring the a b i l i t y of the t i s s u e c u l t u r e to metabolize the x e n o b i o t i c to nontoxic substances. An important example i s the p r i o r mentioned i n v e s t i g a t i o n (22) of m e t r i b u z i n p h y t o t o x i c i t y i n soybean suspension c u l t u r e s where the p r i n c i p l e of r e s i s t a n c e was the a b i l i t y to metabolize an i n h i b i t o r of an enzyme that metabolized m e t r i b u z i n to a nontoxic form.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
M U M M A AND H A M I L T O N
Figure 11.
Plant Tissue and Cell
Cultures
Metabolism of aldrin and dieldrin. The dicarboxylic acid metabolite could not be detected in plant cell suspension cultures.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
68
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The e f f e c t s of organic s o l v e n t s on growth and u l t r a s t r u c t u r e of p l a n t c e l l suspensions have been s t u d i e d r e c e n t l y (75). These s t u d i e s have bearing on most metabolism s t u d i e s . The phytotoxic e f f e c t s of s o l v e n t s i n c r e a s e d i n the f o l l o w i n g order: chloroform, dimethyl s u l f o x i d e , methanol, acetone, i s o p r o p a n o l , and ethanol. At concentrations of 0.25%, chloroform had no apparent a f f e c t on c e l l growth, however, ethanol i n h i b i t e d growth at concentrations as low as 0.05%. These data suggest that small amounts of organic s o l v e n t s , o f t e n used to i n t r o d u c e the x e n o b i o t i c to the p l a n t t i s s u e c u l t u r e , must be c a r e f u l l y considered. P l a n t t i s s u e c u l t u r e techniques have been used to evaluate the b i o l o g i c a l a c t i v i t y of growth s t i m u l a t i n g h e r b i c i d e s and t h e i r m e t a t o l i t e s or p o t e n t i a l m e t a b o l i t e s . Amino a c i d conjugates of 2,4-D (76), 2,4,5-T (77), and IAA (78) were added to c u l t u r e media (10 **-10~ M) of soybea a b i l i t y to s t i m u l a t e c e l crease i n weight. Amino a c i d conjugates of 2,4-D and IAA stimu l a t e d the growth of c a l l u s t i s s u e and some conjugates of 2,4-D stimulated growth even b e t t e r than 2,4-D itself. Surprisingly, the amino a c i d conjugates of 2,4,5-T d i d not s t i m u l a t e growth of soybean root c a l l u s . 8
Production
of Secondary Substances
P l a n t t i s s u e c u l t u r e s have been evaluated as a p o t e n t i a l means of production of p l a n t products sometimes r e f e r r e d to as secondary products. Plant products of p o t e n t i a l commercial use such as s t e r o i d s , a l k a l o i d s , g l y c o s i d e s , vitamins and others have r e c e i v e d some a t t e n t i o n of i n v e s t i g a t o r s , e s p e c i a l l y those of m e d i c i n a l importance (79-83). The metabolism of three a l k a l o i d s , v i n d o l i n e , catharanthine HC1, and v i n c a l e u k o b l a s t i n e s u l f a t e have been examined w i t h suspension c u l t u r e s of Catharanthus roseus (L.) (84). V i n d o l i n e formed two m e t a b o l i t e s , d e s a c e t y l v i n d o l i n e and dihydrovindoline. No m e t a b o l i t e s of catharanthine HC1 were de t e c t e d . V i n c a l e u k o b l a s t i n e s u l f a t e formed three m e t a b o l i t e s . The b i o s y n t h e s i s of a l k a l o i d s by suspension c u l t u r e s may be enhanced g r e a t l y by i n c u b a t i o n of the p l a n t t i s s u e w i t h s e l e c t e d intermediates (85). In t h i s way, the c e l l media would be supple mented w i t h key intermediates that would be metabolized f u r t h e r to the d e s i r e d a l k a l o i d . The b i o s y n t h e s i s of e d u l i n i n e and f u r o q u i n o l i n e a l k a l o i d s from s e l e c t i v e q u i n o l i n e d e r i v a t i v e s by Ruta graveolens c e l l sus pension c u l t u r e s has been s t u d i e d (86). The b i o g e n e t i c s y n t h e s i s of the a l k a l o i d s i n the t i s s u e c u l t u r e experiments were c o n s i s t a n t w i t h pathways discovered i n rutaceous p l a n t s . The metabolic i n termediates d i f f e r e d q u a n t i t a t i v e l y from i n t a c t p l a n t s and v a r i e d w i t h the age of the c u l t u r e and w i t h d i f f e r e n t c u l t u r e i s o l a t e s . Berberine and ρlamatine, two m e d i c i n a l l y important a l k a l o i d s , are produced by p l a n t t i s s u e c u l t u r e s of Coptis j a p o n i c a (79). The production of ginseng g l y c o s i d e s has been s t u d i e d i n p l a n t
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t i s s u e c u l t u r e s of Panax ginseng (79). N i t r o q u a n i d i n e and X-ray treatments were used to o b t a i n mutants that had higher t i t e r s of saponins. These genetic mutation techniques may a l s o be u s e f u l i n the s e l e c t i o n of r e s i s t a n t and n o n r e s i s t a n t c u l t i v a r s by a l t e r ing t h e i r metabolism of x e n o b i o t i c s . So f a r , l a r g e s c a l e commerc i a l drug production using p l a n t t i s s u e c u l t u r e s has not been achieved. I t has been suggested (87) that through proper s e l e c t i o n of p l a n t s p e c i e s , t i s s u e c u l t u r e techniques may be u s e f u l f o r the p r e p a r a t i o n of metabolites of x e n o b i o t i c s i n q u a n t i t i e s needed f o r standards and f o r s t r u c t u r a l c h a r a c t e r i z a t i o n . For example, the glucose e s t e r of 2,4-D can be mass produced w i t h r i c e c a l l u s t i s sue (28) and amino a c i d conjugates of 2,4-D can be mass produced from soybean c a l l u s t i s s u e when c u l t u r e c o n d i t i o n s are s e l e c t e d f o r o p t i m i z a t i o n of amin Advantages and
Disadvantages
In general, a l l s t u d i e s i n d i c a t e that the same metabolic pathways of degradation appear to be operating i n i n t a c t p l a n t s and p l a n t t i s s u e c u l t u r e s . Thus, x e n o b i o t i c metabolism by p l a n t t i s s u e c u l t u r e s i s a d e s i r a b l e model f o r e s t i m a t i n g metabolic pathways i n the i n t a c t p l a n t . However, at the present time, work w i t h i n t a c t p l a n t s (or p a r t s ) i s a l s o necessary to confirm and evaluate the q u a n t i t a t i v e aspects of x e n o b i o t i c metabolism. Plant t i s s u e c u l t u r e s are s t e r i l e , homogenous, r a p i d l y growing and do not r e q u i r e extensive f a c i l i t i e s . Because of ease of s t a n d a r d i z a t i o n of c o n d i t i o n s , r e p r o d u c i b i l i t y should be achieved more readi l y , data are amenable to s t a t i s t i c a l a n a l y s i s and r e s u l t s could be compared d i r e c t l y i n d i f f e r e n t l a b o r a t o r i e s . Use of t i s s u e c u l t u r e s obviates the d i f f i c u l t i e s of poor p e n e t r a t i o n , t r a n s l o c a t i o n , and perhaps there i s l e s s compartmentation of metabolic pools than encountered w i t h i n t a c t p l a n t s . P l a n t t i s s u e c u l t u r e s permit the use of lower l e v e l s of r a d i o l a b e l e d m a t e r i a l and the i s o l a t i o n of metabolites that are f r e e of many i n t e r f e r i n g substances so troublesome w i t h i n t a c t p l a n t s . Metabolism by d i f f e r e n t p l a n t species or by d i f f e r e n t t i s s u e s of the same p l a n t can be compared readily. However, i t i s not at a l l c l e a r that c a l l u s s t a r t e d from d i f f e r e n t t i s s u e s w i l l show major d i f f e r e n c e s i n metabolism. However, one would expect organ c u l t u r e to e x h i b i t c l e a r d i f f e r e n c e s i n the metabolism of x e n o b i o t i c s . P l a n t t i s s u e c u l t u r e s are a l s o u s e f u l f o r studying the mode of a c t i o n of phyto-active x e n o b i o t i c s because of ease of c o n t r o l l i n g the v a r i o u s parameters of growth. C u l t u r e s have been used as a technique f o r the bioassay of chemic a l s that cause p h y s i o l o g i c a l changes such as growth s t i m u l a t i o n , c e l l d i v i s i o n and p h y t o t o x i c i t y . In a d d i t i o n , p l a n t t i s s u e c u l ture techniques o f f e r great p o t e n t i a l i n the mass production of metabolites as w e l l as m e d i c i n a l l y important chemicals. Obvious disadvantages do e x i s t i n the use of p l a n t t i s s u e cultures. The technique does not show the importance of c u t i c u l a r
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penetration,of root a b s o r p t i o n , of microorganisms a s s o c i a t e d with the i n t a c t p l a n t , or of v a s c u l a r t r a n s p o r t . Tissue cultures would not i n d i c a t e the importance of environmental f a c t o r s such as wind, n u t r i t i o n , disease, water s t r e s s , temperature or s u n l i g h t on metabolic products and pool s i z e s . In t i s s u e c u l t u r e s , the photosynthetic apparatus i s o f t e n absent or only marginal. If photosynthesis i s important f o r the metabolism of the x e n o b i o t i c , then t i s s u e c u l t u r e technqiues might be u n d e s i r a b l e . Experiments w i t h t i s s u e c u l t u r e s have shown the importance of c u l t u r e age and the method of x e n o b i o t i c a d m i n i s t r a t i o n i n determining m e t a b o l i t e levels. These c o n d i t i o n s would be d i f f e r e n t w i t h i n t a c t p l a n t s . Conclusions X e n o b i o t i c metabolite q u a l i t a t i v e l y the same with quantitative d i f f e r e n c e s . Therefore, metabolic pathways operating i n whole p l a n t s a l s o seem to be operating p l a n t t i s s u e c u l t u r e s . With some m e t a b o l i t e s , such as g l y c o s i d e s and aglycons, there may be a l a r g e q u a n t i t a t i v e d i f f e r e n c e between the two t e c h niques. This i s e s p e c i a l l y true w i t h p l a n t suspension c u l t u r e technqiues which o f t e n produce q u a n t i t a t i v e l y lower amounts of g l y c o s i d e s and higher amounts of aglycons than s t u d i e s w i t h i n t a c t plants. Apparently the aglycons can be excreted from the c e l l s before the g l y c o s i d e s are formed. Perhaps the l e v e l of carbohydrates i n the media a f f e c t s g l y c o s i d e s y n t h e s i s . Common r e a c t i o n s found i n p l a n t t i s s u e c u l t u r e s are o x i d a t i o n s at both a l i p h a t i c and aromatic carbons, d e a l k y l a t i o n s , h y d r o l y t i c enzymes such as amidases, and e s t e r a s e s , dehydrochlorinations, d e c h l o r i n a t i o n s , hydrogénations and conjugations w i t h sugars, amino a c i d s and a c i d s . Plant t i s s u e c u l t u r e s can r e f l e c t the t o l erance of the parent p l a n t , e.g., c u l t i v a r s showing r e s i s t a n c e to phytotoxic x e n o b i o t i c s a l s o e x h i b i t r e s i s t a n c e i n t i s s u e c u l t u r e . Many f a c t o r s a f f e c t x e n o b i o t i c metabolism by p l a n t t i s s u e c u l t u r e s . The source of the t i s s u e i s important. D i f f e r e n t t i s sues from the same p l a n t show minor v a r i a t i o n s , and t i s s u e from d i f f e r e n t species (or even v a r i e t i e s ) of p l a n t s show greater v a r i a t i o n s (e.g., monocot and d i c o t p l a n t t i s s u e c u l t u r e s may exh i b i t major d i f f e r e n c e s i n metabolism). The t i s s u e c u l t u r e method i s a l s o very important. Suspension c u l t u r e s may give r i s e to s i g n i g i c a n t amounts of m e t a b o l i t e s i n the medium and low r e l a t i v e proportions of g l y c o s i d e s . In the few cases reported, incubations w i t h c a l l u s t i s s u e r e s u l t s i n r a p i d absorption of the x e n o b i o t i c , no s i g n i f i c a n t m e t a b o l i t e s i n the medium and accumulation of g l y goside conjugates s i m i l a r to that found i n i n t a c t p l a n t s . The method of a d m i n i s t r a t i n g the x e n o b i o t i c to the c u l t u r e may a l s o be important. D i r e c t i n j e c t i o n of 2,4-D i n t o the c a l l u s t i s s u e seemingly gave d i f f e r e n t r e s u l t s from c a l l u s t i s s u e incubated i n s o l u t i o n w i t h 2,4-D (26). Another f a c t o r , of some importance, i s the composition of the medium (40). In i n t a c t p l a n t s , i l l u m i n a t i o n
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has been shown to s t i m u l a t e the formation of the glucose e s t e r (60) and enzymes such as phenylalanine ammonia l y a s e (46). This parameter has not been examined thoroughly i n p l a n t t i s s u e c u l tures and needs f u r t h e r study. The p h y s i o l o g i c a l stage or age of the t i s s u e c u l t u r e has been demonstrated to show a s i g n i f i c a n t e f f e c t on metabolism (15). In some cases only o l d e r t i s s u e s demonstrated enzymatic a c t i v i t y (20) and c o n t r o l l e d metabolism (15). In s p i t e of these l i m i t a t i o n s , i n v e s t i g a t i o n s of the metabo l i s m of x e n o b i o t i c s by p l a n t t i s s u e c u l t u r e s provides a reasonable approximation of the metabolism expected w i t h i n t a c t p l a n t s . With so many f a c t o r s a f f e c t i n g metabolism and each research l a b o r a t o r y using d i f f e r e n t t i s s u e s and c u l t u r e c o n d i t i o n s , there i s a need f o r the s t a n d a r d i z a t i o n of some t i s s u e c u l t u r e parameters. Perhaps c e r t a i n w e l l s t u d i e d p l a n t t i s s u e s should be used by a l l l a b o r a t o r i e s as a sues should i n c l u d e som Tobacco t i s s u e has been suggested as at l e a s t one p o s s i b i l i t y f o r r o u t i n e use (38). Perhaps a p l a n t t i s s u e bank, s i m i l a r to those e s t a b l i s h e d f o r microorganisms, could be e s t a b l i s h e d f o r l a b o r a t o r i e s working w i t h plant t i s s u e c u l t u r e s . A d d i t i o n a l research i s needed to determine which of the many f a c t o r s a f f e c t i n g metabolism i n p l a n t t i s s u e c u l t u r e s are most important before we can suggest s t a n d a r d i z a t i o n of these f a c t o r s . R e a l l y , only a few x e n o b i o t i c s and p e s t i c i d e s have been examined, e.g., few compounds from the important organophosphate or t r i a z i n e p e s t i c i d e s have been examined. Fungicides appear to have been neglected. Therefore, a d d i t i o n a l i n v e s t i g a t i o n s w i t h other pesti c i d e s and x e n o b i o t i c s are d e s i r a b l e before we can p r o p e r l y e v a l uate the r e l a t i v e merits of studying metabolism with p l a n t t i s s u e c u l t u r e s and the e x t r a p o l a t i o n of these r e s u l t s to i n t a c t p l a n t s .
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ABSTRACT X e n o b i o t i c metabolism by p l a n t t i s s u e c u l t u r e s are reviewed with an emphasis on p e s t i c i d e metabolism. The r e s u l t s obtained with t i s s u e c u l t u r e techniques are evaluated and compared w i t h r e s u l t s using whole p l a n t s . Some f a c t o r s a f f e c t i n g metabolism s t u d i e s w i t h p l a n t t i s s u e c u l t u r e techniques are a l s o considered. Some advantages o f the p l a n t t i s s u e c u l t u r e techniques include: sterility, a r a p i d l y growing homogenous t i s s u e w i t h low pigment content, moderate cost and space requirements, ease of d u p l i c a t i o n of treatment c o n d i t i o n s , r a p i d uptake and metabol i s m of x e n o b i o t i c s , and f isolation f metabolites It o f f e r s the opportunity t w e l l as evaluate p h y t o t o x i c i t y p h y s i o l o g i c a change p l a n t t i s s u e . Obvious disadvantages of t h i s technique are its failure to evaluate such f a c t o r s as: penetration, absorption, microorganisms, v a s c u l a r t r a n s p o r t and environmental i n f l u e n c e s . Other f a c t o r s a f f e c t i n g x e n o b i o t i c metabolism by p l a n t t i s s u e c u l t u r e s are: c u l t u r e method, source of the t i s s u e , method of treatment and co n c e n t r a t i o n of x e n o b i o t i c , composition of medium and p h y s i o l o g i c a l age of t i s s u e . Although q u a n t i t a t i v e and perhaps q u a l i t a t i v e d i f f e r e n c e s may be found, it is concluded that x e n o b i o t i c metabolism by p l a n t t i s s u e c u l t u r e provides a u s e f u l approximation of metabolic pathways in i n t a c t p l a n t s .
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2. MUMMA AND HAMILTON
Plant Tissue and Cell Cultures
47. Chkanikov, D. I., Makeyev, Α. Μ., Pavlova, Ν. Ν., Grygoryeva, L. V., Dubovoi, V. P., Klimov, O. V., Arch. Environ. Contam. Toxicol. (1976) 5, 97. 48. Hamilton, R. Η., Hurter, J., Hall, J. K., Ercegovich, C. D., J. Agric. Food Chem. (1971) 19, 480. 49. Schneider, Ε. Α., Wightman, F., Ann. Rev. Plant Physiol. (1974) 25, 487. 50. Lee, T. T., Plant Physiol. (1971) 48, 56. 51. Lavee, S., Galston, A. W., Am. J. Bot. (1968) 55, 890. 52. Scandalios, J. G., Ann. Rev. Plant Physiol. (1974) 25, 225. 53. Shannon, L. Μ., Kay, E., Lew, J. Y., J. Biol. Chem. (1966) 241, 2166. 54. Reinert, J., Schraudolf, Η., Tazawa, Μ., Naturwiss (1957) 44, 588. 55. Lipetz, J., Galston 56. Witham, F. Η., Gentile 57. Hamilton, R. Η., Meyer, H. E., Burke, R. Ε., Feung, C.-S., Mumma, R. O., Plant Physiol. (1976) 58, 77. 58. Tuli, V., Moyed, H. S., Plant Physiol. (1967) 42, 425. 59. Feung, C.-S., Hamilton, R. Η., Mumma, R. O., J. Agric. Food Chem. (1975) 23, 1120. 60. Bandurski, R. S., Schulze, Α., Cohen, J. D., Biochem. Biophys. Res. Comm. (1977) 79, 1219. 61. Davies, P. J., Physiol. Plant (1972) 27, 262. 62. Patrick, J. W., Wolley, D. J., J. Exp. Bot. (1973) 24, 949. 63. Zenk, Μ. Η., In "Régulateurs Naturels de la Croissance Végétale," C.N.R.S. Paris, (1964) pp. 241-249. 64. Zenk, M. H., Nature (1961) 191, 493. 65. Kuhr, R. J., Casida, J. E., J. Agric. Food Chem. (1967) 15, 814. 66. Kohli, J . , Weisgerber, I., Klein, W., Pest. Biochem. Physiol. (1976) 6, 91. 67. Diaz-Colon, J. D., Bovey, R. W., Davis, F. S., Baur, J. R., Physiol. Plant (1972) 27, 60. 68. Bovey, R. W., Baur, J. R., Diaz-Colon, J. D., Weed Sci. (1974) 22, 191. 69. Zilkah, S., Gressel, J . , Plant Cell Physiol. (1977) 18, 815. 70. Davis, F. S., Villarreal, Α., Baur, J. R. Goldstein, I. S., Weed Sci. (1972) 20, 185. 71. Zilkah, S., Gressel, J., Plant Cell Physiol. (1977) 18, 641. 72. Barg, R., Umiel, Ν., Z. Pflanzenphysiol. (1977) 83, 437. 73. Zilkah, S., Bocion, P. F., Gressel, J . , Plant Cell Physiol. (1977) 18, 657. 74. Davis, D. G., Hoerauf, R. Α., Dusbabek, Κ. Ε., Dougall, D. Κ., Physiol. Plant (1977) 40, 15. 75. Davis, D. G., Wergin, W. P., Dusababek, Κ. E., Pest. Biochem. Physiol. (1978) 8, 84. 76. Feung, C.-S., Mumma, R. O., Hamilton, R. H., J. Agric. Food Chem. (1974) 22, 307.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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77.
Arjmand, Μ., Hamilton, R. H., Davidonis, G. Η., Mumma, R. O., J. Agric. Food Chem. (1979) (in press). Feung, C.-S., Hamilton, R. Η., Mumma, R. O., Plant Physiol. (1977) 59, 91. Misawa, Μ., In "Plant Tissue Culture and Its Bio-technologi cal Application," Barz, W., Reinhard, Ε., Zenk, Μ. Η., Eds., Springer-Verlag, Berlin, Heidelberg, New York (1977) pp. 17-26. Muhle, E., Hoesel, W., Barz, W., Phytochem. (Oxf.) (1976) 15, 1669. Heeger, V., Leienbach, K.-W., Barz, W., Hoppe-Seyler's Ζ. Physiol. Chem. (1976) 357, 1081. Leienbach, K.-W., Barz, W., Hoppe-Seyler's Ζ. Physiol. Chem. (1976) 357, 1069. Koop, W., Barz, W. Carew, D. P., Drueger Strek, W., In "Plant Tissue Culture Methods," Gamborg, O. L . , Wetter, L. R., Eds., Natl. Res. Council (Canada) Ottawa, Canada (1975), p. 83. Boulanger, D., Bailey, B. K., Steck, N., Phytochem. (1973) 12, 2399 Mumma, R. O., Hamilton, R. Η., In "Bound and Conjugated Pesticide Residues," Kaufman, D. D., Still, C. C., Paulson, G. D., Bandal, S. Κ., Eds., ACS Symposium Series No. 29, Am. Chem. Soc. (1976), pp. 68-85.
78. 79. 80. 81. 82. 83. 84. 85. 86. 87.
RECEIVED
December 20, 1978.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
3 Pesticide Metabolism in Higher Plants: In Vitro Enzyme Studies
1
G. L. LAMOUREUX and D. S. FREAR Metabolism and Radiation Research Laboratory, Agricultural Research, Science and Education Administration, U. S. Department of Agriculture, Fargo, ND 58105
The study of x e n o b i o t i p r i m a r i l y as a r e s u l t o pesticide protec plan crops from damage by weeds, i n s e c t s and other pests. Great stimulus f o r research i n t h i s f i e l d was experienced i n the e a r l y I960 s when increased concern was r a i s e d over the p o s s i b l e hazards of p e s t i c i d e residues to man and h i s environment. The complexity of x e n o b i o t i c metabolism s t u d i e s i s a continuum with i n v i v o s t u d i e s conducted i n the n a t u r a l environment at one end, and the use of i s o l a t e d enzymes to study molecular r e a c t i o n s at the other. Each technique i n t h i s continuum has i t s own p a r t i c u l a r l i m i t a t i o n s , but can be used to great advantage under the proper c o n d i t i o n s . In the previous two r e p o r t s , the use of c e l l c u l t u r e s , t i s s u e c u l t u r e s , i s o l a t e d c e l l s and i s o l a t e d p l a n t organs were considered. This report w i l l examine the use of i s o l a t e d p l a n t enzyme systems i n x e n o b i o t i c metabolism s t u d i e s . The primary t o p i c w i l l be a d i s c u s s i o n of the enzymes involved i n the four b a s i c metab o l i c r e a c t i o n s of x e n o b i o t i c s i n p l a n t s : o x i d a t i o n , r e d u c t i o n , h y d r o l y s i s and conjugation. The l i t e r a t u r e regarding key enzymes w i t h i n these c l a s s e s w i l l be reviewed. S p e c i f i c examples of i n v i t r o p l a n t enzyme systems used to study x e n o b i o t i c metabolism w i l l be presented. A d e t a i l e d d i s c u s s i o n of v a r i o u s techniques w i l l not be attempted. 1
OXIDATION REACTIONS: Oxidations are among the most important r e a c t i o n s i n the metabolism of p e s t i c i d e s because they are f r e q u e n t l y the primary 1 Mention of a trademark or p r o p r i e t a r y product does not cons t i t u t e a guarantee or warranty of the product by the U. S. Department of A g r i c u l t u r e and does not imply i t s approval to the e x c l u s i o n of other products that may a l s o be s u i t a b l e .
This chapter not subject to U.S. copyright. Published 1979 American Chemical Society
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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XENOBIOTIC METABOLISM
r e a c t i o n that r e s u l t s i n d e t o x i c a t i o n o r a c t i v a t i o n o f a p e s t i cide. In mammals and i n s e c t s , o x i d a t i o n r e a c t i o n s have been the subject of many i n v i t r o s t u d i e s . I t i s now evident that mixed f u n c t i o n oxidases are r e s p o n s i b l e f o r many o f the o x i d a t i o n r e a c t i o n s i n these organisms. Although p l a n t s have mixed f u n c t i o n oxidase systems, t h e i r r o l e i n p e s t i c i d e metabolism has not been widely demonstrated. I n a d d i t i o n to mixed f u n c t i o n oxidases, peroxidases, laçasses and polyphenol oxidases are found u n i v e r s a l l y i n the p l a n t kingdom and some o f these enzymes may a l s o p l a y a r o l e i n o x i d a t i v e p e s t i c i d e metabolism. At the present, our knowledge o f the enzyme systems i n v o l v e d i n many of the p e s t i c i d e o x i d a t i o n r e a c t i o n s that occur i n p l a n t s i s l i m i t e d and there i s great need f o r more research. Peroxidases : The p l a n t peroxidases c a t a l y z e two general types o f o x i d a t i o n r e a c t i o n s , the c l a s s i c a l p e r o x i d a t i v e r e a c t i o n that r e q u i r e s hydrogen peroxide and the o x i d a t i v e r e a c t i o n that u t i l i z e s molecular oxygen. The h o r s e r a d i s h peroxidase (HRP) p e r o x i d a t i v e r e a c t i o n normally proceeds by the f o l l o w i n g mechanism (1, 2) : HRP + H2O2 HRP-I HRP-I + AH2 + HRP-I I + AHHRP-II + AH 2 -* HRP + AH2 AH-> Products The o x i d a t i v e r e a c t i o n s are not as w e l l understood as the peroxidative reactions. I n Mn++ i n h i b i t e d r e a c t i o n s , the o x i d a t i v e h y d r o x y l a t i o n of s e v e r a l substrates can be c a t a l y z e d by peroxidase i n the presence o f dihydroxyfumaric a c i d (3). Other o x i d a t i v e r e a c t i o n s r e q u i r e an aromatic c o - f a c t o r such as 2,4d i c h l o r o p h e n o l and Mn++ (4·) . I n some o x i d a t i v e r e a c t i o n s , a c a t a l y t i c amount of hydrogen peroxide i s needed as an i n i t i a t o r , but oxygen i s used i n s t o i c h i o m e t r i c amounts ( 5 ) . Plant peroxidases c a t a l y z e the o x i d a t i o n o f a l a r g e and d i v e r s e c l a s s of endogenous and exogenous substrates such as phenols, aromatic amines, e n e d i o l s , ascorbate, ferrocyanide, cytochrome C., i n d o l e - 3 - a c e t i c a c i d , and the leuco form of many dyes. Phenols and aromatic amines are among the most commonly used substrates and the r e a c t i o n c a t a l y z e d i s g e n e r a l l y an o x i d a t i v e condensation o f the substrate (6). I n a d d i t i o n t o o x i d a t i v e condensations, decarboxylations, s u l f u r o x i d a t i o n s , Ndemethylations, r i n g h y d r o x y l a t i o n s , carbon-halogen bond élevages, and o x i d a t i o n o f aromatic methyl groups have a l l been a t t r i b u t e d to peroxidases (3) (Figure 1). Peroxidases are ubiquitous i n the p l a n t kingdom ( 3 ) . They occur throughout the p l a n t c e l l and have been found i n the cytoplasm, c e l l w a l l , membranes, n u c l e i , mitochondria and ribosomes (7). Peroxidase isozymes have been demonstrated i n h o r s e r a d i s h (4, 8-11) and s e v e r a l other species (4, 8-13). S t r i k i n g d i f f e r e n c e s have been
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
3.
LAMOUREUX
A N D FREAR
Plant
Enzyme
79
Studies
I)
OXIDATIVE CONDENSATIONS Of PHENOLS AND AROMATIC AMINES
2)
DECARBOXYLATION
3)
4)
oca
oca
SULFUR OXIDATIONS
N—DE METHYL AT IONS OH il ;-N-CH
t
Ο H il ι 0-C-N-CH
s
CHÎ^^CH,
CH^^CH,
CH^CHj 5)
CH^%
RING HYDROXYLATIONS COOH
6)
COOH
COOH
ô
û ~ - j ù r
OXIDATIONS OF AROMATIC METHYL GROUPS OH OH
OH
CH, 7Î
5
0
CH OH 2
CARBON —HALOGEN BOND CLEAVAGES F F~ + COMPLEX CONDENSATION PRODUCTS I NH
8)
2
IODIDE OXIDATION I"
Figure 1.
Types of reactions attributed to plant peroxidases ( 3 , 2 2 , 1 8 8 , 1 8 9 , 1 9 0 )
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
80
XENOBIOTIC METABOLISM
noted i n the s u b s t r a t e s p e c i f i c i t y of s e v e r a l HRP isozymes (14). In s p i t e of t h e i r widespread occurrence, the p h y s i o l o g i c a l r o l e of peroxidases i s u n c e r t a i n . T h e i r a b i l i t y to u t i l i z e i n d o l e - 3 - a c e t i c a c i d (15-18) and f l a v o n o i d s (19) as substrates suggests that peroxidases may be important i n the metabolic r e g u l a t i o n of these endogenous s u b s t r a t e s . Peroxidases are a l s o thought to be i n v o l v e d i n the l i g n i f i c a t i o n process (20). The h i g h c o n c e n t r a t i o n of peroxidase a c t i v i t y a s s o c i a t e d w i t h the c e l l w a l l (12) i s c o n s i s t e n t with t h i s theory. Several r e p o r t s have a s s o c i a t e d peroxidase a c t i v i t y w i t h p e s t i c i d e metabolism. The o x i d a t i o n of p a r a t h i o n to paraoxon and the h y d r o l y s i s of both p a r a t h i o n and paraoxon can be c a t a l y z e d by HRP (21) (Figure 2). The r e a c t i o n occurs under both o x i d a t i v e and p e r o x i d a t i v i s o l a t e d from bean hypocoty a c t i v e as HRP i n the o x i d a t i o n and h y d r o l y s i s of parathion (21). T h i s enzyme was equal to HRP i n the o x i d a t i o n of g u i c a c o l . The i n v i t r o r e a c t i o n of ten [l^C-carbonyl]carbamate i n s e c t i c i d e s w i t h hydrogen peroxide i n the presence of HRP has been examined (22). Conversion to e t h e r - s o l u b l e products that could be d i f f e r e n t i a t e d from the r e a c t a n t s by TLC or conversion to waters o l u b l e products were used as the c r i t e r i a f o r a r e a c t i o n . Of the ten substrates t e s t e d (banol, baygon, c a r b a r y l , HRS-1422, i s o l a n , mesurol, UC-10854, m a t a c i l and z e c t r a n ) , only m a t a c i l and z e c t r a n r e a c t e d . The highest percentage of r a d i o a c t i v i t y was i n the form of u n i d e n t i f i e d water-soluble products; however, s e v e r a l e t h e r - s o l u b l e products were i d e n t i f i e d (Figure 3). The e t h e r - s o l u b l e products represented v a r i o u s stages of N-dealkylation. T h i s may suggest a minor r o l e f o r peroxidases i n Nd e a l k y l a t i o n . Mesurol, a carbamate i n s e c t i c i d e that contains an a r y l m e t h y l s u l f i d e , and c y s t e i n e , t h i o g l y c o l i c a c i d , mercaptoethanol, t h i o u r e a and t h i o u r a c i l were not substrates f o r HRP (22, 23); however, chloropromazine, a h e t e r o c y c l i c s u l f i d e , was a s u b s t r a t e ( 3 ) . The p r e c i s e r o l e of peroxidases i n s u l f u r o x i d a t i o n i s thus u n c e r t a i n . A n i l i n e s are known degradation products of phenylcarbamates, phenylureas, and a c y l a n i l i d e h e r b i c i d e s ; t h e r e f o r e , the f a t e of a n i l i n e s i n the environment i s an important c o n s i d e r a t i o n . A number of workers have shown that v a r i o u s a n i l i n e s are converted to azobenzenes and other products by the a c t i o n of HRP and hydrogen peroxide (3). However, i t i s of p a r t i c u l a r note that the c h l o r o a n i l i n e s that formed azobenzenes i n v i t r o as the r e s u l t of HRP and hydrogen peroxide were the same as those that formed azobenzenes i n the s o i l as a r e s u l t of m i c r o b i a l a c t i o n (24) (Figure 4 ) . The HRP system served as a model to p r e d i c t the l i k e l i h o o d of azobenzene formation i n the s o i l . In these s t u d i e s , no e f f o r t was made to i d e n t i f y products other than the azobenzene analogs or to q u a n t i t a t e the r e a c t i o n ; t h e r e f o r e , the f u l l scope of products produced and the y i e l d s are not known.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
3.
LAMOUREUX AND FREAR
0-P(OC H ) 2
5
2
Plant Enzyme
BEAN HYPOCOTYL PEROXlDASE(lOuq) pH4.7 DIHYDROXY MALEIC ACID
81
Studies
0-P(OC H5) 2
2
+ HYDROLYSIS PRODUCTS 14%
36% HYDROLYSIS AND 10% OXIDATION OBTAINED WITH 50jjg HRP Figure 2.
Oxidation and
MATACIL Figure 3. Ν -Demethyhtion of substituted phenylcarbamate insecticides by horse radish peroxidase (22)
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
82
XENOBIOTIC METABOLISM
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
3.
L A M O U R E U X AND FREAR
Plant Enzyme
83
Studies
Although azobenzenes do not appear t o be metabolites of c h l o r o a n i l i n e s i n higher p l a n t s , c h l o r o a n i l i n e s may be i n c o r p o r ated i n t o l i g n i n or converted to l i g n i n - l i k e products (25). In an e f f o r t to determine the chemical nature of the c h l o r o a n i l i n e residue i n the l i g n i n f r a c t i o n , a model system was developed that uses HRP to c a t a l y z e the f r e e r a d i c a l formation of a s y n t h e t i c l i g n i n - l i k e m a t e r i a l (26). I n t h i s system, the ^ C - l a b e l e d p e s t i c i d e metabolite, 3 - c h l o r o a n i l i n e or 3 , 4 - d i c h l o r o a n i l i n e , was co-polymerized w i t h c o n i f e r y l a l c o h o l , a b u i l d i n g b l o c k of n a t u r a l l i g n i n (Figure 5). The p o l y m e r i z a t i o n was accomplished by simultaneously pumping s o l u t i o n s of (a) c o n i f e r y l a l c o h o l and HRP i n pH 7.2 b u f f e r , (b) hydrogen peroxide i n pH 7.2 b u f f e r , and (c) the l ^ C - l a b e l e d c h l o r o a n i l i n e i n pH 7.2 b u f f e r i n t o a b u f f e r e d s o l u t i o n of v a n i l l y l a l c o h o l d HRP Afte 10 h i the dark, the i n s o l u b l removed by c e n t r i f u g a t i o n chromatography. Based upon an average molecular weight of 1,000, 1.19 r e s i d u e s of 3 - c h l o r o a n i l i n e or 1.68 residues of 3,4-di c h l o r o a n i l i n e were incorporated per molecule of polymer formed. In v i v o i n c o r p o r a t i o n i n t o r i c e l i g n i n was a l s o greater f o r 3 , 4 - d i c h l o r o a n i l i n e than f o r 3 - c h l o r o a n i l i n e . When a c e t y l a t e d a n i l i n e s were used i n the model HRP system, the i n c o r p o r a t i o n r a t e was reduced by over 50%. T h i s may i n d i c a t e the involvement of the a n i l i n e n i t r o g e n i n the co-polymerization r e a c t i o n . 1
A number o f techniques were used f o r the a n a l y s i s of the synthetic l i g n i n . The most s u c c e s s f u l method was p y r o l y t i c GC/MS. P y r o l y s i s r e l e a s e d over 80% of the r a d i o a c t i v i t y i n a v o l a t i l e form. Over 60% o f the r e l e a s e d l^C was 3 - c h l o r o a n i l i n e or 3,4dichloroaniline. S i m i l a r r e s u l t s were obtained with n a t u r a l l i g n i n i s o l a t e d from c h l o r o a n i l i n e - t r e a t e d r i c e , but w i t h much lower y i e l d s . I t was concluded that a high percentage of the c h l o r o a n i l i n e i n the l i g n i n - l i k e m a t e r i a l was c o v a l e n t l y bonded between the a n i l i n e n i t r o g e n and the α-carbon of the polymer (Figure 5 ) . HRP was a l s o used to study the metabolism of b o t r a n (27). Botran was not a d i r e c t s u b s t r a t e f o r HRP, but i t s r e d u c t i o n product (2,6-dichlorophenylenediamine) was r e a d i l y converted to at l e a s t 8 products upon treatment with HRP (Figure 6). One of the most abundant products was i d e n t i c a l t o a s o i l metabolite of botran. High r e s o l u t i o n mass spectrometry of the s o i l metabolite i n d i c a t e d a molecular formula of Cl2H6N4Cl6- A botran metabolite, N-(4-amino-3,5-dichlorophenyl)malonamic a c i d , was found i n soy bean p l a n t s and i n soybean c a l l u s c u l t u r e s (28). The presumed intermediate of t h i s metabolite (2,6-dichlorophenylenediamine) was not detected; however, p l a n t s c o n t a i n a system that i s capable o f reducing a r y l n i t r o groups (29). The diamine of botran could thus be formed i n the p l a n t and serve as a s u b s t r a t e f o r a malonyl t r a n s f e r r e a c t i o n , a peroxidase-catalyzed o x i d a t i o n or l i g n i n i n c o r p o r a t i o n .
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
84
XENOBIOTIC M E T A B O L I S M
RAT LIVER MICROSOMES, SOIL BACTERIA* PLANTS
AT LEAST 8 PRODUCTS, INCLUDING C H N CI
HRP/HOg 10 MIN 23°C pH 5.1 2
|2
6
4
4
BOTRAN Figure 6.
Table I.
A possible role for peroxidases in botran metabolism (27,28, 29)
R e l a t i o n s h i p between r e s i s t a n c e capable o f o x i d i z i n g iodide.*
t o i o d i d e and absence o f p e r o x i d a s e
Peroxidase A c t i v i t y
Species
G u a i a c o l donor (units/mg protein)
I o d i d e donor (units/mg protein)
Iodide accumulation (mg/g d r y weight)
Resistance
tomato
2.6
0
70
short
buttercup
2.0
9
13
resistant
cabbage
6.7
26
4
resistant
bean
12.7
28
15
susceptible
mettle
15.3
75
5
susceptible
pea
82
123
8
susceptible
*Adapted from ( 3 2 ) .
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
term
3.
LAMOUREUX A N D FREAR
Plant Enzyme
Studies
85
The f i n d i n g that i o x y n i l l i b e r a t e d i o d i d e when exposed to u l t r a v i o l e t l i g h t or when subjected t o p l a n t or animal metabolism r e s u l t e d i n an examination of the h e r b i c i d a l p r o p e r t i e s of i o d i d e . Subsequently, i o d i d e was shown t o possess some s e l e c t i v i t y as a h e r b i c i d e (30). The a b i l i t y of p l a n t peroxidases to o x i d i z e i o d i d e to i o d i n e (31) v a r i e d g r e a t l y w i t h the p l a n t s p e c i e s (32) and t h i s a b i l i t y was r e l a t e d t o r e s i s t a n c e t o i o d i d e t o x i c i t y (Table I ) . Iodine was shown to be 10^ times more powerful than i o d i d e as an i n h i b i t o r of the H i l l r e a c t i o n (33). T h i s suggested that i o d i d e t o x i c i t y was due, i n p a r t , to i n t r a c e l l u l a r o x i d a t i o n of i o d i d e to i o d i n e . When 30 iodo-benzoic a c i d s were assayed f o r h e r b i c i d a l a c t i v i t y , only those that l i b e r a t e d i o d i d e i n v i v o were t o x i c to Phaseolus v u l g a r i s seedlings. I t was suggested that l i b e r a t i o f i o d i d might b one f a c t o r i n the h e r b i c i d a In a d d i t i o n to i n v i t r o s t u d i e s that suggested a a c t i v e r o l e f o r peroxidases i n p e s t i c i d e metabolism, s e v e r a l s t u d i e s have i n d i c a t e d that c e r t a i n p e s t i c i d e s may i n c r e a s e the l e v e l of peroxidases i n p l a n t s . EPTC was r e p o r t e d to i n c r e a s e peroxidase a c t i v i t y and l i g n i f i c a t i o n i n corn s e e d l i n g s ; these i n c r e a s e s could be a l l e v i a t e d by treatment with a p r o t e c t a n t , N , N - d i a l l y l 2,2-dichloroacetamide (9). S t i m u l a t i o n of peroxidase a c t i v i t y was a l s o observed when Phaseolus r a d i a t u s s e e d l i n g s were t r e a t e d with sodium d i e t h y l d i t h i o c a r b a m a t e (34). A microsomal hydroperoxide-dependent o x i d i z i n g system (hydroperoxidase) was r e c e n t l y i s o l a t e d from pea seeds (35, 36) (Figure 7 ) . T h i s enzyme hydroxylated i n d o l e , phenol, α-naphthol, and a n i l i n e to i n d o x y l , hydroquinone, α-napthylhydroquinone, and N^hydroxyaniline, r e s p e c t i v e l y . Hydrogen peroxide, t e r t - b u t y l hydroperoxide, cumene hydroperoxide, or l i n o l e i c a c i d hydro peroxide served as sources of o x i d i z i n g power. A w e l l - d e f i n e d pH optimum a t pH 7.2 was observed w i t h l i n o l e i c a c i d hydroper oxide, but a broad pH optimum around 8.7 was observed with the other hydroperoxides. An a l l o s t e r i c e f f e c t was noted w i t h l i n o l e i c a c i d hydroperoxide, but not w i t h the other hydroper oxides. I t was speculated that the n a t u r a l hydroperoxides f o r t h i s system were produced by the a c t i o n of lipoxygenases on s u b s t r a t e s such as l i n o l e i c a c i d . Studies with 0^-8-labeled l i n o l e i c a c i d hydroperoxide i n d i c a t e d a d i r e c t oxygen t r a n s f e r from the hydroperoxide to the hydroxylated s u b s t r a t e . No p a r t i c i p a t i o n of molecular oxygen was observed. Pre-treatment w i t h j>-chloromercuriobenzoate r e s u l t e d i n a s l i g h t promotion of the hydroperoxidase r e a c t i o n , i n d i c a t i n g no involvement of P450. Other p r o p e r t i e s of t h i s system a l s o i n d i c a t e d that a c t i v i t y was d i s t i n c t from that of P450. Although none of the i n v i t r o peroxidase s t u d i e s d i s c u s s e d proves that these enzymes p l a y an important r o l e i n p e s t i c i d e metabolism i n p l a n t s , i t i s c l e a r that the spectrum of s u b s t r a t e s u t i l i z e d by these enzymes does encompass many p e s t i c i d e s or p e s t i c i d e m e t a b o l i t e s . The apparent u n i v e r s a l occurrence of
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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Figure 7.
H y droper oxide-dependent enzyme system from pea (35, 36)
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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L A M O U R E U X AND FREAR
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Studies
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peroxidases i n the p l a n t kingdom and throughout the c e l l c o n t r i b u t e s g r e a t l y to the p o s s i b l e involvement of these enzymes i n p e s t i c i d e metabolism. A number of p o s s i b l e r o l e s f o r p e r o x i dases i n the metabolism of s p e c i f i c p e s t i c i d e s or c l a s s e s of p e s t i c i d e s have been pointed out. The u t i l i t y of HRP i n model systems to produce metabolites found i n the s o i l and to a i d i n the study of the p e s t i c i d e residues found i n the p l a n t l i g n i n f r a c t i o n have been c l e a r l y demonstrated. Mixed Function
Oxidases:
Extensive research over the l a s t 28 years has shown that mixed f u n c t i o n oxidases are of great importance i n x e n o b i o t i c metabolism i n i n s e c t s and animal (37) Th f mixed f u n c t i o n oxidases i n highe l a s t 10 years by experiment endogenou substrates (38-42) and x e n o b i o t i c s (43-48). Based upon l i g h t r e v e r s i b l e CO i n h i b i t i o n (39, 44) and s p e c t r a l p r o p e r t i e s (49), s e v e r a l mixed f u n c t i o n oxidase (mfo) systems i n p l a n t s appear to resemble the cytochrome P450 mfo systems found i n animals and insects. Other p l a n t mfo systems, however, appear to be q u i t e d i f f e r e n t (38, 40). Based upon our knowledge of i n s e c t and mammalian mfo systems and our knowledge of metabolites produced i n p l a n t s , a number of r e a c t i o n s i n p l a n t s should be considered as p o s s i b l e mixed f u n c t i o n oxidase-catalyzed r e a c t i o n s . Some of these are N-dea l k y l a t i o n , C)-dealkylation, aromatic h y d r o x y l a t i o n , a l k y l o x i d a t i o n , epoxidation, d e s u l f u r a t i o n , s u l f u r o x i d a t i o n , e s t e r h y d r o l y s i s , and n i t r o g e n o x i d a t i o n (Figure 8). The demonstrated mfo r e a c t i o n s i n p l a n t s are more l i m i t e d . There are s e v e r a l examples of mfo-catalyzed N - d e a l k y l a t i o n s i n p l a n t s . A mfo system from cotton that c a t a l y z e s the N-demethylation of phenylurea h e r b i c i d e s (47, 48, 50) and mfo systems from c a s t o r beans (51) and avocado pear (46) that c a t a l y z e the N-demethylation of _rj-chloro-N-methylaniline have been reported. The O-demethylation of £-nitroanisole has been detected with an ±n v i t r o system from avocado pear (46). A s i m i l a r mfo-catalyzed d e a l k y l a t i o n might e x p l a i n the presence of the phenol of 2,4-D that has been reported as a metabolite i n c e r t a i n p l a n t systems. The formation of the phenol of 2,4-D does not seem to i n v o l v e an i n i t i a l d e c a r b o x y l a t i o n (52). The reported i n v i v o a l k y l o x i d a t i o n of the p l a n t growth r e g u l a t o r f l u r e n o l b u t y l e s t e r might a l s o be explained by a mfo (53). Several mixed f u n c t i o n oxidase systems that c a t a l y z e aromatic r i n g h y d r o x y l a t i o n s have been s t u d i e d jln v i t r o w i t h both x e n o b i o t i c (44-46) and endogenous substrates (38-41, 45, 54-57). Because of the l a r g e number of p o t e n t i a l s u b s t r a t e s , t h i s r e a c t i o n i s of p a r t i c u l a r i n t e r e s t . The epoxidation a l d r i n has been demonstrated by jLn v i t r o systems i s o l a t e d from pea and bean (43, 58-62). The a l d r i n epoxidase systems have not been c l e a r l y
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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L A M O U R E U X AND FREAR
Plant Enzyme
Studies
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demonstrated to be mfo systems. The conversion of organophosphorothioates to phosphates i n p l a n t s has been w e l l e s t a b l i s h e d . T h i s conversion i n v o l v e s mixed f u n c t i o n oxidases i n animals and i n s e c t s (63). Mixed f u n c t i o n oxidases may a l s o c a t a l y z e t h i s r e a c t i o n i n p l a n t s , but the p o s s i b i l i t y that other enzyme systems may be i n v o l v e d should be considered (21). The o x i d a t i o n of a number of t h i o - e t h e r s to s u l f o x i d e s and sulfones has been reported i n p l a n t metabolism s t u d i e s ; again, the enzymes r e s p o n s i b l e f o r these r e a c t i o n s have not been w e l l c h a r a c t e r i z e d . An enzyme that c a t a l y z e s the s u l f o x i d a t i o n of phorate has been i s o l a t e d from soybean root (64). A d d i t i o n a l s t u d i e s are needed to c h a r a c t e r i z e t h i s system. In animals, organophosphate e s t e r h y d r o l y s i s can be c a t a l y z e d by both mixed f u n c t i o n oxidases and g l u t a t h i o n e S-transferase (63) plants t h i r e a c t i o b c a t a l y z e d by esterases o r e a c t i o n mechanism need y In p l a n t s , N-oxide formation does not appear to be a common r e a c t i o n i n p e s t i c i d e metabolism. However, an enzyme that c a t a l y z e s the formation of the N-oxide of morphine has been detected i n Papaver somniferum l a t e x (67). Since peroxidases and polyphenol oxidases are found i n high concentrations i n p l a n t l a t e x , the p o s s i b i l i t y that t h i s r e a c t i o n may be c a t a l y z e d by one of these enzymes should a l s o be considered. The N-demethylase from cotton (47) and the cinnamic a c i d hydroxylase from cucumber (44) are two mfo systems that have been shown to u t i l i z e p e s t i c i d e s as s u b s t r a t e s . The N-demethylase system appears to have l i m i t e d s u b s t r a t e s p e c i f i c i t y , only N-methyl s u b s t i t u t e d phenylureas were found to be a c t i v e substrates. The N-demethylation r e a c t i o n was s t r o n g l y and competi t i v e l y i n h i b i t e d by carbamate i n s e c t i c i d e s that had high e l e c t r o n d e n s i t y at the p o s i t i o n ortho to the carbamate group (50, 68). I n h i b i t i o n by s u b s t i t u t e d phenylureas was a l s o demonstrated. T h i s i n h i b i t i o n was dependent upon the presence of a proton on the a n i l i n e n i t r o g e n atom (50). F i e l d observations (69, 70) and an i n v i t r o study (71) i n d i c a t e d an a n t a g o n i s t i c i n t e r a c t i o n of carbamates w i t h dimethylphenylurea h e r b i c i d e s . A p o s i t i v e c o r r e l a t i o n between r e s i s t a n c e to phenylurea h e r b i c i d e s and the presence of an a c t i v e N-demethylase system was supported by i n v i t r o determinations of N-demethylase a c t i v i t y i n 12 p l a n t s p e c i e s . In c o t t o n , the s p e c i f i c a c t i v i t y of the mfo system v a r i e d over a 1 0 - f o l d range, depending upon the age of the p l a n t and the s p e c i f i c t i s s u e . A recent study has shown that 2,4-D i s hydroxylated by an i n v i t r o mfo system from cucumber leaves (44). T h i s appears to be the f i r s t reported r i n g - h y d r o x y l a t i o n of a p e s t i c i d e c a t a l y z e d by an i s o l a t e d p l a n t mfo system. Cinnamic a c i d was a l s o hydroxylated by t h i s system. Hydroxylase a c t i v i t y was increased 2- to 3 - f o l d by spraying the cucumber leaves with 2,4-D b e f o r e enzyme i s o l a t i o n . Other s t u d i e s have shown that c e r t a i n p l a n t mfo systems were induced or stimulated by l i g h t
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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(39, 72). The NIH s h i f t was observed i n the h y d r o x y l a t i o n of 2,4-D. The primary product was 4-hydroxy-2,5-dichlorophenoxya c e t i c a c i d . The NIH s h i f t was a l s o demonstrated i n the 4h y d r o x y l a t i o n of cinnamic a c i d by a mfo system from pea (73). These s t u d i e s suggest that a d d i t i o n a l i n v e s t i g a t i o n s on the i n d u c t i o n of p l a n t mfo systems may be warranted. They a l s o suggest the need to conduct k i n e t i c s t u d i e s on the cinnamic a c i d 4-hydroxylase r e a c t i o n w i t h 2,4-D and r e l a t e d p e s t i c i d e s as possible inhibitors. Since microsomal mixed f u n c t i o n oxidases p l a y an important r o l e i n the metabolic pathway of some endogenous substrates (39, 41), the i n d u c t i o n or i n h i b i t i o n of these r e a c t i o n s by p e s t i c i d e s could have important i m p l i c a t i o n s . In animals, the microsomal f r a c t i o n has t y p i c a l l y d i s p l a y e d a broad s p e c i f i c i t y rang presence of a s e r i e s o mixed f u n c t i o n oxidases from p l a n t s appear to have a f a i r l y narrow s u b s t r a t e s p e c i f i c i t y range with some t i s s u e s p e c i f i c i t y . The mfo system from c a s t o r bean (51) d i s p l a y e d 4-hydroxylase a c t i v i t y f o r cinnamic a c i d and N-demethylase a c t i v i t y f o r j>chloro-N-methylaniline, N-methylaniline, and Ν,N-dimethylaniline, but d i s p l a y e d no a c t i v i t y f o r 15 other compounds that contained N-, 0-, or S-methyl groups. The system from avocado pear may have one of the broadest s p e c i f i c i t y ranges of the i s o l a t e d p l a n t systems. I t apparently has hydroxylase a c t i v i t y f o r b i p h e n y l and a n i l i n e , 0-demethylase a c t i v i t y f o r j > - n i t r o a n i s o l e , and N-demethylase a c t i v i t y f o r p_-chloro-N-methylaniline (46). Mixed f u n c t i o n oxidase systems i s o l a t e d from p l a n t s t y p i c a l l y d i s p l a y very low l e v e l s of a c t i v i t y . Specific a c t i v i t i e s are f r e q u e n t l y i n the range of 1- to 10-nmol product/mg protein/hr. T h i s low l e v e l of a c t i v i t y can be explained by the r e p o r t that P450 concentrations i n p l a n t s range from 0.007 to 0.02 nmol P450/mg microsomal p r o t e i n as compared to 0.9 to 1.2 nmol P450/mg microsomal p r o t e i n i n r a t l i v e r (49). Because of the low l e v e l s of mfo a c t i v i t y i n p l a n t t i s s u e s , the use of r a d i o a c t i v e s u b s t r a t e s i n assays i s o f t e n necessary. The p r o p e r t i e s of i s o l a t e d p l a n t mfo systems are h i g h l y v a r i a b l e (Table I I ) . Some mfo systems are microsomal, undergo l i g h t r e v e r s i b l e C O - i n h i b i t i o n , r e q u i r e NADPH as a c o - f a c t o r and behave much l i k e mammalian P450 mfo systems (39). Others r e q u i r e i l l u m i n a t e d c h l o r o p l a s t s as a c o - f a c t o r and may be s o l u b l e (38). Some systems r e q u i r e only one c o - f a c t o r (47), but other systems r e q u i r e s e v e r a l (40). A s c o r b i c a c i d , NADH, NADPH, d i t h i o n i t e , i l l u m i n a t e d c h l o r o p l a s t s , and an u n i d e n t i f i e d n a t u r a l product (probably a p t e r i d i n e ) have a l l been u t i l i z e d i n v a r i o u s i n v i t r o p l a n t mfo s t u d i e s (38, 39, 40, 47). The response of these systems to v a r i o u s i n h i b i t o r s i s a l s o q u i t e v a r i a b l e . Mercaptoethanol was an i n h i b i t o r of phenylalanine hydroxylase (40) and g i b b e r e l l i n Αχ hydroxylase (41), but i t was necessary to demonstrate 4-hydroxylase a c t i v i t y w i t h cinnamic a c i d (39). C h e l a t i n g agents i n h i b i t e d an N-demethylase (47) and a
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. 44
CO-light
weak w i t h CO, SKF 525A, CU++, NADH.
mercaptoethanol
s a f r o l e and 3,4-benzopyrene a r e s t i m u l a t o r y t o 2hydroxylation.
39
51
CO-light reversible, azide.
CO l i p a s e s , menadione, 1,4-nap thoquinone, riboflavin.
mercaptoethanol
EDTA
illuminated chloro p l a s t s , or d i t h i o nite
NADPH o r NADH, and an u n i d e n t i f i e d p r o d u c t o r THFA
NADPH, a s c o r b a t e Fe-H-
NADPH
NADPH ( o r NADH w i t h 2nd substrate)
pyroloxygenase
phenylalanine hydroxylase
g i b b e r e l l i n Αχ hydroxylase
cinnamic a c i d hydroxylase
cinnamic a c i d , h y d r o x y l a s e and N-demethylase
ascorbate
NADPH
b i p h e n y l hydroxylase (microsomal)
40
41
aminopteridine, s u l f h y d r y l c o n t a i n i n g compounds; i . e . , mercaptoethanol. mercaptoethanol,
EDTA
38
c h e l a t i n g agents, d i t h i o t h r e i t o l and mercapto ethanol.
45,46
NADPH
2,4-D h y d r o x y l a s e and c i n n a m i c a c i d hydroxylase
reversible.
43
e l e c t r o n a c c e p t o r s , CN, phenols, a n i l i n e .
c h e l a t i n g agents and detergents
NADPH ( a c t i v i t y p r e s e n t a t reduced l e v e l without)
aldrin
epoxidase
47
Reference
CO, i o n i c d e t e r g e n t s s u l f h y d r y l reagents, c h e l a t i n g agents and electron acceptors.
Inhibitors
NaCN, i s o a s c o r b a t e , p o l y c l a r AT
A c t i v a t o r s o r f a c t o r s used t o demonstrate a c t i v i t y
NADH o r NADPH
Co-factors
P r o p e r t i e s o f p l a n t enzymes w i t h mfo t y p e a c t i v i t y .
N-demethylase
Enzyme
Table I I .
SU
«s:
co
92
XENOBIOTIC METABOLISM
pyroloxygenase (38), but s l i g h t l y stimulated an a l d r i n epoxidase (43). S a f r o l e and 3,4-benzopyrene stimulated a b i p h e n y l 2hydroxylase from avocado, but other mfo a c t i v i t i e s were u n a f f e c t ed (46). Castor bean N-demethylase was s t r o n g l y i n h i b i t e d by K p i b u f f e r and was assayed w i t h t r i c i n e b u f f e r (51). In sharp c o n t r a s t , N-demethylase from cotton was i n h i b i t e d by t r i c i n e , but not by K p i (47). Based on d i f f e r e n c e s i n c o - f a c t o r requirements, responses to d i f f e r e n t i n h i b i t o r s and responses to d i f f e r e n t b u f f e r s , i t would appear that a v a r i e t y of d i f f e r e n t p l a n t mfo systems e x i s t . Although most mfo systems i n p l a n t s are a s s o c i a t e d w i t h p a r t i c u l a t e (microsomal) f r a c t i o n s , a g i b b e r e l l i n Ai hydroxylase (41) and a b i p h e n y l hydroxylase (45, 46) are s o l u b l e systems. The nature of s e v e r a l othe The s o l u b i l i z a t i o n of Swed T r i t o n X-100 has been reported (75) and both s o l u b l e and p a r t i c u l a t e a l d r i n epoxidase (60, 61) and b i p h e n y l hydroxylase (45, 46) have been i s o l a t e d from the same t i s s u e s . I t appears that i s o l a t e d a c t i v e f r a c t i o n s may not always be a true i n d i c a t i o n of the nature and l o c a t i o n of the enzyme i n the n a t i v e s t a t e , but may a l s o be a f u n c t i o n of the combination of techniques and t i s s u e used. A number of d i f f e r e n t procedures and s p e c i a l precautions were used to i s o l a t e mfo a c t i v i t y from p l a n t sources (Table I I I ) . In the i s o l a t i o n of N-demethylase a c t i v i t y from c o t t o n , the t i s s u e was ground under l i q u i d n i t r o g e n (47, 48) and i n the i s o l a t i o n of a l d r i n epoxidase from pea (43), t o t a l l y anaerobic c o n d i t i o n s were used. Most e x t r a c t i o n and i s o l a t i o n procedures u t i l i z e d pH 7.5 b u f f e r s that contained e i t h e r p o l y v i n y l p y r r o l i d i n e or p o l y c l a r AT. The b u f f e r used to e x t r a c t N-demethylase a c t i v i t y from c o t t o n contained i s o a s c o r b a t e , p o l y c l a r AT and sodium cyanide. Mercaptoethanol, c h e l a t i n g agents, sucrose, and bovine serum albumin have a l s o been used w i t h v a r y i n g frequency to help p r o t e c t mfo systems during i s o l a t i o n . Differential centrifugation has been the most commonly used f r a c t i o n a t i o n method, but DEAE chromatography, sephadex chromatography, ammonium s u l f a t e f r a c t i o n a t i o n and d e n s i t y gradient c e n t r i f u g a t i o n have a l s o been employed. Based on t h i s b r i e f survey, i t i s apparent that the number of x e n o b i o t i c s shown to be metabolized by i n v i t r o p l a n t mixed f u n c t i o n oxidases i s very l i m i t e d . Some of these o x i d a t i v e systems have not been w e l l d e f i n e d . Considerable e f f o r t i s needed to i s o l a t e and c h a r a c t e r i z e these systems. The methods used f o r enzyme i s o l a t i o n have been h i g h l y v a r i a b l e . Because of the extremely low l e v e l s of enzyme a c t i v i t y a s s o c i a t e d w i t h many of the mfo systems, they are f r e q u e n t l y d i f f i c u l t to study and s p e c i a l methods are o f t e n needed to measure r e a c t i o n r a t e s . This i s f u r t h e r complicated by the presence of endogenous i n h i b i t o r s and the i n s t a b i l i t y of many of these systems.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. diff
diff
75
76
EDTA, sucrose, mercaptoethanol
mercaptoethanol, EDTA, serum albumin, p o l y c l a r AT, mannitol.
homogenize i n pH 7.5 b u f f e r , f i l t e r , d i f f e r e n t i a l centrifug.
homogenize i n pH 7.5 b u f f e r , f i l t e r , d i f f e r e n t i a l c e n t r i f u g . and d e n s i t y gradient c e n t r i f u g .
aged Swede r o o t
wounded Jerusalem a r t i c h o k e tuber
cinnamic a c i d hydroxylase (microsomal)
cinnamic a c i d hydroxylase (microsomal)
*Also i s o l a t e d from other sources.
51
EDTA, sucrose
p u l v e r i z e i n pH 7.5 b u f f e r , f i l t e r , d i f f e r e n t i a l c e n t r i f u g . or d e n s i t y gradient c e n t r i f u g .
c a s t o r bean
41
cinnamic a c i d hydroxylase (microsomal)
PVP
sucrose,
homogenize decoated imbibed seeds (pH 6.5), d i f f e r e n t i a l c e n t r i f u g .
snap bean
40
38
45,46
43
47
g i b b e r e l l i n Αχ hydroxylase
none apparent
remove high inhibitor
m.w.
isolation, AT
none apparent
PVP
anaerobic polyclar
AT,
Reference
p u l v e r i z e f r o z e n t i s s u e , watere x t r a c t , f i l t e r , acetone f r a c t i o n a t i o n , DEAE-cellulose and calcium phosphate g e l a d s o r p t i o n .
water-extract, (NH4)2SO4 f r a c t i o n a t i o n , DEAE and Sephadex G-100
e x t r a c t (pH 7.4), f i l t e r , erential centrifug.
e x t r a c t (pH 7.5), f i l t e r , erential centrifug.
e x t r a c t (pH 7.5), f i l t e r , Sephadex G-100 and d i f f e r e n t i a l c e n t r i f u g .
NaCN, p o l y c l a r isoascorbate
S p e c i a l Precautions
spinach leaves
*wheat germ
avocado pear
*cucumber leayes
pea root
Procedure
p u l v e r i z e d i n l i q u i d N2» e x t r a c t (pH 7.5), f i l t e r , d i f f e r e n t i a l and d e n s i t y g r a d i e n t c e n t r i f u g .
General I s o l a t i o n
phenylalanine hydroxylase
pyroloxygenase
b i p h e n y l hydroxy l a s e (microsomal and s o l u b l e )
2,4-D and cinnamic A. hydroxylase (microsomal)
a l d r i n epoxidase (microsomal)
* c o t t o n leaves
T i s s u e Source
Methods used to I s o l a t e mixed f u n c t i o n o x i d a s e - l i k e a c t i v i t y from higher p l a n t s .
N-demethylase (microsomal)
Enzyme
Table I I I .
94
XENOBIOTIC METABOLISM
REDUCTION REACTIONS: Although r e d u c t i v e r e a c t i o n s have not been demonstrated t o p l a y a major r o l e i n x e n o b i o t i c metabolism i n higher p l a n t s , s e v e r a l i n v e s t i g a t o r s have reported the r e d u c t i o n of aromatic n i t r o groups i n p l a n t s (77-82) and c a l l u s c u l t u r e (82). A r e d u c t i v e dehalogenation (83) and the r e d u c t i o n o f a s u l f o x i d e to a s u l f i d e (84) have a l s o been reported. The reported a r y l n i t r o reductions of pentachloronitrobenzene (PCNB) (77) and f l u o r o d i f e n (j>-nitrophenyl-a,a,a-trifluoro-2-nitrophenyl-£-tolyl ether) (7£, 80, 85, 86) were i n competition w i t h g l u t a t h i o n e conjugation. In studies with f l u o r o d i f e n , a r y l n i t r o reduction was a minor metabolic pathway. However, i n s t u d i e s w i t h pentachloronitrobenzene, about 28% of the p e s t i c i d e was converted to pentachloroaniline. The only r e d u c t i v g pesticide appear to have been studied i n d e t a i l i n p l a n t s a r e those c a t a l y z e d by a r y l n i t r o r e d u c t a s e s . A r y l nitroreductase a c t i v i t y has been i s o l a t e d from peanut (77) and pea (78). The s o l u b l e a r y l n i t r o r e d u c t a s e system from peanut was i s o l a t e d i n c o n j u n c t i o n with s t u d i e s on GSH S-transferase a c t i v i t y . Magnesium c h l o r i d e was used i n the i s o l a t i o n procedure to p r e c i p i t a t e the microsomal f r a c t i o n (87) and to avoid h i g h speed c e n t r i f u g a t i o n . With PCNB as the s u b s t r a t e , reductase a c t i v i t y was detected w i t h enzyme p r e p a r a t i o n s from r o o t s and hypocotyls o f 7-day-old e t i o l a t e d peanut seedings. A r y l n i t r o r e d u c t a s e a c t i v i t y was detected only when the r e a c t i o n was run under a n i t r o g e n atmosphere i n the presence of both FAD and NADPH. The a r y l n i t r o r e d u c t a s e i s o l a t e d from soybean s e e d l i n g r o o t s c a t a l y z e d the r e d u c t i o n of dinoben ( 2 , 5 - d i c h l o r o - 3 - n i t r o b e n z o i c acid) when the system was incubated under n i t r o g e n a t pH 8.2 w i t h e i t h e r NADPH or NADH (78). When t h i s enzyme system was p u r i f i e d f u r t h e r , FAD or FMN was r e q u i r e d i n a d d i t i o n t o NADH or NADPH. The f a c t that i n v i t r o r e d u c t i o n could be demonstrated only under a n i t r o g e n atmosphere suggests that these r e a c t i o n s may become more important i n v i v o under c o n d i t i o n s o f low oxygen t e n s i o n . In many cases i n which r e d u c t i o n r e a c t i o n s have been reported, p l a n t s have been grown and t r e a t e d i n such a manner that m i c r o b i a l a c t i o n could have accounted f o r the r e a c t i o n . This p o s s i b i l i t y should be considered i n any study i n which r e d u c t i v e r e a c t i o n s a r e reported i n x e n o b i o t i c metabolism i n p l a n t s . The r e d u c t i o n of the a r y l n i t r o groups of PCNB and dinoben with i n v i t r o enzyme systems i s o l a t e d from p l a n t s and the apparent r e d u c t i o n of the a r y l n i t r o group of 2 , 6 - d i c h l o r o - 4 - n i t r o a n i l i n e i n soybean c a l l u s c u l t u r e suggests, however, that a r y l n i t r o reductions may r e s u l t from p l a n t metabolism. HYDROLYTIC REACTIONS: A r y l Acylamidases:
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
3.
LAMOUREUX AND FREAR
Plant Enzyme
Studies
95
A r y l acylamidases that hydrolyze the h e r b i c i d e p r o p a n i l ( 3 , 4 - d i c h l o r o p r o p i o n a n i l i d e ) have been i s o l a t e d from b a c t e r i a (88), f u n g i (89), b i r d s (90), mammals (91, 92) and p l a n t s (93110) (Figure 9). Of the h y d r o l y t i c enzymes that p l a y important r o l e s i n the metabolism of p e s t i c i d e s i n p l a n t s , the a r y l a c y l amidases appear to be the most thoroughly s t u d i e d . Studies w i t h homogenates of r i c e p l a n t s (101, 105, 106) e s t a b l i s h e d the enzymatic nature of p r o p a n i l h y d r o l y s i s . H y d r o l y t i c a c t i v i t y was a s s o c i a t e d w i t h a p a r t i c u l a t e f r a c t i o n (93, 94). The enzyme was s t a b l e , r e q u i r e d no c o - f a c t o r s , and had a pH optimum of 7.5-7.9. A comparison of the enzyme a c t i v i t y i n r i c e and barnyard grass showed a 60-fold higher l e v e l of enzyme a c t i v i t y i n r i c e (94). T h i s suggested that p r o p a n i l r e s i s t a n c e was based on the presence of the a r y l acylamidas the h e r b i c i d e before s e n s i t i v a c t i v i t y i n r i c e s e e d l i n g s was a f u n c t i o o the developmenta stage of the p l a n t and maximum a c t i v i t y was reached at the f o u r t h l e a f stage. In f i e l d s t u d i e s , however, seedlings i n the t h i r d and f o u r t h l e a f stages were more s e n s i t i v e to p r o p a n i l than were younger seedlings (98). A r y l acylamidase a c t i v i t y was a l s o reported i n r i c e r o o t c a l l u s suspension c u l t u r e s (98), but i n v i t r o a c t i v i t y was demonstrated only i n c u l t u r e s that were at l e a s t 120 hr o l d . T h i s d i d not correspond w i t h the much e a r l i e r appearance of 3 , 4 - d i c h l o r o a n i l i n e i n p r o p a n i l - t r e a t e d c u l t u r e s . f
f
I n h i b i t o r s t u d i e s showed that carbamate i n s e c t i c i d e s were powerful competitive i n h i b i t o r s of the a r y l acylamidase from r i c e (94). The phosphorothioate i n s e c t i c i d e s , p a r a t h i o n and sumithion were much weaker i n h i b i t o r s of the enzyme, but t h e i r o x i d i z e d analogs, paraoxon and sumioxon, were r e s p e c t i v e l y 100X and 200X more e f f e c t i v e as i n h i b i t o r s (95). Phosphorothioate i n s e c t i c i d e s are p a r t i a l l y converted to t h e i r oxygen analogs i n p l a n t s (103). The s t r e n g t h of paraoxon as an i n h i b i t o r of the h y d r o l y s i s r e a c t i o n i n v i t r o was c o r r e l a t e d w i t h i t s s y n e r g i s t i c e f f e c t i n reducing the f r e s h weight of p r o p a n i l - t r e a t e d r i c e seedlings (95). The s y n e r g i s t i c e f f e c t between p r o p a n i l and the organophosphorthioate and carbamate i n s e c t i c i d e s (101, 107) can thus be explained on the b a s i s of competitive i n h i b i t i o n of the a r y l acylamidase enzyme necessary f o r the d e t o x i c a t i o n of propanil. A p a r t i c u l a t e a r y l acylamidase enzyme has a l s o been i s o l a t e d from red r i c e (108) and s o l u b l e a r y l acylamidases have been i s o l a t e d from t u l i p (99) and dandelion (109). Substrate s p e c i f i c i t i e s of these enzymes are shown i n Tables IV and V. Significant and p o t e n t i a l l y u s e f u l d i f f e r e n c e s e x i s t i n the s u b s t r a t e preferences of these enzymes f o r d i f f e r e n t halogenated propionanilides. 2, 3 D i c h l o r o p r o p i o n a n i l i d e was the p r e f e r r e d s u b s t r a t e f o r the amidase from c u l t i v a t e d r i c e , but i t was the poorest s u b s t r a t e f o r red r i c e . In c o n t r a s t , p r o p a n i l was the best s u b s t r a t e f o r red r i c e , but had a r e l a t i v e r a t e of metabolism of only 42% i n c u l t i v a t e d r i c e . Substrate preferences f o r f
L
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
96
XENOBIOTIC METABOLISM
*
,
0
CI - ^ J V N - C - C H
Cl'
ARYL ACYLAMIDASE 2
-CH
»»
3
PRORANIL
Figure 9.
. . C.,vO/~ Cl'
mz
Hydrolysis of 3' ,4'-dichloropropionanilide amidase
"**
M
0
0
~
C
C
H
2~
C
H
3
(propanil) by an aryl acyl-
d i f f e r e n t a l k y l s i d e chains were s i m i l a r f o r these enzymes, each had a marked preference f o r the p r o p i o n i c s i d e c h a i n . These a r y l acylamidases were not capable o f h y d r o l y z i n g carbamate o r urea herbicides. Some d i f f e r e n c e s were found i n the response o f these enzymes to i n h i b i t o r s (Table V I ) . These d i f f e r e n c e s between the a r y l acylamidases suggest that s u b s t r a t e s p e c i f i c i t y t e s t s might be used t o develop more s e l e c t i v e compounds o r compounds with more d e s i r a b l e b i o l o g i c a l s t a b i l i t y . Differences i n response to s u l f h y d r y l i n h i b i t o r s suggests that i n h i b i t o r s t u d i e s w i t h i s o l a t e d enzyme systems could provide an e f f e c t i v e means f o r the development of s e l e c t i v e p e s t i c i d e s y n e r g i s t s .
Table IV.
Substrate s p e c i f i c i t y of a r y l acylamidase from red r i c e (108), r i c e (94), t u l i p (99) and dandelion (109): the e f f e c t of c h l o r i n e r i n g s u b s t i t u t i o n . R e l a t i v e A c t i v i t y (%)
Substrate
1
1
1
1
2 ,3 -dichloropropionanilide 2 ,4 -dichloropropionanilide 2 -chloropropionanilide 3 -chloropropionanilide 3 ^'-dichloropropionanilide 3 ,5 -dichloropropionanilide 2 ,5 -dichloropropionanilide 4 -chloropropionanilide 2 ,6 -dichloropropionanilide propionanilide 1
1
1
1
1
1
1
1
1
1
Red Rice
29 47 29 73 100 33 27 38
—
58
Rice
Tulip
Dandelion
100 84 60 42 42 30 27 21 1
41 100 66 27 100
14 49 37 51 100
— —
— —
—
—
—
99 2
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
54 0
L A M O U R E U X AND FREAR
3.
Table V.
Fiant Enzyme
97
Studies
Substrate s p e c i f i c i t y of a r y l acylamidases from red r i c e (108), r i c e (94), t u l i p (99) and dandelion (109): the e f f e c t of v a r i o u s 3, 4 d i c h l o r o a n i l i d e a l k y l analogs. f
L
Relative A c t i v i t y Substrate 1
1
3 ,4 -dichloroacetanilide 3 ,4'-dichloropropionanilide 3 ,4 -dichlorobutyranilide 3 ,4 -dichlorovaleranilide 3 ,4 -dichloro-2-methyl propionanilide 3 ,4 -dichloro-2-methy acrylanilide 3 ,4 -dichloro-3-methyl butyranilide f
f
T
1
1
f
1
1
1
1
1
Red
Rice
82 100 18 40
— —
(%)
Rice
Tulip
Dandelion
59 100 32 39 2
49 100 18 3
49 100 37 12
—
—
—
—
0
T h i r t y - e i g h t d i f f e r e n t h o r t i c u l t u r a l and agronomic crop species r e p r e s e n t i n g 10 d i f f e r e n t p l a n t f a m i l i e s were assayed f o r a r y l acylamidase a c t i v i t y with p r o p a n i l as the substrate (96). Enzyme a c t i v i t y was reported i n over h a l f of the s p e c i e s . Only one f a m i l y , leguminosae, was devoid of a c t i v i t y . A s i m i l a r study was conducted with 19 genera of weeds. Enzyme a c t i v i t y was assayed w i t h p r o p a n i l , 1,l-dimethyl-3-phenylurea (fenuron), and i s o p r o p y l c a r b a n i l a t e (propham) (97). P r o p a n i l was hydrolyzed at widely v a r y i n g r a t e s by approximately 70% of the s p e c i e s . Fenuron and propham, however, were hydrolyzed by the enzyme p r e p a r a t i o n from only one s p e c i e s , w i l d cucumber. The d i s t r i b u t i o n of a r y l acylamidases i n other members of the p l a n t kingdom has a l s o been reported (100, 110). Extensive s t u d i e s w i t h a r y l acylamidases have shown that these enzymes are widely d i s t r i b u t e d i n the p l a n t kingdom. A c t i v i t y v a r i e s widely w i t h i n d i f f e r e n t p l a n t t i s s u e s and among d i f f e r e n t plant species. Resistance to p r o p a n i l i s dependent upon the presence of these enzymes. D e t a i l e d substrate s p e c i f i c i t y s t u d i e s w i t h four a r y l acylamidases revealed s u b t l e d i f f e r e n c e s i n s u b s t r a t e s p e c i f i c i t y and response to i n h i b i t o r s . I n h i b i t o r s t u d i e s with carbamate and organophosphate i n s e c t i c i d e s have c l e a r l y shown that these compounds are strong competitive i n h i b i t o r s of the a r y l acylamidases from r i c e . Interactions observed i n the f i e l d between p r o p a n i l and these i n s e c t i c i d e s can be a t t r i b u t e d to the i n h i b i t i o n of a r y l acylamidases.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. 29
— —
0.2mM
0.2mM
0.2mM
— —
iodoacetate
2,4-dichlorophenoxyacetic
pyrocatecol
3
6
35
**Catechol used instead of pyrocatechol.
FeCl
*CuCl2 used instead of CUSO4
2
0.2mM
N-ethylmaleimide
C0CI2
6
0.2mM
j>-benzoquinone
4
0.2mM
j>-chloromercuribenzoate
60
0.2mM
2
— — —
CUSO4
HgCl
Na2As02
— — —
Red Rice Cone. % Inhib.
74
O.lmM
—
— — — — —
18
44
47
50
0.5mM
0.5mM
0.5mM
0.5mM
59
74
l.OmM
0.2mM
74
Rice % Inhib.
1 0
0.50mM
— —
2
0.50mM
— —
0.50mM
9
— —
— — 0.50mM
16
16 6
l.OmM
73 l.OmM
0.5mM**
—
2
l.OmM
—
23
50
61
l.OmM
0.50mM
0.25mM
86
89
0.5mM
100
0.25mM
0.25mM 0.5mM*
—
—
—
—
5
0.25mM
27
Dandelion Cone. % Inhib
0.25mM
Tulip Cone. % Inhib.
Source of Enzyme
(108), r i c e (94), t u l i p (99) and dandelion (109).
0.5mM
Cone.
I n h i b i t i o n of a r y l acylamidases from red r i c e
_o-iodosobenzoate
Inhibitor
Table VI.
3.
L A M O U R E U X A N D FREAR
Plant Enzyme
Studies
99
Esterases: Many c a r b o x y l i c a c i d e s t e r p e s t i c i d e s are r e a d i l y hydrolyzed to f r e e a c i d s i n v i t r o . In most cases, the f r e e a c i d form of the p e s t i c i d e i s presumed or known to be the a c t i v e agent. Esterases that hydrolyze c a r b o x y l i c a c i d e s t e r p e s t i c i d e s are important, not only because they p l a y a r o l e i n the degradation of the p e s t i c i d e , but a l s o because they may be i n v o l v e d i n a c t i v a t i o n , s e l e c t i v i t y or d e t o x i c a t i o n . Some esterases may be i n d u c i b l e (111). Common i n h i b i t o r s of these enzymes i n c l u d e a number of the organophosphate and carbamate i n s e c t i c i d e s (112-114). The p o s s i b i l i t y of p e s t i c i d e i n t e r a c t i o n between c a r b o x y l i c a c i d e s t e r h e r b i c i d e s and c e r t a i n i n s e c t i c i d e s e x i s t s . Research on the i n d u c t i o n , a c t i v a t i o n , i n h i b i t i o n , and s u b s t r a t e s p e c i f i c i t y of esterases should hav improvement and b e t t e r us Esterases are probably ubiquitous and have been i s o l a t e d from many p l a n t s p e c i e s . The s t a b i l i t y of p l a n t esterase pre p a r a t i o n s v a r i e s with the source and may be r e l a t e d to the presence of phenol oxidases and polyphenols. Gel e l e c t r o p h o r e s i s has been a v a l u a b l e t o o l i n studying p l a n t e s t e r a s e s and has shown that these enzymes are a complex f a m i l y of isozymes with d i f f e r e n c e s i n s u b s t r a t e preference and s u s c e p t i b i l i t y to i n h i b i t o r s (115-118). P o l y a c r y l a m i d e - g e l e l e c t r o p h o r e s i s separated 7 esterases from pea and 14 esterases from green bean (114). Separated isozymes responded d i f f e r e n t l y to the assay s u b s t r a t e s , a-naphtylacetate, α-naphthylbutyrate, and a-naphthylpropionate. D i f f e r e n t i a l response to i n h i b i t o r s such as p a r a t h i o n , paraoxon and d i i s o p r o p y l p h o s p h o r o f l u o r i d a t e was a l s o observed. Gel e l e c t r o p h o r e s i s of enzyme p r e p a r a t i o n s from cabbage i n d i c a t e d the presence of 6 esterases (113). With 2naphthylacetate as the s u b s t r a t e , i n h i b i t i o n was demonstrated w i t h carbofuran, e s e r i n e and s e v e r a l other compounds. The p a r t i a l l y p u r i f i e d e s t e r a s e from cabbage had a pH optimum of approximately 7, and an estimated molecular weight of 69,000. T h i s enzyme l o s t 50% of the o r i g i n a l a c t i v i t y when i t was s t o r e d f o r 22 days a t -22°. A few s t u d i e s have reported the h y d r o l y s i s of p e s t i c i d e s by i n v i t r o esterase systems (Figure 10). Recent s t u d i e s showed that apple and cucumber leaves c o n t a i n esterases that hydrolyze f u n g i c i d a l n i t r o p h e n y l e s t e r s (115). Esterase a c t i v i t y was detected w i t h 18 d i f f e r e n t s u b s t r a t e s . Hydrolysis rates varied 100-fold, depending upon the s u b s t r a t e . Selective i n h i b i t i o n with paraoxon and s e v e r a l other compounds was a l s o demonstrated. E s e r i n e and EDTA were not i n h i b i t o r y . D i f f e r e n c e s i n isozyme patterns were observed between the two s p e c i e s . The c a r b o x y l i c a c i d e s t e r h e r b i c i d e chlorfenprop-methyl (methyl 2-chloro-3-(4-chlorophenyl)propionate) i s hydrolyzed i n v i v o and i n v i t r o to an a c t i v e h e r b i c i d e (119). A c t i v e esterases were i s o l a t e d from 2 c u l t i v a r s of oat, w i l d oat, wheat and beet
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
100
XENOBIOTIC M E T A B O L I S M
0
O-c'-R
NOo
OH
(FUNGICIDE)
NOo
(ACTIVE)
Ο OH NO,
N0
O
v.
(FUNGICIDE)
(ACTIVE)
Ο CH -ÇH-C-OCH I Cl CHLORFENPROP-METHYL (HERBICIDE) 2
CH.-CH-COOH I Cl (ACTIVE)
3
Figure 10. Hydrolysis of fungicides and herbicides by plant esterases (115, 119)
S (CH 0) P 3
(CH OÎ P-OH
2
3
2
S
+ CH3O-P-OH + CH3O-P-OH
S i
ÇH
S I
CH
2
I C-N-CH3 ' 1
S S I CH I -OH II 0 2
2
I _ _ II H 0 2
C
H
0
N
z
C
H
C
DIMETHOATE
S 0 Il II (CH 0) P-S-CH-C-OC H 3
2
2
5
CH^-C-OC^H. II 0 2
2
S II (CH 0) P-OH 3
2
S + (CH 0) P-SH 3
2
5
MALATHION Figure 11.
Hydrolysis of organophosphorus insecticides by plant esterases (127, 128)
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
3.
L A M O U R E U X AND FREAR
Vlant Enzyme
Studies
101
(119). Estimates of i n v i v o r a t e s of h y d r o l y s i s based upon i n v i t r o data i n d i c a t e d that h e r b i c i d e s e l e c t i v i t y was not r e l a t e d to esterase a c t i v i t y . Two r e l a t e d h e r b i c i d e s , flamprop-isopropyl [(±)-N-benzoyl-N-(3-chloro-4-fluorophenyl)alanine i s o p r o p y l e s t e r ] and benzoylprop-ethyl [(±)-N-benzoyl-N-(3,4-dichloropheny1) a l a n i n e e t h y l e s t e r J are a l s o hydrolyzed to a c t i v e h e r b i c i d e s , presumably by p l a n t esterases (120, 121). Very low l e v e l s of a c t i v i t y have been reported f o r the i n v i t r o h y d r o l y s i s of b i f e n o x [methyl 5-(2,4-dichlorophenoxy)-2-nitrobenzoate] i n homogenates of v e l v e t g r a s s (122). These i n v i t r o and i n v i v o s t u d i e s have shown that p l a n t esterases can c a t a l y z e the h y d r o l y s i s of a number of p e s t i c i d e s . These s t u d i e s do not, however, answer important questions regarding the l o c a l i z a t i o d i s t r i b u t i o n of the enzyme the case of h e r b i c i d e s , y play importan in selectivity. A number of organophosphorous compounds are metabolized i n higher p l a n t s to products that are c o n s i s t e n t w i t h the i n v o l v e ment of h y d r o l y t i c enzymes (123-129). In v i v o and i n v i t r o s t u d i e s showed that wheat and sorghum g r a i n s r a p i d l y degrade dimethoate [(),O-dimethyl-S-(N-methylcarbamoylmethyl)phosphorot h i o l o t h i o n a t e ] to a number of products i n c l u d i n g j),0-dimethylphosphoro thionate, mono-C^-me thy 1 JS-N-me t h y l - c a r b amo y lme t hy l p ho s p h o r o t h i o l o t h i o n a t e and mono-O-methyl-S-carboxymethyl-phosphorot h i o l o t h i o n a t e (127) (Figure 11). The apparent h y d r o l y s i s of malathion (J3,0-dimethyl-S-bis(carboethoxy)ethyl phosphorodit h i o a t e ) to dimethylphosphorothionate and dimethylphosphorothiolt h i o n a t e with crude e x t r a c t s from wheat germ was a l s o demonstrated (128). Unfortunately, these s t u d i e s do not e s t a b l i s h the s i g n i f i c a n c e of esterases or phosphohydrolyases i n the h y d r o l y s i s of organophosphorous p e s t i c i d e s . In animals and i n s e c t s , g l u t a t h i o n e S-transferases and mixed f u n c t i o n oxidases p l a y important r o l e s i n the metabolism of organophosphorus compounds (126). These enzymes may form some of the same products produced by e s t e r a s e s . Unless experiments are conducted p r o p e r l y , i t may not be p o s s i b l e to d i s c e r n whether GSH S - t r a n s f e r a s e , esterase or mixed f u n c t i o n o x i d a s e - r e l a t e d r e a c t i o n s are r e s p o n s i b l e f o r e s t e r hydrolysis. In a d d i t i o n , p l a n t peroxidases may a l s o c a t a l y z e the cleavage of organophosphorous compounds (21). In v i v o and jLn v i t r o s t u d i e s have shown that chloramben (3amino-2-5-dichlorobenzoate) i s r a p i d l y metabolized to a s t a b l e N-glucoside i n r e s i s t a n t p l a n t species (130). In a d d i t i o n to the N-glucoside, s u s c e p t i b l e species a l s o form the chloramben glucose e s t e r (130, 131). The glucose e s t e r was not s t a b l e i n v i v o and appeared to a c t as a chloramben r e s e r v o i r (Figure 12). Crude homogenates from cucumber and b a r l e y t i s s u e s hydrolyzed the chloramben glucose e s t e r q u i t e r a p i d l y . Since other benzoic a c i d or phenoxy h e r b i c i d e s may form glucose e s t e r conjugates, these h y d r o l y t i c enzymes could p l a y an important r o l e i n r e g u l a t i n g
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
102
XENOBIOTIC METABOLISM
CHLORAMBEN (ACTIVE)
TRANSIENT PRODUCT Figure 12.
Chloramben conjugation and hydrolysis (ISO, 131)
α — CHLOROACETAMIDES (2 EXAMPLES) Figure 13.
OIPHENYL ETHERS (I EXAMPLE)
4—HYDROXYANILINOS (2 EXAMPLES)
Agricultural chemicals known to be enzymatically conjugated to GSH in phnts
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
3.
LAMOUREUX A N D FREAR
Phnt
Enzyme
103
Studies
the l e v e l s of'these h e r b i c i d e s i n v i v o . CONJUGATION: Glutathione
S-Transferase:
In animals, g l u t a t h i o n e (GSH) conjugation i s an important r e a c t i o n i n the metabolism o f a wide range o f x e n o b i o t i c s and has been the subject of a number o f reviews (132-136). HN-CH2-C00H
HN-CH -C00H 2
C=0 R-X
+
I CHo-CH
I
C=0 GS
S-transferas
| ^
I
I I
SH HN-C=0
COOH
S
CH2-CH -CH
R
2
NH GSH
COOH
I I I
I I Substrate
HN-C=0
CH -CH -CH 2
2
NH
2
2
GSH Conjugate
Recent f i n d i n g s (137-146) have e s t a b l i s h e d that GSH conjugation i s a l s o important i n p l a n t s . Although the range of x e n o b i o t i c compounds that have been demonstrated to undergo GSH conjugation i n p l a n t s i s l i m i t e d (Figure 13), i t can be assumed that GSH conjugation o f a d d i t i o n a l c l a s s e s of compounds w i l l be shown as more s t u d i e s a r e conducted. Glutathione conjugation should be considered as a p o s s i b l e metabolic r e a c t i o n with any x e n o b i o t i c that possesses an e l e c t r o p h i l i c center and an a p p r o p r i a t e l e a v i n g group, o r with x e n o b i o t i c s that can be a c t i v a t e d by o x i d a t i o n or by some other means to a f f o r d an a c t i v e s u b s t r a t e . Glutathione conjugation i s of p a r t i c u l a r importance i n p l a n t s (a) because of the wide range of p o t e n t i a l s u b s t r a t e s , (b) because i t may determine the nature of the t e r m i n a l r e s i d u e s i n the p l a n t , and (c) because i t may be a major f a c t o r i n p e s t i c i d e d e t o x i c a t i o n or h e r b i c i d e s e l e c t i v i t y . Glutathione S^-transferases have been found i n 21 p l a n t s p e c i e s (Table VII) and appear to be widespread i n the p l a n t kingdom. G l u t a t h i o n e S-transferases from animals (133) and p l a n t s (140, 142, 143, 145, 146) appear to be s o l u b l e enzymes. G l u t a t h i o n e ^ - t r a n s f e r a s e a c t i v i t y may not be detected i n crude enzyme p r e p a r a t i o n s u n t i l a f t e r g e l chromatography (140) or may have l i m i t e d s t a b i l i t y when p a r t i a l l y p u r i f i e d (142).
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
104
XENOBIOTIC METABOLISM
Table V I I .
Species which have been examined i n v i t r o f o r GSH a c t i v i t y and c y s t e i n e S - t r a n s f e r a s e a c t i v i t y .
S-transferase
Substrate Used to Assay f o r A c t i v i t y
Plant
Atrazine (143)
barley
N. D
corn
H
Fluorodifen (142)
PCNB (146)
EPTC (144)
4-hydroxy chlorpropham
C191)
cotton crabgrass foxtail lambsquarter johnsongrass
H
oat
Ν. D.
okra pea peanut pigweed soybean sorghum sudangrass sugarcane squash tomato wheat cucumber rice
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
3.
LAMOUREUX A N D FREAR
Plant Enzyme
Studies
105
The f i r s t demonstrated GSH conjugation of a p e s t i c i d e i n p l a n t s was reported i n sorghum (137). A t r a z i n e (2-chloro-4ethylamino-6-isopropylamino-j3-triazine) was i s o l a t e d as a GSH conjugate. The conjugation r e a c t i o n was enzymatic (143) and a c t i v e enzyme systems were subsequently i s o l a t e d from a t r a z i n e r e s i s t a n t corn, sorghum, johnsongrass, sudangrass and sugarcane. A t r a z i n e - s u s c e p t i b l e species (pea, oats, wheat, b a r l e y and pigweed) contained no d e t e c t a b l e enzyme a c t i v i t y . The f i n d i n g that GSH S - t r a n s f e r a s e a c t i v i t y was e a s i l y i s o l a t e d from r e s i s t a n t s p e c i e s , but not from s u s c e p t i b l e s p e c i e s , i n d i c a t e d that r e s i s t a n c e to a t r a z i n e was based on the presence of t h i s enzyme. Subsequent s t u d i e s w i t h an inbred corn l i n e showed that GSH JS-transferase a c t i v i t y was present i n high concentrations i n the r e s i s t a n t l i n e , bu c o r r e l a t e d to the productio l a c k of photosynthetic i n h i b i t i o n i n the r e s i s t a n t l i n e (147) (Table V I I I ) . I t was f u r t h e r shown that a number of r e s i s t a n t corn l i n e s contained h i g h concentrations of GSH S - t r a n s f e r a s e activity. These s t u d i e s confirmed the importance of GSH St r a n s f e r a s e a c t i v i t y i n the s e l e c t i v i t y of a t r a z i n e . In the t o l e r a n t s p e c i e s , corn and sorghum, GSH JS-transferase a c t i v i t y was concentrated i n the f o l i a r t i s s u e . Partially p u r i f i e d enzyme p r e p a r a t i o n s from corn leaves were s t a b l e and could be s t o r e d w i t h l i t t l e l o s s i n a c t i v i t y . Unfortunately, attempts to f u r t h e r p u r i f y the enzyme were not s u c c e s s f u l (148). Substrate s p e c i f i c i t y s t u d i e s with s e v e r a l 2 - c h l o r o - s — t r i a z i n e , 2-methoxy-s-triazine, and 2-methylmercapto-_s-1 r i a z ine h e r b i c i d e s showed that the 2 - c h l o r o - s - t r i a z i n e s were the only e f f e c t i v e s u b s t r a t e s . A l a t e r study showed that the s u l f o x i d e of a 2methylmercapto-j5-triazine was a good s u b s t r a t e f o r GSH conjugation (149). The i s o l a t e d corn enzyme was s p e c i f i c f o r GSH. D i t h i o t h r e i t o l , mercaptoethanol, 2,3-dimercaptopropanol or Lc y s t e i n e d i d not f u n c t i o n as s u l f h y d r y l s u b s t r a t e s . R e s u l t s from s u b s t r a t e s p e c i f i c i t y s t u d i e s w i t h the corn enzyme were comparable to those obtained w i t h e x c i s e d sorghum leaves (Table I X ) . Although i n v i t r o s t u d i e s i n d i c a t e d that 2-chloro-4amino-6-isopropyl-amino-s-triazine was a poor s u b s t r a t e f o r GSH jS-transferase i n corn, i n v i v o s t u d i e s with sorghum (150) showed that t h i s s u b s t r a t e was conjugated with GSH, T h i s d i f f e r e n c e may be a t t r i b u t e d to d i f f e r e n c e s i n the s u b s t r a t e s p e c i f i c i t y of the GSH ^ - t r a n s f e r a s e s from sorghum and corn. The GSH jS-transferase mediated cleavage of f l u o r o d i f e n (p_n i t r o p h e n y l α,α,α-trifluoro-2-nitro-p-tolyl ether) to S - ( 4 - t r i f l u o r o - 2 - n i t r o p h e n y l ) g l u t a t h i o n e and 4-nitrophenol appears to be the f i r s t demonstrated cleavage of a diphenylether by a GSH t r a n s f e r a s e system (142). When j v - ( 4 - t r i f l u o r o m e t h y l - 2 - n i t r o p h e n y l ) g l u t a t h i o n e was f i r s t detected i n v i v o , the nature of t h i s metabolite was not understood. A crude enzyme that produced the same product i n the presence of GSH was i s o l a t e d from pea epicotyl tissue. Subsequent l a r g e - s c a l e enzyme i n c u b a t i o n s
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
106
XENOBIOTIC METABOLISM
Table VIII.
Relationship between GSH-transferase a c t i v i t y , formation of GSH conjugates, and i n h i b i t i o n of photosynthesis i n atrazine resistant and susceptible corn.
GSH transferase* activity in vitro
% GSH conjugate i n leaf disc after 5 hours
% Inhibition of photosynthesis i n leaf discs after 2 hours
Corn l i n e
Response to atrazine
GT112RfRf
resistant
1.63
43.1
8.6
GT112
susceptible
0.03
0.4
64.9
6 other resistant
resistant
2.62 ±
1.0
*nmoles/mg protein/hour. **adapted from (147).
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
3.
LAMOUREUX AND FREAR
Plant Enzyme
107
Studies
produced S - ( 4 - t r i f l u o r o m e t h y l - 2 - n i t r o p h e n y l ) g l u t a t h i o n e i n good y i e l d and g r e a t l y f a c i l i t a t e d the development of a n a l y t i c a l techniques that were used i n the i s o l a t i o n and i d e n t i f i c a t i o n of the i n v i v o metabolite (141, 142).
Table IX.
Comparison of the r e l a t i v e s u b s t r a t e s p e c i f i c i t y of excised sorghum leaves (138, 150) w i t h the s u b s t r a t e s p e c i f i c i t y of the g l u t a t h i o n e S-transferase from corn (143).
Substrate
atrazine GS-13529 cyprazine propazine simazine 2-chloro-4-amino-6i s ο ρ ropylamino-s_triazine 2-hydroxy-4-ethylamino 6-isopropylamino-striazine
Relative activity in vitro (corn)
1.00 0.52 0.42 0.05 0.01
Relative a c t i v i t y i n vivo (sorghum)
0.93 0.92 0.90 0.62 active*
0.01
* G l u t a t h i o n e - r e l a t e d conjugates were the primary products produced, but experimental c o n d i t i o n s were d i f f e r e n t from those used w i t h the other j>-triazines. The i s o l a t e d GSH jS-transferase from pea was s t a b l e , but crude enzyme preparations from corn, peanut and c o t t o n underwent i r r e v e r s i b l e i n h i b i t i o n when s t o r e d f o r s e v e r a l hours a t 4 ° . Enzyme a c t i v i t y was detected i n higher concentrations i n r e s i s t a n t species ( c o t t o n , corn, peanut, pea, soybean and okra) than i n s u s c e p t i b l e s p e c i e s (cucumber, tomato and squash). F l u o r o d i f e n s e l e c t i v i t y appeared to be based on enzyme d i s t r i b u t i o n and c o n c e n t r a t i o n . A broad s u b s t r a t e s p e c i f i c i t y study was not conducted w i t h t h i s enzyme, but two other diphenylether h e r b i c i d e s were t e s t e d and found to be i n a c t i v e . I t was hypo t h e s i z e d that only diphenylethers that were h i g h l y a c t i v a t e d a t the C - l p o s i t i o n would a c t as s u b s t r a t e s . Mercaptoethanol, 2,3-dimercaptopropanol, d i t h i o t h r e i t o l and c y s t e i n e would not f u n c t i o n as the s u l f h d r y l s u b s t r a t e with the enzyme from pea. Recent s t u d i e s have shown that the f u n g i c i d e PCNB (pentachloronitrobenzene) i s a l s o converted to GSH conjugates w i t h an enzyme system i s o l a t e d from pea (145). A d d i t i o n a l s t u d i e s w i l l be needed to determine i f the same enzyme i s i n v o l v e d i n the metabolism of both PCNB and f l u o r o d i f e n . A unique f e a t u r e of the
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC M E T A B O L I S M
108
PCNB GSH S - t r a n s f e r a s e assay system was the i n c l u s i o n of t e r t ^ butanol to i n c r e a s e PCNB s o l u b i l i t y and enzymatic a c t i v i t y (Figure 14). The use of t e r t - b u t a n o l i n enzyme systems has been p r e v i o u s l y reported (151). T h i s system has not been completely s t u d i e d w i t h r e s p e c t to the PCNB-GSH conjugation r e a c t i o n . D e t a i l e d i n h i b i t o r s t u d i e s were conducted w i t h the GSH S~ t r a n s f e r a s e s i s o l a t e d from both corn and pea (Table X ) . Both enzymes gave s i m i l a r responses to s u l f h y d r y l compounds and to the c l a s s i c a l i n h i b i t o r s of mammalian GSH S - t r a n s f e r a s e a c t i v i t y , sulfobromophthalein and 1,2-dichloronitrobenzene. I n h i b i t i o n by sulfobromophthalein was competitive i n mammalian systems and a l s o appeared to be competitive i n both p l a n t systems, Propachlor ( 2 - c h l o r o - N - i s o p r o p y l a c e t a n i l i d e ) and barban (4-chloro~2-butynyl*m-chlorocarbanilate) were i n h i b i t o r s of the enzymatic r e a c t i o n s with a t r a z i n e and f l u o r o d i f e n was a l s o observed. The competitive i n h i b i t o r of both enzymes suggested some commonality of the a c t i v e s i t e ( s ) . On the other hand, the f a c t that a t r a z i n e was n e i t h e r a s u b s t r a t e nor an i n h i b i t o r of the pea enzyme suggested important d i f f e r e n c e s . I n h i b i t i o n of the pea enzyme by other diphenylether compounds, phenylureas and acetamide h e r b i c i d e s suggested the p o s s i b i l i t y of p e s t i c i d e i n t e r a c t i o n s w i t h these compounds. A number of s - t r i a z i n e s were t e s t e d as i n h i b i t o r s of the corn enzyme. These s t u d i e s suggested that the bis(alkylamino)-methoxy-s-1riaz ines and the methylmercapto analogs were probably competitive i n h i b i t o r s capable of b i n d i n g at the a c t i v e s i t e , but i n c a p a b l e of undergoing r e a c t i o n , Hydrox y t r i a z i n e s and the d e a l k y l a t e d t r i a z i n e s were not e f f e c t i v e inhibitors. I t i s of p a r t i c u l a r i n t e r e s t to note that a known s y n e r g i s t o f a t r a z i n e , 2 , 3 , 6 - t r i c h l o r o p h e n y l a c e t i c a c i d , was an i n h i b i t o r of the GSH S - t r a n s f e r a s e from corn. EPTC ( S - e t h y l dipropylthiocarbamate) does not appear to be a GSH S - t r a n s f e r a s e s u b s t r a t e . However, a f t e r o x i d a t i o n to the s u l f o x i d e , i t r e a d i l y undergoes conjugation i n the presence of GSH S - t r a n s f e r a s e s i s o l a t e d from r a t l i v e r (152) or corn r o o t (144). 0 tt
0 0 f C2H5-S-C-NCC3H7)2 M
C H5-S-C-N(C3H )2 2
7
EPTC
EPTC S u l f o x i d e
0 It GSH-transferase EPTC S u l f o x i d e
;-cS-Carbamyl^-GSH
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
3.
LAMOUREUX A N D FREAR
Plant Enzyme
109
Studies
14 -,
12 -I
ίο 1
φ ε
6 J
4 J
2Η
ImM GSH 9.2 μΜ PCNB 120 mM Pi 0.32 mg enzyme
TO
Τ 15
—ι 20
% (v/v) tert-butanol in system Figure 14.
Effect of tert-butanol on GSH conjugation of PCNB catalyzed by an enzyme system isolated from pea (192)
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. 72 0
29 29 34 41 54 —
86 0 3 —
—
28 60 88 79 55 57 68
1 1 0.1 0.06
0.1 0.1 0.1 0.1 1 0.03 0,1 0.1 1.0
2,3-dimercaptopropanol
cysteine
atrazine
2,4-bis(isopropylamino-6-methylmercapto-s-triazine*
2,3,6-trichlorophenyl acetic acid
propachlor
barban
sulfobromophthalein
1,2-dichloro-4-nitrobenzene
nitrofen*
diuron*
propanil
l-chloro-3-tosylamldo-7-amino-L-2 2-heptane«HCl* ( a l k y l a t i n g agents)
*Related compounds were t e s t e d w i t h s i m i l a r r e s u l t s .
58
11
6
1
S-methyl g l u t a t h i o n e
61
N.A.
40
14
1
dithiothreitol
Inhibitor
% I n h i b i t i o n of GSH-transferase (corn)
% I n h i b i t i o n of GSH-transferase (pea)
I n h i b i t o r s t u d i e s w i t h the g l u t a t h i o n e S-transferases i s o l a t e d from corn (143) and pea (142). Inhibitor concentration (mM)
Table X.
3.
L A M O U R E U X AND FREAR
Phnt
Enzyme
Studies
111
T h i s i s comparable to a previous observation that a methylm e r c a p t o - s - t r i a z i n e would not undergo conjugation u n t i l a f t e r o x i d a t i o n t o the s u l f o x i d e (149). Recent s t u d i e s have a l s o shown that chlorpropham ( i s o p r o p y l m-chlorocarbanilate) i s not a s u b s t r a t e f o r GSH S-transferase a c t i v i t y i n oat, but i t s o x i d a t i o n product, 4-hydroxychlorpropham, r e a d i l y undergoes an enzymatic r e a c t i o n w i t h c y s t e i n e or GSH (139, 140). These observations i l l u s t r a t e the need to consider the p o s s i b l e r o l e of a c t i v a t i n g r e a c t i o n s i n GSH conjugation. When corn i s t r e a t e d w i t h Ν,N-diallyl-2,2-dichloroacetamide (R-25788), h e r b i c i d a l i n j u r y due to EPTC i s g r e a t l y reduced (153). Glutathione J3-transferase a c t i v i t y and the c o n c e n t r a t i o n o f GSH were increased 2- to 3 - f o l d by treatment w i t h 0.3 to 30 ppm of R-25788. I t was concluded that decreased h e r b i c i d a l i n j u r y was due to an increased r a t elevated l e v e l s of GSH EPTC-susceptible o a t s e e d l i n g s , the l e v e l s o f GSH and GSH j>t r a n s f e r a s e d i d not i n c r e a s e i n response t o R-25788. The a c t i o n of R-25788 appears t o be s e l e c t i v e . No i n c r e a s e i n a c t i v i t y was noted when the i s o l a t e d corn system was t r e a t e d w i t h R-25788; t h e r e f o r e , R-25788 does not appear to be a simple a c t i v a t o r . Increased l e v e l s o f GSH ^ - t r a n s f e r a s e a c t i v i t y were observed i n both crude and p a r t i a l l y p u r i f i e d enzyme preparations a f t e r treatment w i t h R-25788. Although i t was not proven, the r e s u l t s suggest that enzyme i n d u c t i o n o r p o s s i b l e removal o f endogenous i n h i b i t o r s may be r e s p o n s i b l e f o r the observed increases i n enzyme a c t i v i t y . Twenty-eight compounds were compared to R-25788 f o r t h e i r e f f e c t i v e n e s s i n i n c r e a s i n g GSH S-transferase a c t i v i t y and GSH content i n corn s e e d l i n g r o o t s . Although s i g n i f i c a n t exceptions were noted, the e f f e c t i v e n e s s of these compounds as a n t i d o t e s g e n e r a l l y c o r r e l a t e d w i t h increased GSH and GSH Sr-transferase l e v e l s . Chlorpropham ( i s o p r o p y l m-chlorocarbanilate) and c i s a n i l i d e ( c i s - 2 , N - p h e n y l - l - p y r r o l i d i n e c a r b o x a n i l i d e ) a r e metabolized to hydroxylated d e r i v a t i v e s i n c e r t a i n p l a n t species (139, 154). Recent evidence i n d i c a t e s that the 4-hydroxylated d e r i v a t i v e s of chlorpropham and c i s a n i l i d e a r e converted to GSH and c y s t e i n e conjugates i n oat shoot s e c t i o n s (139, 140) (Figure 15). The s o l u b l e enzyme complex that c a t a l y z e s conjugate formation was i s o l a t e d from oat. When c y s t e i n e and 4-hydroxychlorpropham were incubated with the enzyme, a p o l a r metabolite was formed. When GSH was s u b s t i t u t e d f o r c y s t e i n e , a more p o l a r product was formed. The i n v i t r o enzyme system was used to produce s u f f i c i e n t metabolite from the r e a c t i o n w i t h c y s t e i n e and 4-hydroxychlor propham to allow i s o l a t i o n and p a r t i a l c h a r a c t e r i z a t i o n of the product (139). Because o f the low y i e l d o f t h i s product i n the i n v i v o system and d i f f i c u l t i e s encountered i n i t s i s o l a t i o n , the use of an i n v i t r o system f o r product formation g r e a t l y f a c i l i t a t e d the c h a r a c t e r i z a t i o n of t h i s product. In the c h a r a c t e r i z a t i o n o f the c y s t e i n e conjugate, c y s t e i n e C-S_ l y a s e
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC
112
METABOLISM
SG
Figure 15.
Reactions thought to he catalyzed by a GSH/cysteine from oat (139,140)
S-transferase
R-S-CH -C-C00H 2
NH
2
-[c-s (ORGANIC)
LYASE]
(H 0) 2
0 0
RSH CH
II II -C-C-OH ^ - N A D H
MASS
f [LACTIC \~~[ DEHYDROGENASE |
SPECTROMETER
^^NAD OH I CH,-C-COOH I H 3
Δ Α 340nm Figure 16.
Cysteine C-S lyase cleavage of cysteine conjugates (139,150)
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
3.
L A M O U R E U X AND FREAR
Plant Enzyme
Studies
113
was used to cleave the c y s t e i n e moiety from the a r y l group to y i e l d p y r u v i c a c i d and a thiophenol (Figure 16). The r e a c t i o n was q u a n t i t a t i v e and the l i b e r a t e d p y r u v i c a c i d was measured by c o u p l i n g the c y s t e i n e £-S l y a s e r e a c t i o n to a l a c t i c a c i d dehydrogenase r e a c t i o n . These coupled r e a c t i o n s were p r e v i o u s l y used i n the c h a r a c t e r i z a t i o n of a GSH-related conjugate of a t r a z i n e (150). Glutathione jS-transferase a c t i v i t y w i t h GSH and 4-hydroxychlorpropham was not demonstrated u n t i l the crude enzyme was p a r t i a l l y p u r i f i e d by Sephadex g e l chromatography. This behavior suggested the presence of endogenous i n h i b i t o r s . D e t a i l e d i n h i b i t o r s t u d i e s showed that s e v e r a l n a t u r a l l y o c c u r r i n g aromatic compounds and 3-chloro-4-hydroxyaniline were powerful i n h i b i t o r s of th c y s t e i n S-transferas activit (155) I t was suggested that thes o c c u r r i n g hydroxylated aromati x e n o b i o t i c s as s u b s t r a t e s . Two t r a n s f e r a s e enzyme systems were apparently present i n oat shoots. One e x h i b i t e d n e a r l y comparable a c t i v i t y with e i t h e r c y s t e i n e or GSH and the other d i s p l a y e d much greater a c t i v i t y w i t h c y s t e i n e . The l a t t e r enzyme a l s o functioned as a GSH S-transferase when the e t h y l e s t e r of c y s t e i n e was added to the r e a c t i o n mixture (140). The nature and the s i g n i f i c a n c e of t h i s a c t i v a t i o n i s not understood. These are the f i r s t s t u d i e s to suggest that c y s t e i n e may be u t i l i z e d as a s u b s t r a t e much l i k e GSH i n a t r a n s f e r a s e r e a c t i o n (139, 140, 155). T h i s system should be studied i n greater d e t a i l to b e t t e r evaluate i t s s i g n i f i c a n c e to x e n o b i o t i c metabolism. Glutathione j>-transferase a c t i v i t y was r e c e n t l y i s o l a t e d from 10 a g r i c u l t u r a l l y important p l a n t species and screened f o r a c t i v i t y with 8 d i f f e r e n t p e s t i c i d e substrates (146). Of the substrates examined, GSH S-transferase a c t i v i t y was demonstrated i n a l l species w i t h PCNB, propachlor and CDAA ( N , N - d i a l l y l chloroacetamide). The r e s u l t s suggested that c e r t a i n types of GSH j>-transferase a c t i v i t y may be widely d i s t r i b u t e d i n higher plants. These l i m i t e d s t u d i e s have c l e a r l y shown that GSH S-transf e r a s e s p l a y an important r o l e i n x e n o b i o t i c metabolism i n p l a n t s . Some GSH S-transferases appear to be widely d i s t r i b u t e d i n the p l a n t kingdom, but others appear to be more l i m i t e d i n t h e i r distribution. Glutathione ^ - t r a n s f e r a s e enzymes p l a y an important r o l e i n the s e l e c t i v i t y of c e r t a i n h e r b i c i d e s , such as the 2 - c h l o r o - s - t r i a z i n e s , f l u o r o d i f e n and EPTC s u l f o x i d e , but t h e i r r o l e i n the s e l e c t i v i t y of h e r b i c i d e s such as the a - c h l o r o acetamides i s u n c e r t a i n . The p o s s i b i l i t y that h e r b i c i d a l s e l e c t i v i t y may be increased by s e l e c t i v i t y s t i m u l a t i n g or inducing GSH ^ - t r a n s f e r a s e l e v e l s has been r a i s e d . A d d i t i o n a l s t u d i e s are needed to determine the d i s t r i b u t i o n of GSH St r a n s f e r a s e s i n higher p l a n t s and to b e t t e r determine the p r o p e r t i e s of the i n d i v i d u a l t r a n s f e r a s e s .
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC
114 Glucose
METABOLISM
Conjugation:
The r e p o r t s that ethylenechlorohydrin was converted to a 3-0-D-glucoside and a g e n t i o b i o s i d e i n wheat (156) and tomato (157T were among the f i r s t i n d i c a t i o n s that p l a n t s had the a b i l i t y to convert c e r t a i n x e n o b i o t i c s to g l u c o s i d e s . I t was l a t e r shown that most higher p l a n t s (158, 159) had the a b i l i t y to convert exogenous phenols to $-j)-D-glueosides. T h i s a b i l i t y was apparently l a c k i n g i n algae, fungT and c e r t a i n aquatic p l a n t s (158). The formation of 0-, N-, and ^ - g l u c o s i d e s , a c y l a t e d g l u c o s i d e s , g e n t i o b i o s i d e s , and glucose e s t e r s have a l l been demonstrated e i t h e r i n v i t r o or i n v i v o with x e n o b i o t i c s or n a t u r a l substrates (160). The formation of glucosides i s extremely important i n p e s t i c i d e biochemistry f o r the f o l l o w i n g reasons: there i s a wid conjugation, g l u c o s i d e formatio terminal r e s i d u e , and g l u c o s y l a t i o n may play a r o l e i n p e s t i c i d e s e l e c t i v i t y or d e t o x i c a t i o n . The most common types of g l y c o s i d e r e a c t i o n s encountered i n p e s t i c i d e metabolism appear to i n v o l v e an i n i t i a l UDPG-dependent g l u c o s y l t r a n s f e r r e a c t i o n (Figure 17), The formation of simple 0-glucosides from polyhydroxylated phenols was demonstrated w i t h an i n v i t r o system from wheat germ (161). Substrate s p e c i f i c i t y t e s t s showed that the wheat germ g l u c o s y l t r a n s f e r a s e could use a number of polyhydroxy phenols as s u b s t r a t e s , but was not a c t i v e with simple phenols. A f t e r p u r i f i c a t i o n of t h i s enzyme, a c t i v i t y f o r c e r t a i n substrates was l o s t ; thus, the presence of more than one t r a n s f e r a s e was i n d i c a t e d . The i n v i t r o synthesis of 14 phenolic glucosides by crude enzymes from wheat germ and bean was compared with the i n v i v o s y n t h e s i s i n bean (162). The only products detected i n v i t r o were g e n e r a l l y the primary products formed i n v i v o . The enzyme systems from wheat germ and bean could not u t i l i z e simple mono-hydroxylated phenols as substrates; i t i s , t h e r e f o r e , questionable whether these enzymes are i n v o l v e d i n the formation of 3-0-D-glucosides from p e s t i c i d e s or p e s t i c i d e m e t a b o l i t e s . A number of UDPG:sterol g l u c o s y l t r a n s f e r a s e s have been i s o l a t e d from v a r i o u s p l a n t sources (163-166). These enzymes are u s u a l l y a s s o c i a t e d with the p a r t i c u l a t e f r a c t i o n (164). For some phenolic x e n o b i o t i c s , the p o s s i b i l i t y should be considered that UDPG:glucosyltransferase a c t i v i t y may be membrane bound. A UDPG-dependent enzyme that c a t a l y z e s the formation of β-0-D-glucosides with a v a r i e t y of phenols, a l k y l a l c o h o l s and other substrates has been i s o l a t e d from germinating mung bean (167). Attempts to demonstrate the presence of t h i s enzyme i n seedlings were not s u c c e s s f u l . The ammonium s u l f a t e f r a c t i o n a t e d enzyme from germinating mung beans could be stored i n l i q u i d n i t r o g e n with l i t t l e l o s s i n a c t i v i t y , but the more h i g h l y p u r i f i e d enzyme l o s t a l l a c t i v i t y upon f r e e z i n g . T h i s enzyme u t i l i z e d UDPG as the g l u c o s y l donor, had an estimated M.W. of 62,000 and had a pH optimum of approximately 10, The pH optimum
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
3.
LAMOUREUX A N D FREAR
Plant Enzyme
115
Studies
3) 1-0 GLUCOSE ESTERS
4) Ν-GLUCOSIDES
Figure 17.
(SOYBEAN)
Glucosyl transferase systems that use UDPG
as the glucosyl donor
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
116
XENOBIOTIC METABOLISM
f o r most other g l u c o s y l t r a n s f e r a s e s i s between 6.5 and 9. D e t a i l e d s u b s t r a t e s t u d i e s showed that r e a c t i o n r a t e s with the mung bean enzyme were p r i m a r i l y dependent upon the s i z e o f the acceptor s u b s t r a t e . n-Butanol was the most a c t i v e a l k y l acceptor and phenol was the most a c t i v e a r y l acceptor. The values observed w i t h a number of low molecular weight s u b s t r a t e s suggested that t h i s enzyme was n o n - s p e c i f i c and could play an important r o l e i n the metabolism of p e s t i c i d e s and other xenobiotics. A crude enzyme system from wheat germ was shown to c o n t a i n an enzyme that u t i l i z e d a r b u t i n as à s u b s t r a t e i n the formation of the corresponding g e n t i o b i o s i d e (168). T h i s enzyme was separated from UDPG:polyhydroxyphenol g l u c o s y l t r a n s f e r a s e a c t i v i t y and was p a r t i a l l y p u r i f i e d A broad range o f p h e n o l i c 3-glucosides were s u b s t r a t e a c t i v e w i t h f r e e phenols i n the formation of p e s t i c i d e g e n t i o b i o s i d e conjugates such as diphenamid (169). A s i m i l a r enzyme that c a t a l y z e s the formation of an JD-a-L-rhamnosyl- (1-0)-3-D-glucosyl conjugate o f q u e r c e t i n has been i s o l a t e d from mung beans (170). P e s t i c i d e conjugates of t h i s type have not been reported. The i n v i t r o formation of the 1-0-glucose e s t e r of anthrani l i c a c i d was demonstrated w i t h a UDPG-dependent t r a n s f e r a s e from l e n t i l s (171, 172). The formation o f 1-0-glucose e s t e r s o f s e v e r a l 4-hydroxy cinnamic a c i d d e r i v a t i v e s (p-coumaric, c a f f e i c , f e r u l i c , and s i n a p i c a c i d s ) and s e v e r a l hydroxybenzoic a c i d s ( v a n i l l i c , i s o v a n i l l i c and s y r i n g i c acid) was demonstrated w i t h an acetone powder from geranium leaves (173). The crude enzyme system from geranium leaves r e q u i r e d UDPG f o r a c t i v i t y , had a pH optimum of approximately 8.5 f o r e s t e r formation, and was a l s o capable of forming β-0-glucosides w i t h phenols a t pH 7.4. The 1-0-glucose e s t e r s of both chloramben (131) and naphthelenea c e t i c a c i d (174) have been reported i n p l a n t s . These products may a l s o be formed by UDPG-dependent systems such as those from l e n t i l s or geranium l e a v e s . The formation of 1-0-glucose e s t e r conjugates o f h e r b i c i d e s may not be a permanent d e t o x i c a t i o n mechanism s i n c e these products are r e a d i l y hydrolyzed jin v i v o and i n v i t r o as p r e v i o u s l y d i s c u s s e d . The formation of 2-0, 4-0 and 6-0 glucose e s t e r s of c a r b o x y l i c a c i d appears to i n v o l v e a d i f f e r e n t mechanism (Figure 18). A s o l u b l e enzyme from sweet corn k e r n e l s was shown to c a t a l y z e the formation of 2-0, 4-0 and 6-0 glucose e s t e r s of i n d o l e - 3 - a c e t i c a c i d i n the presence of ATP, CoA, and Mg " " (175) . Products such as these may a l s o be important i n p e s t i c i d e metabolism i n higher p l a n t s (160). The i n v i t r o formation o f s u b s t i t u t e d a n i l i n e N-glucosides was demonstrated w i t h enzyme systems from soybean (176, 177) and P (178) (Figure 17). The UDPG:arylamine g l u c o s y l t r a n s f e r a s e from soybean u t i l i z e d UDPG o r TDPG as the g l u c o s y l donor f o r 15 d i f f e r e n t arylamine acceptors (176). During the i s o l a t i o n of t h i s 4
1
e a
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
3.
LAMOUREUX AND FREAR
Figure 18.
Plant Enzyme
Studies
Formation of 2-0,4-0, and 6-0 glucose ester conjugates (175)
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
117
XENOBIOTIC
118
METABOLISM
enzyme, a c t i v i t y was demonstrated w i t h arylamines and hydroquinones; however, the r a t i o of these a c t i v i t i e s changed d r a m a t i c a l l y during p u r i f i c a t i o n . I t appears that more than one t r a n s f e r a s e was o r i g i n a l l y present. In v i v o s t u d i e s i n d i c a t e d a d i r e c t r e l a t i o n s h i p between chloramben N-glucoside formation and r e s i s t a n c e (179). Several arylamine h e r b i c i d e s are metabolized d i r e c t l y to N-glucosides i n p l a n t s and a number of other h e r b i c i d e s have been reported to be metabolized to arylamines by p l a n t s (160). These metabolites might a l s o be expected to form N-glucosides. Based upon our knowledge of the metabolism of h e r b i c i d e s to a n i l i n e d e r i v a t i v e s and the broad s p e c i f i c i t y of the UDPG:arylamine g l u c o s y l t r a n s ~ f e r a s e from soybean, i t would appear that t h i s enzyme plays an important r o l e i n x e n o b i o t i c metabolism The formation of 6-0-malonyl-$-D-glucoside substrates (180-182) a and flamprop (184) have been reported. The r e a c t i o n , with flavone g l y c o s i d e s as s u b s t r a t e s , has been studied i n v i t r o with an enzyme i s o l a t e d from i l l u m i n a t e d c e l l suspension c u l t u r e s of p a r s l e y (185). The enzyme i s a malonyl coenzyme A:flavone g l y c o s i d e malonyl-transferase. The enzyme d i d not appear to be s p e c i f i c f o r the few g l y c o s i d e substrates tested and no cof a c t o r s other than malonyl coenzyme A were r e q u i r e d . In a d d i t i o n to the UDPG-dependent g l u c o s y l t r a n s f e r a s e systems, an enzyme that u t i l i z e s an endogenous g l u c o s i d e , i s o succinimide β-glucoside, as the g l u c o s y l donor has been c h a r a c t e r i z e d from pea (186) (Figure 19). T h i s s o l u b l e enzyme has a pH optimum of 5.5 and converts ethanol and isopropanol to t h e i r corresponding 3-0-D-glucosides. Isotope s t u d i e s showed that the r e a c t i o n was e s s e n t i a l l y i r r e v e r s i b l e . Various phenolic g l u c o s i d e s a l s o functioned as g l u c o s y l donors, but at a reduced r a t e . UDPG, glucose-l-P and r e l a t e d compounds d i d not f u n c t i o n i n t h i s c a p a c i t y . A high K f o r ethanol, 0.5 M, suggested that ethanol and isopropanol were probably not the n a t u r a l substrates f o r t h i s enzyme. A s i m i l a r or i d e n t i c a l system from pea was r e c e n t l y shown to u t i l i z e polyethoxylated a l k y l p h e n o l detergents as substrates (187). An u n i d e n t i f i e d endogenous glucoside and s e v e r a l exogenous phenolic glucosides functioned as g l u c o s y l donors. A d d i t i o n a l s t u d i e s on the substrate s p e c i f i c i t y , d i s t r i b u t i o n , and k i n e t i c s of t h i s enzyme are needed to p r o p e r l y evaluate i t s importance i n the metabolism of detergents and other x e n o b i o t i c s . m
SUMMARY: Based on the enzyme s t u d i e s considered i n t h i s r e p o r t , i t can be concluded that i n v i t r o enzyme techniques can be used to great advantage to study p e s t i c i d e metabolism i n p l a n t s . Important advantages that may be o f f e r e d by these techniques are as f o l l o w s :
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Advantages : (1) S p e c i f i c metabolic r e a c t i o n s can be i s o l a t e d from competing r e a c t i o n s and studied i n a s i m p l i f i e d system. D e t a i l s of the r e a c t i o n mechanism can be examined. T r a n s i t o r y or unstable intermediates can be detected or i s o l a t e d , (2) Extensive i n h i b i t o r and a c t i v a t o r s t u d i e s can be e f f i c i e n t l y conducted. P o s s i b l e p e s t i c i d e i n t e r a c t i o n s may be i d e n t i f i e d and n o n p e s t i c i d a l agents can be screened f o r use i n combinations with p e s t i c i d e s to change t h e i r s e l e c t i v i t y or improve t h e i r e f f i c a c y . (3) Broad s t r u c t u r e - a c t i v i t y s t u d i e s can be conducted e a s i l y to determine the s t r u c t u r a l requirements f o r a s p e c i f i c metabolic r e a c t i o n . (4) Screening s t u d i e i s o l a t e d from many d i f f e r e n o b t a i n b a s i c information on genetic d i f f e r e n c e s i n metabolism or the mechanism of h e r b i c i d e r e s i s t a n c e . (5) Enzyme i n d u c t i o n s t u d i e s can help i n the development of p r o t e c t a n t s and other u s e f u l chemicals that f u n c t i o n by s t i m u l a t i n g the formation of key enzymes involved i n metabolism, (6) I s o l a t e d enzymes may be used e f f e c t i v e l y f o r the b i o s y n t h e s i s of s p e c i f i c metabolites i n q u a n t i t i e s that s i m p l i f y t h e i r i s o l a t i o n and i d e n t i f i c a t i o n . (7) S p e c i f i c enzymes can be used to c h a r a c t e r i z e and degrade more complex metabolites. (8) Model enzyme systems can be developed to help solve d i f f i c u l t problems such as those a s s o c i a t e d w i t h the bound residues of p e s t i c i d e s . (9) C e r t a i n metabolic r e a c t i o n s may be studied i n v i t r o without the need f o r r a d i o a c t i v e s u b s t r a t e s . (10) Enzyme concentrations as a f u n c t i o n of age or t i s s u e type can be s t u d i e d . (11) Enzyme k i n e t i c s t u d i e s can be used to determine r e a c t i o n mechanisms and to evaluate the importance of competing metabolic r e a c t i o n s . The use of i s o l a t e d enzymes to study x e n o b i o t i c metabolism i s not without c e r t a i n disadvantages. In a d d i t i o n to s p e c i f i c problems a s s o c i a t e d with the i s o l a t i o n of enzymes from p l a n t s , such as low l e v e l s of enzyme a c t i v i t y w i t h i n the p l a n t and the presence of phenolics and t h e i r o x i d a t i v e enzymes that can r e s u l t i n enzyme i n a c t i v a t i o n or degradation during e x t r a c t i o n and i s o l a t i o n , there are a number of disadvantages or problems a s s o c i a t e d w i t h jLn v i t r o enzyme techniques i n general. Those most r e l e v a n t to x e n o b i o t i c metabolism are as f o l l o w s : Disadvantages : (1) Results of i s o l a t e d enzyme s t u d i e s must u s u a l l y be v e r i f i e d by i n v i v o s t u d i e s .
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(2) The enzyme may be extremely difficult to isolate in an active form or may be labile and require reisolation for each study. (3) The nature of the enzyme system may be unknown and i t may be difficult to determine the proper co-factors. (4) If the desired enzyme activity is found in several fractions, the researcher must decide whether to study only one fraction, study each fraction separately, or study the crude system. (5) Endogenous inhibitors may be present. If not properly handled, they can yield misleading results. (6) The system may be susceptible to substrate or product inhibition or the kinetics may be dependent upon the substrate concentration. (7) The solubilit reliable kinetic data an or solvents to solubilize the substrate. These agents may alter the kinetics or inhibit the enzyme. (8) The methods necessary to isolate a specific enzyme activity from different plant species may vary sufficiently to make i t difficult to conduct broad screening studies. (9) It may be difficult to obtain the enzyme in sufficiently pure form to conduct meaningful kinetic studies. (10) Low enzymatic activity generally makes necessary the use of radioactive substrates or other more sensitive assay methods. This brief survey has documented the role played by various isolated enzyme studies in answering important questions related to pesticide metabolism in plants. It has shown that when proper consideration is given, in vitro techniques can be used to great advantage. Currently, our ability to use isolated enzymes to study certain areas of pesticide metabolism is limited by our basic knowledge of the enzymes involved. As this knowledge becomes available, our ability to use ±a vitro enzyme techniques to study additional areas of pesticide metabolism will be increased. Abstract The four basic biochemical reactions commonly involved in pesticide metabolism in higher plants (oxidation, reduction, hydrolysis and conjugation) are discussed in relation to the enzyme systems capable of catalyzing these reactions. The literature regarding the use of enzymes from these classes to study pesticide metabolism is reviewed. The following enzymes are considered: peroxidases, mixed function oxidases, hydroperoxidases, aryl nitroreductases, aryl acylamidases, esterases, glutathione S-transferases, cysteine S-transferases and various glucose-conjugating enzyme systems. The advantages and disadvantages of in vitro enzyme techniques as they related to pesticide metabolism studies are also discussed.
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140. Rusness, D. G. and S t i l l , G. G., Pest. Biochem. Physiol. (1977) 7, 220. 141. Shimabukuro, R. Η., Lamoureux, G. L., Swanson, H. R., Walsh, W. C., Stafford, L. E. and Frear, D. S., Pest. Biochem. Physiol. (1973) 3, 483. 142. Frear, D. S. and Swanson, H. R., Pest. Biochem. Physiol. (1973) 3, 473. 143. Frear, D. S. and Swanson, H. R., Phytochem. (1970) 9, 2123. 144. Lay, M. M. and Casida, J. E., Pest. Biochem. Physiol. (1977) 6, 442. 145. Lamoureux, G. L. and Rusness, D. G. (1976) 172nd National ACS Meeting, Aug. 29-Sept. 3, San Francisco, Calif. 146. Burkholder, R. R. S., Plant Glutathione S-Transferase, M. S. Thesis, N. D State University Fargo N D (1978) 147. Shimabukuro, R. Η. W. C., Plant Physiol (1971) , 148. Frear, D. S. Unpublished data. 149. Bedford, C. T., Crawford, M. J. and Hutson, D. Η., Chemosphere (1975) 5, 311. 150. Lamoureux, G. L., Stafford, L. Ε., Shimabukuro, R. H. and Zaylskie, R. Ε., J. Agric. Food Chem. (1973) 21, 1021. 151. Tan, Κ. H. and Lovrien, R., J. Biol. Chem. (1972) 247, 3278. 152. Lay, M. M. and Casida, J. E., Science (1975) 189, 287. 153. Lay, M. M. and Casida, J. Ε., In: "Chemistry and Action of Herbicide Antidotes," (Edited by Pallos, F. M. and Casida, J. Ε.), Academic Press Inc., New York (1978). 154. Frear, D. S. and Swanson, H. R., Pest. Biochem. Physiol. (1976) 6, 52. 155. Rusness, D. G. and S t i l l , G. G., Pest. Biochem. Physiol. (1977) 7, 232. 156. Miller, L. P., Contrib. Boyce Thompson Inst. (1941) 12, 25. 157. Miller, L. P., Contrib. Boyce Thompson Inst. (1941) 12, 15. 158. Pridham, J. B., Phytochem. (1964) 3, 493. 159. Glass, A. D. M. and Bohm, Β. Α., Phytochem. (1970) 9, 2197. 160. Frear, D. S., In: "Pesticide Conjugates-Glycosides, ACS Symposium Series 29 (Edited by Kaufmann, D. D., S t i l l , G. G., Paulson, G. D. and Bandal, S. Κ.), American Chemical Society, Washington, D. C. (1976). 161. Yamaha, T. and Cardini, C. Ε., Arch. Biochem. Biophys. (1960) 86, 127. 162. Pridham, J. B. and Saltmarch, M. J., Biochem. J. (1963) 87, 218. 163. Wojciechowski, Α. Ζ., Phytochem. (1974) 13, 2091. 164. Fang, T. Y. and Baisted, D. Β., Phytochem. (1976) 15, 273. 165. Baisted, D. Β., Phytochem. (1978) 17, 435. 166. Adler, G. and Kasprzyk, Z., Phytochem. (1975) 14, 627. 167. Storm, D. L. and Hassid, W. C., Plant Physiol. (1974) 54, 840. 168. Yamaha, T. and Cardini, C., Arch. Biochem. Biophys. (1960) 86, 133.
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169. Hodgson, R. Η., Dusbabek, Κ. E. and Hoffer, B. L., Weed Sci. (1973) 21, 542. 170. Barber, G. Α., Biochem. (1962) 1, 463. 171. Tabone, D., Compt. Rend. (1955) 241, 1521. 172. Jacobelli, G., Tabone, M. J. and Tabone, D., Bull. Soc. Chim. Biol. (1958) 40, 955. 173. Corner, J. J. and Swain, T., Nature (1965) 207, 634. 174. Shindy, W. W., Jordan, L. S. and Jolliffe, V. Α., J. Agric. Food Chem. (1973) 21, 629. 175. Kopcewicz, J., Ehmann, A. and Bandurski, R. S., Plant Physiol. (1974) 54, 846. 176. Frear, D. S., Phytochem. (1968) 7, 381. 177. Frear, D. S., Swanson, C. R. and Kadunce, R. Ε., Weeds (1967) 15, 101. 178. Murakoshi, I., Ikegama Phytochem. (1975) , 179. Colby, S. R., Science (1965) 150, 619. 180. Minale, L., Piatelli, M., DeStefano, S. and Nicolaus, R. Α., Phytochem. (1966) 5, 1037. 181. Beck, A. B. and Knox, J. R., Aust. J. Chem. (1971) 24, 1509. 182. Kruezaler, F. and Hahlbrock, Κ., Phytochem. (1973) 12, 1149. 183. Shimabukuro, R. Η., Walsh, W. C., Stolzenberg, G. E. and Olson, P. A. (1975) Weed Sci. Soc. Amer. Meetings, Washington, D. C. 184. Dutton, A. J., Roberts, T. R. and Wright, Α. Ν., Chemosphere (1976) 3, 195. 185. Hahlbrock, Κ., FEBS Letters (1972) 28, 65. 186. Liu, T. Y. and Castelfranco, P., Plant Physiol. (1970) 45, 424. 187. Frear, D. S., Swanson, H. R. and Stolzenberg, G. E. (1977) 174th National ACS Meeting, Aug. 28-Sept. 4, Chicago, Ill. 188. Hideaki, S. and Noguchi, Μ., Phytochem. (1975) 14, 1255. 189. Machackova, I., Ganceva, K. and Zmrhal, Ζ., Phytochem. (1975) 14, 1251. 190. Wright, B. J., Dowsett, J. R., Rubery, P. H., Baillie, A. C. and Corbett, J. R., Pestic. Sci. (1973) 191. S t i l l , G. G., Rusness, D. G., Mansager, E. R., In: "Mechanism of Pesticide Action," ACS Symposium Series 2, ACS, Washington, D. C., (1974), 117. 192. Lamoureux, G. L. and Rusness, D. G., Unpublished data. RECEIVED
December 20, 1978.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
4 Techniques for Studying the Metabolism of Xenobiotics by Intact Animal Cells, Tissues, and Organs In Vitro ROBERT E. MENZER Department of Entomology, University of Maryland, College Park, M D 20742
Researchers studying t h e f a t e o f x e n o b i o t i c s i n mammals have tended t o concentrate t h e i r e f f o r t s e i t h e r on the whole animal o r on l i v e r homogenate system has i t s advantages. Th respons y represents what i s l i k e l y t o occur upon exposure o f man t o a compound. L i v e r homogenates are u s e f u l i n that the l i v e r i s t h e p r i n c i p a l organ r e s p o n s i b l e f o r the degradation and e l i m i n a t i o n of x e n o b i o t i c s from mammalian systems, and l i v e r microsomes appear t o be the p r i n c i p a l s i t e w i t h i n the mammal where metabolic conversions take p l a c e . These systems a l l o w a r a p i d , inexpensive e v a l u a t i o n o f metabolic events which a r e l i k e l y to take p l a c e i n the mammal f o r any x e n o b i o t i c . The combination o f s t u d i e s i n whole animals and l i v e r microsomal systems g e n e r a l l y provides a good understanding o f the f a t e o f a x e n o b i o t i c i n a mammalian system. O c c a s i o n a l l y , however, a compound w i l l be i n v e s t i g a t e d i n which the events t a k i n g p l a c e i n a l i v e r microsomal system do not e n t i r e l y m i r r o r what occurs i n the whole organism. Furthermore, the use o f microsomal systems does not p r o v i d e i n f o r m a t i o n on t h e pharmacodynamics i n v o l v e d i n t h e a b s o r p t i o n , d i s t r i b u t i o n , and e l i m i n a t i o n o f a x e n o b i o t i c from an organism. Other techniques are a v a i l a b l e which w i l l p r o v i d e a d d i t i o n a l i n f o r m a t i o n on t h e f a t e o f a x e n o b i o t i c i n a mammal that would be d i f f i c u l t to o b t a i n e i t h e r w i t h l i v e r microsomal systems o r i n whole organisms. In t h i s paper we w i l l examine the use o f perfused organ systems, w i t h p a r t i c u l a r emphasis on l i v e r and lung p e r f u s i o n ; t i s s u e s l i c e s , p a r t i c u l a r l y from lung and l i v e r ; c e l l c u l t u r e systems, both primary c e l l c u l t u r e s and e s t a b l i s h e d c e l l l i n e s ; and f i n a l l y , the promising new technique o f i s o l a t e d hepatocyte preparat i o n s . For each technique we w i l l examine the methodology c u r r e n t l y i n use and evaluate the ease by which an i n v e s t i g a t o r inexperienced i n the use o f t h e technique would be a b l e t o adopt i t f o r s t u d i e s on s p e c i f i c compounds. The a p p l i c a t i o n o f each o f these techniques t o the study o f x e n o b i o t i c s , w i t h p a r t i c u l a r emphasis on p e s t i c i d e s , w i l l be i l l u s t r a t e d . 0-8412-0486-l/79/47-097-131$05.00/0 © 1979 American Chemical Society
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Organ P e r f u s i o n As i s the case with most in vitro mammalian p r e p a r a t i o n s , organ p e r f u s i o n was o r i g i n a l l y developed to study the physiology and biochemistry o f the organ i t s e l f and the f u n c t i o n a l p o s i t i o n o f the organ i n the metabolism o f the normal animal. Organ perf u s i o n s t u d i e s are an intermediate step between s t u d i e s i n the whole animal and experiments with i s o l a t e d s u b c e l l u l a r preparations. By observing the response o f a v i a b l e organ i s o l a t e d from the system i n which i t r e s i d e s , one i s a b l e to assess the p a r t which the organ p l a y s i n the metabolism o f any given system. I t i s f r e q u e n t l y p o s s i b l e to observe s p e c i f i c steps i n metabolic processes i n an i s o l a t e d organ when i n the whole animal only sub s t r a t e and f i n a l produc o f the r e s e a r c h e r to i s o l a t organs have t h e i r p r i n c i p a l u t i l i t y . Another advantage o f perfused organ p r e p a r a t i o n s i s that blood flow, gas exchange, and temperature are under d i r e c t c o n t r o l . T h i s enables the r e s e a r c h e r to d e l i b e r a t e l y manipulate these parameters to assess the e f f e c t of, f o r example, reduced blood flow, anoxia, hypothermia, changes i n blood pH, or o s m o l a r i t y on the metabolic process being s t u d i e d . For background i n f o r m a t i o n and d e s c r i p t i o n s o f the methodology a v a i l a b l e f o r organ p e r f u s i o n one may r e f e r to a number o f books d e a l i n g with the s u b j e c t . Two examples are R i t c h i e and Hardc a s t l e (11) and Ross (12). P e r f u s i o n may be d e f i n e d as the passage of a f l u i d medium or blood through the v a s c u l a r bed o f an organ. I t i s easy to see how one can u t i l i z e a p e r f u s i n g organ to study the metabolism o f a x e n o b i o t i c by the simple i n t r o d u c t i o n o f the chemical i n t o the p e r f u s a t e and o b s e r v a t i o n o f the e f f e c t which the organ has on the chemical. The p o s s i b i l i t i e s f o r continuous sampling of the p e r f u s a t e , continuous a d d i t i o n or p u l s e a d d i t i o n o f the chemical to the p e r f u s a t e , and s t u d i e s of the i n t e r a c t i o n s o f more than one chemical i n organs are r e a d i l y apparent. For workers studying x e n o b i o t i c s the two most important organ p e r f u s i o n systems are those f o r the l i v e r and the lungs. The l i v e r i s probably the most f r e q u e n t l y perfused organ and i s used f o r a wide v a r i e t y o f s t u d i e s . The techniques f o r l i v e r p e r f u s i o n p r e s e n t l y most f r e q u e n t l y used are those developed by M i l l e r et al. (7) as i l l u s t r a t e d i n F i g u r e 1. The apparatus designed by M i l l e r and co-workers i s now commercially a v a i l a b l e . Techniques f o r p e r f u s i o n o f the lung have been more r e c e n t l y developed, the system o f Niemeier and Bingham (8) being most f r e quently r e f e r r e d to by workers i n t h i s area (9). Niemeier and Bingham developed a system f o r the p e r f u s i o n of r a b b i t lungs which allows the use o f u n d i l u t e d autologous whole blood as the p e r f u sate. Lung p e r f u s i o n i s more complicated than l i v e r s i n c e one i s able to p r o v i d e both a c i r c u l a t i n g l i q u i d p e r f u s a t e as w e l l as being able to v e n t i l a t e the i s o l a t e d lung with a gas system. In
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Figure 1. Liver perfusion apparatus. The apparatus is enclosed in a temperatureregulated cabinet and is composed of a system for pumping the perfusate (blood) at constant hydrostatic pressure and a system for oxygenating the blood (7, 12).
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a lung, t h e r e f o r e , one can introduce a x e n o b i o t i c to the system e i t h e r through the p e r f u s i o n system i t s e l f or through the gas v e n t i l a t i n g system, thus approximating the i n t r o d u c t i o n o f the x e n o b i o t i c e i t h e r through the c i r c u l a t o r y system or the r e s p i r a t o r y system i n the whole animal. The use o f lung p e r f u s i o n i s p a r t i c u l a r l y advantageous because the e s t i m a t i o n o f s u b s t r a t e u t i l i z a t i o n by the lung i s very d i f f i c u l t in vivo. Lung t i s s u e s l i c e s or other in vitro p r e p a r a t i o n s do not seem to approximate the p h y s i o l o g i c a l s t a t e o f the whole organ as w e l l as comparable preparations from the l i v e r ; the need f o r oxygen and the problem o f d i f f u s i o n are p a r t i c u l a r problems i n the lung. I s o l a t e d perfused lung systems overcome these problems and w i l l be p a r t i c u l a r l y u s e f u l i n the f u t u r e f o r the study o f the metabolism by the lung o f x e n o b i o t i c s introduced by i n h a l a t i o n . X e n o b i o t i c metabolis with p a r a t h i o n . Both p a r a t h i o assess the metabolic r e l a t i o n s h i p s o f these compounds i n the l i v e r ( 3 ) . I t was shown that 68% o f the administered p a r a t h i o n was metabolized to water s o l u b l e compounds. These water s o l u b l e compounds were found to be conjugates o f p - n i t r o p h e n o l , the bulk o f which was i n the c i r c u l a t i n g p e r f u s a t e , not a s s o c i a t e d with the l i v e r t i s s u e or excreted v i a the b i l e . An a d d i t i o n a l 2.5% was paraoxon and there were traces o f unconjugated p - n i t r o p h e n o l . Administered paraoxon was degraded almost e n t i r e l y (98.5%) to water s o l u b l e compounds, which again were conjugates o f p - n i t r o phenol . Another type of experiment p o s s i b l e using perfused organ systems i s i l l u s t r a t e d by a study o f mirex-induced suppression o f b i l i a r y e x c r e t i o n o f p o l y c h l o r i n a t e d biphenyls ( 5 ) . In t h i s study i t was shown t h a t 50 mg/kg/day o f mirex-pretreatment o f the r a t s whose l i v e r s were perfused suppressed the b i l i a r y e x c r e t i o n o f 4-chlorobiphenyl and i t s metabolites by 92%. Furthermore, the r a t e o f metabolism o f 4-chlorobiphenyl was decreased s l i g h t l y by mirex pretreatment. The reason f o r t h i s phenomenon was t h e o r i z e d to be that t r a n s p o r t o f otherwise r e a d i l y e x c r e t a b l e metabolites from the hepatocytes i n t o the b i l e c a n a l i c u l i was a f f e c t e d by mirex. The f a c t that mirex causes changes i n the a b i l i t y o f the l i v e r to excrete x e n o b i o t i c s has i m p l i c a t i o n s f o r the p o s s i b l e e f f e c t o f t h i s compound on the t o x i c o l o g y o f other compounds. A number o f recent s t u d i e s o f x e n o b i o t i c s i n perfused lung systems have been reported: a l d r i n and d i e l d r i n (6), p a r a t h i o n , methadone, imipramine, c h l o r c y c l i z i n e , and p e n t o b a r b i t a l ( 4 ) , t r i c h l o r o e t h y l e n e (2), and c a r b a r y l (1). These s t u d i e s i l l u s t r a t e w e l l the p o t e n t i a l f o r important r e s u l t s which can be obtained from organ p e r f u s i o n s t u d i e s . Perfused r a b b i t lungs were used t o study the metabolism and b i n d i n g p r o p e r t i e s o f a l d r i n and d i e l d r i n . The compounds were added to the system v i a the p e r f u s i o n medium and samples were withdrawn at s e v e r a l i n t e r v a l s . I t was noted that a l d r i n was epoxidized t o d i e l d r i n , but d i e l d r i n was not f u r t h e r metabolized
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i n the system; no epoxide hydrase a c t i v i t y could be detected. The uptake of a l d r i n and d i e l d r i n was by d i f f u s i o n . The r a t e of uptake was b i p h a s i c , c o n s i s t i n g of an i n i t i a l r a p i d phase f o l l o w ed by a slower one, r e l a t e d to conversion o f a l d r i n to d i e l d r i n i n the case of a l d r i n . These s t u d i e s show that the lungs are not a s i g n i f i c a n t storage s i t e f o r e i t h e r compound (6). The metabolism o f p a r a t h i o n , methadone, imipramine, c h l o r c y c l i z i n e , and p e n t o b a r b i t a l was compared i n a r a b b i t perfused lung p r e p a r a t i o n with r a b b i t lung and l i v e r microsomal preparations. In the perfused lung p a r a t h i o n , methadone, and pentob a r b i t a l were o x i d a t i v e l y metabolized. Parathion was e x t e n s i v e l y metabolized to paraoxon and water s o l u b l e m e t a b o l i t e s , which were not f u r t h e r i d e n t i f i e d . No accumulation of p a r a t h i o n was observed i n the system. With i n c r e a s i n g p e r f u s i o n time there was a decrease i n the appearanc b a r b i t a l metabolites. Thi decreasing s u b s t r a t e or d e p l e t i o n o f c o f a c t o r than to denatura t i o n or d e s t r u c t i o n o f the lung system. No s i g n i f i c a n t d i f f e r ences were observed between the drug m e t a b o l i z i n g a c t i v i t i e s o f the microsomes o f lung or l i v e r and the perfused lung system ( 4 ) . An i n t e r e s t i n g example o f the use o f the perfused lung system to study the metabolism o f a x e n o b i o t i c by the i n h a l a t i o n route i s given by Dalbey and Bingham (2). They s t u d i e d the metabolism of t r i c h l o r o e t h y l e n e i n a r a b b i t perfused lung system. T r i c h l o r o e t h y l e n e was generated i n t o the a i r s u p p l i e d to the i s o l a t e d perfused lungs, and the compound and i t s metabolites were measured p e r i o d i c a l l y i n the p e r f u s a t e and i n the lung t i s s u e f o l l o w i n g a three-hour p e r f u s i o n p e r i o d . T r i c h l o r o e t h y l e n e was e x t e n s i v e l y metabolized to t r i c h l o r o e t h a n o l , t r i c h l o r o e t h a n o l glucuronide, and t r i c h l o r o a c e t i c a c i d . I t was p o s t u l a t e d that c h l o r a l hydrate was an intermediate i n the metabolism o f t r i chloroethylene but i t was not i s o l a t e d i n t h i s system. C a r b a r y l metabolism i n the perfused r a b b i t lung was shown to be r a p i d . The pharmacokinetics of c a r b a r y l uptake demonstrated simple d i f f u s i o n . A f t e r 30 minutes o f p e r f u s i o n 1-naphthol was seen i n the p e r f u s a t e e x t r a c t s . Since i t s c o n c e n t r a t i o n i n the p e r f u s a t e decreased during the course o f the experiment, i t was concluded that the 1-naphthol which was taken up by the lungs was formed by nonenzymatic h y d r o l y s i s of c a r b a r y l i n the p e r f u s a t e . 4-Hydroxycarbaryl appeared i n the p e r f u s a t e at 30 minutes and i n creased i n c o n c e n t r a t i o n u n t i l 60 minutes, a f t e r which i t decreased. Other metabolites were i s o l a t e d but no attempt was made to i d e n t i f y them ( 1 ) . Comparative s t u d i e s (10) have shown that perfused organs, e s p e c i a l l y the l i v e r , p a r a l l e l changes which occur i n the whole organism. Thus, the technique can be a u s e f u l b r i d g e between other in vitro s t u d i e s and in vivo s t u d i e s .
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Slices
The techniques f o r t i s s u e s l i c e s t u d i e s o f metabolism were probably f i r s t introduced by Otto Warburg i n 1923 (19). The method caught on r a p i d l y and was used by many workers so extens i v e l y that Krebs and H e n s e l e i t , w r i t i n g i n 1932 (15), noted that between the two methods a v a i l a b l e f o r studying metabolism i n animal t i s s u e s , they p r e f e r r e d t i s s u e s l i c e s over p e r f u s i o n . T h i s judgment probably represented a r e a c t i o n to the lack o f r e p r o d u c i b i l i t y o f p e r f u s i o n techniques and the complicated systems then i n use. By comparison, t i s s u e s l i c e s coupled with manometry o f f e r e d a simple, p r o d u c i b l e , f l e x i b l e method f o r studying metabolism. While the judgment made by Krebs and H e n s e l e i t would probably not be v a l i d today, the i n t e r v e n i n g years have seen a great deal o f f i n e wor Again, the l i v e r i s t u d i e d using t i s s u e s l i c e s . However, many other organs have a l s o been used i n a v a r i e t y o f s t u d i e s . Of p a r t i c u l a r note f o r x e n o b i o t i c metabolism s t u d i e s i n a d d i t i o n to the l i v e r a r e kidneys, lungs, and i n t e s t i n e s . The key to the p r e p a r a t i o n o f v i a b l e t i s s u e s l i c e s i s to o b t a i n r e p r o d u c i b l e , t h i n s l i c e s , g e n e r a l l y l e s s than 0.5 mm t h i c k . T h i s i s most f r e q u e n t l y done at present using a microtome or s i m i l a r instrument. The v i a b i l i t y o f the t i s s u e and s t a n d a r d i z a t i o n to determine r e p r o d u c i b i l i t y are f r e q u e n t l y evaluated using Warburg respirometry (16). Thin s l i c e techniques have been used e x t e n s i v e l y f o r the study o f p e s t i c i d e metabolism, and examples o f a v a r i e t y o f s t u d i e s f o l l o w . One o f the e a r l i e s t establishments o f the need f o r conversion of an organophosphorus i n s e c t i c i d e t o an a c t i v e a n t i c h o l i n e s t e r a s e metabolite used the l i v e r s l i c e technique i n combination with Warburg respirometry (14) . L i v e r s l i c e s were used t o demonstrate the conversion o f dimefox, a phosphoramidate, to an a c t i v e i n h i b i t o r using r a t b r a i n c h o l i n e s t e r a s e as the substrate. I n h i b i t i o n o f c h o l i n e s t e r a s e was measured i n the Warburg apparatus, and i n h i b i t i o n o f c h o l i n e s t e r a s e was taken as i n d i r e c t evidence f o r metabolism o f dimefox by the l i v e r s l i c e . Liver s l i c e s 0.5 mm t h i c k and 5 mm square, washed twice with 0.9% sodium c h l o r i d e , were used i n each Warburg f l a s k . Substrate and enzyme sources were added from s i d e arms, and c h o l i n e s t e r a s e i n h i b i t i o n was assayed by standard methodology. The h e r b i c i d e s propham and chloropropham were s t u d i e d i n the r a t in vivo and metabolism was compared i n l i v e r s l i c e s and kidney s l i c e s (13) . These h e r b i c i d e s were metabolized in vivo to two major and three minor metabolites; both o x i d a t i v e and h y d r o l y t i c mechanisms were evident. L i v e r and kidney s l i c e s , however, d i d not hydrolyze the c h a i n moiety as observed in vitro. Only l i v e r s l i c e s converted the h e r b i c i d e s to t h e i r o x i d a t i v e m e t a b o l i t e s . Rat r e n a l c o r t i c a l s l i c e s and r a t l i v e r s l i c e s were used t o assess the mechanism f o r e x c r e t i o n o f d i c h l o r o d i p h e n y l a c e t i c a c i d (DDA) from animals (18) . I t was hypothesized that DDA i s excreted
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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v i a the organic a c i d system. I t was shown that DDA was a v i d l y accumulated by l i v e r and by kidney s l i c e s . DDT o r organic bases d i d not i n h i b i t the accumulation of DDA, i n d i c a t i n g that the organic a c i d e x c r e t i o n system i s indeed r e s p o n s i b l e f o r the e l i m i n a t i o n o f DDA from the mammalian system. This u l t i m a t e l y , o f course, i s the method f o r the e l i m i n a t i o n o f DDT from the system s i n c e DDT i s f i r s t converted t o DDA before i t i s excreted. An i n t e r e s t i n g study o f c a r b a r y l metabolism has been reported using an i s o l a t e d i n t e s t i n a l t i s s u e (17) . For t h i s study the small i n t e s t i n e between the b i l e duct and the cecum was removed from male r a t s , r i n s e d i n i s o t o n i c s a l i n e , and d i v i d e d i n t o three approximately equal p a r t s . The s e c t i o n s were everted and maint a i n e d over i c e i n f r e s h s a l i n e plus glucose s o l u t i o n u n t i l the s e r o s a l compartment was f i l l e d with s e r o s a l f l u i d The f i l l e d sacs were then t r a n s f e r r e and incubated with a g i t a t i o the products o f c a r b a r y l i n c u b a t i o n i n d i c a t e d the p r o d u c t i o n o f 1-naphthol, again apparently by nonenzymatic mechanisms. At l e a s t seven metabolites were i d e n t i f i e d . The p r i n c i p a l water s o l u b l e metabolite (60%) was 1-naphthol glucuronide. Although much u s e f u l i n f o r m a t i o n about x e n o b i o t i c metabolism has been obtained with l i v e r s l i c e techniques, most workers today p r e f e r to use other methods, probably because of d i f f i c u l t i e s o f r e p r o d u c i b i l i t y u s i n g the technique. C e l l Culture Mammalian c e l l s i n c u l t u r e have been used f o r over one-half century to study v a r i o u s aspects of b i o l o g y . Since H a r r i s o n f i r s t s u c c e s s f u l l y propagated medullary t i s s u e in vitro i n 1907, c e l l c u l t u r e s have been u t i l i z e d i n the study o f r a d i o b i o l o g y , c e l l d i v i s i o n , and g e n e t i c cytology as w e l l as other areas o f c e l l b i ology. Mammalian c e l l c u l t u r e s have a l s o been used to study the c o r r e l a t i o n o f c y t o t o x i c i t y of drugs with other pharmacological a t t r i b u t e s . The primary use o f c e l l c u l t u r e s has been to p r o v i d e a method f o r i n v e s t i g a t i n g the d i r e c t a c t i o n o f drugs and other chemicals on c e l l s i n the absence o f the complex i n t e r a c t i o n s which apply i n the whole animal. A high c o r r e l a t i o n , f o r example, has been found between in vitro c y t o t o x i c i t y and in vivo antitumor a c t i v i t y o f a number of chemotherapeutic agents screened f o r a n t i cancer a c t i v i t y . A wide v a r i e t y o f c e l l types have been c u l t u r e d and maintained in vitro. Both normal c e l l s from many d i f f e r e n t organs and t i s s u e s of many d i f f e r e n t animal species and abnormal c e l l s i s o l a t e d from tumors or other abnormal t i s s u e s have been u t i l i z e d . C e l l s have been s u c c e s s f u l l y explanted from animals i n a l l stages of development, from the embryo to the a d u l t . One of the problems which must be r e c o g n i z e d i n working with c e l l c u l t u r e s i s the d i f f e r e n c e which i s l i k e l y to e x i s t between these c e l l s t r a i n s and the t i s s u e from which they o r i g i n a t e d .
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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C e l l s t r a i n s or c e l l l i n e s , so c a l l e d , c o n s i s t o f c e l l s which may have been growing in vitro f o r a c o n s i d e r a b l e length o f time and which have undergone many subcultures or d i l u t i o n s o f c e l l numbers. G e n e r a l l y they have a d i f f e r e n t p a t t e r n o f metabolism, are capable o f supporting the growth o f a wide v a r i e t y of v i r u s e s and microorganisms, and are f r e q u e n t l y p o l y p l o i d . Such d i v e r gences from normal t i s s u e must be considered when e v a l u a t i n g experimental r e s u l t s . Some o f the d i f f i c u l t i e s i n working with c e l l l i n e s are e l i m i n a t e d by working i n primary c e l l c u l t u r e s . These are c e l l s which have not undergone even a s i n g l e passage or subculture s i n c e having been explanted from the donor animal. Such c u l t u r e s have been shown to r e t a i n enzymatic a c t i v i t i e s s i m i l a r to those of the in vivo donor t i s s u e The a c t i v i t y u s u a l l y l a s t s through a few i n i t i a l subculture C e l l s o f both types are commercially a v a i l a b l e . The techniques f o r maintaining and using c e l l s are simple, although r i g o r o u s s t e r i l e technique i s a b s o l u t e l y necessary t o avoid contamination o f c e l l c u l t u r e s by invading microorganisms o f a l l types. The media used f o r c e l l c u l t u r e s are i d e a l f o r the growth of microorganisms, which, i f they contaminate the c u l t u r e s , lead to a r t i f a c t s i n the experimental r e s u l t s . Rigorous a t t e n t i o n to s t e r i l e technique i s a p r e r e q u i s i t e to accurate i n t e r p r e t a t i o n o f the r e s u l t s o f r e search u s i n g c e l l c u l t u r e s . Only a few papers appear i n the l i t e r a t u r e r e p o r t i n g the r e s u l t s of s t u d i e s o f p e s t i c i d e metabolism i n c u l t u r e d mammalian cells. The v a r i o u s s t u d i e s have used human embryonic lung L-132 c e l l s , HeLa S c e l l s , mouse f i b r o b l a s t L-929 c e l l s , and mouse L-5178 lymphoma c e l l s . A l l o f these are e s t a b l i s h e d c e l l l i n e s . In a d d i t i o n , s t u d i e s have been conducted using primary human embryonic lung c e l l s . .DDT metabolism has been s t u d i e d i n HeLa S c e l l s (22), mouse L-5178 lymphoma c e l l s (29), and primary human embryonic lung c e l l s (28). The v a r i a t i o n i n the s u s c e p t i b i l i t y o f a compound to metabolism by v a r i o u s c e l l types i s i l l u s t r a t e d i n these s t u d i e s . Mouse lymphoma c e l l s grown f o r 72 hours i n the presence o f DDT f a i l e d to metabolize the compound. At the other end of the spectrum, HeLa S c e l l s metabolized DDT to DDD, DDE, DBM, and DBP. DDE was the metabolite present i n the h i g h e s t q u a n t i t y a f t e r 24 hours o f i n c u b a t i o n and was p o s t u l a t e d t o be the t e r m i n a l metabo l i t e i n t h i s system. I t i s noted i n the paper, however, that the conversion o f DDT t o DDE could have been enhanced by the i r o n p o r p h y r i n complexes i n the medium. Primary human embryonic lung c e l l s metabolized DDT only to DDD (38%) and DDA ( 4 % ) . No other metabolites were found i n t h i s c e l l c u l t u r e system. C a r b a r y l has been s t u d i e d i n both a human embryonic lung c e l l l i n e , the L-132 s t r a i n (20, 21) and i n primary human embryonic lung c e l l s (23). The HEL c e l l l i n e was apparently a c t i v e i n conj u g a t i n g c a r b a r y l m e t a b o l i t e s . The water s o l u b l e aglycones r e -
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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s u i t i n g from beta-glucuronidase and a r y l s u l f a t a s e treatment were 4- hydroxycarbaryl and 5,6-dihydro-5,6-dihydroxycarbaryl. In a d d i t i o n , h y d r o x y l a t i o n a t the C-4 p o s i t i o n i n c o n j u n c t i o n with h y d r o l y s i s of the carbamate group r e s u l t e d i n the formation o f naphthalene-1,4-diol. The i n t e r e s t i n g m e t a b o l i t e , 1-naphthyl methylcarbamate-tf-glucuronide, was a l s o r e p o r t e d i n t h i s study. The r e s u l t s obtained i n primary HEL c e l l s agreed v e r y c l o s e l y with those from the HEL c e l l l i n e . In the primary c e l l c u l t u r e s 1-naphthol was the p r i n c i p a l m e t a b o l i t e i s o l a t e d . Others i n c l u d ed n a p h t h a l e n e - l , 4 - d i o l , naphthalene-1,5-diol, 4-hydroxycarbary1, 5- hydroxycarbaryl, and 5,6-dihydro-5,6-dihydroxycarbaryl. In a d d i t i o n s i g n i f i c a n t amounts o f the administered c a r b a r y l were present i n e x t r a c t s as conjugates. A c i d h y d r o l y s i s f r e e d 4hydroxycarbary1, naphthalene-1,4-diol, and 5,6-dihydro-5,6-dihydroxycarbaryl. On th o f the aqueous m a t e r i a i n agreement with the e a r l i e r work i n which Baron and Locke post u l a t e d the formation of an #-glucuronide i n the c e l l c u l t u r e system. The use o f both primary and e s t a b l i s h e d HEL c e l l s i s an important l i n k i n understanding the metabolism o f c a r b a r y l i n mammalian systems. The u t i l i t y and value o f c e l l c u l t u r e s t u d i e s i s i l l u s t r a t e d w e l l by a study o f dimethoate metabolism i n primary human embryo n i c lung c e l l s (27). In t h i s study the c e l l s were shown t o o x i d i z e dimethoate with no i n t e r f e r e n c e from competing h y d r o l y t i c r e a c t i o n s . Thus, the p r o g r e s s i o n o f dimethoate metabolism from the phosphorodithioate to the phosphorothioate, concomitant with o x i d a t i v e #-dimethylation o f both the phosphorodithioate and phosphorothioate, c o u l d be demonstrated (Figure 2 ) . The r e l a t i o n ships e x i s t i n g between these three metabolites and t h e i r parent compound could not be e s t a b l i s h e d so w e l l i n a system i n which h y d r o l y t i c r e a c t i o n were competing with the o x i d a t i v e ones. S t u d i e s o f the a c a r a c i d e chlorphenamidine (24), and the r e l a t e d phenylurea h e r b i c i d e s , c h l o r o t o l u r o n , fluometuron, and metobromuron, (25) revealed s t r i k i n g d i f f e r e n c e s i n the suscept i b i l i t y o f these m a t e r i a l s to metabolism by the c e l l s . On the one hand, chlorphenamidine was very s u s c e p t i b l e to o x i d a t i v e metabolism i n HEL c e l l s with the formation o f n e a r l y 82% o f the iV-formyl m e t a b o l i t e and 2% o f 4 - c h l o r o - o - t o l u i d i n e as w e l l as small q u a n t i t i e s o f other m e t a b o l i t e s . While the h e r b i c i d e s were very r e s i s t a n t t o metabolism ( l e s s than 2% o f the a p p l i e d compound was metabolized i n 72 hours i n each c a s e ) , the small q u a n t i t i e s of m e t a b o l i t e s that were formed were the r e s u l t o f o x i d a t i v e reactions i n c e l l s . I t was i n t h i s c e l l c u l t u r e system that the formation o f #-formyl d e r i v a t i v e s o f c h l o r o t o l u r o n was f i r s t observed, a r e s u l t l a t e r corroborated i n l i v e r microsomal prepa r a t i o n s and i n r a t s in vivo (26). The chromatographic behavior o f the formyl d e r i v a t i v e s made t h e i r d e t e c t i o n d i f f i c u l t i n systems which more a c t i v e l y metabolized the compound because o f i n t e r f e r i n g m a t e r i a l s on t h i n l a y e r chromatographic p l a t e s .
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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I N C U B A T I O N Pesticide Biochemistry and Physiology
Figure 2. Dimethoate disappearance, organoextractable metabolite formation, and percentage of protein increase in HEL cell cultures incubated with C-dimethoate. Disappearance of dimethoate corresponds with the appearance of the des-N-methyl metabolite and the oxygen analog, which in turn disappears corresponding with the increase of its des-N-methyl derivative (27). 14
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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C e l l c u l t u r e techniques are u s e f u l i n the study o f xenob i o t i c metabolism. They cannot be used t o e s t a b l i s h q u a n t i t a t i v e r e l a t i o n s h i p s between x e n o b i o t i c s and t h e i r m e t a b o l i t e s , p a r t i c u l a r l y when r e f e r e n c e to the whole animal i s d e s i r e d , but they can be i n v a l u a b l e i n studying the mechanisms o f metabolism and i n c h a r a c t e r i z i n g metabolites which may appear i n only minor q u a n t i t i e s i n other mammalian systems. Isolated
Hepatocytes
The p r e p a r a t i o n and use o f i s o l a t e d hepatocytes i s a r e c e n t i n n o v a t i o n i n the study o f x e n o b i o t i c metabolism and should prove to be most u s e f u l . The technique i s b a s i c a l l y a simple one. L i v e r c e l l s are d i s s o c i a t e d from the p r o t e i n matrix o f the organ and are i s o l a t e d i n v i a b l isolated c e l l s i n cultur methods have been developed f o r the i s o l a t i o n of hepatocytes, although the techniques have not yet reached the r o u t i n e stage so that a l l r e s e a r c h e r s agree on the same b a s i c methodology f o r t h e i r p r e p a r a t i o n . E a r l i e r techniques i n v o l v e d e i t h e r ( l ) t h e perf u s i o n of l i v e r with calcium c h e l a t o r s or a l k a l i n e hyperosmolar s a l t s o l u t i o n s , (2) the d i g e s t i o n of l i v e r p i e c e s i n t e t r a p h e n y l boron, a potassium c h e l a t o r , (3) the use o f enzymes as d i s s o c i a t ing agents, n o t a b l y t r y p s i n f o r f e t a l o r neonatal m a t e r i a l , and (4) collagenase/hyaluronidase d i g e s t i o n . Various combinations o f these techniques have a l s o been t r i e d (40). C u r r e n t l y there appear t o be b a s i c a l l y two methods f o r the p r e p a r a t i o n o f hepatocytes i n general use. One i n v o l v e s the p e r f u s i o n of i s o l a t e d l i v e r with Hank's b u f f e r f o r approximately 15 minutes f o l l o w e d by the a d d i t i o n of collagenase to the p e r f u s a t e f o r an a d d i t i o n a l 10 to 15 minutes. T h i s treatment c o l l a p s e s the l i v e r , a f t e r which i t i s minced, c e n t r i f u g e d , washed, and resuspended i n Hank's b u f f e r f o r immediate use (45, 46, 47). The other technique e l i m inates the n e c e s s i t y f o r p e r f u s i o n and uses enzymatic d i s s o c i a t i o n by treatment o f l i v e r with c o l l a g e n a s e / h y a l u r o n i d a s e i n Hank's balanced s a l t s o l u t i o n f o l l o w e d by c e n t r i f u g a t i o n , washing, and resuspension (32, 40). The l a t t e r technique has the advantage o f s i m p l i c i t y and low cost s i n c e no s p e c i a l p e r f u s i o n apparatus i s needed. Furthermore, i t would be p o s s i b l e to prepare hepatocytes from p i e c e s of f r e s h l i v e r which might, f o r example, be a v a i l a b l e from b i o p s i e s without the need f o r the whole organ as r e q u i r e d i n the p e r f u s i o n technique. Both techniques appear to g i v e good y i e l d s o f v i a b l e c e l l s which can be used f o r metabo l i s m s t u d i e s . As f o r most in vitro methods, most o f the e a r l y developmental work on i s o l a t i o n o f v i a b l e hepatocytes has been done with r a t l i v e r s . However, there i s no reason why the t e c h niques developed cannot be a p p l i e d to the l i v e r s o f other s p e c i e s as w e l l , perhaps with very l i t t l e m o d i f i c a t i o n . In f a c t chicken hepatocytes have been i s o l a t e d using the r a t l i v e r techniques and were shown to c a r r y out normal biochemical f u n c t i o n s (37, 38).
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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The c r i t e r i a o f v i a b i l i t y o f i s o l a t e d l i v e r c e l l s are a matter o f c o n s i d e r a b l e concern f o r metabolism s t u d i e s s i n c e r e p r o d u c i b i l i t y demands a technique f o r s t a n d a r d i z a t i o n o f prep a r a t i o n s made at d i f f e r e n t times or by d i f f e r e n t methods. The most u n i v e r s a l l y used technique to assess v i a b i l i t y o f i s o l a t e d hepatocytes i s the trypan blue e x c l u s i o n t e s t (31, 45). The t e s t depends on the f a c t that the i n t a c t plasma membrane excludes dyes such as trypan b l u e , but damaged c e l l s are s t a i n e d , p a r t i c u l a r l y i n t e n s e l y i n the nucleus. U n f o r t u n a t e l y , t h i s a c t u a l l y measures s t r u c t u r a l i n t e g r i t y , not v i a b i l i t y per se. Other t e s t s have been used i n a d d i t i o n , i n c l u d i n g e l e c t r o n microscopy, the content of adenine n u c l e o t i d e s , the a c t i v i t i e s o f v a r i o u s enzymes, and the a b i l i t y to s y n t h e s i z e v a r i o u s compounds. I t seems to t h i s reviewer that one must e s t a b l i s h f o r h i s own type o f experiments whether the c e l l s are v i a b l c a b l e to the type o f r e s e a r c a metabolism study one might choose a p a r t i c u l a r s u b s t r a t e which could be used as a p o s i t i v e c o n t r o l i n a l l p r e p a r a t i o n s , whose metabolism could be c o n v e n i e n t l y measured and q u a n t i f i e d f o r comparison between v a r i o u s p r e p a r a t i o n s . I t i s somewhat s u r p r i s i n g to r e p o r t that no s t u d i e s have yet been p u b l i s h e d i n v o l v i n g the metabolism o f a p e s t i c i d e i n i s o l a t e d hepatocytes. However, the technique has been widely ap p l i e d i n s t u d i e s of drug metabolism and s e v e r a l r e p o r t s on the metabolism o f a i r p o l l u t a n t s and i n d u s t r i a l chemicals have now appeared. Examples o f drug metabolism s t u d i e s are the r e p o r t s o f B i l l i n g s et al. (33) on α-1-acetylmethadol, propoxyphene, butamoxane, ethinimate, 8-methoxybutamoxane, and p - n i t r o p h e n o l ; E r i c k s o n and Holtzman (39) on ethylmorphine; Hayes and Brendel (42) on q u i n i n e s u l f a t e , dansylamide, and a n t i p y r i n e ; and Aarbakke et al. (30) on a n t i p y r i n e . In g e n e r a l , the drugs stud i e d were metabolized by the i s o l a t e d hepatocytes by N- and 0demethylation, aromatic and a l i p h a t i c h y d r o x y l a t i o n , and s u l f a t e and g l u c u r o n i c a c i d conjugation. These s t u d i e s show that the metabolism of drugs i n i s o l a t e d hepatocytes c o r r e l a t e s with in vivo drug metabolism b e t t e r than does the l i v e r homogenate 9000^ supernatant or microsomal f r a c t i o n . The r e s u l t s obtained from hepatocytes were more comparable t o l i v e r p e r f u s i o n s than t o sub c e l l u l a r f r a c t i o n s i n terms o f the r e l a t i v e r a t e s of i n d i v i d u a l r e a c t i o n s , which were sometimes f a s t e r and sometimes slower i n hepatocytes than i n microsomes. Aromatic hydrocarbons are r e a d i l y hydroxylated by i s o l a t e d hepatocytes (34, 35, 43). Bock and co-workers showed that naph thalene was converted to 1-naphthol and to 1 , 2 - d i h y d r o - l , 2 - d i hydroxynaphthalene and i t s s u l f a t e and g l u c u r o n i c a c i d conjugates (34). The i s o l a t e d hepatocytes were more e f f i c i e n t i n c a r r y i n g out these conversions than microsomes, the reason being that the enzymes r e s p o n s i b l e , mixed f u n c t i o n oxygenase, epoxide hydratase, and g l u c u r o n y l t r a n s f e r a s e , are a l l l o c a t e d i n the same mem branes. Benzo(a)pyrene was converted i n t o arene oxides, phenols,
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
4.
MENZER
Intact Animal
Cells,
Tissues, and
Organs
143
quinones, and d i h y d r o d i o l s as i n i t i a l products and l a t e r s u l f a t e , glucuronide, and g l u t a t h i o n e conjugates were i s o l a t e d (35). The monohydroxylated compounds and s u l f a t e e s t e r s accumulated i n t r a c e l l u l a r ^ , the 4,5- and 7,8-dihydrodiols were d i s t r i b u t e d evenl y between the c e l l s and the medium, and the 9,10-dihydrodiol accumulated i n the medium. In these experiments s i g n i f i c a n t amounts o f r a d i o a c t i v i t y were bound i r r e v e r s i b l y to c e l l u l a r macromolecules (43). The s e q u e n t i a l formation o f metabolites by i s o l a t e d hepatocytes i s i l l u s t r a t e d by s t u d i e s on the 4-hydroxylation o f b i phenyl and the subsequent conjugation o f the metabolite (44). F i g u r e 3 i l l u s t r a t e s the f a c t that h y d r o x y l a t i o n preceded g l u curonide formation and that the removal o f 4-hydroxybiphenyl by conjugation was necessary to s t i m u l a t e a second phase o f hydroxylation. Fry et at. (41) hav and kidney i n terms o f t h e i r a b i l i t y to metabolize ethoxycoumarin, b i p h e n y l , benzo(a)pyrene, 4-methylumbelliferone, and benzoic a c i d . The l e v e l o f metabolism i n the kidney c e l l suspension was extremely low compared with l i v e r c e l l s f o r i n i t i a l o x i d a t i v e r e a c t i o n s , but the p a t t e r n and extent o f conjugations were very s i m i l a r between the two types o f c e l l s . The d i f f e r e n c e i n the a b i l i t y o f these c e l l s t o metabolize these compounds may r e l a t e to a d i f f e r e n t s p e c i f i c i t y o f the r e n a l cytochrome P-450, which i s not adapted to x e n o b i o t i c metabolism. The a b i l i t y t o r e t a i n a c t i v e cytochrome P-450 i n i s o l a t e d hepatocytes i s c r u c i a l t o the use o f t h i s system to study xenob i o t i c metabolism. Cytochrome P-450 d e c l i n e s r a p i d l y i n i s o l a t e d hepatocytes, but r e c e n t l y i t was discovered that the a d d i t i o n o f c e r t a i n hormones t o a primary hepatocyte c u l t u r e r e t a i n e d the cytochrome P-450 a c t i v i t y at optimum l e v e l s f o r up to 24 hours (36). These workers used primary*hepatocyte c u l t u r e s der i v e d from collagenase p r e p a r a t i o n with added hormones t o study the metabolism o f a f l a t o x i n B^. Although i s o l a t e d hepatocytes have not as yet been extens i v e l y used i n metabolism s t u d i e s , they c l e a r l y o f f e r great promise i n t h i s area. Conclusion When one compares the v a r i o u s in vitro techniques used t o study the f a t e o f x e n o b i o t i c s i n mammals, one must be impressed by the f a c t that no one o f the methods a v a i l a b l e i s adequate to a complete understanding o f the metabolism o f a compound. However, when a l l methods are taken together, a r a t h e r complete p i c t u r e can be assembled. Using c a r b a r y l , one o f the most e x t e n s i v e l y studied p e s t i c i d e s , as an example, one can see that the t o t a l in vitro r e s u l t s r a t h e r completely m i r r o r in vivo metabolism. In f a c t , c e r t a i n metabolites found in vitro have not been i s o l a t e d f o l l o w i n g in vivo s t u d i e s , the N-glucuronide noted from c e l l
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC METABOLISM
144
«
Biochimica et Biophysica Acta
Figure 3. Rate of formation of 4-hydroxy biphenyl and its glucuronide conjugate in viable isolated hepatocytes. The rates of formation of (O), 4-hydroxy biphenyl and (U)> Ms glucuronide conjugate are average rates for the respective 5-min intervab. Each point represents the mean of three values obtained from different experiments (± S.D.) (44).
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
MENZER
4.
Intact Animal
Cells,
Tissues, and
145
Organs
c u l t u r e s . The p r i n c i p a l m e t a b o l i t e , 1-naphthol, i s found i n a l l systems s t u d i e d . The 5,6-dihydro-5,6-diol, 5-hydroxy, 1,5-diol sequence i s observed i n c e l l c u l t u r e s , while the 4-hydroxy metab o l i t e i s observed i n perfused organs and c e l l c u l t u r e s . The 1,4d i o l was a l s o i s o l a t e d from c e l l c u l t u r e s as were s e v e r a l g l y c o s i d e conjugates. One might compare the r e l a t i v e merits o f the f o u r techniques considered i n t h i s paper, adding microsomes to complete the p i c t u r e , from f o u r p o i n t s o f view: (1) ease of p r e p a r a t i o n , (2) f l e x i b i l i t y o f experimental use, (3) r e p r o d u c i b i l i t y o f r e s u l t s , and (4) the extent to which the r e s u l t s obtained m i r r o r in vivo r e s u l t s (Table I ) . Table I. R e l a t i v e merits o f in vitro f o r studying th
techniques
r+
Ο
2ί H'
Ο
Η ο (Λ Ο
Β
CD (Λ
Η
3
(Λ (Λ
"Ο Φ Η
Favorable, p o s i t i v e aspect o f t h i s Neutral Negative aspect o f t h i s technique
C Φ
tn
t-h
t—»
C (Λ Η· Ο 3
Ease o f P r e p a r a t i o n Requirement f o r S p e c i a l Equipment Requires s p e c i a l t r a i n i n g o f personnel Time f o r p r e p a r a t i o n Expense F l e x a b i l i t y o f Experimental Use R e p r o d u c i b i l i t y o f Results M i r r o r s in vivo r e s u l t s + ο -
CD Ο*
OQ
-
Η· Ο Φ «Λ
Φ t—' h-*
η
c
Κ-* Γι C
Η Φ
+
0
SC CD »Ί3 Ρ Γ+
ο Ο Χ
Γ+
Φ (Λ
0
ο
+ +
ο +
+
+
-
+
ο +
ο
+ +
+ +
ichnique
Although the assignment o f values f o r each category i s a r b i t r a r y and s u b j e c t i v e , one must conclude from t h i s e x e r c i s e that each technique w i l l be u s e f u l f o r some purposes i n some r e s e a r c h e r ' s hands. U l t i m a t e l y our understanding o f the f a t e o f x e n o b i o t i c s i n mammals w i l l be enhanced by accumulating data from a l l sources and a p p l y i n g each b i t o f data to o b t a i n the complete p i c t u r e . Acknowledgement S c i e n t i f i c A r t i c l e No. A2519 C o n t r i b u t i o n No. 5551 o f the Maryland A g r i c u l t u r a l Experiment S t a t i o n , Department of Entomology.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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XENOBIOTIC METABOLISM
The author thanks Miss Figen Un111 f o r her a s s i s t a n c e i n the pre p a r a t i o n o f t h i s paper and Dr. Judd 0. Nelson f o r h i s h e l p f u l advice i n i t s development.
Abstract Techniques considered include isolated whole living cells; cell, organ, and tissue culture; organ slices; and isolated per fused organs. Whole cell techniques serve as an important link between studies using purified enzymes and subcellular fractions and studies using whole organisms. The use of cells, tissues, and organs in culture is growing since they allow the researcher to explore the nature of metabolites and to research an under standing of the mechanisms of metabolism taking place within the cells or organs without the whole organism. Bot ed cell lines have been used to study xenobiotic degradation. Conversely, the effects of xenobiotics on the cell can also be conveniently studied. The combination of the two types of stud ies allows one to ascertain whether the cell's metabolism of a xenobiotic is accomplished by a healthy cell or as the result of or in combination with some cellular defect. The use of whole organs, such as perfused liver, provides the opportunity to ex tend a metabolism experiment over a longer period of time than is possible with either subcellular fractions or isolated cells. Whole organs or organ slices allow the introduction of a higher degree of cellular organization and differentiation than the single cell, but without the complications of external regulation. The use of isolated hepatocytes is a recent innovation in the study of xenobiotic metabolism and should be most useful. Literature Cited Perfused Organs 1.
Blase, B. W., and Loomis, T. A. Toxicol. Appl. Pharmacol. (1976) 37, 481. 2. Dalbey, W., and Bingham, E. Toxicol. Appl. Pharmacol. (1978) 43, 267. 3. Fuhremann, T. W., Lichtenstein, E. P., Zahlten, R. Ν., Stratman, F. W., and Schnoes, H. K. Pestic. Sci. (1974) 5, 31. 4. Law, F. C. P., Eling, T. W., Bend, J. R., and Fouts, J. R. Drug Metab. Disp. (1974) 2, 433. 5. Mehendale, H. M. Toxicol. Appl. Pharmacol. (1976) 36, 369. 6. Mehendale, H. M., and El-Bassiouni, Ε. A. Drug Metab. Disp. (1975) 3, 543. 7. Miller, L. L., Bly, C. G., Watson, M. L., and Bale, W. F. J. Exp. Med. (1951) 94, 431.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
4.
MENZER
Intact Animal
Cells,
Tissues, and
Organs
147
8. Niemeier, R. W., and Bingham, E. Life Sci. (1972) 11, part II, 807. 9. Orton, T. C., Anderson, M. W., Pickett, R. D., Eling, T. C., and Fouts, J. R. J. Pharmacol. Exp. Therap. (1973) 186, 482. 10. Popov, Τ. Α., and Kagan, Y. S. Gig. Sanit. (1977) 40. [CA 87:904] 11. Ritchie, H. D., and Hardcastle, J. C. (eds.) "Isolated Organ Perfusion", University Park Press, Baltimore, 1973. 12. Ross, B. D., "Perfusion Techniques in Biochemistry", Clarendon Press, Oxford, 1972. Tissue
Slices
13. Fang, S. C., Fallin Pharmacol. (1973 14. Fenwick, M. L., Barron, J. R. and Watson, W. A. Biochem. J. (1957) 65, 58. 15. Krebs, Η. Α., and Henseleit, K. Hoppe-Seyler's Ζ. Physiol. Chem. (1932) 210, 33. 16. O'Neil, J. J., Sanford, R. L., Wasserman, S., and Tierney, D. F. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. (1977) 43, 902. 17. Pekas, J. C. Am. J. Physiol. (1971) 220, 2008. 18. Pritchard, J. B. Toxicol. Appl. Pharmacol. (1976) 38, 621. 19. Warburg, O. Biochem. Z. (1923) 142, 317. Cell Culture 20. Baron, R. L., and Locke, R. K. Bull. Environ. Contam. Toxicol. (1970) 5, 287. 21. Locke, R. K., Bastone, V. B., and Baron, R. L. J. Agr. Food Chem. (1971) 19, 1205. 22. Huang, Ε. Α., Lu, J. Y., and Chung, R. A. Biochem. Pharmac. (1970) 19, 637. 23. Lin, Τ. Η., North, Η. Η., and Menzer, R. E. J. Agr. Food Chem. (1975) 23, 253. 24. Lin, Τ.Η.,North, Η. Η., and Menzer, R. E. J. Agr. Food Chem. (1975) 23, 257. 25. Lin, Τ. Η., Menzer, R. Ε., and North, H. H. J. Agr. Food Chem. (1976) 24, 756. 26. Muecke, W., Menzer, R. Ε., Alt, K. O., Richter, W., and Esser, H. O., Pestic. Biochem. Physiol. (1976) 6, 430. 27. North, Η. Η., and Menzer, R. E. Pestic. Biochem. Physiol. (1972) 2, 278. 28. North, Η. Η., and Menzer, R. E. J. Agr. Food Chem. (1973) 21, 509. 29. Spalding, J. W., Ford, E., Lane, D., and Blois, M. Biochem. Pharmacol. (1971) 20, 3185.
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Isolated Hepatocytes 30. Aarbakke, J., Bessesen, Α., and Morland, J. Acta Pharmacol. Toxicol. (1977) 41, 225. 31. Baur, Η., Kasperek, S., and Pfaff, E. Hoppe-Seyler's Ζ. Physiol. Chem. (1975) 356, 827. 32. Bellemann, P., Gebhardt, R., and Mecke, D. Anal. Biochem. (1977) 81, 408. 33. Billings, R. Ε., McMahon, R. Ε., Ashmore, J., and Wagle, S. R. Drug Metab. Disposition (1977) 5, 518. 34. Bock, K. W., VanAckeren, G., Lorch, F., and Birke, F. W. Biochem. Pharmacol. (1976) 25, 2351. 35. Burke, M. D., Vodi, H., Jernström, B., and Orrenius, S. J. Biol. Chem. (1977) 252, 6424. 36. Decad, G. M., Hsieh Biophys. Res. Commun 37. Dickson, A. J., and Langslow, D. R. Biochem. Soc. Trans. (1975) 3, 1034. 38. Dickson, A. J., and Langslow, D. R. Biochem. Soc. Trans. (1977) 5, 983. 39. Erickson, R. R., and Holtzman, J. L. Biochem. Pharmacol. (1976) 25, 1501. 40. Fry, J. R., Jones, C. Α., Wiebkin, P., Bellemann, P., and Bridges, J. W. Anal. Biochem. (1976) 71, 341. 41. Fry, J. R., Wiebkin, P., Kao, J., Jones, C. Α., Gwynn, J., and Bridges, J. W. Xenobiotica (1978) 8, 113. 42. Hayes, J. S., and Brendel, K. Biochem. Pharmacol. (1976) 25, 1495. 43. Jones, C. Α., Moore, B. P., Cohen, G. Μ., Fry, J. R., Biochem. Pharmacol. (1978) 27, 693. 44. Jones, R. S., Mendis, D., and Parke, D. V. Biochim. Biophys. Acta (1977) 500, 124. 45. Seglen, P. O. "Methods in Cell Biology", D. M. Prescott (ed.), Vol. 8, Academic Press, New York, 1976, pp. 29-83. 46. Wagle, S. R. Life Sci. (1975) 17, 827. 47. Wagle, S. R., and Ingebretsen, Jr., W. R. Methods in Enzymol. (1975) 35, 579. RECEIVED
December 2 0 , 1978.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
5 The Use of Animal Subcellular Fractions to Study Type I Metabolism of Xenobiotics R O N A L D W. ESTABROOK, JURGEN and J U L I A N A . P E T E R S O N
1
WERRINGLOER,
Department of Biochemistry, Southwestern Medical School, University of Texas Health Science Center, Dallas, TX 75235
The genius of man ha r e s u l t e d i th synthesi f t number of organic compound to seek new drugs, s y n t h e t i polymer , i n s e c t i c i d e s and p e s t i c i d e s — to name but a few areas of chemical development — has provided both b e n e f i c i a l as w e l l as d e t r i m e n t a l r e s u l t s which touch the l i v e s of each of us. The l a s t decade has seen a surge of a c t i v i t y d i r e c t e d toward the e v a l u a t i o n of a wide a r r a y of organic chemicals which have the p o t e n t i a l of n e g a t i v e l y modifying the homeostasis of c e l l s and organs. Nearly every day the popular press i d e n t i f i e s yet another agent which can be shown i n experimental animals or b a c t e r i a l t e s t systems t o cause mutagenesis and c e l l u l a r dysf u n c t i o n or n e o p l a s i a . I t i s now w e l l e s t a b l i s h e d that many of these chemicals are merely precursors of metabolic products which a r e the true causative agents of c e l l u l a r changes. Therefore considerable e f f o r t i s now being expended to i d e n t i f y and c h a r a c t e r i z e the r e q u i s i t e enzyme systems r e s p o n s i b l e f o r the metabolic transformation of such chemicals. The purpose of t h i s p r e s e n t a t i o n i s to review the methodology employed and the r e s u l t s obtained when studying one such enzyme system — an enzyme complex which has a broad s p e c i f i c i t y f o r the o x i d a t i v e a l t e r a t i o n of a v a r i e t y of both n a t u r a l and s y n t h e t i c organic chemicals. C e n t r a l to t h i s enzyme complex i s a f a m i l y of hemeproteins c a l l e d cytochromes P-450. I t i s the p r o p e r t i e s and r e a c t i o n s of t h i s type of hemeprotein and i t s a s s o c i a t e d e l e c t r o n transport c a r r i e r p r o t e i n s which w i l l serve as the primary emphasis of the present r e p o r t .
1
Supported in part by a research grant from the United States P u b l i c Health S e r v i c e N a t i o n a l I n s t i t u t e s of Health (GM-16488).
0-8412-0486-l/79/47-097-149$07.75/0 © 1979 American Chemical Society
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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The
C h a r a c t e r i z a t i o n of Cytochrome P-450
Cytochrome P-450 i s widely d i s t r i b u t e d i n nature spanning the phylogenetic t r e e from b a c t e r i a to the human organism. I t has been observed i n yeast, f l o w e r i n g p l a n t s , i n s e c t s , rept i l e s , b i r d s and animals (1,2). Indeed, i t i s i n t e r e s t i n g that the f i r s t r e p o r t of cytochrome P-450 occurred as a consequence of studying the r e s p i r a t o r y pigments of the mid-gut of the silkworm (3). The ease of d e t e c t i n g cytochrome P-450, using spectrophotometric methods to assess the c h a r a c t e r i s t i c absorbance band of the carbon monoxide adduct of the reduced hernep r o t e i n , has g r e a t l y f a c i l i t a t e d the q u a n t i t a t i v e e v a l u a t i o n of t h i s c e l l u l a r pigment (4,5_). In organisms other than some s p e c i a l i z e d b a c t e r i a , cytochrome P-450 i s a membrane bound p r o t e i n — a property whic r e f r a c t o r y to i s o l a t i o now a v a i l a b l e , however, which permit the s o l u b i l i z a t i o n and s t a b i l i z a t i o n of the hemeprotein p e r m i t t i n g i t s c h a r a c t e r i z a t i o n as a homogeneous p r o t e i n s p e c i e s . I t i s p o s s i b l e to g e n e r a l i z e and i d e n t i f y two b a s i c types of cytochrome P-450 depending on the a s s o c i a t e d e l e c t r o n transport c a r r i e r proteins responsible f o r t r a n s f e r r i n g electrons (reducing e q u i v a l e n t s ) from reduced p y r i d i n e n u c l e o t i d e s to the hemeprotein. As shown i n F i g u r e 1, we have designated these as C l a s s A o r B. The C l a s s A type of cytochrome P-450 i s found a s s o c i a t e d w i t h the mitochrondria of many c e l l types where i t f u n c t i o n s together with an i r o n - s u l f u r p r o t e i n and a f l a v o p r o t e i n (6). The r e a c t i o n s of c h o l e s t e r o l metabolism i n s t e r o i d o g e n i c organs such as the adrenal, t e s t i s , and placenta are c a t a l y z e d by t h i s c l a s s of cytochromes P-450.
ClQSS Reduced Pyridine Nucleotide
h
A
Flavonrntein _Jron-Sulfur ^ Protein ,
a
v
o
ÇlQSS
p
r
o
t
e
m
^ Cytochrome P-450
Β
P y r i ^ N u c l e o t i d e - - Flavoprofin (FMN.FAD) V
* /
Cytochrome bs Figure 1. Two general classes of cytochrome F-450 based on the type of electron carriers functional in the transfer of reducing equivalents from reduced pyridine nucleotides
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
5.
ESTABROOK ET A L .
Type I
Metabolism
151
Best c h a r a c t e r i z e d (_7,8) are the r e a c t i o n s f o r the o x i d a t i v e degradation of camphor by the bacterium Pseudomonas putida where t h i s c l a s s of cytochrome P-450 p l a y s a key r o l e i n the i n i t i a t i o n of the metabolism of t h i s complex molecule. The Class Β type of cytochromes P-450 i s g e n e r a l l y assoc i a t e d with the endoplasmic r e t i c u l u m (or i t s membrane equiva l e n t ) where i t f u n c t i o n s i n concert with a f l a v o p r o t e i n which contains both FMN and FAD as p r o s t h e t i c groups (9-11). This c l a s s of cytochrome P-450 i s g e n e r a l l y found a s s o c i a t e d with a second hemeprotein, cytochrome b . Although the r o l e of cytochrome b<- i n the c y c l i c f u n c t i o n of cytochrome P-450 (see below) i s c o n t r o v e r s i a l , the p a r a l l e l i s m of c e l l u l a r a s s o c i a t i o n lends credence to i t s p o s s i b l e a c t i o n as an e l e c t r o n transport c a r r i e r required f o reductio f intermediat formed during the cours The d e s c r i p t i o n provide pres e n t a t i o n w i l l emphasize those r e a c t i o n s of cytochrome P-450 which occur during i t s i n t e r a c t i o n with the s u b s t r a t e to be metabolized as w e l l as molecular oxygen. A common p a t t e r n has emerged f o r the sequence of r e a c t i o n steps i n v o l v i n g cytochrome P-450 whether i t i s the pigment of e i t h e r Class A or B. The p r i n c i p a l d i f f e r e n c e r e s i d e s i n the manner of donation of e l e c t r o n s transported from reduced p y r i d i n e n u c l e o t i d e r a t h e r than the r e a c t i o n intermediates formed during oxygen a c t i v a t i o n and substrate transformation. s
Methodologies of Study An e v a l u a t i o n of the r e a c t i o n s o c c u r r i n g during the mixed f u n c t i o n o x i d a t i o n of many x e n o b i o t i c s r e q u i r e s a s u i t a b l e source of the enzyme system as w e l l as appropriate instrumental apparatus. The most f r e q u e n t l y employed enzyme system which has been studied i n g r e a t e s t d e t a i l i s the one a s s o c i a t e d with the microsomal f r a c t i o n of l i v e r . T h i s organ source i s r i c h i n the e l e c t r o n t r a n s p o r t c a r r i e r s which f u n c t i o n i n the a c t i v a t i o n of oxygen f o r x e n o b i o t i c metabolism, i n part because of the r o l e of the l i v e r i n s e r v i n g as the primary r e c i p i e n t of chemicals absorbed from the i n t e s t i n a l t r a c t . Further the r e q u i s i t e enzyme c o n s t i t u e n t s , notably cytochrome P-450, are r a p i d l y synthesized i n l i v e r as a r e s u l t of exposure of animals to v a r i o u s inducing agents such as b a r b i t u r a t e s or p o l y c y l i c hydrocarbons. Thus, the content and composition of cytochromes and f l a v o p r o t e i n s a s s o c i a t e d with l i v e r microsomes can be r e a d i l y a l t e r e d to permit b i o l o g i c a l l y d i f f e r i n g c o n d i t i o n s which a s s i s t i n the d e l i n e a t i o n of the p a t t e r n of enzymatic reactions occurring. Studies with l i v e r microsomes have served as the p a t t e r n f o r comparable s t u d i e s of x e n o b i o t i c metabolism i n other t i s s u e s such as the lung, kidney, i n t e s t i n e , etc.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC METABOLISM
152
The P r e p a r a t i o n of L i v e r Microsomes. Rather standard techniques of d i f f e r e n t i a l c e n t r i f u g a t i o n of l i v e r homogenates have been developed f o r r o u t i n e use i n the p r e p a r a t i o n of microsomal f r a c t i o n s f o r study. Most important i s the need to recognize the multitude of f a c t o r s which can i n f l u e n c e the subsequent experimental r e s u l t s obtained when u s i n g t h i s source of the enzyme system. The i n v e s t i g a t o r should be cognizant of the v a r i a t i o n s introduced when using animals that are not c o n t r o l l e d f o r a l t e r a t i o n s r e s u l t i n g from d i e t , inadvertent exposure to inducers (such as i n s e c t i c i d e s commonly used i n d i s c r i m i n a t e l y i n animal q u a r t e r s ) and s t r e s s to name but a few. F u r t h e r , the choice of age, sex, s p e c i e s , and g e n e t i c c h a r a c t e r i s t i c s a l l introduce v a r i a b l e s which l e a d t d i f f e r e n c e i th enzymati charac t e r i s t i c s measured. In general animal decapitate perfuse i n s i t u with c o l d i s o t o n i c s a l i n e to remove as much blood as p o s s i b l e . A f t e r homogenization i n the presence of a polyhydroxy compound, such as 0.25 M sucrose, the microsomal f r a c t i o n i s i s o l a t e d by d i f f e r e n t i a l c e n t r i f u g a t i o n a f t e r f i r s t removing unbroken c e l l s , n u c l e i , and mitochondria. I f precautions are taken, the enzyme a c t i v i t y of microsomes i s o l a t e d i n t h i s way remains r e l a t i v e l y s t a b l e f o r 48 to 72 h r s . The Spectrophotometric A n a l y s i s of Microsomal Pigments. The microsomal f r a c t i o n i s composed of membrane v e s i c l e s o r i g i n a t i n g from d i s r u p t i o n of the endoplasmic r e t i c u l u m . Therefore s p e c i a l instrumentation should be employed to accur a t e l y evaluate the spectrophotometric p r o p e r t i e s of the hemeproteins a s s o c i a t e d with these t u r b i d membrane suspensions. A number of d i f f e r e n t types of commercial spectrophotometers are a v a i l a b l e f o r the measurement of d i f f e r e n c e s p e c t r a when using samples that have h i g h l i g h t s c a t t e r i n g c h a r a c t e r i s t i c s . A schematic diagram f o r one such instrument i s shown i n F i g u r e 2. B a s i c a l l y , the p r i n c i p a l i n v o l v e d r e q u i r e s l i g h t from a tungsten f i l a m e n t lamp to be a p p r o p r i a t e l y attenuated and d i s p e r s e d by a s p l i t g r a t i n g to generate two beams of monochromatic l i g h t which can impinge on a p a i r of cuvettes cont a i n i n g the membrane fragments to be analyzed. These cuvettes are p o s i t i o n e d near the s u r f a c e of the l i g h t d e t e c t o r to i n c l u d e the g r e a t e s t s o l i d angle of s c a t t e r e d transmitted light. S u i t a b l e e l e c t r o n i c demodulation permits the accurate e v a l u a t i o n of d i f f e r e n c e s i n l i g h t a b s o r p t i o n between the pigments i n a sample cuvette minus those i n the r e f e r e n c e cuvette. Coupled together with wavelength scanning c a p a b i l i t i e s , the d i f f e r e n c e absorbance spectrum can be r e a d i l y obtained. In a d d i t i o n to the c a p a b i l i t y of r e c o r d i n g d i f ference s p e c t r a , Using the s p l i t - b e a m mode, such an i n s t r u ment can be m o d i f i e d to measure the k i n e t i c s of absorbance changes at a predetermined wavelength, r e l a t i v e to a M
,!
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
5.
ESTABROOK ET A L .
Type
I
Metabolism
DUOCHROMATOR
TELESCOPE MIRROR „
COLLIGATING MIRROR
GRATING
V
EXIT SLIT\ CUVETTES
ENTRANCE SLIT
V P. M. TUBE
Ë^Ûf
CELL « COMP ARTMENT
LIGHT SOURCE
OPTICAL^---
" l \ M M
I BENCH
5
LIGHT BEAM ALTERNATOR
M fv~ OPTICAL ATTENUATOR
American Instrument Company Figure
2. Representation of the duochromator optical configuration of the Aminco-DW2a spectrophotometer in the split-beam mode (12)
reference wavelength ( u s u a l l y an i s o s b e s t i c p o i n t ) , as a f u n c t i o n of time. A number of a r t i c l e s have been w r i t t e n d e s c r i b i n g the a p p l i c a t i o n of t h i s methodology and many of the p i t f a l l s and l i m i t s which may i n f l u e n c e the i n t e r p r e t a t i o n of such spectrophotometric techniques (13,14). Determination of Reaction Products. Measurements of the o v e r a l l r a t e s of x e n o b i o t i c metabolic transformations have been l i m i t e d i n the past to the c o l o r i m e t r i c e v a l u a t i o n of products that can be r e a d i l y d e r i v a t i z e d o r made r e a c t i v e i n coupled enzymatic o x i d a t i o n - r e d u c t i o n r e a c t i o n s . For example, formaldehyde i s e a s i l y measured c o l o r i m e t r i c a l l y during the Nor O-demethylation of many drug substrates (15). Other compounds, such as p o l y c y c l i c hydrocarbons, form f l u o r e s c e n t products during metabolism and these can be r e a d i l y q u a n t i t a t i v e l y measured (16,17,18). In other cases r a d i o a c t i v e compounds can be employed and use of d i f f e r e n t i a l s o l u b i l i t y , as a consequence of the more h y d r o p h i l i c c h a r a c t e r i s t i c s of the metabolic products, provides an a d d i t i o n a l means of e v a l u a t i n g r e a c t i o n r a t e s (19).
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
154
XENOBIOTIC METABOLISM
In recent years the i n t r o d u c t i o n of high pressure l i q u i d chromatography has opened a new and powerful method f o r product identification. T h i s method i s r a p i d , s e n s i t i v e , and, w i t h proper precautions, q u a n t i t a t i v e . One of the major new cont r i b u t i o n s to our knowledge of x e n o b i o t i c metabolism, which has r e s u l t e d from the a p p l i c a t i o n of HPLC methods, i s the r e c o g n i t i o n that m u l t i p l e products are formed and that i n i t i a l products formed may be r a p i d l y f u r t h e r metabolized to secondary or t e r t i a r y products (20,21,22). The more general a p p l i c a t i o n of t h i s methodology w i l l undoubtedly have a profound i n f l u e n c e on the current r a t h e r s i m p l i s t i c viewpoint which p r e v a i l s regarding the o x i d a t i v e transformation of many organic compounds. The
C y c l i c Function
of Cytochrome P-450
The primary f u n c t i o with molecular oxygen i n a manner that permits the cleavage of the oxygen molecule to form an atom of oxygen with e l e c t r o philic characteristics. This " a c t i v a t e d oxygen" i s then presumed to r e a c t with a molecule of the organic s u b s t r a t e , which i s at or near the s i t e of oxygen bound to the hemeprot e i n , so that the substrate molecule i s transformed to a higher o x i d a t i o n s t a t e . T h i s may be evaluated, f o r example, by the formation of an epoxide w i t h aromatic compounds, by the Nor 0 - d e a l k y l a t i o n of secondary or t e r t i a r y amines as w e l l as e t h e r s , or by the i n c o r p o r a t i o n of oxygen i n t o alkanes. Many of the r e a c t i o n steps f o r the mixed f u n c t i o n o x i d a t i o n of s u b s t r a t e s have been d e l i n e a t e d and the r e a c t i v e intermediates characterized. The f o l l o w i n g d e s c r i p t i o n b r i e f l y summarizes our current s t a t e of knowledge of these r e a c t i o n s . R
Figure 3
The I n t e r a c t i o n of Substrate. The i n i t i a l step i n the f u n c t i o n of cytochrome P-450 i s the binding of the organic s u b s t r a t e to be metabolized to the o x i d i z e d form of the hemep r o t e i n , as i l l u s t r a t e d i n F i g u r e 3. Associated with t h i s i n t e r a c t i o n i s a change i n the p r o p e r t i e s of the heme of c y t o chrome P-450. In the absence of a substrate molecule, the heme i s i n the low-spin, hexacoordinate form and i t i s c h a r a c t e r i z e d by an o p t i c a l absorbance spectrum with a major absorbance band whose maximum i s about 418 nm (Figure 4). The presence of a s i n g l e unpaired e l e c t r o n i n the f e r r i c i r o n of low s p i n c y t o -
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
5.
ESTABROOK E T A L .
Cy».P-450p.
Type
I
Metabolism
155
puli-e
- - - CAMPHOR - FREE , CAMPHOR-BOUND (PetefSon,J.A. Arch.Biochtm. "4:678(1971» V
Ο
0.00
Figure 4. Comparison of the optical spectral characteristics of two forms of ferric Cytochrome P-450. PB microsomes: 4 μ Μ Cyt.P-450 Ι50μΜ BENZPHE Τ AMINE — S 400
500 WAVELENGTH
600 {nm)
Acta Biologica et Medica Germanica
(Top) The absolute spectra of soluble Cyto chrome P-450 isolated from Pseudomonas putida in the presence and absence of the substrate camphor (23). (Bottom) The dif ference spectra obtained when benzphetamine interacts with Cytochrome P-450 of liver microsomes (24).
AS PREPARED
PLUS METYRAPONE
3000 GAUSS
Annals of the New York Academy of Sciences Figure 5. EPR spectra of Cytochrome P-450 of liver microsomes and the influ ence of ethanol and the dipyridyl derivative, metyrapone. Rat liver microsomes from phénobarbital treated animals were examined using an E-4 Varian EPR spectrometer (29).
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
156
XENOBIOTIC METABOLISM
chrome P-450 renders i t paramagnetic. This i s r e f l e c t e d by an e l e c t r o n paramagnetic resonance spectrum (shown i n i t s f i r s t d e r i v a t i v e form) of the type i l l u s t r a t e d i n F i g u r e 5. Cons i d e r a b l e i n t e r e s t and debate has centered on the i n t e r p r e t a t i o n of t h i s EPR spectrum and the p o s s i b l e presence of unique l i g a n d s , such as s u l f u r , coordinated to the heme of cytochrome P-450 (25^26,27,28). The a d d i t i o n of an excess of s u b s t r a t e (R) to be metabo l i z e d , to cytochrome P-450 r e s u l t s i n a marked change i n the absorbance spectrum of the f e r r i c hemeprotein (Figure 4) l e a d i n g to a pigment w i t h i t s major absorbance band l o c a t e d at about 390 nm. The b a s i s f o r t h i s hypsochromic e f f e c t of the s u b s t r a t e on the s p e c t r a l p r o p e r t i e s of o x i d i z e d cytochrome P450 i s r e l a t e d to the s p i n s t a t e change of the heme i r o n This i s confirmed by the appearanc of about 8 when the s u b s t r a t chrome P-450 i s examined by low temperature EPR spectroscopy methods (30,31). One i n t e r p r e t a t i o n of these changes i n the p h y s i c a l p r o p e r t i e s of the heme p r o t e i n i s the b i n d i n g of the organic s u b s t r a t e molecule i n the h y d r o p h i l i c heme pocket of the hemeprotein, thereby p e r t u r b i n g the e l e c t r o n d e n s i t y d i s t r i b u t i o n of the heme to form the pentacoordinate, h i g h - s p i n complex (32,33). Such an i n t e r p r e t a t i o n (supported by s t u d i e s u s i n g s p i n - l a b e l e d organic compounds (34)) would p l a c e the s u b s t r a t e molecule i n c l o s e proximity to the heme i r o n and thus i n a s p a t i a l c o n f i g u r a t i o n where i t i s a p p r o p r i a t e l y l o c a t e d f o r i n t e r a c t i o n w i t h the " a c t i v e oxygen" generated during the c y c l e of cytochrome P-450 f u n c t i o n . The o p t i c a l s p e c t r a l changes a s s o c i a t e d w i t h s u b s t r a t e b i n d i n g provide a r a t h e r simple technique to measure t h i s f i r s t step i n cytochrome P-450 a c t i o n . Since many systems under study i n v o l v e the membrane bound form of cytochrome P-450, the technique of d i f f e r e n c e absorbance spectrophotometry has been a p p l i e d (see d i s c u s s i o n i n S e c t i o n I I ) . As i l l u s t r a t e d i n F i g u r e 6, the a d d i t i o n of i n c r e a s i n g c o n c e n t r a t i o n s of a subs t r a t e ( i n t h i s example, the s t e r o i d androstanedione has been added to a cytochrome P-450 c o n t a i n i n g p r e p a r a t i o n of l i v e r microsomes) r e s u l t s i n a p r o g r e s s i v e decrease i n absorbance at about 420 nm concomitant with an i n c r e a s e i n absorbance at about 385 nm. This form of absorbance change has been termed "Type I " (36) and many s u b s t r a t e s metabolized by cytochrome P-450 can be shown to cause t h i s a l t e r a t i o n i n the s p e c t r a l p r o p e r t i e s of the hemeprotein. In a number of i n s t a n c e s , the magnitude of s p e c t r a l change observed can be r e l a t e d to the c o n c e n t r a t i o n of s u b s t r a t e added to the r e a c t i o n mixture and one can then estimate the a f f i n i t y of cytochrome P-450 f o r each type of s u b s t r a t e examined. However, a s a t i s f a c t o r y i n t e r p r e t a t i o n of s u b s t r a t e i n t e r a c t i o n w i t h f e r r i c cytochrome P-450 (based s o l e l y on o p t i c a l absorbance d i f f e r e n c e spectrophotometry of the membrane
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
5.
ESTABROOK ET A L .
Type I
Metabolism
157
.387
CONCENTRATION ANDROSTANEDIONE
ο
6.6/JM
Liver microsomes from phénobarbital treated suspended
19.8 330 "
350
400
Figure 6. Effect of increasing concen trations of substrate on the optical den sity changes observed during the spin state transition of ferric Cytochrome P-450.
tents of the sample cuvette and the resultant difference spectra recorded.
450
WAVELENGTH(nm)
enzyme) i s sometimes l a c k i n g . This i s due i n part t o complic a t i o n s a r i s i n g from the presence of "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 membrane fragments (24, 37-40), extraneous s o l v e n t e f f e c t s which presumably a l t e r the hydrophobic heme environment of the hemeprotein (41,42), as w e l l as temperature dependent changes i n the p r o p e r t i e s of the membrane (31). The general concept that o x i d i z e d cytochrome P-450 r e a c t s w i t h the s u b s t r a t e t o be metabolized as the f i r s t step i n the f u n c t i o n of the hemeprotein appears to be g e n e r a l l y accepted. The d e t a i l e d i n t e r p r e t a t i o n of the changes i n the p h y s i c a l p r o p e r t i e s o f the hemeprotein accompanying the b i n d i n g o f s u b s t r a t e remains l a r g e l y s p e c u l a t i v e and serves as an area of research c u r r e n t l y under a c t i v e i n v e s t i g a t i o n .
R
+2
Fe -R Figure 7
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
158
XENOBIOTIC
METABOLISM
Reduction o f cytochrome P-450. The complex of f e r r i c cytochrome P-450 w i t h substrate undergoes a one e l e c t r o n reduc t i o n (Figure 7) to form the f e r r o u s hemeprotein. In r e a c t i o n s i n v o l v i n g the C l a s s A type of cytochrome P-450, t h i s e l e c t r o n i s donated by a reduced i r o n - s u l f u r p r o t e i n . The i n i t i a l source of t h i s e l e c t r o n i s from reduced p y r i d i n e n u c l e o t i d e s ( e i t h e r NADPH or NADH depending on the system under study) which i n t e r a c t s w i t h a s p e c i f i c f l a v o p r o t e i n that serves a l s o as an i r o n - s u l f u r p r o t e i n reductase (6,43, 44). In the case of the C l a s s Β type of cytochrome P-450, the f l a v o p r o t e i n , NADPHcytochrome P-450 reductase, mediates the t r a n s f e r of the e l e c t r o n from NADPH to the f e r r i c hemeprotein (45). This f l a v o p r o t e i n has a t t r a c t e d a great deal of i n t e r e s t because of i t s unusual property o (FMN and FAD) and the a b i l i t forms of the enzyme during oxidatio (9,10,11). Recent experiments have i n d i c a t e d that the sequence of e l e c t r o n t r a n s port by t h i s f l a v o p r o t e i n i n v o l v e s f i r s t the r e d u c t i o n of FAD followed by the r e d u c t i o n of FMN (46), i . e . , a form of reduced FMN i s envisioned as the agent donating an e l e c t r o n to reduce cytochrome P-450. However, the a b i l i t y to form a number of d i f f e r e n t s t a t e s of r e d u c t i o n of t h i s f l a v o p r o t e i n (Figure 8) and the need to t r a n s f e r o n l y one e l e c t r o n f o r the r e d u c t i o n of cytochrome P-450 (47,48), leaves unresolved the p o s s i b l e r o l e of other f r e e r a d i c a l forms of the f l a v o p r o t e i n from p a r t i c i p a t i n g i n t h i s or other r e a c t i o n s . NADPH
NADP
/"~"sîô
®
FH 2
e
Λ >,H
2
1
NADPH
F,HF H* 2
NADP
Figure 8. Schematic proposal for the various states of reduction of the flavo protein, NADPH-Cytochrome P-450 reductase. F, is presumed to represent FAD and F to represent FMN. The existence of various free radical species is indi cated. 2
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
5.
ESTABROOK E T A L .
Type
I Metabolism
159
Table I
Concentration o f Microsomal Electron Transfer Components FMN P-450 FAD nmoles/mg protein RATS control phénobarbital pretreatment MICE C57BL/6J DBA/2J
RATIO P-450/FMN
0.71
0.075
0.15
2.1
0.08
0.13
26.3
0.51 0.53
0.021 0.013
0.094 0.090
24.3 40.8
9.5
Recently i n t e r e s t has developed i n the area of membrane s t r u c t u r e and the s p a t i a l r e l a t i o n s h i p of t h i s f l a v o p r o t e i n to cytochrome P-450 (49). A c o n s i d e r a t i o n of the stoichiometry (Table I ) of the f l a v o p r o t e i n to cytochrome P-450 r e v e a l s a s u r f e i t of hemeprotein molecules r e l a t i v e to i t s e l e c t r o n donating partner. The problem becomes even more complex when c o n s i d e r i n g the amphipathic p r o p e r t i e s of the f l a v o p r o t e i n and i t s p h y s i c a l l o c a t i o n on the surface of the membrane (50-54). The p o t e n t i a l r o l e of membrane f l u i d i t y f a c i l i t a t i n g the " p i n b a l l " l i k e m o t i l i t y of the f l a v o p r o t e i n as i t s e r v i c e s the dispersed pool of hemeprotein molecules i s a hypothesis (55, 56) which stands i n c o n t r a s t to the proposal (49,57-59) of c l u s t e r s or patches of multienzyme complexes based on hydrophobic i n t e r a c t i o n s i n the membrane. C l e a r l y , a more d e t a i l e d understanding o f membrane s t r u c t u r e i s a p r e r e q u i s i t e to the d e l i n e a t i o n of the process of r e d u c t i o n of the high s p i n form of o x i d i z e d cytochrome P-450 bound by substrate. R
Figure 9
Reaction with Carbon Monoxide. Reduced cytochrome P-450 r e a c t s r a p i d l y with carbon monoxide (Figure 9) to form a complex which serves as the hallmark f o r t h i s c l a s s of c e l l u l a r pigment (60-62). A c h a r a c t e r i s t i c absorbance band (Figure 10)
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
160
XENOBIOTIC METABOLISM
at about 450 nm (hence the name P-450) i s a s s o c i a t e d w i t h t h i s complex. The chemical b a s i s f o r the unique absorbance band a t 450 nm f o r the carbon monoxide adduct of the reduced hemeprotein i s unknown although numerous s t u d i e s have been c a r r i e d out, i n p a r t i c u l a r using model compound complexes of heme, which suggest that a form of s u l f u r p a r t i c i p a t e s as one of the l i g a n d s of the heme of cytochrome P-450 (25-28) and that t h i s " s o f t l i g a n d " may i n p a r t be r e s p o n s i b l e f o r t h i s unusual property of t h i s c l a s s of hemeproteins. The bathochromic s h i f t i n absorbance of reduced cytochrome P-450, when i t r e a c t s w i t h carbon monoxide, stands i n sharp c o n t r a s t to other known hemeproteins where a hypsochromic s p e c t r a l s h i f t i s observed ( 6 ) . The a b i l i t y of reduced cytochrome P-450 to form complexes w i t h absorbance bands i n the v i c i n i t y of 450 nm i s not r e s t r i c t e d to i t s a c t i o n of reduced cytochrom t i v e , metyrapone, r e s u l t s i n the formation of an absorbance a t about 445 nm (63). Recently, i t was shown that a number of other organic compounds (such as s a f r o l e , the c l a s s of amphetamines, SKF-525A, e t c . ) r e a c t with reduced cytochrome P-450 to form d e r i v a t i v e s , c a l l e d product adducts or m e t a b o l i t e complexes, with absorbance bands i n the s p e c t r a l r e g i o n from 450 to 460 nm (64). The d e t a i l s of the chemistry of these types of r e a c t i o n s remains to be f u r t h e r s t u d i e d . S u f f i c e i t to say that a l l of these s t u d i e s s t r o n g l y suggest the existance of an environment i n the heme pocket of cytochrome P-450 which i s a t y p i c a l of other known hemeproteins (59).
448
0.2 —(
Figure 10. Optical absorbance properties of the carbon monoxide complex of reduced Cytochrome P-450. Cytochrome P-450 associated with liver microsomes prepared from animals pretreated with phénobarbital (PB) or 3-methylchohnthrene (3-MC) as well as Cytochrome P-450 isohted and purified from Pseudomonas putida (camphor) were reduced by sodium dithionite and examined by difference spectrophotometry. After gassing the contents of the sample cuvette with carbon monoxide, the various spectra were recorded. Differences in the location of the absorbance band maxima are indicated.
ο 0.0
-0.H
- 0 . 2 H 1
1
I
1
400
1
]—ι—ι—ι—r 4 50
Wavelength (nm)
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
5.
ESTABROOK ET A L .
Type I
161
Metabolism
Although the most commonly observed property f o r the reduced cytochrome P-450 complex w i t h carbon monoxide i s an absorbance band w i t h a maximum a t 450 nm, the exact locus of t h i s absorbance maximum can be s u b t l y s h i f t e d depending on the source of the hemeprotein. As shown i n F i g u r e 10, the absor bance band maximum i s l o c a t e d at about 446 nm f o r the cytochrome P-450 i s o l a t e d and p u r i f i e d from the camphor grown bacterium, Pseudomonas p u t i d a (43,65). The hemeprotein formed i n l i v e r microsomes as a r e s u l t of exposure of animals to p o l y c y c l i c hydrocarbons such as 3-methylcholanthrene has an absorbance band at 448 nm (66). These d i f f e r e n c e s undoubtedly r e s u l t from changes i n the environment of the heme f o r each of these hemep r o t e i n s as a consequence of s y n t h e s i s of p r o t e i n s with m o d i f i e d amino a c i d composition has been a p l e t h e r a of which f r e q u e n t l y confuse i n d i v i d u a l s not f a m i l i a r with the v a g a r i e s of a developing nomenclature which s t i l l seeks a common base.
1.0 4-
Λ
ι \
Science
0.54-
Figure 11. Photochemical action spec trum for the light reversibility of carbon monoxide inhibition of Cytochrome P450 catalyzed reactions.
1
400
Η
1
h
480 440 Wavelength (ητψ)
Rat liver microsomes were incubated with (Φ), codeine; (O), aminopyrine; or (Δ), acetanilide with various mixtures of carbon monoxide and oxygen and NADPH and irradiated with light of selected wavelengths (69j.
The property of reduced cytochrome P-450 r e a c t i n g w i t h carbon monoxide served as the primary c h a r a c t e r i s t i c f o r d e l i n e a t i n g the f u n c t i o n of t h i s hemeprotein i n the o x i d a t i v e metabolism of many d i f f e r e n t s u b s t r a t e s (68,69). L i k e other hemeproteins, the r e a c t i o n of carbon monoxide w i t h the reduced pigment i s an e q u i l i b r i u m r e a c t i o n which i s p h o t o s e n s i t i v e (70). The f i r s t d e f i n i t i o n of a b i o l o g i c a l f u n c t i o n f o r c y t o chrome P-450 was achieved by a p p l y i n g the photochemical a c t i o n spectrum methodology of Warburg during a study of the 21 h y d r o x y l a t i o n of progesterone as c a t a l y z e d by the microsomal f r a c t i o n i s o l a t e d from the a d r e n a l cortex (68). Extension of these s t u d i e s to an examination of many 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 c a t a l y z e d by l i v e r microsomes (Figure 11)
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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XENOBIOTIC M E T A B O L I S M
confirmed the general r o l e of t h i s hemeprotein i n the oxygen a c t i v a t i o n r e q u i r e d f o r the o x i d a t i v e conversion of a broad spectrum o f organic compounds. The f a c t that the carbon monoxide complex of reduced cytochrome P-450 has an absorbance band i n the Soret r e g i o n of the spectrum, which i s s i g n i f i c a n t l y d i s p l a c e d from the absorbance bands of comparable complexes of other reduced hemeproteins, serves as a u s e f u l property i n e v a l u a t i n g s p e c t r o p h o t o m e t r i c a l l y changes i n the c e l l u l a r content of t h i s pigment f o l l o w i n g exposure of animals to a v a r i e t y of chemicals. As shown i n F i g u r e 12, treatment of animals with a b a r b i t u r a t e , phénobarbital, r e s u l t s i n a marked i n c r e a s e i n the content of cytochrome P-450 i n the l i v e r of these animals. T h i s property of "enzyme i n d u c t i o n " can be a c c u r a t e l y and r a p i d l y monitored spectrophotometrically type presented i n F i g u r
HOURS Figure 12. Induction of liver microsomal Cytochrome P-450 following treatment of rats with phénobarbital. Male rats (150-200 gm) were injected intraperitoneally daily with 80 mg of phénobarbital /kg body weight. Animals were sacrificed at the times indicated and the content of Cytochrome P-450 in (O O), isolated liver microsomes as well as ( Ο — Ο ), liver homogenates (IS).
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
5.
ESTABROOK ET A L .
Type
I
Metabolism
163
cytochrome P-450 i n an organ such as the l i v e r . Subsequent to treatment of the animals f o r f i v e days w i t h phénobarbital, approximately 15 percent of the p r o t e i n of the endoplasmic r e t i c u l u m of l i v e r i s composed of t h i s one c l a s s of p r o t e i n (71). Likewise, as much as 5 percent of the p r o t e i n of the l i v e r can be cytochrome P-450. T r u l y , cytochrome P-450 i s not a minor c o n s t i t u e n t i n t h i s and other organs - i t i s the dominant species of hemeprotein which i s present a t a c o n c e n t r a t i o n e q u i v a l e n t to that of myoglobin i n some muscle t i s s u e .
R
+2
Fe -R
Figure 13
Reaction w i t h Oxygen. In the presence of molecular oxygen reduced cytochrome P-450 r e a c t s r a p i d l y to form an intermediate (Figure 13) termed oxycytochrome P-450. The e x i s t e n c e of an oxygenated form of reduced cytochrome P-450 was p r e d i c t e d from the e a r l y experiments designed to determine those f a c t o r s which i n f l u e n c e the extent of carbon monoxide i n h i b i t i o n of cytochrome P-450 c a t a l y z e d r e a c t i o n s (68). These s t u d i e s had c l e a r l y shown that carbon monoxide was an i n h i b i t o r which was competit i v e w i t h oxygen, i . e . , the magnitude of i n h i b i t i o n observed i s d i c t a t e d by the r a t i o of carbon monoxide to oxygen r a t h e r than simply the c o n c e n t r a t i o n of carbon monoxide i n the r e a c t i o n system. Studies by Ishimura £t a l . (72,73) as w e l l as Gunsalus et a l . (74,75), using the s o l u b l e and p u r i f i e d cytochrome P-450 i s o l a t e d from Pseudomonas p u t i d a , c l e a r l y demonstrated the presence of a reasonably s t a b l e d e r i v a t i v e of the reduced hemeprotein when exposed to oxygen. T h i s complex was 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 , as shown i n F i g u r e 14, and
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC METABOLISM
164
^
1 400
ι
1
1
500
1 600
1
Γ 700
Wavelength (nm) Figure 14. Absorption spectra of oxidized, reduced, and Oxycytochrome P-450. Samples of purified Cytochrome P-450 isolated from Pseudomonas putida were examined spectrophotometrically in the presence of the substrate camphor (73).
WAVELENGTH(nm)
Figure 15. Repetitive scan difference spectral measurements of liver microsomal Cytochrome P-450 during the NADPH supported steady state metabolism of hexobarbital. Liver microsomes from phénobarbital treated male rats were diluted to 1 mg of protein/mL in a buffer mixture containing 50mM TRIS chloride (pH 7.5), 150mM KCl, 10 mM MgCk, and 2mM hexobarbital. NADPH (0.5mM final concentration) was added to initiate the reaction and the difference spectra recorded every 30 sec. The absorbance band at about 440 nm is attributed to Oxycytochrome P-450.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
5.
ESTABROOK
χ. F e i
°2
Type
ET AL.
X-Fe
+2
l
-
°i
+3
I
165
Metabolism
• X· Fe
+3
+ 01 r SOD
H 0 2
2
Academic Press Figure 16.
Possible valence states of Oxycytochrome P-450 and its dissociation to form superoxide
was recognized to resemble the oxygenated form o f other hemep r o t e i n s (76-79). The a b i l i t y to demonstrate the presence o f oxycytochrome P-450 during the f u n c t i o n i n g of the i n t e g r a t e d and membrane bound e l e c t r o n transport system of microsomes (the C l a s s Β type of cytochrome P-450) i s more complex and subject to many v a r i a b l e s . R e p e t i t i v e scan spectrophotometric measure ments during the NADPH supported aerobic steady s t a t e o x i d a t i o n of a v a r i e t y o f substrates by l i v e r microsomes r e v e a l s (Figure 15) the presence o f an absorbance band with a r a t h e r broad maximum a t about 440 nm i n the d i f f e r e n c e spectrum (80,81). This absorbance band i s s i m i l a r to the s p e c t r a l intermediate seen with the p u r i f i e d cytochrome P-450 from Pseudomonas putida. The magnitude o f t h i s absorbance band of oxycytochrome P-450, when studied using l i v e r microsomes, i s markedly i n fluenced by the type o f substrate studied, the a s s o c i a t e d a c t i v i t y of NADPH-cytochrome P-450 reductase, i . e . , the balance of a d d i t i o n of the f i r s t e l e c t r o n versus the second e l e c t r o n (see below) f o r the f u n c t i o n of cytochrome P-450, and the presence of excess NADPH and oxygen. As shown i n Figure 15, the concentration of oxycytochrome P-450 p r o g r e s s i v e l y de creases as the oxygen concentration i n the r e a c t i o n medium decreases. Oxycytochrome P-450 can be considered to be an e q u i l i b r i u m between two ternary complexes. In one case (Figure 16), the f e r r o u s hemeprotein can be envisioned as complexed with a molecule o f superoxide. The l a t t e r can be v i s u a l i z e d as de caying g i v i n g r i s e to the high s p i n f e r r i c hemeprotein sub s t r a t e complex and the superoxide anion (or i t s protonated form, the perhydroxyl r a d i c a l ) . The presence of a d v e n t i t i o u s superoxide dismutase a s s o c i a t e d with the microsomal f r a c t i o n
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
166
XENOBIOTIC METABOLISM
would then c a t a l y z e the formation of hydrogen peroxide. It i s now w e l l e s t a b l i s h e d that hydrogen peroxide i s formed during NADPH o x i d a t i o n by l i v e r microsomes (82,83). Although the source of t h i s hydrogen peroxide remains s p e c u l a t i v e , the decomposition of oxycytochrome P-450 seems a very l i k e l y possi b i l i t y and recent s t u d i e s (83-85) have supported the hypothesis that superoxide i s generated during the f u n c t i o n of cytochrome P-450. Thus, one can consider an "oxidase type" c y c l e f o r cytochrome P-450 (Figure 17) whereby e l e c t r o n s o r i g i n a t i n g from NADPH are d i v e r t e d to the r e d u c t i o n of oxygen f o r the formation of hydrogen peroxide r a t h e r than f o r the mixed f u n c t i o n o x i d a t i v e metabolism of x e n o b i o t i c s . In v i t r o s t u d i e s using i s o l a t e d l i v e r microsomes have revealed the a b i l i t y of many compounds to s t i m u l a t e the r a t e of formation of hydrogen peroxide formation concomitan tochrome P-450 (86,87) genated hydrocarbons (88,89) i t i s apparent that a s i g n i f i c a n t d i v e r s i o n of e l e c t r o n s occurs r e s u l t i n g i n an uncoupling of R
Figure 17.
Dissociation of superoxide from Oxycytochrome F-450 for the formation of hydrogen peroxide
oxygenation r e a c t i o n s . T h i s may be of p a r t i c u l a r i n t e r e s t to those concerned w i t h p e s t i c i d e s because of the frequent use of compounds, such as Lindane. The Second E l e c t r o n . Oxycytochrome P-450, to which a molecule of s u b s t r a t e i s bound, undergoes f u r t h e r r e d u c t i o n to an intermediate termed peroxycytochrome P-450 (Figure 18). L i k e oxycytochrome P-450, the proposed intermediate peroxycytochrome P-450 can be w r i t t e n i n a v a r i e t y of equivalent e l e c t r o n valence forms. The i n a b i l i t y to i s o l a t e and chara c t e r i z e t h i s proposed intermediate has impeded f u r t h e r understanding of i t s chemistry and p h y s i c a l p r o p e r t i e s
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
5.
ESTABROOK ET A L .
Type
I
Metabolism
167
R
Fe +3
X
e
Fe -R +2
0
2
Τ e
Figure 18
although i t s e x i s t e n c e as a t r a n s i e n t intermediate i n the f u n c t i o n o f cytochrome P-450 has been g e n e r a l l y assumed. The s t o i c h i o m e t r y of 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 r e q u i r e s the p a r t i c i p a t i o n of two e l e c t r o n s ; the r e d u c t i o n of the f e r r i c cytochrome P-450 complex has been e s t a b l i s h e d (47,48) to be a one e l e c t r o n t r a n s f e r r e a c t i o n . Studies with the p u r i f i e d b a c t e r i a l cytochrome P-450 have shown (7) the transformation of oxycytochrome P-450 by the a d d i t i o n of the reduced i r o n s u l f u r p r o t e i n , p u t i d a r e d o x i n . F u r t h e r , the a b i l i t y to observe s p e c t r o p h o t o m e t r i c a l l y intermediates formed during the r e a c t i o n of f e r r i c cytochrome P-450 with organic hydroperoxides, such as cumene hydroperoxide, which d i f f e r from oxycytochrome P-450 s u b s t a n t i a t e s the e x i s t e n c e of a d d i t i o n a l oxygen c o n t a i n i n g complexes of cytochrome P-450 (90). P r o t o n a t i o n of peroxycytochrome P-450 and the d i r e c t r e l e a s e of hydrogen peroxide concomitant with the formation of the f e r r i c cytochrome P-450 complex with s u b s t r a t e (Figure 19) i s an a l t e r n a t i v e means of e x p l a i n i n g the formation of hydrogen peroxide during NADPH o x i d a t i o n by l i v e r microsomes. Consid e r a b l e work has centered on d i s t i n g u i s h i n g the two a l t e r n a t i v e s f o r the formation of hydrogen peroxide, i . e . , the decomposition of oxycytochrome P-450 with the r e l e a s e of superoxide which then undergoes dismutation or the p r o t o n a t i o n of peroxycytochrome P-450 and the d i r e c t formation of hydrogen peroxide. An examination of the r e a c t i o n scheme shown i n F i g u r e 19 r e v e a l s a major d i f f e r e n c e between these two options which should d e l i n eate the dominant pathway f o r hydrogen peroxide formation. As discussed above, two e l e c t r o n t r a n s f e r steps are r e q u i r e d to form peroxycytochrome P-450 while only one e l e c t r o n t r a n s f e r
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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XENOBIOTIC METABOLISM
step i s needed to generate oxycytochrome P-450. Previous s t u d i e s of drug metabolism by the cytochrome P-450 c o n t a i n i n g l i v e r microsomes revealed a " s y n e r g i s t i c e f f e c t " of NADH
R
e Figure 19.
Proposed formation of hydrogen peroxide from Peroxycytochrome P-450
X I NADH
Figure 20. Scheme showing the proposed role of reduced Cytochrome h as the donor of the electron required for the reduction of Oxycytochrome P-450 and the formation of Per oxycytochrome P-450 5
when supplementing the NADPH dependent mixed f u n c t i o n o x i d a t i o n of v a r i o u s s u b s t r a t e s . This " s y n e r g i s t i c e f f e c t " e l i c i t e d by NADH (91-94) was a t t r i b u t e d to a r o l e f o r reduced cytochrome b^ as the donor of the second e l e c t r o n r e q u i r e d f o r the c y c l i c f u n c t i o n of cytochrome P-450. This i s i l l u s t r a t e d i n F i g u r e 20. The presence of NADH would spare reducing e q u i v a l e n t s o r i g i n a t i n g from NADPH needed f o r the r e d u c t i o n of
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
5. ESTABROOK E T A L .
Type
I
Metabolism
169
cytochrome b^ and i t would a l s o i n c r e a s e the extent of steady s t a t e r e d u c t i o n of t h i s hemeprotein. Comparable s t u d i e s were c a r r i e d out to determine whether a " s y n e r g i s t i c e f f e c t " by NADH on the NADPH dependent formation of hydrogen peroxide a l s o occurred. These r e s u l t s were negative (83,85). This suggests that the p r i n c i p a l pathway f o r the formation of R
ι I e
Figure 21. Proposed formation of an oxene-type intermediate during the cyclic function of Cytochrome P-450.
hydrogen peroxide was the decay of oxycytochrome P-450 r a t h e r than the p r o t o n a t i o n of peroxycytochrome P-450. Obviously, more d e f i n i t i v e experiments w i l l have to be c a r r i e d out but the present l i m i t a t i o n i n methodologies precludes the design of such unequivacol s t u d i e s . Other P o s s i b l e Intermediates. The sequence of r e a c t i o n s i n v o l v e d i n the " a c t i v a t i o n of oxygen" from peroxycytochrome P-450 a r e unknown. Much has been w r i t t e n and many s p e c u l a t i v e hypotheses have been proposed but no experimental v e r i f i c a t i o n e x i s t s to b e t t e r d e f i n e p o s s i b l e intermediates. One such hypothesis, which i s very a t t r a c t i v e , proposes the formation of an oxene complex of cytochrome P-450 r e s u l t i n g from the p r o t o n a t i o n of peroxycytochrome P-450 and the r e l e a s e of a molecule of water (95,96). This i s i l l u s t r a t e d i n F i g u r e 21. The e x i s t e n c e of an oxenoid species of oxygen would f u l f i l l the requirement of a h i g h l y e l e c t r o p h i l i c atom which could p a r t i c i p a t e i n h y d r o x y l a t i o n r e a c t i o n s . Recently, the p r e s ence of f r e e - r a d i c a l species has a l s o been deduced from deuterium i s o t o p e experiments (97). However, the f a i l u r e to detect any measureable c o n c e n t r a t i o n of f r e e r a d i c a l s during the microsomal c a t a l y z e d o x i d a t i v e metabolism of any s u b s t r a t e yet examined i n d i c a t e s the absence of a steady s t a t e concen t r a t i o n of such proposed f r e e r a d i c a l s . Additional inter mediates such as the carbanion form of some s u b s t r a t e s , the
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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XENOBIOTIC METABOLISM
e x i s t e n c e of a carbene form of the s u b s t r a t e , and the generat i o n of epoxides should a l l be considered as p o s s i b i l i t i e s u n t i l our l e v e l of knowledge i s s u f f i c i e n t l y expanded to permit choices based on experimental r a t h e r than t h e o r e t i c a l considerations. The
Role of the Membrane
Cytochrome P-450 present i n mammalian t i s s u e s i s recogn i z e d to be i n t i m a t e l y a s s o c i a t e d with membranes. Studies of membrane bound enzymes have the disadvantage that s p e c i a l methodologies, such as the use of d i f f e r e n c e spectrophotometry as described e a r l i e r , as w e l l as the presence of high concent r a t i o n s of l i p i d can introduc v a r i a b l e that difficult to f u l l y evaluate. Th can perturb the p a t t e r sequestering of the h i g h l y l i p o p h i l i c substrates that are o x i d a t i v e l y transformed during the f u n c t i o n of cytochrome P450 (31,98). Recently considerable i n t e r e s t has centered on the s p a t i a l and s t r u c t u r a l o r g a n i z a t i o n of cytochrome P-450 and i t s a s s o c i a t e d e l e c t r o n t r a n s f e r p r o t e i n s w i t h i n the mileau of the microsomal membrane. The unique stoichiometry of f l a v o p r o t e i n s and cytochromes ( c f . Table I) have provided the opportunity to evaluate the r o l e of the membrane i n regul a t i n g and modifying the types of r e a c t i o n s c a t a l y z e d and the i n f l u e n c e of 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 w i t h i n the l i p i d mosaic of the membrane. I t has been proposed (49,58,59) that cytochrome P-450 e x i s t s as c l u s t e r s (Figure 22) which
K" 200A—H Figure 22. Schematic of the microsomal membrane showing clusters of molecules of Cytochrome P-450 surrounding the flavoprotein reductase
can form patches of e l e c t r o n t r a n s p o r t complexes w i t h i n or withon the membrane. Much controversy surrounds t h i s i n t e r -
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p r e t a t i o n (55,56) and the extent of membrane f l u i d i t y , as i t i n f l u e n c e s the cytochrome P-450 dependent metabolism of subs t r a t e s , remains as an area r e q u i r i n g f u r t h e r study. The s o l u t i o n of t h i s i n t r i g u i n g problem w i l l undoubtedly i n f l u e n c e our f u r t h e r understanding of the r e a c t i o n s i n v o l v e d and the r e l a t i o n s h i p of s t u d i e s c a r r i e d out i n v i v o to those that have been experimentally a s c e r t a i n e d by i n v i t r o s t u d i e s . The importance of the membrane to s t u d i e s of the metabolism of x e n o b i o t i c s cannot be underestimated. Most of the compounds which are o x i d a t i v e l y transformed are h i g h l y l i p o p h i l i c . Further, the l i p i d composition of the microsomal membrane can be r e a d i l y modified by the type of d i e t employed as w e l l as the age and sex of the experimental animal system under study. M u l t i p l e Types of Cytochrom Any c o n s i d e r a t i o n of the f a c t o r s which i n f l u e n c e the r o l e of cytochrome P-450 i n the metabolism of x e n o b i o t i c s must account f o r the ever i n c r e a s i n g body of evidence which shows the m u l t i p l e types of cytochrome P-450 that e x i s t i n a s i n g l e c e l l type, such as the hepatocyte. I t has been known f o r many years that v a r i o u s inducing agents can cause a p r e f e r e n t i a l s t i m u l a t i o n i n the metabolism of drug s u b s t r a t e s . The b a s i s f o r t h i s d i f f e r e n c e was c l e a r l y d e l i n e a t e d by the observation (66) that treatment of animals with the inducing agent, 3methylcholanthrene, r e s u l t e d i n the s y n t h e s i s of a hemeprotein a s s o c i a t e d with l i v e r microsomes which had o p t i c a l absorbance p r o p e r t i e s d i s t i n c t from a s i m i l a r pigment induced upon t r e a t ment of animals with phénobarbital ( c f . Figure 10). The l a s t decade has seen major advances i n the i s o l a t i o n and p u r i f i c a t i o n of d i f f e r e n t types of cytochromes P-450. D i f ferences i n response to s p e c i f i c a n t i b o d i e s , p r o t e i n s t r u c t u r e , and molecular weights are a l l subjects of current research (99-104). A d i r e c t demonstration of the change i n the p a t t e r n of types of cytochrome P-450 a s s o c i a t e d with l i v e r microsomes i s o l a t e d from animals subjected to d i f f e r e n t inducing agents i s shown by the polyacrylamide g e l e l e c t r o p h o r e s i s r e s u l t s presented i n F i g u r e 23. Dramatic a l t e r a t i o n s i n the magnitude as w e l l as m o b i l i t y of p r o t e i n s i n the molecular weight range between 45,000 and 50,000 can be seen a f t e r exposure of animals to the inducing agents, phénobarbital or pregnenolong-16alpha-carbonitrile. Thus one must consider, when d i s c u s s i n g the i n v i v o enzymatic a c t i v i t y of cytochrome P-450, not only the i n f l u e n c e of the membrane and the a v a i l a b i l i t y of cof a c t o r s such as reduced p y r i d i n e n u c l e o t i d e s and oxygen but a l s o the presence of unique types of cytochrome P-450. A l o g i c a l extension of these s t u d i e s i s to p o s t u l a t e the presence of unique types of cytochrome P-450 which possess a s p e c i f i c i t y f o r v a r i o u s c l a s s e s of s u b s t r a t e s . As of t h i s w r i t i n g , the demonstration of s u b s t r a t e s p e c i f i c i t y has not
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Figure 23. PAGE patterns of proteins present in liver microsomes from untreated animals (control), pregnenolone-16 a-carbonitrile (PCN)-, or phénobarbital (PB)treated male rats. Microsomes were isolated and treated with SDS prior to electrophoresis and staining with Coomassie blue.
p r e t r e a t e d with a p o l y c y c l i c hydrocarbon has a high a c t i v i t y i n the metabolism of benzo(a)pyrene but a l s o c a t a l y z e s the o x i d a t i v e metabolism of drugs such as benzphetamine. This puzzle becomes even more i n t r i g u i n g when e v a l u a t i n g the recognized competitive e f f e c t s of v a r i o u s types of substrates when r a t e s of metabolism a r e measured using the membrane bound form of the enzyme. C l e a r l y , the i n t e g r a t i o n of knowledge gained from s t u d i e s with v a r i o u s types of p u r i f i e d cytochrome P-450 as w e l l as the e l e c t r o n transport complexes e x i s t e n t i n the membrane w i l l be r e q u i r e d before a meaningful assessment of the i n v i v o transformation of x e n o b i o t i c s can be accomplished. Concluding Remarks The d e l i n e a t i o n of the sequence o f events which occurs as the hemeprotein, cytochrome P-450, r e a c t s s e q u e n t i a l l y with a molecule of s u b s t r a t e to be metabolized, an e l e c t r o n d e r i v e d from NADPH and t r a n s f e r r e d v i a the f l a v o p r o t e i n , NADPH-cytochrome P-450 reductase, a molecule of oxygen, and then a second e l e c t r o n has been b r i e f l y discussed i n the foregoing s e c t i o n s .
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The r e s u l t i n g o v e r a l l scheme shown i n F i g u r e 24 attempts to s y n t h e s i z e our present understanding of t h i s c y c l i c process. One must r e c o g n i z e , however, that much remains to be r e v e a l e d i f we a r e to g a i n s u f f i c i e n t knowledge which may permit the development of agents that can p r e d i c t a b l y perturb t h i s
Figure 24. Scheme of intermediates formed during the cyclic reaction of Cytochrome P-450 associated with the metabolism of many xenobiotics. The possible sites of formation of hydrogen peroxide as well as the pathway of metabolism initiated by organic hydroperoxides are illustrated.
r e a c t i o n system. Indeed, achievement of t h i s knowledge i s a necessary p r e r e q u i s i t e to attainment of the goal of p r e d i c t i n g the o x i d a t i v e transformation of x e n o b i o t i c s to h i g h l y t o x i c agents (many w i t h the known p o t e n t i a l of s e r v i n g as carcinogens) or to products that a r e not d e t r i m e n t a l to c e l l u l a r f u n c t i o n . The i n v e n t o r y of questions which remain unanswered i s long and i n c l u d e s such items as f o l l o w s : a) What i s " a c t i v e oxygen" and how does i t perform the necessary chemistry to i n t e r a c t with such a broad spectrum of chemicals as those known to be metabolized by c y t o chrome P-450? b) What i s the unique chemistry of cytochrome P-450 that permits i t to r e a c t with oxygen and the s u b s t r a t e molec u l e s i n a manner that permits the c a t a l y s i s of x e n o b i o t i c transformation?
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c) Do the m u l t i p l e forms of cytochrome P-450, which are s e l e c t i v e l y induced by v a r i o u s chemicals, r e f l e c t more than a s i n g l e mechanism f o r the a c t i v a t i o n of oxygen? d) What c o n s t r a i n t s are imposed by the hydrophobic environs of the membrane and a s s o c i a t e d problems of p r o t e i n p o l a r i t y , f l u i d i t y , or existence as c l u s t e r s ? e) What f a c t o r s d i c t a t e the p a t t e r n of e l e c t r o n t r a n s p o r t from reduced p y r i d i n e n u c l e o t i d e s and how are these r e f l e c t e d i n the case of s u b s t r a t e metabolism? f ) Is the concomitant formation of hydrogen peroxide, known to occur during the cytochrome P-450 metabolism of s u b s t r a t e s , of an These and many more questions are under i n t e n s i v e i n v e s t i g a t i o n i n many l a b o r a t o r i e s . Undoubtedly, new r e s u l t s w i l l r e v e a l the f r a g i l e foundation on which our current hypotheses are developed. The importance of knowning how t h i s enzyme system f u n c t i o n s i s at the f o r e f r o n t of s c i e n t i f i c concern during the present time when a great d e a l of a t t e n t i o n has been focused on the wide spread d i s s e m i n a t i o n i n our environment of xenobiotics. The f u t u r e holds great promise to b r i n g f o r t h new ideas and new concepts. I t w i l l be i n t e r e s t i n g to r e f l e c t back on the meager beginning where we stand now.
Abstract The oxidative conversion of many lipophilic chemicals which are xenobiotics occurs by means of an enzyme system where the hemeprotein, cytochrome P-450, functions as an oxygenase. The importance of these types of reactions is now recognized as the primary step in the metabolic formation of many toxic agents including carcinogens. The details of our current understanding of cytochrome P-450 interaction with the organic substrate molecule, oxygen, and electrons derived from reduced pyridine nucleotides is described in the present paper. Particular attention is directed toward the sequence of reactions occurring and the attendent problems of defining the parameters which regulate the functionality of the membrane bound enzyme complex. The methodologies employed for studies to define the role of cytochrome P-450 in the metabolism of drugs, steroids, and chemicals which can be converted to carcinogens, have been described and typical results illustrated. The extension of these studies to better define the specificity of the reaction system as well as the properties of reactive intermediates poses the direction for future experimentation.
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Drug Oxidations, edited by Ullrich, V., Roots, I., Hildebrandt, A. G., Estabrook, R. W., and Conney, A. H., p. 748, Pergamon Press, Oxford (1977). Ullrich, V. and Diehl, Η., Eur. J. Biochem. (1971) 2 0 , 509. Staudt, Η., Lichtenberger, F., and Ullrich, V., Eur. J. Biochem. (1974) 46, 99. Rahimtula, Α., O'Brien, P. J., Hrycay, E. G., Peterson, J. Α., and Estabrook, R. W., Biochem. Biophys. Res. Commun. (1974) 60, 695. Cohen, B. S. and Estabrook, R. W., Arch. Biochem. Biophys. (1971) 143, 46. Hildebrandt, A. G. and Estabrook, R. W., Arch. Biochem. Biophys. (1971) 143, 66. Mannering, G. J., Kuwahara, S., and Omura, T., Biochem. Biophys. Res. Commun Werringloer, J. an Res. Commun. (1976) 71, 834. Ullrich, V. and Staudinger, Hj., in Biochemie des Sauerstoffs, edited by Hess,Β. and Staudinger, Hj., p. 229, Springer-Verlag, Berlin (1968). Ullrich, V., in Biological Reactive Intermediates, edited by Jollow, D. J., Kocsis, J. J., Snyder, R., and Vainio, H., p. 65, Plenum Press, New York (1977). Groves, J. T., McClusky, G. Α., White, R. A. and Coon, M. J., Biochem. Biophys. Res. Commun. (1978) 81, 154. Parry, G., Palmer, D. N., and Williams, D. J., FEBS Lett. (1976) 67, 123. Welton, A. F. and Aust, S. D., Biochim. Biophys. Acta (1974) 373, 197. Haugen, D. Α., Coon, M. J., and Nebert, D. W., J. Biol. Chem. (1976) 251, 1817. Haugen, D. Α., van der Hoeven, Τ. Α., and Coon, M. J., J. Biol. Chem. (1975) 250, 3567. Thomas, P. Ε., Lu, A. Y. H., Rayan, D., West, S. Β., Kawalek, J. and Levin, W., J. Biol. Chem. (1976) 251, 1385. Johnson, E. F. and Muller-Eberhard, U., in Drug Metabolism Concepts, edited by Jerina, D., p. 72, American Chemical Soc., Washington, D. C. (1977). Ryan, D.. Ε., Thomas, P. Ε., and Levin, W., Mol. Pharmacol. (1977) 13, 521.
RECEIVED December 20, 1978.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
6 The Use of Animal Subcellular Fractions to Study Type II Metabolism of Xenobiotics MAUREEN
J. C R A W F O R D and D A V I D H . H U T S O N
Shell Research Ltd., Shell Toxicology Laboratory (Tunstall), Sittingbourne Research Centre, Sittingbourne, Kent, M E 9 8AG, U . K .
Type II biotransformations or conjugation reactions are enzyme -catalysed energy-requiring biosyntheses. The ultimate reaction requires a high-energy dono and an appropriate transferase contain the endogenous conjugating agent (conjugand) (e.g. glucuronic acid, as in UDPGA) or i t may contain the xenobiotic (e.g. 4-chlorobenzoic acid, as in 4-chlorobenzoyl-CoA). If the xenobiotic is to be activated to become a donor substrate, other enzymes and high energy substrates (e.g. ATP) are called into action. If we assume for the moment that the low molecular weight substrates for these reactions have equal access to every component in the cell, then the subcellular location of the transferring enzyme will be the controlling factor in the subcellular distribution of the conjugation reaction. History of the Technique The metabolism of foreign compounds has been studied in various subcellular fractions from about 1950. Brodie and coworkers (1) presented the first overview of the enzyme -catalysed metabolism of these compounds in 1958. In the intervening years, the properties of microsomes and other subcellular fractions in relation to the metabolism and toxicity of drugs, pesticides and, more recently, 'environmental' chemicals have received an enormous amount of study. However, i t is interesting to note that the best of the earlier reviews of the enzymology of foreign compound metabolism by J. R. Gillette in 1963 (2) contains the essential details of each of the conjugation reactions referred to below. Progress has not been even: i t has proved difficult to keep our treatment of glucuronyltransferase within reasonable limits, yet amino acid conjugation, despite some very interesting species differences, has received very little attention at the enzyme level. For our practical purposes, the animal cell can be regarded as composed of nucleus, endoplasmic reticulum, mitochondria 0-8412-0486-l/79/47-097-181$12.25/0 © 1979 A m e r i c a n C h e m i c a l Society
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and lysosomes suspended together i n water c o n t a i n i n g d i s s o l v e d p r o t e i n s and small molecules. One o f the values o f studying type I I r e a c t i o n s at the s u b c e l l u l a r l e v e l l i e s i n the informa t i o n gained about the r o l e of these components and the mechanism of the r e a c t i o n s . When the c e l l i s fragmented and the v a r i o u s o r g a n e l l e s are separated, the conjugation r e a c t i o n under i n v e s t i g a t i o n may not be observed i n any o f the f r a c t i o n s because enzymes have been separated from mandatory c o f a c t o r s . For example, g l u c u r o n i d a t i o n r e a c t i o n s are not observed i n the i s o l a t e d endoplasmic r e t i c u l u m (which contains the t r a n s f e r a s e ) because the high-energy donor s u b s t r a t e , u r i d i n e diphosphog l u c u r o n i c a c i d (UDPGA), i s l o c a t e d i n the s o l u b l e f r a c t i o n . Thus the e f f o r t r e q u i r e d to r e c o n s t i t u t e the conjugation r e a c t i o n s at the s u b c e l l u l a r l e v e l has been v i t a l to the f u l l understanding of t h e i r mechanisms None of t h i s coul biochemists who have developed an understanding of intermediary metabolism and s u b c e l l u l a r f r a c t i o n a t i o n procedures. D i f f e r e n t i a l c e n t r i f u g a t i o n i s s t i l l the main method used and t h e r e f o r e the steady development of p r e p a r a t i v e - s c a l e c e n t r i f u g e s has a l s o been very important. The
Preparation
of S u b c e l l u l a r F r a c t i o n s
S u b c e l l u l a r f r a c t i o n a t i o n i s u s u a l l y c a r r i e d out by d i f f e r e n t i a l c e n t r i f u g a t i o n . However, other methods such as p r e c i p i t a t i o n and chromatography have been i n v e s t i g a t e d f o r the i s o l a t i o n of s p e c i f i c f r a c t i o n s . 2.1 D i f f e r e n t i a l C e n t r i f u g a t i o n D i f f e r e n t i a l c e n t r i f u g a t i o n i s by f a r the most widely used technique. I t i s e f f e c t i v e , clean and gentle and although i t could have been d i s p l a c e d at one time by a f a s t e r technique, the a v a i l a b i l i t y of p r e p a r a t i v e u l t r a - c e n t r i f u g e s capable of up to 500,000 g has cut down p r e p a r a t i o n times to a very competitive l e v e l . The machines now a l s o operate at acceptable noise l e v e l s . Fresh t i s s u e i s homogenised i n b u f f e r e d s a l t s o l u t i o n and the r e s u l t a n t homogenate i s c e n t r i f u g e d at about 600 g to remove unbroken c e l l s and c e l l d e b r i s . The supernatant i s then c e n t r i fuged at 8000-10,000 g f o r about 20 min to sediment the mitochondria. Some lysosomes are sedimented at t h i s stage. The supernatant i s then c e n t r i f u g e d at about 200,000 g f o r 20 min to sediment the fragmented endoplasmic r e t i c u l u m (microsomal f r a c t i o n ) together with some lysosomes. The r e s u l t i n g supernatant i s the s o l u b l e f r a c t i o n ( c y t o s o l ) . Each of the p a r t i c u l a t e f r a c t i o n s may be f u r t h e r p u r i f i e d by washing and r e c e n t r i f u g a t i o n . They may be i n d i v i d u a l l y f u r t h e r p u r i f i e d by d e n s i t y gradient c e n t r i f u g a t i o n . The i s o l a t i o n o f microsomes has r e c e i v e d a l o t of a t t e n t i o n from drug metabolism researchers because of the importance of the microsomal mono-oxygenase system i n x e n o b i o t i c metabolism (see preceding Chapter). When
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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the r o u t i n e procedure o u t l i n e d above i s c a r r i e d out there i s i n e v i t a b l y some contamination of one f r a c t i o n by another. This i s best seen by c o n s i d e r i n g a f r a c t i o n a t i o n of r a t l i v e r c a r r i e d out i n our l a b o r a t o r y by Wright and coworkers (3) during s t u d i e s on the e f f e c t of the i n g e s t i o n of d i e l d r i n on hepatocytes. The f r a c t i o n a t i o n procedure was monitored by measuring the a c t i v i t i e s of s e v e r a l enzymes i n each f r a c t i o n , i n c l u d i n g s u c c i n i c dehydrogenase ( m i t o c h o n d r i a l ) , and glucose-6-phosphatase and c h l o r f e n vinphos d e a l k y l a s e (microsomal). Some of these r e s u l t s are i l l u s t r a t e d i n Figure 1. Often we r e q u i r e samples of only the microsomal and c y t o s o l i c f r a c t i o n s , i n which case we r o u t i n e l y prepare 20% homogenates i n 0.1 M potassium phosphate b u f f e r pH 7.4, c e n t r i fuge at 10,000 g f o r 20 min and use t h i s supernatant to prepare microsomes (190,000 g/3 (4). P r o t e i n concentration s p e c i f i c a c t i v i t i e s can be c a l c u l a t e d . These are measured u s i n g e i t h e r the s e n s i t i v e procedure d e s c r i b e s by Lowry et a l (5) or the simple m o d i f i c a t i o n of the b i u r e t r e a c t i o n d e s c r i b e d by Robinson and Hodgen (6). Precipitation Rapid methods f o r the p r e p a r a t i o n of microsomal f r a c t i o n s have been looked f o r i n recent y e a r s . I s o e l e c t r i c p r e c i p i t a t i o n of l i v e r microsomes from p o s t - m i t o c h o n d r i a l supernatant at pH 5.4 i s a u s e f u l such method (_7) . Mono-oxygenase c h a r a c t e r i s t i c s i n the product compare very w e l l with those of conventional microsomes but the status of the g l u c u r o n y l t r a n s f e r a s e was not reported. These preparations contain about 45% more p r o t e i n than do conventional preparations and t h e i r p o s s i b l e contamina t i o n by c y t o s o l enzymes, such as the g l u t a t h i o n e t r a n s f e r a s e s , r e q u i r e s f u r t h e r i n v e s t i g a t i o n . A calcium i o n sedimentation method was a c c i d e n t l y discovered during a p r e p a r a t i o n of plasma membranes which became contaminated with aggregated microsomes. The method was then developed s p e c i f i c a l l y f o r microsomes (8). Mono-oxygenase c h a r a c t e r i s t i c s were s i m i l a r to those i n conventionally-prepared microsomes but again g l u c u r o n y l t r a n s f e r a s e was not t e s t e d (9)(10). The method has been a p p l i e d to r a t and r a b b i t kidney and lung t i s s u e . Microsomal y i e l d s from the l a t t e r d i f f e r from those obtained by c e n t r i f u g a t i o n i n that there i s a higher y i e l d of p r o t e i n with lower s p e c i f i c a c t i v i t y ^
}
'
Gel F i l t r a t i o n Chromatography Microsomes and c y t o s o l of r a t l i v e r 13,000 g supernatant have been separated by g e l f i l t r a t i o n through Sepharose 2B; the microsomes were c o l l e c t e d i n the e x c l u s i o n volume (Vo) and the c y t o s o l , between Vo and Vt (12). The method has been a p p l i e d to r a t lung with remarkable success g i v i n g microsomes with very high s p e c i f i c a c t i v i t i e s ( f o r o x i d a t i v e r e a c t i o n s ) (13). An example of one of our own separations using t h i s technique i s shown i n Figure 2 (10 ml of 10,000 g supernatant from 40% r a t
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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glucose—6— phosphatase
succinic dehydrogenase
I I
5 0
METABOLISM
chlorfenvinphos dealkylase
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In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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II
Metabolism
Protein mg.ml
-1
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15 Fraction number
20
25
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cytochrome P450
Haemoglobin
2 Figure 2.
Gel filtration of rat liver 10,000 g supernatant on Sepharose 2B
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
30'
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METABOLISM
l i v e r homogenate a p p l i e d to a 2.5 cm χ 25 cm column run at upward flow, 1 ml/min; 5 ml f r a c t i o n s c o l l e c t e d ) .
4-5°,
Advantages of the S u b c e l l u l a r F r a c t i o n s I n d i v i d u a l s u b c e l l u l a r f r a c t i o n s , supplemented with the appropriate c o f a c t o r s , u s u a l l y a f f o r d information on d i s c r e t e steps i n metabolic pathways i n a way that i s not p o s s i b l e u s i n g animals, organs or c e l l s . The study of c o f a c t o r requirements provides information on the mechanism of each r e a c t i o n , as do studies w i t h i s o l a t e d p u r i f i e d enzymes. The conditions f o r the r e a c t i o n to be studied can be optimised and f u r t h e r metabolism can, i f necessary, be blocked so that an intermediate of i n t e r e s t can be i s o l a t e d . In a converse sense mechanisms of the more conjugatio , y of the s u b c e l l u l a r l o c a t i o n and c o f a c t o r requirements of the b i o t r a n s f o r m a t i o n of a x e n o b i o t i c o f f e r s u s e f u l information on the metabolism of that compound. I t i s important to know each d i s c r e t e step i n the b i o t r a n s f o r m a t i o n of a x e n o b i o t i c because we then have a chance of i d e n t i f y i n g intermediates that may prove hazardous under c e r t a i n circumstances. I t i s important a l s o to know the e f f e c t of t o x i c a n t s on the f u n c t i o n of a p a r t i c u l a r c e l l o r g a n e l l e . This requires the study of s u b c e l l u l a r f r a c t i o n s . Another important advantage of s u b c e l l u l a r techniques i s that the f r a c t i o n s are r e l a t i v e l y easy to prepare and they are reasonably robust provided that c o r r e c t conditions are used. Conditions are described i n some d e t a i l i n the i n d i v i d u a l s e c t i o n s below. Disadvantages of the Technique The d i f f i c u l t i e s that may be experienced i n working with s u b c e l l u l a r f r a c t i o n s are d e t a i l e d below i n the sections on the i n d i v i d u a l conjugation r e a c t i o n s . However, some u s e f u l g e n e r a l i s a t i o n s may be made. The e x t r a p o l a t i o n back to the s i t u a t i o n i n v i v o from s u b c e l l u l a r systems i s a long one. We tend to base our comparisons on enzyme a c t i v i t y measured under conditions favouring good k i n e t i c s (e.g. zero order f o r c o f a c t o r s , r a t e l i n e a r with time and p r o t e i n c o n c e n t r a t i o n s ) . However, i n v i v o , c o f a c t o r s and/or substrate concentration may be r a t e - l i m i t i n g ; p e n e t r a t i o n of substrate to enzyme may be r e s t r i c t e d or f a c i l i t a t e d ; n a t u r a l m o d i f i e r s may be present; i n t e r a c t i o n between metabolic processes may occur and a f f e c t r e a c t i o n r a t e s . The values KJQ (a measure of enzyme-substrate a f f i n i t y ) and V (the c a p a b i l i t y of the enzyme when saturated with substrate) are v a l u a b l e , p a r t i c u l a r l y i n a comparative sense. T h e i r true relevance to a p a r t i c u l a r s i t u a t i o n i n v i v o however i s d i f f i c u l t to assess. M
A
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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Another t r a p , but of our own making, must be considered. A r a p i d assay system i s very important i n enzyme p u r i f i c a t i o n and i s very convenient i n t i s s u e and species-comparisons. I t a l s o h e l p s , but i s l e s s easy to arrange, i n s t r u c t u r e - a c t i v i t y s t u d i e s . There has been a tendency i n x e n o b i o t i c enzymology f o r convenience to dominate i n the s e l e c t i o n of substates. The newcomer to the science of x e n o b i o t i c metabolism must s u r e l y be puzzled at the c e n t r a l r o l e occupied by £-nitrophenol. Unsuitable or r e s t r i c t e d s u b s t r a t e s e l e c t i o n has l e d to the p e r p e t r a t i o n of some q u i t e unwarranted g e n e r a l i s a t i o n s . In a d d i t i o n c e r t a i n convenient subs t r a t e s have proved u n s u i t a b l e i n a physico-chemical sense and t h e i r use has l e d to some very complex k i n e t i c s dominated by the p r o p e r t i e s of the s u b s t r a t e r a t h e r than those of the enzyme. Another d i f f i c u l t y rathe potential failure f approaches u s i n g separate miss an i n t e r a c t i o n betwee processes example of t h i s i s the production of a metabolite by o x i d a t i o n ( i . e . a type I process) followed by i t s conjugation (type I I p r o c e s s ) . The two r e a c t i o n s may be more than simply consecutive. S p e c i f i c examples w i l l be d i s c u s s e d below. Use
i n X e n o b i o t i c Metabolism
Studies
S u b c e l l u l a r f r a c t i o n s have been used e x t e n s i v e l y to study the d i s c r e t e steps i n x e n o b i o t i c metabolism. They are a l s o very u s e f u l i n a comparative sense. Conjugation r e a c t i o n s , l i k e other b i o t r a n s f o r m a t i o n s , may be a f f e c t e d by a number of f a c t o r s . These i n c l u d e s p e c i e s , t i s s u e , sex, s t r a i n , s t r e s s , age, time, chemicals ( i n d u c t i o n , i n h i b i t i o n , a c t i v a t i o n ) and pregnancy. The e f f e c t s of these on a p a r t i c u l a r b i o t r a n s f o r m a t i o n are conv e n i e n t l y s t u d i e d at the s u b c e l l u l a r l e v e l . For example, i f species comparisons i n v i t r o are shown to be v a l i d i n terms of i n v i v o r e s u l t s across a range of experimental animals, they can be u s e f u l l y extended to human biopsy samples, thus f u r n i s h i n g some metabolic data f o r man. The r e l a t i o n s h i p between chemical s t r u c t u r e and metabolism i s a l s o very conveniently i n v e s t i g a t e d in vitro. These v a r i o u s aspects are e x e m p l i f i e d below f o r the i n d i v i dual conjugation r e a c t i o n s . The r e a c t i o n s r e q u i r i n g a c t i v a t e d conjugand (glucuronide formation, s u l p h a t i o n , phosphorylation, a c e t y l a t i o n and methylation) are d i s c u s s e d f i r s t followed by those i n v o l v i n g a c t i v a t i o n of the x e n o b i o t i c (amino a c i d conj u g a t i o n ) . Glutathione conjugation, which depends upon the mutual i n t r i n s i c r e a c t i v i t y of both s u b s t r a t e s , i s d i s c u s s e d l a s t . Conjugation w i t h Glucuronic A c i d Mechanism and l o c a t i o n . Glucuronic a c i d conjugation i s probably the most q u a n t i t a t i v e l y important of the Type I I processes, both i n terms of p r o p o r t i o n of a p a r t i c u l a r x e n o b i o t i c being so conjugated, and i n the v a r i e t y of compounds t a k i n g p a r t
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i n the r e a c t i o n . Phenols, a l c o h o l s , c a r b o x y l i c acids and N-hydroxy compounds a l l form O-glucuronides and a number of S- and Nglucuronides have been detected. The process has r e c e n t l y been reviewed i n some d e t a i l by Dutton and co-workers (14)(15). This process i s an example of one i n which the endogenous conjugand ( g l u c u r o n i c acid) i s a c t i v a t e d to a high energy donor (uridine-5 -diphospho-a-D-glucuronic a c i d , UDPGA). The t r a n s f e r of the α-D-glucopyranuronyl group from UDPGA to the acceptor, forming £-D-glucopyranuronosides, i s c a t a l y s e d by UDPGAglucuronyl transferase (EC 2.4.1.17). The enzyme i s l o c a t e d i n the endoplasmic r e t i c u l u m of mammalian c e l l s and t h e r e f o r e appears, on s u b c e l l u l a r f r a c t i o n a t i o n , i n the microsomes. I s o l a t i o n , p r o p e r t i e s and use. The components r e q u i r e d f o r an i n v i t r o study: microsomes UDPGA x e n o b i o t i c and b u f f e r are r e a d i l y a v a i l a b l e . UDPG simple system i s p e r f e c t l metabolism. I t i s i n a p p r o p r i a t e to review assay methods i n great d e t a i l i n t h i s chapter because, i n x e n o b i o t i c metabolism, we are so o f t e n i n t e r e s t e d i n a s p e c i f i c compound or s e r i e s of compounds and the assay procedure w i l l be based on the conversion of that compound. There are, however, some recent methods that may be g e n e r a l l y u s e f u l . A method i n v o l v i n g UDP[1^C]GA u t i l i s e s an Amberlite XAD-2 column to separate unchanged UDPGA, conjugate and unchanged substrate (16). A continuous assay based on the enzymatic assay of the UDP r e l e a s e d during the t r a n s f e r has a l s o been reported (17). The i s o l a t i o n of the enzyme i s achieved by the p r e p a r a t i o n of microsomes as described above. The enzyme i s r e l a t i v e l y s t a b l e i n f r o z e n 10,000 g supernatant and i n f r o z e n microsomal p e l l e t . I f s t o r e d at -196°C ( l i q u i d n i t r o g e n ) , r a t and r a b b i t enzymes can be kept f o r many days (18).Because the enzyme i s c l o s e l y a s s o c i a t e d with the l i p o p r o t e i n microsomal membranes, f u r t h e r p u r i f i c a t i o n must be preceded by s o l u b i l i s a t i o n . A recent method (19) i n v o l v e s treatment of the microsomes with 1% Lubrol 12A9 (a condensate of dodecyl a l c o h o l with approx. 9.5 mol of ethylene oxide per mol) f o r 20 min at 4°C. The enzyme remained i n the supernatant a f t e r f u r t h e r c e n t r i f u g a t i o n i . e . s o l u b i l i s a t i o n apparently occurred. I t was then p r e c i p i t a t e d w i t h ammonium sulphate and f u r t h e r p u r i f i e d i n the presence of 0.05% L u b r o l by DEAE c e l l u l o s e chromatography. The product contained only 3 p o l y p e p t i d e s ; enzyme a c t i v i t i e s towards 2-aminophenol and 4 - n i t r o phenol were increased 43- and 4 6 - f o l d r e s p e c t i v e l y . The p u r i f i e d enzyme had much improved s t a b i l i t y i n comparison with the microsomally-bound enzyme. Although there i s much k i n e t i c evidence f o r the existence of s e v e r a l glucuronyl t r a n s f e r a s e s (15), very few p a i r s of aglycone spécificités have been separated p h y s i c a l l y . One such separation i s that of the a c t i v i t i e s towards 4-nitrophenol and morphine by D e l l V i l l a r et a l (20). I t i s i n t h i s area where p u r i f i c a t i o n of the enzymes i s important. P u r i f i c a t i o n , however d e s i r a b l e i n theory and however necessary f
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to the study of the enzyme, i s r a r e l y c a r r i e d out i n s t u d i e s of p e s t i c i d e conjugation. Our main concern i s the f a t e of the p e s t i c i d e i n the whole animal and the r o l e that the conjugation may p l a y i n the d i s p o s i t i o n of the p e s t i c i d e . Nevertheless we have to be aware of some p r a c t i c a l complications consequent on the p a r t i c u l a t e nature of t h i s enzyme. UDPGA-glucuronyl t r a n s f e r a s e i s ' l a t e n t i n f r e s h l y p r e pared microsomes. Membrane-perturbing processes such as aging, f r e e z i n g and thawing, and treatment with detergents, chaotropes, organic s o l v e n t s , a l k a l i , t r y p s i n or phospholipases may a c t i v a t e the enzyme by a f a c t o r of 40 or more (15). T h i s property, together with evidence d e r i v e d from k i n e t i c s t u d i e s , some i n v o l v i n g competitive s u b s t r a t e s , i n d i c a t e s that the enzyme i s deeply b u r i e d , p o s s i b l th inne surfac f th microsomal v e s i c l e . For t h i s reaso with T r i t o n X-100 (overal ) g x e n o b i o t i c s u b s t r a t e . This treatment should f u l l y a c t i v a t e the enzyme and remove e r r o r s due to unknown amounts of a c t i v a t i o n caused by age, storage, mechanical e f f e c t s or even an e f f e c t of the x e n o b i o t i c substrate i t s e l f . The treatment should a l s o allow access of both UDPGA and the x e n o b i o t i c to the enzyme. T h i s may be r e s t r i c t e d , p a r t i c u l a r l y i f an i o n i c substrate (e.g. a c a r b o x y l i c acid) i s being i n v e s t i g a t e d . There are two f u r t h e r problems a s s o c i a t e d with i t s use however. The amount of a c t i v a t i o n i s species-dependent. For example, the g l u c u r o n i d a t i o n of 7-hydroxychlorpromazine (21) i s a c t i v a t e d twice as much i n r a t l i v e r microsomes as i t i s i n guinea-pig l i v e r microsomes. A c t i v a t i o n i s a l s o dependent on the c o n c e n t r a t i o n of T r i t o n X100 (see Figure 3). 1
Another problem i s the d e s t r u c t i o n of UDPGA by pyrophosphorylase. This i s p a r t l y i n h i b i t e d by EDTA but a b e t t e r s o l u t i o n i s apparently the use of c i t r a t e b u f f e r which v i r t u a l l y abolishes the pyrophosphorylase a c t i o n (15). The enzyme has a pH optimum of about 7.4 though t h i s may vary somewhat with i o n i s a b l e s u b s t r a t e s . Reaction rates are l i n e a r f o r at l e a s t an hour at 37°C. Examples of use. One of the most frequent uses of the g l u c u r o n i d a t i o n system i n our l a b o r a t o r y i s i n the b i o s y n t h e s i s of glucuronides of f a e c a l m e t a b o l i t e s . These are then chromatog r a p h i c a l l y compared with suspected glucuronides found i n the u r i n e or i n the b i l e of t r e a t e d animals. Figure 4 i l l u s t r a t e s the formation of the glucuronide of a n t i - 1 2 - h y d r o x y - [ l ^ C l e n d r i n under the f o l l o w i n g c o n d i t i o n s : volume, 5 ml; hydroxyendrin, 0.004 mM; UDPGA, 0.75 mM; washed r a b b i t l i v e r microsomes (4.2 mg/ml) i n 0.1 M TRIS-HC1 b u f f e r (pH 7.4 at 37°C); i n c u b a t i o n temperature, 37°C. P o r t i o n s (1 ml) were withdrawn at i n t e r v a l s and p a r t i t i o n e d between benzene and water which were then r a d i o assayed to a f f o r d the proportions of unchanged substrate and conjugate r e s p e c t i v e l y (22). T r i t o n X-100 had no e f f e c t on t h i s r e a c t i o n , p o s s i b l y because the very l i p o p h i l i c a n t i - 1 2 -
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC METABOLISM
0.1
0.2
0.3
0.4
0.5
Concentration of Triton X-100 (%) ure 3.
Effect of Triton X-100 on glucuronidation: ( ), rat liver microsomes; ( ), guinea pig liver microsomes.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
CRAWFORD AND HUTSON
Figure 4.
Type
II
Metabolism
Time course of the glucuronidation of C anti-12-hydroxyendrin by rabbit liver microsomes 14
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XENOBIOTIC METABOLISM
hydroxyendrin was r e a d i l y a c c e s s i b l e to the enzyme. The common experimental animals, e.g. guinea-pig, r a t , mouse, hamster and dog, a l l possess reasonable amounts of h e p a t i c microsomal g l u c u r o n y l t r a n s f e r a s e . L i t t e r s t e t a l (23) have compared the h e p a t i c enzyme i n rhesus monkey ( c u r r e n t l y i n short supply) with species which may be used as a l t e r n a t i v e t e s t animals. A c t i v i t i e s towards 4-nitrophenol (nmol per min per mg p r o t e i n ) were: rhesus monkey, 7; s q u i r r e l monkey, 8-11 (sex d i f f e r e n c e ) ; common t r e e shrew (9-12); miniature p i g (6-9); Sprague-Dawley r a t (2-3). These measurements were made without the a d d i t i o n of detergent. Conditions were: s u b s t r a t e , 0.2 mM; UDPGA, 3.3 mM; p r o t e i n , 1 mg/ml; T r i s b u f f e r pH 7.4, 1 mM. The cat and other F e l i d a e excrete l i t t l e o r no glucuronide conjugates of x e n o b i o t i c s (24). The h i g h K value f o r 4-nitrophenol with cat l i v e r g l u c u r o n y l t r a n s f e r a s t r a n s f e r a s e (25) i s i n s i m i l a r l y do not excrete many glucuronide conjugates and have very low enzyme a c t i v i t i e s i n l i v e r . These are examples of the i n v i t r o s u b c e l l u l a r r e s u l t s comparing w e l l w i t h the s i t u a t i o n i n v i v o . The occurrence of g l u c u r o n y l t r a n s f e r a s e i n b i r d s g e n e r a l l y has not been s t u d i e d . F i s h excrete glucuronides (26) and have been shown to possess h e p a t i c g l u c u r o n y l t r a n s f e r a s e a c t i v i t y towards 2-aminophenol (27) 3 - t r i f l u o r o m e t h y l - 4 - n i t r o phenol (26), and 4-nitrophenol (28). Glucuronide formation i s important i n man but the h e p a t i c enzyme has not been examined in detail. Another important use of i n v i t r o techniques i s f o r the comparison of b i o t r a n s f o r m a t i o n i n d i f f e r e n t t i s s u e types. However, a f t e r a survey u s i n g a s e n s i t i v e me thylumb e l l i f e r o n e assay, A i t i o (29) concluded that l i v e r i s the most important organ of glucuronide s y n t h e s i s . I t was estimated that the whole g a s t r o i n t e s t i n a l t r a c t possessed only 15-20% of the enzyme a c t i v i t y found i n l i v e r . Some r e s u l t s are summarised i n Table I. Low enzyme a c t i v i t y has a l s o been found i n the p l a c e n t a of s e v e r a l s p e c i e s , i n c l u d i n g man (30). The r e l a t i v e a c t i v i t i e s found f o r v a r i o u s t i s s u e s are p a r t l y a f u n c t i o n of the choice of s u b s t r a t e . For example, b r a i n , h e a r t , f a t and diaphragm possess very low a c t i v i t i e s towards methylumbelliferone (30) but q u i t e h i g h activités towards the c a r b a r y l m e t a b o l i t e , 1-naphthol (31). This demonstrates that model s u b s t r a t e s serve only as a guide; f o r r e l e v a n t i n f o r m a t i o n on a s p e c i f i c chemical, only s t u d i e s on that chemical w i l l r e a l l y s u f f i c e . T o x i c o l o g i c a l s i g n i f i c a n c e . The value of g l u c u r o n i d a t i o n l i e s i n the dramatic change i n p o l a r i t y that the process confers. The glucuronides are also very r e a d i l y secreted i n b i l e or v i a the kidneys and thus removed from the body. G l u c u r o n i d a t i o n can a l s o prevent c e r t a i n types of metabolite from being f u r t h e r b i o a c t i v a t e d to r e a c t i v e species such as the quinones which may be formed from aromatic d i h y d r o d i o l s (32). The process i s almost always a d e t o x i f i c a t i o n , however i n some important
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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cases the reverse i s observed. The g l u c u r o n i d a t i o n o f Ν-hydroxyarylacetamides (e.g. N-hydroxyphenacetin, Figure 5) a f f o r d s chemically r e a c t i v e molecules capable of i n t e r a c t i n g with t i s s u e macromolecules (33). The demonstration of t h i s type o f b i o a c t i v a t i o n i s a valuable use of i n v i t r o t e s t systems.
Table I. Glucuronidation o f methylumbelliferone in rat tissues
Enzyme a c t i v i t y Tissue
Liver Duodenal mucosa Adrenal glands Kidneys Spleen Lungs Thymus Heart Brain
nmol product p e r g
460 260 170 150 30 28 19 1.4 0.8
nmol product
3200 96 63 120 35 34 9 1.1 1.3
The use of separated s u b c e l l u l a r f r a c t i o n s may f a i l to r e v e a l p o s s i b l e i n t e r a c t i v e e f f e c t s or f u n c t i o n a l r e l a t i o n s h i p s between the f r a c t i o n s . For example, the a d d i t i o n of UDPGA to r a t l i v e r microsomes increases t h e i r rate of 12-hydroxylation of d i e l d r i n (34). The r a t i o n a l e f o r adding UDPGA t o an o x i d a t i v e system was that sjn-12-hydroxydieldrin (Figure 6), although more h y d r o p h i l i c than d i e l d r i n , i s s t i l l a very l i p o p h i l i c molecule, p a r t i c u l a r l y i n view of the hydrogen bonding of the hydroxy1 group to the epoxide oxygen. Thus, when formed as a metabolite v i a the a c t i o n o f microsomal mon o-oxygen as e, i t would remain near i t s s i t e of formation, p o s s i b l y i n h i b i t i n g f u r t h e r h y d r o x y l a t i o n of d i e l d r i n . This e f f e c t was f i r s t noted by von Bahr and B e r t i l s s o n (35) with demethylimipramine. The f u n c t i o n a l r e l a t i o n s h i p between microsomal monooxygenase, epoxide hydratase and glucuronyl t r a n s f e r a s e has r e c e n t l y been i n v e s t i g a t e d i n l i v e r microsomes and i n i s o l a t e d hepatocytes (32). The r e s u l t s provide a good i l l u s t r a t i o n of one of the l i m i t a t i o n s of the s u b c e l l u l a r approach. In the hepatocyte and i n microsomes f o r t i f i e d with NADPH and UDPGA, naphtha lene i s oxygenated to i t s 1,2-oxide which i s cleaved t o a d i h y d r o d i o l (by epoxide hydratase) which, i n t u r n , i s conjugated with g l u c u r o n i c a c i d (Figure 7). However, microsomes a f f o r d the d i h y d r o d i o l as the major metabolite; hepatocytes a f f o r d the glucuronide. Only by using e i t h e r more microsomal p r o t e i n o r the
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
194
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Figure 5.
Conjugation in the formation of reactive metabolites from phenacetin
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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CRAWFORD AND HUTSON
Cl
Type
Cl
Figure 6.
II
195
Metabolism
Cl
Hydroxyhtion and glucuronidation of dieldrin
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
196
XENOBIOTIC
mo no-oxygenase plus 0
2
METABOLISM
epoxide hydratase + H 0 2
microsomal glucuronyl transferase + UDPGA
Figure 7.
Rehtionship between the enzymes affecting the metabolism of naphthalene
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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a l l o s t e r i c e f f e c t o r of g l u c u r o n y l t r a n s f e r a s e , UDP-N-acetylglucosamine, could microsomes be f o r c e d to y i e l d reasonable q u a n t i t i e s of glucuronide. T h i s e f f e c t o r may a l s o c o m p e t i t i v e l y i n h i b i t the d e s t r u c t i v e e f f e c t of the pyrophosphorylase on UDPGA in vitro. Conjugation with Sulphate Mechanism and l o c a t i o n . Conjugation with sulphate i s mediated by the s u l p h o t r a n s f e r a s e enzymes ( h i s t o r i c a l l y sulphok i n a s e s ) . The donor s u b s t r a t e , which contains the a c t i v a t e d sulphate group, i s 3'-phosphoadenosine-S -phosphosulphate (PAPS). Acceptor groups are p r i n c i p a l l y p h e n o l i c but an a l c o h o l i c or primary amine f u n c t i o n can a l s o be sulphated v i a the same mechanism. I r r e s p e c t i v e of the chemical nature of the f i n a l sulphated product, sulphoconjugatio pathway: a c t i v a t i o n of sine 5 -phosphosulphate (APS) and then 3 -phosphoadenosine 5 phosphosulphate (PAPS), followed by t r a n s f e r of the sulphate group from PAPS to s u i t a b l e acceptors: 1
1
f
2
ATP
+ S0 ~
*
APS
+ ATP
*
4
PAPS + Acceptor
PAP
APS
f
+ PP£
(1)
PAPS + ADP
(2)
+ sulphated acceptor
(3)
The enzymes c a t a l y s i n g these r e a c t i o n s are (1) ATP-sulphate adenylyl t r a n s f e r a s e (ATP-sulphurylase); (2) ATP-adenylyl sulphate 3 -phosphotransferase (APS-kinase) and (3) an appropriate s u l p h o t r a n s f e r a s e (EC 2.8.2). In mammalian c e l l s the enzymes r e s p o n s i b l e f o r the p r o d u c t i o n of PAPS from sulphate are l o c a l i s e d i n the c y t o s o l . Sulphotransferases are l o c a t e d i n both the microsomal and the c y t o s o l i c f r a c t i o n s of a wide v a r i e t y of t i s s u e s ; t h e i r general t i s s u e d i s t r i b u t i o n resembles that of the UDP-glucuronyltransferases, except that they are a l s o abundant i n p l a c e n t a . Boundaries of t h e i r s p e c i f i c i t i e s are more c l e a r than those of the g l u c u r o n y l t r a n s f e r a s e s , and d i s t i n c t i v e phenol, s t e r o i d and arylamine s u l p h o t r a n s f e r a s e a c t i v i t i e s have been detected. Other t r a n s f e r a s e s deal apparently s o l e l y with endogenous compounds, e.g. cerebroside and p o l y s a c c h a r i d e sulphot r a n s f erases. Simple endogenous sulphates i n c l u d e s those of s t e r o i d s , a d r e n a l i n e , t r i - i o d o t h y r o n i n e and s e r o t o n i n . The p i c t u r e emerges ( c f . the g l u c u r o n y l t r a n s f e r a s e s ) of the sulphot r a n s f e r a s e s r e s p o n s i b l e f o r the s u l p h a t i o n of small molecules being f r e e l y s o l u b l e i n the c y t o s o l , whereas those that are i n v o l v e d i n the assembly of l a r g e r molecules are arranged, together with other r e q u i s i t e b i o s y n t h e t i c enzymes, i n assemblyl i n e f a s h i o n on the membranes of the endoplasmic r e t i c u l u m and the G o l g i apparatus. I t i s extremely d o u b t f u l whether any of these bound enzymes p l a y any r o l e i n the metabolism of xenob i o t i c s (36). f
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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I s o l a t i o n , p r o p e r t i e s and use. The u n c e r t a i n t y i n the t o t a l number, p r e c i s e r o l e s and s p e c i f i c i t i e s of the s u l p h o t r a n s f e r a s e s r e f l e c t s the f a c t that they are exceedingly d i f f i c u l t to p u r i f y and to separate from one another. Moreover, some of them aggre gate and/or change conformation under c e r t a i n c o n d i t i o n s ; m u l t i p l e peaks of s i m i l a r a c t i v i t y , which may o r may not be due to the same enzyme, appear on chromatography columns and are a constant hindrance to the e x p e r i m e n t a l i s t . A s u l p h o t r a n s f e r a s e has been p a r t i a l l y p u r i f i e d from bovine kidney (acetone powder) using 4-nitrophenol as the assay sub s t r a t e (37). The a c t i v i t y of the enzyme was measured using [35s]PAPS £ the assay mixture. Substrate s p e c i f i c i t y was i n v e s t i g a t e d and i t was found to be r e l a t i v e l y s p e c i f i c f o r simple a r y l sulphate formation as shown i n Table I I n
Table I I . R e l a t i v bovine kidney s u l p h o t r a n s f e r a s e
Substrate
Relative specificity
4-Nitrophenol 4-Hydroxybenzaldehyde 4-Chlorophenol 1-Naphthol Phenol o-Cresol m-Cresol 3-Nitrophenol 3-Hydroxybenzaldehyde
100 75.9 79.7 32.5 6.5 21.7 8.3 49.8 21.0
No a c t i v i t y could be demonstrated towards e t h a n o l , propan-Ι οί, b u t a n - l - o l , or towards a range of s t e r o i d s . The i s o l a t i o n of N-hydroxy-2-acetylaminofluorene (N-0H-2AAF) s u l p h o t r a n s f e r a s e has r e c e n t l y been achieved from the c y t o s o l f r a c t i o n s of male and female r a t l i v e r s (the l a t t e r possess very low a c t i v i t y ) (38). A 2000-fold p u r i f i c a t i o n with a y i e l d of over 12% was achieved using the f o l l o w i n g procedure: ammonium sulphate f r a c t i o n a t i o n , DEAE-cellulose column chromatography, hydroxya p a t i t e column chromatography, sephadex G-200 g e l f i l t r a t i o n , i s o e l e c t r i c f o c u s s i n g and, f i n a l l y , more sephadex G-200 g e l f i l t r a t i o n . The f i n a l p r e p a r a t i o n was homogenous on a n a l y t i c a l d i s c g e l e l e c t r o p h o r e s i s . The p u r i f i e d enzyme had a c t i v i t y towards 4-nitrophenol with an approximately 1600-fold i n c r e a s e i n s p e c i f i c a c t i v i t y over the crude homogenate, but i t had very low a c t i v i t y towards endogenous s t e r o i d s and s e r o t o n i n . PAPS was used as the sulphate donor i n these assay mixtures and was synthesised e n z y m a t i c a l l y (39). The pure enzyme was very u n s t a b l e , e s p e c i a l l y i n d i l u t e s o l u t i o n s . T h i o l compounds were found to have a s t a b i l i s i n g e f f e c t and t h i o l b l o c k i n g reagents were potent i n h i b i t o r s .
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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The i n v o l v e d nature of these p u r i f i c a t i o n s suggests that the study of phase I I sulphoconjugations u s i n g p u r i f i e d sulphotrans ferase from v a r i o u s animal species and animal t i s s u e s i s impract i c a l at present. An i n v i t r o assay system f o r studying these r e a c t i o n s u s i n g crude s u b c e l l u l a r f r a c t i o n s has been developed by Mulder et a l (33). I t comprises c y t o s o l (600 μg p r o t e i n / m l ) , x e n o b i o t i c (0.5 mM), T r i s - H C l b u f f e r (100 mM, pH 8.0) and a PAPSgenerating system (PAPS-GS): 3 5 - a d e n o s i n e diphosphate (PAP, 10 μΜ) and 4-nitrophenyl sulphate (10 mM). In t h i s assay mixture 4-nitrophenyl sulphate i s used to convert PAP i n t o PAPS, a r e a c t i o n presumably c a t a l y s e d by a phenolsulphotransferase i n the c y t o s o l . The r a t e of formation of 4-nitrophenol i s spectrophotom e t r i c a l l y determined and i s c a l c u l a t e d by s u b t r a c t i n g the amount of 4-nitrophenol r e l e a s e d i n a c o n t r o l i n c u b a t i o n mixture ( x e n o b i o t i c absent) fro x e n o b i o t i c . The r a t e o c u l a t e d from the amount of 4-nitrophenol r e l e a s e d u s i n g a molar e x t i n c t i o n c o e f f i c i e n t of 17,500 M" cm~l (at pH 8.0). This assay procedure makes an important c o n t r i b u t i o n to the f i e l d of xeno b i o t i c metabolism i n that i t provides a cheap and simple method f o r studying the mechanisms and r a t e s of phase I I sulphate con j u g a t i o n r e a c t i o n s using a PAPS-GS and c y t o s o l . H i t h e r t o PAPS has been commercially a v a i l a b l e i n the l a b e l l e d form only and i t s enzymic s y n t h e s i s i s a tedious process (39)(40). A more t r a d i t i o n a l assay procedure i s that described by Wu and Straub (38) where PAPS i s used at a c o n c e n t r a t i o n of approximately 0.3 mM when substrate concentration i s 0.2 mM. f
1
1
Examples of use. Harmol (Figure 8) i s a good substrate f o r phenol sulphotransferase i n r a t l i v e r 600 g f r a c t i o n (41) but harmalol (Figure 8) i s a very poor s u b s t r a t e . The reason f o r the d i f f e r e n c e i n r a t e of s u l p h a t i o n of these two substrates i s unknown, but the f i n d i n g s agree with the d i f f e r e n t r a t e s and modes of conjugation ( s u l p h a t i o n and g l u c u r o n i d a t i o n ) of the two compounds found i n v i v o . The metabolism of harmol i l l u s t r a t e s a problem that o f t e n a r i s e s when d i s c u s s i n g the phase I I conjugation of x e n o b i o t i c s with sulphate. Sulphation of a x e n o b i o t i c or i t s metabolite i s p r a c t i c a l l y always accompanied by conjugation with g l u c u r o n i c a c i d . Glucuronic a c i d conjugation o f t e n predominates and t h i s has been assumed to be due to a l i m i t e d supply of sulphate i n v i v o . Mulder and coworkers have developed a method f o r the simultan eous measurement of UDP-glucuronyltransferase and phenolsulphot r a n s f e r a s e from r a t l i v e r i n v i t r o , using harmol as substrate and 600 g supernatant as the enzyme source (42). T r i t o n X-100 was used to a c t i v a t e g l u c u r o n y l t r a n s f e r a s e ( S e c t i o n 5.1) and was shown to have no e f f e c t on the a c t i v i t y of phenolsulpho t r a n s f erase. The amount of the conjugates formed was measured f l u o r o m e t r i c a l l y and the a c t i v i t i e s of the enzymes were expres sed as a r b i t r a r y u n i t s of fluorescence recovered from a t i c a n a l y s i s of the incubates. At low substrate concentrations
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC
METABOLISM
glucuronidation sulphation phosphorylation (?)
,οχ COCH
3
(reactive) Figure 9.
Acetyhtion and conjugation in the activation of aromatic amines
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
6.
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Metabolism
201
(<40 μΜ) harmol was p r e f e r e n t i a l l y sulphated but with i n c r e a s i n g substrate concentration, g l u c u r o n i d a t i o n became more important. These r e s u l t s agree with e a r l i e r i n v i t r o r e s u l t s (41) when the 1^ value f o r harmol of UDP-glucuronyl t r a n s f e r a s e was found to be 150 μΜ and of phenolsulphotransferase was <15 uM. Therefore, i n some circumstances, the a f f i n i t i e s of the enzymes f o r the sub s t r a t e may c o n t r o l the r e l a t i v e amounts of conjugation. 2,6-Dic h l o r o - 4 - n i t r o p h e n o l may be used as a s e l e c t i v e i n h i b i t o r of s u l p h a t i o n i f g l u c u r o n i c a c i d conjugation needs to be s t u d i e d i n the 10,000 g f r a c t i o n of l i v e r (43). Thus, using i n v i t r o sub c e l l u l a r f r a c t i o n s c o n t a i n i n g even crude enzyme p r e p a r a t i o n s , a greater understanding of i n v i v o f i n d i n g s i s p o s s i b l e . A second important f a c e t of i n v i t r o s t u d i e s i s the assess ment of the r e l a t i v e importance of various animal organs i n x e n o b i o t i c metabolism. f i c a n t s u l p h a t i o n of 4-nitropheno been demonstrated (44). The former was sulphated by lung c y t o s o l at about one t h i r d the r a t e found f o r l i v e r c y t o s o l (per mg p r o t e i n ) . In these experiments PAPS was i n c l u d e d i n the i n c u b a t i o n mixtures. This may account f o r the apparent d i s p a r i t y between these r e s u l t s and those of Gram et a l (45) who could not detect any sulphotransferase a c t i v i t y towards 4-nitrophenol i n r a b b i t lung supernatant. These authors u t i l i s e d a PAPS-generating system i n t h e i r experiments. Sulphation of s t e r o i d s has been s t u d i e d i n the c y t o s o l of b r a i n and l i v e r from guinea-pigs and r a t s (46). [35s]PAPS was used as the sulphate donor. 17-£-0estradiol (E) and d i e t h y l s t i l b e s t r o l (DES) were used as s u b s t r a t e s . The f o l l o w i n g a c t i v i t i e s (pmol conjugate/mg tissue/20 min) were measured with b r a i n pre p a r a t i o n s : r a t : Ε, 0.6; DES, 2.0; guinea-pig: Ε, 0.1; DES, 4.1. A c t i v i t i e s with guinea-pig l i v e r were: E, 98; DES 333. Thus, with these s u b s t r a t e s , the a c t i v i t y of the t r a n s f e r a s e i n b r a i n i s only a small f r a c t i o n of that of l i v e r . T o x i c o l o g i c a l s i g n i f i c a n c e . Conjugation of a x e n o b i o t i c with a completely i o n i s e d and h i g h l y h y d r o p h i l i c moiety such as s u l phate markedly a l t e r s i t s physico-chemical p r o p e r t i e s and t h i s u s u a l l y leads, l i k e g l u c u r o n i d a t i o n , to a complete l o s s of pharmacological or p e s t i c i d a l a c t i v i t y . This and a r a p i d r a t e of e x c r e t i o n are the b e n e f i c i a l consequences of sulphoconjugation. Several x e n o b i o t i c s exert t h e i r t o x i c e f f e c t s i n mammals though the formation of r e a c t i v e metabolites that combine with c e l l u l a r macromolecules. The 0-sulphation of N-0H-2-AAF has been p o s t u l a t e d as an a c t i v a t i o n r e a c t i o n (Figure 9) l e a d i n g to the c a r c i n o g e n i c a c t i o n of t h i s compound (47). There i s no d i r e c t evidence f o r the formation of these NO-sulphates i n v i v o ; t h e i r i n s t a b i l i t y precludes t h e i r i s o l a t i o n and c h a r a c t e r i s a t i o n . However, t h e i r t r a n s i e n t e x i s t e n c e has been demonstrated i n v i t r o by the use of i n d i r e c t methods, such as trapping the conjugates i n s i t u by r e a c t i o n with n u c l e o p h i l i c compounds (48). There i s a l s o i n d i r e c t evidence that the sulphate conjugate of N-0H-2-AAF
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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i s formed i n r a t l i v e r i n v i v o (49). Mulder et a l (33) have r e c e n t l y used the PAPS generating system described e a r l i e r i n t h i s s e c t i o n , to i n d i r e c t l y measure the r a t e of s u l p h a t i o n of N-0H-2AAF. The r e a c t i o n r a t e was l i n e a r with c y t o s o l i c p r o t e i n concentration up to 1.2 mg/ml and was about 2.7 nmoles 4 - n i t r o phenol formed per ml per mg s o l u b l e p r o t e i n (at 31°C). These r e s u l t s suggest that the NO-sulphate of N-0H-2-AAF had been formed, but again i s o l a t i o n of the metabolite was not p o s s i b l e because of i t s i n s t a b i l i t y . Evidence that an NO-sulphate conjugate was formed i n the i n c u b a t i o n mixtures was obtained by showing that the covalent b i n d i n g of N-0H-2-AAF to macromolecules increased dramatically during i n c u b a t i o n with 4-nitrophenyl sulphate and PAP. N-Hydroxyphenacetin, N-hydroxyacetanilide, N-hydroxy-4-chloroacetanilide and N-hydroxy-2-acetylaminonaphthalene also formed sulphat that of N-0H-2-AAF. N-Hydroxyphenaceti aminonaphthalene a l s o became r a p i d l y c o v a l e n t l y bound a f t e r s u l p h a t i o n of t h e i r N-hydroxy groups, but the O-sulphates of N-hydroxy-4-chloroacetanilide and N-hydroxyacetanilide d i d not r e a c t (Table I I I ) . DeBaun et a l (48) have a l s o e x t e n s i v e l y s t u d i e d t h i s e f f e c t of s u l p h a t i o n on covalent b i n d i n g of N-0H-2AAF, and the work i s a good example of the use of s u b c e l l u l a r f r a c t i o n s i n the i n v e s t i g a t i o n s of mechanisms of c a r c i n o g e n e s i s . Table I I I . Conjugation rates and e f f e c t of conjugation on covalent b i n d i n g of some N-hydroxy-N-arylacetamides
Substrate
Sulphation rate (nmo l e s /min /mg soluble protein)
Amount c o v a l e n t l y bound (nmoles/ml) with without 4-nitrophenylsulphate
N-hy dr oxyphenacet i n
2.6
25.0
0.2
N-hydroxyacetanilide
1.6
0.2
0.2
N-hydroxy-4-chloroacetanilide
2.4
0.4
0.2
N-hydroxy-2-AAF
2.7
21.8
0.6
N-hydroxy-2-acetylamino naphthalene
2.0
1.9
0.2
This study shows c l e a r l y that the NO-sulphate of N-hydroxyphenacetin binds c o v a l e n t l y to p r o t e i n as soon as i t i s synt h e s i s e d . Since phenacetin i s N-hydroxylated by microsomal enzymes (50)(51), i t seems l i k e l y that the s u l p h a t i o n and g l u c u r o n i dation pathways provide a r e a c t i v e intermediate f o r covalent b i n d i n g . At the present time i t i s not c l e a r whether e i t h e r r e a c t i o n p l a y s a r o l e i n causing the short-term t o x i c e f f e c t s of
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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II
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Metabolism
high doses of phenacetin i n v i v o ( l i v e r n e c r o s i s ) ; however, t h i s work demonstrates the value of s u b c e l l u l a r s t u d i e s i n the charac t e r i s a t i o n of enzymatic determinants of t o x i c i t y . Phosphorylation The e x c r e t i o n of di(2-amino-1-naphthy1)hydrogen phosphate by dogs dosed w i t h 2-naphthylamine (52) suggests that phosphoryl a t i o n occurs i n x e n o b i o t i c metabolism. However, only very few examples have been found i n the intervening 17 y e a r s . The process has been p o s t u l a t e d as a b i o a c t i v a t i o n step i n c a r c i n o g e n e s i s by 2-acetylaminofluorene (53). Monophenyl phosphate i s excreted i n the u r i n e of cats dosed w i t h phenol (54). Rat l i v e r mitochondria c o n t a i n an enzyme that c a t a l y s e s the ATP-dependent p h o s p h o r y l a t i o n of l-aminopropan-2-ol (55). These f a c t s summarise most of our knowledge on the phosphorylatio here to emphasise the apparentl j u g a t i o n . The c e n t r a l r o l e of p h o s p h o r y l a t i o n i n energy meta b o l i s m i s presumably, of n e c e s s i t y , c o n t r o l l e d by very s u b s t r a t e s p e c i f i c processes i n which x e n o b i o t i c s cannot r e a d i l y participate. Acetylation Mechanism and l o c a t i o n . A c e t y l a t i o n i s of r e l a t i v e l y minor importance i n p e s t i c i d e metabolism i n comparison with g l u c u r o n i d a t i o n , s u l p h a t i o n and g l u t a t h i o n e conjugation. However, the r e a c t i o n must be expected whenever an aromatic amine i s under i n v e s t i g a t i o n per se or l i b e r a t e d d u r i n g metabolism. The t r a n s f e r of acetate occurs from acetyl-coenzyme A and i s c a t a l y s e d by arylamine N - a c e t y l t r a n s f e r a s e (EC 2.3.1.5). The enzyme i s l o c a t e d i n the c y t o s o l of mammalian c e l l s . The r e a c t i o n i s general f o r aromatic amines, sulphonamides, hydrazino compounds and some nonaromatic amines (56). The enzyme operates v i a a simple ping-pong mechanism of two consecutive s t e p s : a c e t y l a t i o n of the enzyme by acetyl-CoA followed by t r a n s f e r of acetate from a c e t y l - N a c e t y l - t r a n s f e r a s e to the acceptor s u b s t r a t e . This has been confirmed by the i s o l a t i o n of a [ ] a c e t y l - N - a c e t y l t r a n s f e r a s e p r o t e i n capable of donating i t s a c e t y l group to i s o n i a z i d (57). The r e a c t i o n i s important i n the metabolism of endogenous amines, i n c l u d i n g s e r o t o n i n , tryptamine, histamine and phenylethylamine. Man and r a b b i t e x h i b i t a g e n e t i c polymorphism i n N - a c e t y l a t i o n (58) ( f o r example, of 4-aminobenzoic a c i d and i s o n i a z i d ) and i n d i v i d u a l s can be c l a s s i f i e d as ' r a p i d or s l o w acetylators. The l o c a t i o n of the N - a c e t y l t r a n s f e r a s e s i s not always c y t o s o l i c . Those i n v o l v e d i n the l a s t stages of mercapturic a c i d formation are l o c a t e d i n the l i v e r microsomal f r a c t i o n (59). In p r i n c i p l e , O - a c e t y l a t i o n and S - a c e t y l a t i o n of f o r e i g n compounds may occur (as they do i n the formation of the endogenous s u b s t r a t e s 0 - a c e t y l c h o l i n e and j>-acetyl-CoA); however, they have not yet been demonstrated. I s o l a t i o n , p r o p e r t i e s and use. N-Acetyl t r a n s f e r a s e (to aromatic amines etc.) i s best used and measured i n c y t o s o l p r e pared by u l t r a - c e n t r i f u g a t i o n . T h i s may be d i a l y s e d before use. 1
f
f
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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Rabbit c y t o s o l enzyme i s s t a b l e f o r at l e a s t a month when f r o z e n , and f o r 48 hours i f s t o r e d at 4° or 25°. I t a l s o withstands repeated f r e e z i n g and thawing. I t s pH optimum v a r i e s with sub s t r a t e but l i e s between 6 and 8.5. A p u r i f i c a t i o n procedure from r a b b i t l i v e r c y t o s o l has been described by Weber and Cohen (60). T h i s i n v o l v e d c e n t r i f u g a t i o n to a f f o r d a 100,000 g supernatant, ammonium sulphate p r e c i p i t a t i o n , Sephadex G-100 and DEAEc e l l u l o s e chromatography. T y p i c a l c o n d i t i o n s f o r use are as f o l l o w s : substrate (0.1 fimole) , acetyl-CoA (0.5 μπιοίε), 0.3 mM phosphate b u f f e r pH 6.8 (perhaps h i g h e r ) , c y t o s o l (0.5-5 mg p r o t e i n ) i n a volume of 1 ml. As u s u a l , the assay system w i l l depend on the substrate under i n v e s t i g a t i o n . However, a u s e f u l general method i n v o l v e s [l^C-l-acetyl]-acetyl-CoA the products from which can be e x t r a cted i n t o organic solven chromatographic separatio Examples of use. In v i t r o measurements (4-aminobenzoic acid) have been used to show a wide species d i s t r i b u t i o n of N - a c e t y l t r a n s f e r a s e (23). For example, the a c t i v i t y of the h e p a t i c enzymes l i e s i n the order: rhesus monkey = p i g > s q u i r r e l monkey = t r e e shrew > r a t . The i n d u s t r i a l intermediate 2,4-toluenediamine i s N-acetylated p r e f e r e n t i a l l y at the 4-amino group. The enzyme a c t i v i t y ( i n c y t o s o l ) i s found i n l i v e r > kidney > i n t e s t i n a l mucosa > lung. The a c t i v i t y of the l i v e r enzyme v a r i e s with s p e c i e s : hamster > guinea-pig > r a b b i t > mouse > r a t > human (trace) > dog (zero) (62). The genetic polymorphism noted above was discovered at the i n v i v o l e v e l i n man and r a b b i t s . I t i s of the same type i n both species ( c o n t r o l l e d as a simple autosomal Mendelian character with r a p i d a c e t y l a t i o n dominant to slow (56)). I t has been understood v i a s u b c e l l u l a r s t u d i e s . I t i s probably due to v a r y i n g amounts of the same enzyme r a t h e r than to d i f f e r e n t enzymes (56). At l e a s t two N - a c e t y l t r a n s f e r a s e s e x i s t i n the r a b b i t . They d i f f e r i n t h e i r t i s s u e d i s t r i b u t i o n s , substrate s p e c i f i c i t i e s and p H - a c t i v i t y p r o f i l e s . The one r e s p o n s i b l e f o r the genetic polymorphism has a wide substrate s p e c i f i c i t y and i s found mainly i n the l i v e r and gut (63). T o x i c o l o g i c a l s i g n i f i c a n c e . J H A c e t y l a t i o n i s very important i n the i n a c t i v a t i o n of the pharmacological and b i o c i d a l p r o p e r t i e s of many amine drugs; however, t h i s property i s of minor importance i n the p e s t i c i d e f i e l d . Of greater i n t e r e s t and p o t e n t i a l importance i s the r o l e that N - a c e t y l a t i o n plays i n the b i o a c t i v a t i o n of c a r c i n o g e n i c aromatic amines. 2-Aminofluorene, 4-aminob i p h e n y l and 2-aminonaphthalene (Figure 9 ) , f o r example, are a l l thought to be N - a c e t y l a t e d i n the f i r s t step on t h e i r metabolic routes to u l t i m a t e carcinogens. The enzyme a c t i v i t y i s of course r e a d i l y a v a i l a b l e i n the l i v e r c y t o s o l of v a r i o u s s p e c i e s . I t i s of considérable i n t e r e s t that dog l i v e r c y t o s o l contains no d e t e c t a b l e a c t i v i t y (Figure 10) (nor towards toluenediamine above) and that dog l i v e r i s not s u s c e p t i b l e to the c a r c i n o g e n i c a c t i o n of these compounds (64). When a v a r i e t y of arylamines are
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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XENOBIOTIC METABOLISM
administered to dogs, only u r i n a r y bladder tumours are noted. However, the a d m i n i s t r a t i o n of c a r c i n o g e n i c arylacetamides causes both bladder and l i v e r tumours. C l e a r l y h e p a t i c N-acety1 t r a n s f e r a s e i s a determinant of l i v e r carcinogenesis by arylamines. The N - a c e t y l a t i o n of i s o n i a z i d may a l s o be a t o x i c a t i n g r e a c t i o n . An increased incidence of ' i s o n i a z i d h e p a t i t i s has been noted i n r a p i d l y a c e t y l a t i n g humans (65). Methylation Mechanism and l o c a t i o n . The methylation of phenols, t h i o l s and amines i n v o l v e s a methyl donor OS-adenosylmethionine) and methyltransferases. Thus the mechanism i s t y p i c a l of type I I r e a c t i o n s although the r e s u l t , i n terms of i n c r e a s e i n p o l a r i t y , i s much l e s s or even the reverse of the other type I I processes. O-Methylation. Catechol 0-methyl t r a n s f e r a s e (66) (EC 2.1.1.6) i s the most commo chemistry. I t i s h i g h l y dihydroxybenzene) c o n f i g u r a t i o n , but given t h a t , i t i s undemandi n g i n i t s other requirements (67). I t i s probably encountered most commonly i n x e n o b i o t i c metabolism at the and of the sequence: arene •> arene oxide -> d i h y d r o d i o l -> catechol monomethylcatechol. Most of the enzyme a c t i v i t y i s present i n the c y t o s o l of v a r i o u s t i s s u e s . Phenol 0-methy1transferase, a microsomal enzyme, has been detected i n v i t r o (68) but i t s s i g n i f i c a n c e i n v i v o i s unknown because the 0-methylation of monohydric phenols apparently does not occur i n v i v o . A p o t e n t i a l l y u s e f u l general assay method i n v o l v i n g the use of the t r i t i a t e d cosubstrate S-adenosy1-L[methyl-3H]methionine has been described (69). The r a d i o a c t i v e methylated product i s e x t r a c t e d i n t o organic solvent f o r r a d i o assay. S-Methylation. ^ - M e t h y l a t i o n i s a general pathway of metabo l i s m of the thiopyrimidone a n t i t h y r o i d drugs such as 6-propyl2 - t h i o u r a c i l . T e t r a h y d r o f u r f u r y l mercaptan i s a l s o S-methylated i n an S-adenosylmethionine-dependent enzymatic r e a c t i o n . The enzyme was found i n the microsomal f r a c t i o n of l i v e r , kidney and small i n t e s t i n e (70). Thiophenol l i b e r a t e d during the metabolism of dyfonate (Figure 11) i s methylated (71). This r e a c t i o n has not been s t u d i e d i n v i t r o . N-Methylation. P y r i d i n e d e r i v a t i v e s are N-methylated but the r e a c t i o n s are q u a n t i t a t i v e l y of minor importance. The most widely s t u d i e d N-methylation r e a c t i o n s are those i n v o l v i n g the b i o g e n i c amines. For example, phenylethanolamine N-methyItransferase (EC 2.1.1) (involved i n the b i o s y n t h e s i s of epinephrine) has been s t u d i e d r e c e n t l y and found to methylate non-aromatic substrates as w e l l as aromatic ones (e.g. Figure 12, R = p r o p y l , c y c l o h e x y l , c y c l o h e x - 3 - e n y l , c y c l o - o c t y l ) . The enzyme has an absolute r e q u i r e ment f o r a hydroxy1 group at the 3 - p o s i t i o n on an e t h y l s i d e chain (72). Methylation r e a c t i o n s have not featured widely i n p e s t i c i d e metabolism; however, there i s enough information d e r i v e d from endogenous biochemistry and drug metabolism to be u s e f u l should 1
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
CRAWFORD AND i i u T S O N
Type
II
Metabolism
K3
Et
EtO
Ο
OH
Me S
MeS
<-
Ο Figure 11.
Methylation of thiophenol in the metabolism of dyfonate
R
Figure 12.
CH CH NH I OH 3
2
Substrate for phenylethanohmine
N-methyltransferase
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
208
XENOBIOTIC METABOLISM
i t be necessary to study these r e a c t i o n s with p a r t i c u l a r pesticides. Conjugation with Amino Acids — Mechanism and l o c a t i o n . Amino a c i d conjugation i n p e s t i c i d e metabolism has r e c e n t l y been reviewed (73). Substrates f o r amino a c i d conjugation are u s u a l l y e i t h e r aromatic c a r b o x y l i c a c i d s or a r y l a c e t i c a c i d s . The most important conjugating amino a c i d i s g l y c i n e and, together with glutamine, accounts f o r the m a j o r i t y of α-amino a c i d conjugates formed i n mammals. α-Amino a c i d conjugation occurs with the formation of an amide bond. Thus, both the substrate and the metabolite possess an i o n i s a b l e carboxyl group. The f i r s t step of the r e a c t i o n occurs with the i n t e r a c t i o n s of the x e n o b i o t i c w i t h coenzyme A ( I ) . Conjugation of the i n the second step and i 2.3.1.13) (II) (Figure 13) , glycin jugates may be c o n t r a s t e d with the mechanism of the other con j u g a t i o n r e a c t i o n s d i s c u s s e d so f a r . I t i s the x e n o b i o t i c r a t h e r than the endogenous conjugating moiety that i s a c t i v a t e d p r i o r to the t r a n s f e r a s e a c t i o n . A c t i v a t i o n of benzoate i n t o benzoyl-CoA i s thought to be c a t a l y s e d by butyryl-CoA synthetase (EC 6.2.1.2) (74), which i s found i n the m i t o c h o n d r i a l matrix (75). G l y c i n e N-acylase has been prepared from mitochondria (76)(77) and has r e c e n t l y a l s o been found by Gatley et a l (78) to be l o c a t e d i n the m i t o c h o n d r i a l matrix. These authors have also shown that h y d r o l y s i s of b e n z o y l CoA occurs i n s i t u and they p o s t u l a t e that the f u n c t i o n of the simultaneous a c t i v i t y of benzoyl-CoA synthetase and benzoyl-CoA hydrolase i s to prevent the accumulation of too much benzoyl-CoA. T h i s would occur i f the supply of g l y c i n e , or i t s rate of con j u g a t i o n , were l i m i t i n g f o r hippurate s y n t h e s i s and could thus impair other CoA-requiring r e a c t i o n s (79). I t i s thought that u n d i s s o c i a t e d benzoic a c i d and h i p p u r i c a c i d cross the i n n e r membrane because of t h e i r l i p i d s o l u b i l i t y . F u r t h e r , H a i l i n g et a l (80) conclude that g l y c i n e crosses the inner membrane as a z w i t t e r i o n . Therefore the l o c a t i o n of the enzyme does not apparently impair the access of these s u b s t r a t e s . In a d d i t i o n to the conjugation of x e n o b i o t i c s with amino a c i d s , endogenous compounds, notably the b i l e a c i d s , conjugate with g l y c i n e and t a u r i n e . Recently, a r g i n i n e conjugates of b i l e acids have also been i d e n t i f i e d (81). Choloyl-CoA synthetase (EC 6.2.1.7), the enzyme r e s p o n s i b l e f o r the formation of the a c t i v a t e d b i l e a c i d intermediate, has been l o c a t e d i n the microsomal f r a c t i o n of guinea-pig l i v e r (82). The i n t r a c e l l u l a r l o c a t i o n of the amino a c i d N-acyItransferase enzymes r e s p o n s i b l e f o r the conjugation of the a c t i v a t e d sub s t r a t e with t a u r i n e or g l y c i n e has been i n dispute f o r some time. S i p e r s t e i n and Murray (83) l o c a l i s e d the enzyme(s) i n the s o l u b l e f r a c t i o n of l i v e r c e l l s . Bremer (84) and E l l i o t t (85) concluded that they were l o c a t e d i n the microsomal f r a c t i o n and Schersten
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
6.
CRAWFORD AND HUTSON
Type II
Figure 13.
Metabolism
Amino acid conjugation
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
209
210
XENOBIOTIC METABOLISM
(86), i n the lysosomal f r a c t i o n . Vessey et a l (87) have r e c e n t l y found enzyme a c t i v i t y i n the s o l u b l e f r a c t i o n of bovine l i v e r c e l l s and they have presented evidence f o r two d i s t i n c t enzymes: g l y c i n e N - a c y l t r a n s f e r a s e and t a u r i n e N - a c y l t r a n s f e r a s e . K i l l e n b e r g and Jordan (88) on the other hand have i s o l a t e d a s i n g l e a c y l t r a n s f e r a s e from r a t l i v e r which i s capable of con j u g a t i n g chemically synthesised b i l e a c i d - CoA d e r i v a t i v e s with glycine or taurine. I s o l a t i o n , p r o p e r t i e s and use. There have been no recent p u b l i c a t i o n s on the i s o l a t i o n o f m i t o c h o n d r i a l acyl-CoA syn thetases r e s p o n s i b l e f o r the a c t i v a t i o n of x e n o b i o t i c s o r t h e i r metabolites. We have t o turn to p u r e biochemistry f o r inform a t i o n . Choloyl-CoA synthetase, as s t u d i e d by Vessey and Zakim (82), was s t a b l e f o r at l e a s t one month when the microsomal f r a c t i o n of guinea-pig l i v e assay procedure containe M g , 5 mM ATP, 0.8 mg o f microsomal protein/ml and 100 mM T r i s / HC1 b u f f e r , pH 7.3 i n an assay volume o f 0.1 ml. The i d e n t i f i c a t i o n of [!^C]choloyl-CoA was based on i t s comparison with chemically synthesised choloyl-CoA on paper and t h i n - l a y e r chromatography. The pH optimum f o r i t s r e a c t i o n was pH 7.2-7.3 and the r e a c t i o n had an absolute requirement f o r b i v a l e n t c a t i o n s , Mg and M n being the most e f f e c t i v e . High concentrations of ATP appeared to cause substrate i n h i b i t i o n . The enzyme could be s o l u b i l i s e d from the microsomal membrane i n an a c t i v e form by treatment with T r i t o n N-101. I n h i b i t i o n studies were c o n s i s t e n t with the hypothesis that one enzyme i s r e s p o n s i b l e f o r the synthesis o f the CoA-derivatives o f a l l the major b i l e a c i d s , but that t h i s enzyme i s d i f f e r e n t from that metabolising f a t t y acids to t h e i r CoA d e r i v a t i v e s . These conclusions are supported by the f i n d i n g s o f S i p e r s t e i n and Murray (83) that a p r e p a r a t i o n of palmitoyl-CoA synthetase does not c a t a l y s e the synthesis o f choloyl-CoA. f
f
2+
2 +
2 +
The enzyme r e s p o n s i b l e f o r the second step i n the formation of amino a c i d conjugates, i s amino a c i d N - a c y l t r a n s f e r a s e . I t i s i n t h i s r e a c t i o n that amino a c i d s p e c i f i c i t y would be expected to i n f l u e n c e the spectrum o f conjugates seen i n v i v o . Glycine N-acylase has been i s o l a t e d from an acetone powder of beef l i v e r mitochondria (76). When t e s t e d with benzoyl-CoA as the a c y l donor, the enzyme was s p e c i f i c f o r g l y c i n e . A f t e r p u r i f i c a t i o n , a 53f o l d increase i n the s p e c i f i c a c t i v i t y of the enzyme was r e a l i s e d with an 11% recovery of the o r i g i n a l a c t i v i t y . No l o s s of a c t i v i t y was found when the enzyme was s t o r e d at -25°C f o r 5 months. D i l u t e s o l u t i o n s of the enzyme were s t a b l e at 0 C f o r s e v e r a l hours. Enzyme a c t i v i t y was measured spectrophotometrically, based on the decrease i n absorbance at 208 nm due t o the u t i l i s a t i o n o f benzoyl-CoA during the r e a c t i o n (89). James and Bend (90) have shown from s u b c e l l u l a r f r a c t i o n a t i o n s t u d i e s that g l y c i n e N - a c y l t r a n s f e r a s e a c t i v i t y was mainly l o c a t e d i n the m i t o c h o n d r i a l f r a c t i o n s of r a t and r a b b i t t i s s u e s .
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
6.
CRAWFORD AND HUTSON
Type II
211
Metabolism
Cytosol a l s o contained s i g n i f i c a n t l e v e l s of the enzyme but t h i s f r a c t i o n may have contained p r o t e i n from the matrix of the m i t o chondrion. The enzyme i s b e l i e v e d to be l o c a t e d i n the mitochond r i a l matrix, but i s probably not membrane bound. As p r e v i o u s l y mentioned, Vessey et a l (87) have concluded from t h e i r data that separate enzymes are r e s p o n s i b l e f o r the formation of t a u r o c h o l i c and g l y c o c h o l i c a c i d s . Assays contained 50 mM phosphate b u f f e r (pH 8.0) 0.8 mg of s o l u b l e f r a c t i o n protein, • ^ C - l a b e l l e d amino a c i d and 25 μΜ choloyl-CoA i n a t o t a l volume of 0.1 ml. At t h i s concentration of choloyl-CoA, the r e a c t i o n with [•^C]taurine had a K f o r t a u r i n e of 0.75 mM and a V x of 5.9 mol of conjugate synthesised/min/mg of c y t o s o l p r o t e i n . The r e a c t i o n w i t h [ ^ C ] g l y c i n e had a K f o r g l y c i n e of 0.4 mM and V of 2.0 nmol/min/mg p r o t e i n . Thus, i n the bovine l i v e r the maximum p o t e n t i a l f o r t a u r i n e conjugatio although g l y c i n e has greate technique of a l t e r n a t i v e - s u b s t r a t e i n h i b i t i o n , the authors con cluded that there were separate enzymes f o r the conjugation of c h o l i c a c i d with g l y c i n e and t a u r i n e . In a d i f f e r e n t approach to the problem, K i l l e n b e r g and Jordan (88) have p u r i f i e d an enzyme from r a t l i v e r 20,000 g supernatant e x h i b i t i n g amino a c i d N - a c y l t r a n s f e r a s e a c t i v i t y . A 200-fold p u r i f i c a t i o n of the enzyme was achieved with a 6.5% recovery of the o r i g i n a l a c t i v i t y . Polyacrylamide g e l e l e c t r o p h o r e s i s l o c a l i s e d the g l y c i n e and t a u r i n e a c t i v i t i e s of the enzyme to a s i n g l e band. Both a c t i v i t i e s were optimal at pH 7.8. K i l l e n b e r g and Dukes (91) have p u r i f i e d chemically synthesised coenzyme A t h i o e s t e r s of c h o l i c , chenodeoxycholic, deoxycholic and l i t h o c h o l i c a c i d s . The b i o l o g i c a l a c t i v i t y of these t h i o e s t e r s was greater than 94%. In the presence of these substrates and at p h y s i o l o g i c a l concentration of t a u r i n e and g l y c i n e , t a u r i n e was shown to be the favoured s u b s t r a t e . This i s c o n s i s t e n t with the almost e x c l u s i v e production of t a u r i n e conjugates seen i n the r a t in vivo. A s p e c i f i c radiochemical method f o r the i n v i t r o assay of the g l y c i n e conjugation of c a r b o x y l i c acids has been developed u s i n g phenylacetyl-CoA-[carboxy-l^C] as the a c t i v a t e d c a r b o x y l i c a c i d acceptor (90). This compound i s a commercially a v a i l a b l e radiochemical. The paper i s noteworthy i n that i t describes the f i r s t attempt to r e a l l y study g l y c i n e N - a c y l t r a n s f e r a s e f r e e from the complications of other ( c o f a c t o r - s y n t h e s i s i n g ) enzymes, e.g. an acyl-CoA synthetase. Examples of use. In r a b b i t the s p e c i f i c a c t i v i t y of g l y c i n e N - a c y l t r a n s f e r a s e (90) was found to be ten times higher i n kidney mitochondria than i n l i v e r mitochondria. The r e l a t i v e q u a n t i t a t ive r o l e s of the two t i s s u e s i n v i v o i s u n c e r t a i n . In kidney mito<* chondria l e s s than 1% of the adult a c t i v i t y was detected i n the foetus at 20 days g e s t a t i o n and about 10% of the a d u l t a c t i v i t y at 28 and 30 days g e s t a t i o n . A f t e r b i r t h , a c t i v i t y rose very slowly to adult l e v e l s (10-14 weeks) (92). Using a s i m i l a r m
m a
m
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
m a x
212
XENOBIOTIC METABOLISM
s e n s i t i v e radiochemical procedure, enzyme a c t i v i t y was found t o be undetectable i n r a b b i t lung (44). Caldwell e t a l (93) have reported a r a p i d and s e n s i t i v e i n v i t r o semi-micro method f o r the determination o f h i p p u r i c a c i d formation by human and r a t cadaver t i s s u e samples. T y p i c a l i n c u b a t i o n mixtures (1 ml) contained [ l ^ C ] b e n z o i c a c i d (200 nmol; 0.5 μΟί), CoA (100 nmol), ATP ( f o r kidney 2.5 μπιοί; f o r other organs, 5 μπιοί), g l u t a t h i o n e (20 μπιοί), MgCl2 (3 μπιοί) and g l y c i n e (60 μιηοΐ) with t i s s u e homogenate (equivalent to 20 mg t i s s u e ) i n 0.2 M T r i s b u f f e r , pH 8.0. Human cadaver l i v e r and kidney samples formed h i p p u r i c a c i d conjugates, whereas b r a i n , i n t e s t i n e , heart and lung d i d not. Tissues from human and r a t corpses s t o r e d f o r 72 h at 4°C were s t i l l capable o f forming h i p p u r i c a c i d . H i p p u r i c a c i d formation i n l i v e r and kidney samples of a 31 week o l t h i r d of that seen i n comparabl to that reported f o r l i v e r by I r j a l a (94). The most s t r i k i n g f e a t u r e s o f amino a c i d conjugation are the v a r i e t y of amino acids used and how these vary w i t h species and w i t h s t r u c t u r e o f the x e n o b i o t i c . Hirom e t a l (95) have r e c e n t l y reviewed amino a c i d conjugations i n mammals; these and other authors have noted the involvement o f g l y c i n e , glutamine, glutamic a c i d , s e r i n e , alanine and t a u r i n e i n mammals, o f o r n i t h i n e i n b i r d s and o f s e v e r a l others i n p l a n t s . Two examples w i l l serve t o i l l u s t r a t e the e f f e c t s o f s u b s t r a t e and s p e c i e s . 1- and 2-Naphthylacetic a c i d s are conjugated with g l y c i n e t o equal extents i n the r a t , but i n the f e r r e t a g r e a t e r p r o p o r t i o n o f the 2-isomer i s conjugated w i t h g l y c i n e . The 1-isomer i s 63% conjugated with t a u r i n e by the f e r r e t whereas the r a t excretes only a t r a c e o f t h i s metabolite. The 2-isomer i s con jugated with t a u r i n e e q u a l l y i n the two species (96) f i g u r e 14). 3-Phenoxybenzoic a c i d , l i b e r a t e d during the metabolism o f the p y r e t h r o i d i n s e c t i c i d e s permethrin and cypermethrin, i s excreted i n d i f f e r e n t forms by four species so f a r s t u d i e d (97)(98)(99). I t s major metabolite i n cows i s the glutamic a c i d conjugate, i n mice, the t a u r i n e conjugate and i n dogs, the g l y c i n e conjugate; i n r a t s i t i s Dj-hydroxylated and sulphated p r i o r t o e x c r e t i o n (Figure 15). These i n t e r s p e c i e s v a r i a t i o n s may be a consequence of amino a c i d a v a i l a b i l i t y or of N-acylase s p e c i f i c i t y . The s i t u a t i o n i n v i v o i s complicated by the f a c t that c a r b o x y l i c acids are a l s o conjugated w i t h g l u c u r o n i c a c i d . Εί ^ (100) have used a s i m i l a r i n v i t r o assay system t o that p r e v i o u s l y d e s c r i b e d (93) to study the physico-chemical, s t r u c t u r a l and b i o l o g i c a l f a c t o r s i n f l u e n c i n g the p a t t e r n of conjugation r e a c t i o n s o f a r y l a c e t i c a c i d s . Using p h e n y l a c e t i c a c i d , 1-naph thy1ace t i c a c i d , d i p h e n y l a c e t i c a c i d and h y d r a t r o p i c a c i d (methylphenylacetic acid) as s u b s t r a t e s , they concluded that the main f a c t o r i n f l u e n c i n g conjugation w i t h g l y c i n e i s the chemical s t r u c t u r e o f the s u b s t r a t e . For example, a - s u b s t i t u t i o n of the methylene group of p h e n y l a c e t i c acids r e s u l t s i n a χ ο η
e
t
a
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
6.
CRAWFORD A N D HUTSON
RAT
Type
II
Metabolism
213
FERRET
glycine
23%
taurine
0-1%
glycine > taurine
glutamine 0%
glutamine
glycine
glycine
23%
taurine
6% 63% 0%
16%
38% <·
glutamine 8% Figure 14.
Figure 15.
glutamine 19% Amino acid conjugation of naphthylacetic acids
Species differences in the conjugation of 3-phenoxybenzoic acid
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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XENOBIOTIC METABOLISM
b l o c k i n g of amino a c i d conjugation i n v i t r o . By c o n t r a s t , g l u c u r o n i c a c i d conjugation of a r y l a c e t i c a c i d seems to be favoured by extensive n o n - s p e c i f i c entrapment by the endoplasmic r e t i c u l u m , a process r e l a t e d more to l i p i d s o l u b i l i t y than to chemical s t r u c t u r e . These f i n d i n g s support i n v i v o metabolic p r o f i l e s obtained when these acids are o r a l l y dosed to r a t s (101-106). The e x p l a n a t i o n of these v a r i o u s d i f f e r e n c e s w i l l be found v i a the use of s u b c e l l u l a r f r a c t i o n s , yet to date v i r t u a l l y no work d i r e c t e d to t h i s end has been reported. T o x i c o l o g i c a l s i g n i f i c a n c e . The change i n p h y s i c a l pro p e r t i e s effected by the amino a c i d conjugation of a c a r b o x y l i c a c i d i s not dramatic (although there i s an obvious d i f f e r e n c e i n the extreme case o f , f o r example, 3-phenoxybenzoic a c i d to 3-phenoxybenzoyltaurine) However one may expect a somewhat more e f f i c i e n t e x c r e t i o n of a parent a c i d . In the cas with respect to mammals i n p e s t i c i d e chemistry) conjugation i s a l s o l i k e l y to a l t e r the molecular s t r u c t u r e to d i m i n i s h the a c t i v i t y . The species d i f f e r e n c e s noted above are u n l i k e l y to have great consequences because amino a c i d conjugation i s a r e l a t i v e l y minor molecular change and i t occurs u s u a l l y near the end of a metabolic pathway. Conjugation with Glutathione Mechanism and l o c a t i o n . Glutathione conjugation i s the i n i t i a t i n g step i n mercapturic a c i d b i o s y n t h e s i s i n mammals. The f i r s t step i s the most important i n d e t o x i f i c a t i o n because i t confers h y d r o p h i l i c character on the ( u s u a l l y ) l i p o p h i l i c xeno b i o t i c and, i n a d d i t i o n , the g l u t a t h i o n e conjugates are r e a d i l y secreted i n b i l e and are thus r e a d i l y e l i m i n a t e d from the animal. The second stage (γ-glutamyltransferase a c t i o n ) and t h i r d stage (peptidase a c t i o n ) are n e g l e c t e d areas of study i n mercapturic a c i d b i o s y n t h e s i s , though some recent work has been p u b l i s h e d (107). They seem to occur i n both l i v e r and kidney. Both enzymes, f o r example, have been found i n kidney microsomal f r a c t i o n (108). The f o u r t h stage i s d e s c r i b e d i n the A c e t y l a t i o n s e c t i o n above. Glutathione conjugation i s unique among the Type I I pro cesses i n that a d i s c r e t e high-energy s u b s t r a t e i s not r e q u i r e d . The chemical energy f o r the r e a c t i o n i s d e r i v e d from the mutual r e a c t i v i t y of the n u c l e o p h i l i c sulphur of g l u t a t h i o n e f o r an e l e c t r o p h i l i c centre i n the x e n o b i o t i c s u b s t r a t e . The e l e c t r o p h i l i c centre may be present i n the molecule per se or i t may be generated by type I metabolism (see preceeding chapter). The con j u g a t i o n step i s c a t a l y s e d by one or more of a number of g l u t a thione t r a n s f e r a s e s (EC 2.5.1.18) which are l o c a t e d i n the c y t o s o l of mammalian c e l l s . I s o l a t i o n , p r o p e r t i e s and use. A very simple method of p r e p a r a t i o n from l i v e r t h e r e f o r e c o n s i s t s o f : homogenisation, c e n t r i f u g a t i o n at 200,000 g f o r 30 min and d i a l y s i s against pH 7.4 b u f f e r . These enzymes can be s t o r e d at -25°C f o r months. The demethylation of phosphoric a c i d t r i e s t e r s (with which we have
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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most experience) i s r e t a i n e d a f t e r f r e e z e - d r y i n g and storage at 4°C f o r months. However, the p u r i f i e d enzyme i s , as u s u a l , much l e s s s t a b l e . Whether f r e s h samples are d i a l y s e d or not, they must be f o r t i f i e d with g l u t a t h i o n e (to about 5 mM) f o r f u l l a c t i v i t y . The endogenous g l u t a t h i o n e i s l o s t during l i v e r p r o c e s s i n g i n three ways: catabolism, o x i d a t i o n and d i l u t i o n . The a c i d i t y of g l u t a t h i o n e i s sometimes overlooked by newcomers i n t h i s f i e l d . I t i s e s s e n t i a l to adjust the pH back up to 7.4 when preparing the stock s o l u t i o n , even i n 0.1 M phosphate b u f f e r . The h i s t o r y of the g l u t a t h i o n e t r a n s f e r a s e s i s a c l a s s i c example of the dominant e f f e c t of substrate s e l e c t i o n on research, nomenclature and t h i n k i n g . The e l e c t r o p h i l i c centres that are subject to attack by g l u t a t h i o n e i n c l u d e those i n a l k y l h a l i d e s , a r a l k y l h a l i d e s , a r y l h a l i d e s epoxides alkenes c h l o r o t r i a z i n e s nitrocompounds and compound sulphur atoms. The s i t u a t i o y y Chasseaud (109)(110) and Hutson (111), was one i n which the number of enzymes was i n c r e a s i n g with the i n c r e a s i n g number of substrates i n v e s t i g a t e d . These enzymes (Figure 16) were not shown to be d i s c r e t e p r o t e i n s but e f f o r t s were being made to separate the v a r i o u s a c t i v i t i e s . Jakoby and coworkers then entered the f i e l d and used the approach of s e p a r a t i n g r a t l i v e r c y t o s o l p r o t e i n s by column chromatography and subsequently d e f i n i n g the a c t i v i t i e s of the separated p r o t e i n s . T h e i r c l a s s i f i c a t i o n system (112)(113) i s based on the reverse order of e l u t i o n from a carboxymethylc e l l u l o s e column which was the major step i n the s e p a r a t i o n of the t r a n s f e r a s e s from one another. Seven p r o t e i n s were i s o l a t e d : t r a n s f e r a s e s AA, A, B, C., D, E, and M. Enzymes A, B. C and Ε have molecular weights of 45,000 and are d i s s o c i a b l e i n t o two approxi mately equal sub-units. There i s not much i n f o r m a t i o n a v a i l a b l e on t r a n s f e r a s e s AA (114), D or M. Whilst c l a r i f y i n g the s i t u a t i o n with respect to the p r o t e i n , t h i s work has not allowed a neat c o r r e l a t i o n between p r o t e i n and substrate type. There i s con s i d e r a b l e overlap i n substrate s p e c i f i c i t y (112)(115) which i s i l l u s t r a t e d i n general terms i n Figure 16. An e a r l i e r p u r i f i c a t i o n , aimed s p e c i f i c a l l y at 'dimethyl phosphoric a c i d t r i e s t e r g l u t a t h i o n e methyl t r a n s f e r a s e ' from r a b b i t l i v e r , used a combin a t i o n of ammonium sulphate p r e c i p i t a t i o n , z i n c s a l t formation, calcium phosphate g e l treatment and g e l f i l t r a t i o n on Sephadex G150. A 4 5 - f o l d p u r i f i c a t i o n was achieved (116) but the f i n a l product, which a l s o r e t a i n e d i t s a c t i v i t y towards methyl i o d i d e , was probably a mixture of a l l of the t r a n s f e r a s e s (which form about 10% of the l i v e r c y t o s o l ) . Four organophosphorus i n s e c t i c i d e -degrading enzymes have r e c e n t l y been separated from r a t l i v e r c y t o s o l by chromatography on hydroxyapatite (117). Transferase I c a t a l y s e d the demethylation of methylparathion; t r a n s f e r a s e s I I I and IV c a t a l y s e d the demethylation and the d e - a r y l a t i o n of methyl p a r a t h i o n and the d e p y r i m i d i n y l a t i o n of d i a z i n o n . When d i f f e r e n t groups of workers use d i f f e r e n t p u r i f i c a t i o n methods and assay substrates i t i s d i f f i c u l t to c o r r e l a t e the r e s u l t s of the v a r i o u s
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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Jakoby's protein classification (AA,A,B C,D,E,M)
'Enzymes' classified by substrate
f
Glutathione — 5—alkyl transferase S— aralkyl transferase S— aryl transferase S-epoxide (alkyl) transferase S-epoxide(aryl) transferase S— alkene transferase S—triazinyl transferase nitro transferase mercaptoalkyl transferase Figure 16.
Ε > AA > Β A = C > E > M > B = AA A>AA>B=C>E E>A A =C = E > B > A A C>A>B
7 C>AA>B>A C>A=AA>B
Glutathione transferases
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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s t u d i e s . Recently a study of the a c t i v i t y of t r a n s f e r a s e s A, B, C and Ε (115) i n the demethylation of dimethyl 1-naphthylphosphate revealed that t h i s was an a c t i v i t y of t r a n s f e r a s e Ε (118). There fore i t i s p o s s i b l e that t r a n s f e r a s e I (117) and t r a n s f e r a s e Ε (115) are i d e n t i c a l . Some encouraging r e s u l t s have been gained r e c e n t l y by a f f i n i t y chromatography on agarose to which g l u t a t h i o n e was attached by 1,6-diaminohexane spacer molecules. Transferases AA, A and C were r e t a i n e d while t r a n s f e r a s e Β appeared i n the v o i d volume. The bound t r a n s f e r a s e s could be recovered by a f f i n i t y e l u t i o n with KC1/glutathione gradients (119). Bromosulphophthalein -sepharose has also been used s u c c e s s f u l l y f o r the p i g c y t o s o l enzyme (120). The s e l e c t i o n of assay method depends, as u s u a l , on whether the p r i o r i t y f o r study i s the enzyme a c t i v i t y or the r e a c t i o n i t c a t a l y s e s . Jakoby et a methods. Clarke et a l (121 methylparathion methyl t r a n s f e r a s e . A reasonably convenient method a p p l i c a b l e to r a d i o a c t i v e substrates (4) involves p a r t i t i o n of the r e a c t i o n mixture between water and toluene and r a d i o a n a l y s i s f o r product and substrate r e s p e c t i v e l y . A p o t e n t i a l l y u s e f u l p a r t i t i o n procedure employing [ ^ s ] g l u t a t h i o n e has been described by Hayakawa et a l (122). I t i s rather cumbersome but i t i s very sen s i t i v e and, of course, a p p l i c a b l e to non-radioactive x e n o b i o t i c s u b s t r a t e s , thereby allowing much wider substrate s e l e c t i o n f o r structure-activity studies. I t i s l i k e l y that high-pressure l i q u i d chromatography w i l l be used i n c r e a s i n g l y i n assays of g l u t a t h i o n e conjugation. When operated i n the reverse-phase mode i t i s very s u i t a b l e f o r the a n a l y s i s of p o l a r conjugates, as has r e c e n t l y been demonstrated (123). This method could operate with u l t r a - v i o l e t , fluorescence or r a d i o a c t i v i t y d e t e c t i o n , depending on circumstances. A t e c h n i c a l problem, p a r t i c u l a r l y prevelant i n g l u t a t h i o n e conjugation, i s the tendency of the x e n o b i o t i c to react spontan eously with g l u t a t h i o n e . This feature may give very high blank rates which sometimes a l t e r d r a m a t i c a l l y as the pH i s increased above 8. Therefore great care i s needed i n making allowances f o r t h i s i n q u a n t i t a t i v e work. Examples of use. A combination of methods, u s u a l l y i n v i v o s t u d i e s supported by studies with s u b c e l l u l a r f r a c t i o n s , has been used to demonstrate the involvement of g l u t a t h i o n e conjugation i n the metabolism of s e v e r a l c l a s s e s of p e s t i c i d e . These were reviewed i n some d e t a i l r e c e n t l y i n t h i s s e r i e s (111) and only more recent key references are given i n the l i s t of such r e a c t i o n s shown i n Table IV. Most of these are important d e t o x i f i c a t i o n modes because they e f f e c t the a l t e r a t i o n of the b i o a c t i v e molecule. An exception i s the metabolism of the a l k y l thiocyanates; i n the r e a c t i o n between g l u t a t h i o n e and the e l e c t r o p h i l i c sulphur atom, cyanide ion i s l i b e r a t e d and i s probably r e s p o n s i b l e f o r the b i o a c t i v i t y of t h i s c l a s s of compound. I t should be noted that t h i s r e a c t i o n i s an example of glutathione r e a c t i n g with an
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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218
e l e c t r o p h i l i c atom other than carbon. Glutathione conjugation a l s o occurs f r e q u e n t l y i n the f u r t h e r metabolism o f the primary b i o transformation products o f v a r i o u s p e s t i c i d e s . Some examples are given below. Table IV. Glutathione Pesticide class Organophosphorus t r i e s t e r s
DDT Hexachlorocyclohexanes Diphenyl ethers Alkylthiocyanates Propachlor Haloacetamides Chloro-s-triazines Alkylmercapto-s-triazines Thiocarbamates
conjugation
i n p e s t i c i d e metabolism
Reaction
Reference
de-arylation de-py imi d iny1at i
(117) (116) (126) (117) (127)
dehydrochlorinatio de-arylation de-thiocyanylation de-chlorination de-chlorination de-chlorination de-alkylsulphoxylation de-alky1sulphoxy1ation
(128) (129) (130)(131) (129) (129) (132) (124) (125)
de-alkylation
Two of the compound c l a s s e s shown i n Table IV, the a l k y l mercapto-js-1riazines h e r b i c i d e s and the thiocarbamate h e r b i c i d e s , are good examples where the metabolism was s t u d i e d at the subc e l l u l a r l e v e l i n order to e x p l a i n the b i o t r a n s f o r m a t i o n pathways observed i n v i v o . Moreover, two r e a c t i o n c l a s s e s , i n d i f f e r e n t s u b c e l l u l a r f r a c t i o n s , used i n a s p e c i f i c sequence, were needed to r e c o n s t r u c t and e x p l a i n the o v e r a l l r e a c t i o n s . A h e r b i c i d a l methylmercapto-s-triazine was e l i m i n a t e d i n r a t u r i n e as the s ^ - t r i a z i n y l mercapturic a c i d and i n r a t b i l e as the s - t r i a z i n y l g l u t a t h i o n e conjugate. As the methylmercapto-s_-triazine itself was not a substrate f o r c y t o s o l i c g l u t a t h i o n e t r a n s f e r a s e , c l e a r l y an e x t r a step was o c c u r r i n g i n v i v o . Incubation of the h e r b i c i d e with r a t l i v e r microsomes (+ NADPH) a f f o r d e d a new product, the h e r b i c i d e Sj-oxide. When incubated w i t h g l u t a t h i o n e , the jS-oxide reacted spontaneously t o a f f o r d the same s - t r i a z i n e conjugate as that observed i n the b i l e (124). A s i m i l a r sequence was discovered f o r the h e r b i c i d e s EPTC and b u t y l a t e (N,Ndialky1-S-alky1 thiocarbamates). The thiocarbamates were o x i d i s e d by microsomes to thiocarbamate sulphoxides. These compounds (which are reasonably s t a b l e ) were substrates f 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 - c a t a l y s e d formation of S-(N_,N-dialky 1 carbamoyl) g l u t a t h i o n e s (125). These sequences, i l l u s t r a t e d i n Figure 17, could not have been demonstrated e a s i l y be the use of a higher l e v e l of o r g a n i s a t i o n such as whole c e l l s . Glutathione has been shown to p a r t i c i p a t e i n
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
6.
CRAWFORD A N D HUTSON
I —* ?
Pr-nT^O
Figure 17.
Type
II
Metabolism
1 —* JL — ?
Pr-N'^^O
S
a
>
mercapturic acid
Pr-N^^O
Glutathione in the metabolism of thiocarbamate herbicides
and thiotriazine
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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b i o t r a n s f o r m a t i o n without being obvious i n the f i n a l products. Even with the well-known glutathione-dependent demethylation of dimethylphosphate t r i e s t e r i n s e c t i c i d e s , most of the S-methylg l u t a t h i o n e formed i s metabolised to CO2 and very l i t t l e me t h y 1 mercapturic a c i d i s excreted. C l e a r l y the use of s u b c e l l u l a r f r a c t i o n s i s i n d i c a t e d i n d i s c o v e r i e s of t h i s type. The f a t e of the v i n y l phosphate i n s e c t i c i d e , dimethylvinphos, may be used to i l l u s t r a t e t h i s p o i n t f u r t h e r . The main r a d i o a c t i v e metabolites d e r i v e d from l^C-phenyl l a b e l l i n g i n v i v o were de-methyldimethylvinphos and a metabolite derived from the phenylvinyloxy group, 1-(2,4-dichlorophenyl)ethanol (as glucuronide) (4). The demethylation, p r e d i c t a b l y , was e f f e c t e d by ( d i a l y s e d ) c y t o s o l and g l u t a t h i o n e . 2,4-Dichlorophenylethanol was assumed to be d e r i v e d from 2,4-dichloroacetophenon by the r e d u c t i v e d e c h l o r i n a t i o dimethylvinphos (2,4-dichlorophenacyl c h l o r i d e ) . The mechanism of the r e d u c t i v e d e c h l o r i n a t i o n was unknown u n t i l we s t u d i e d the metabolism of the phenacyl c h l o r i d e i n r a t l i v e r f r a c t i o n s . The f o l l o w i n g sequence of reactions was d i s c o v e r e d u s i n g c y t o s o l : ( i ) spontaneous r e a c t i o n of phenacyl h a l i d e with g l u t a t h i o n e to a f f o r d 2 , 4 - d i c h l o r o p h e n a c y l - g l u t a t h i o n e , ( i i ) enzyme-catalysed r e a c t i o n of the l a t t e r with another molecule of g l u t a t h i o n e to form o x i d i s e d g l u t a t h i o n e and the phenacyl anion which rearranged to 2,4-dichloroacetophenone (133). Microsomes and NADPH simply reduced the keto group of 2,4-dichlorophenacyl c h l o r i d e to give the c h l o r o h y d r i n (not observed i n the i n v i v o metabolism) . Thus g l u t a t h i o n e enters the metabolic pathways of dimethylvinphos at three p o i n t s (Figure 18) but i t i s s c a r c e l y observed i n the excreted m e t a b o l i t e s . In t h i s s e r i e s of experiments we a l s o used the i n v i t r o technique to demonstrate that the a d m i n i s t r a t i o n of phénobarbital to r a t s (which p r o t e c t e d them 1 0 - f o l d against the acute o r a l t o x i c i t y of dimethylvinphos) induced the a c t i v i t y of the c y t o s o l demethylating enzyme 2 - f o l d . Dimethylvinphos e x h i b i t s a large d i f f e r e n c e i n acute t o x i c i t y to r a t and dog ( r a t > dog); dog l i v e r c y t o s o l was shown to demethylate dimethylvinphos at about twice the rate of r a t l i v e r c y t o s o l (4). Thus, the enzyme could have some r o l e i n the s e l e c t i v e t o x i c i t y of dimethylvinphos. This example i l l u s t r a t e s s e v e r a l important f e a t u r e s : d i r e c t g l u t a t h i o n e conjugation of parent molecule ( e f f e c t i n g detoxification) ( i i ) g l u t a t h i o n e conjugation a f t e r b i o a c t i v a t i o n by primary metabolism ( h y d r o l y s i s i n t h i s case) ( i i i ) g l u t a t h i o n e conjugation by attack on an e l e c t r o p h i l i c atom other than carbon (sulphur i n t h i s case) ( i v ) only t r a c e s of the glutathione-conjugated products (mercapturic a c i d s ) appearing i n the excreted metabolites (v) use of s u b c e l l u l a r f r a c t i o n s to i n v e s t i g a t e a species d i f f e r e n c e ( r a t versus dog) (i)
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
6.
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GSH
II
Metabolism
GSH
CI Figure 18.
Type
221
GSH
CI
Glutathione in the metabolism of dimethylvinphos
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XENOBIOTIC M E T A B O L I S M
( v i ) use of s u b c e l l u l a r f r a c t i o n s to i n v e s t i g a t e the of drug-metabolising enzymes of an animal.
alteration
Studies of the l i v e r enzyme have shown that g l u t a t h i o n e t r a n s f e r a s e s occur i n many other animals i n c l u d i n g r a b b i t (118), p i g (118), (23), monkey (23)(134)(135), t r e e shrew (23), sheep (136), guinea-pig (122), horse (122), cow (122), mouse (122), chicken (126), and man (137). Where d e t a i l e d s t u d i e s have been c a r r i e d out (135)(136) (137) the enzymes, i n c l u d i n g those of man, have been shown to be very s i m i l a r i n p h y s i c a l p r o p e r t i e s . L i v e r i s the r i c h e s t source of the enzymes. For example, a recent study of Japanese monkey (Macaca fuscata) (134) was t y p i c a l i n showing the f o l l o w i n g a r y l t r a n s f e r a s e ( 1 , 2 - d i c h l o r o 4-nitrobenzene) a c t i v i t i e s (μιιιοΐ/min/mg p r o t e i n ) : l i v e r , 18; spleen, 2.6; kidney, 2.1 p l a c e n t a , 0.3; pancreas has r e c e n t l y been reported i n small i n t e s t i n e s (138)(139) and i n leucocytes (140). Enzyme measurements have a l s o been used to assess glutathione conjugation during development of the neonate. A l k y l t r a n s f e r a s e a c t i v i t y i n the r a t neonate i s very low f o r about 6 days and then r i s e s s t e a d i l y f o r about 40 days to the adult l e v e l (141). Trans f e r a s e Β (measured as a r y l t r a n s f e r a s e ) i s present at b i r t h at about one f i f t h of the adult l e v e l and r i s e s s t e a d i l y f o r about 40 days (142). However, i n a l l general statements about the presence of g l u t a t h i o n e t r a n s f e r a s e i n various s p e c i e s , t i s s u e s or a l t e r e d s t a t e s , the s u b s t r a t e used f o r assay must be noted before the relevance to one's own work i s assessed. The importance of g l u t a t h i o n e conjugation i n l i m i t i n g the c y t o t o x i c , mutagenic and c a r c i n o g e n i c a c t i o n of e l e c t r o p h i l i c compounds (143) i s gener a t i n g much research i n t o the species and t i s s u e d i s t r i b u t i o n of the enzyme(s). However, too much r e l i a n c e on r e s u l t s d e r i v e d from t e s t s using substrates (e.g. p o l y c y c l i c aromatic hydrocarbon epoxides) unrelated to one's own problem may w e l l prove to be only of l i m i t e d v a l u e . T o x i c o l o g i c a l s i g n i f i c a n c e . Glutathione conjugation r e s u l t s i n a dramatic change i n the p h y s i c a l p r o p e r t i e s of a molecule, u s u a l l y l e a d i n g to a l o s s of b i o a c t i v i t y . The conjugate i s i d e a l l y s t r u c t u r e d f o r b i l i a r y s e c r e t i o n and t h e r e f o r e i t i s e f f i c i e n t l y removed from the l i v e r . Other enzymes e f f i c i e n t l y convert the conjugate i n t o a mercapturic a c i d that i s r e a d i l y excreted v i a the u r i n e . However, perhaps the most important f u n c t i o n of t h i s conjugation process i s the p r o t e c t i o n i t a f f o r d s against e l e c t r o p h i l i c compounds, be they ingested as such or generated w i t h i n the organism v i a metabolism. Without t h i s p r o t e c t i o n mammals would be much more s u s c e p t i b l e than they are to low doses of teratogens, mutagens, carcinogens and c y t o t o x i c compounds.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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Other Uses of S u b c e l l u l a r F r a c t i o n s i n X e n o b i o t i c Metabolism Studies A r e l a t i v e l y recent use of s u b c e l l u l a r f r a c t i o n s i s i n b a c t e r i a l t e s t systems f o r mutagenicity of the type developed by Ames and coworkers (144). The f r a c t i o n commonly used i s a 9000 g f r a c t i o n (S-9 f r a c t i o n ) from the l i v e r s of r a t s t r e a t e d with an A r a c h l o r (to induce microsomal enzymes). I t s r o l e i n the t e s t system i s the p r o v i s i o n of a mammalian metabolism c a p a b i l i t y f o r the ( p o s s i b l e ) a c t i v a t i o n of i n t r i n s i c a l l y i n a c t i v e compounds. This may not commonly be seen as a use i n metabolism s t u d i e s ; but i f the b a c t e r i a are regarded as a bioassay technique f o r the d e t e c t i o n of mutagens, the system i s a u s e f u l a d d i t i o n to the techniques used f o r the study of metabolism i n v i t r o The main component of the b i o a c t i v a t i o microsomal mono-oxygenas a more 'potent S-9 f r a c t i o n ) . However, i t w i l l be c l e a r from the v a r i o u s r e a c t i o n s discussed above that some of the type I I r e a c t i o n s e f f e c t the b i o a c t i v a t i o n of c e r t a i n m e t a b o l i t e s . They a l s o e f f e c t the d e a c t i v a t i o n of many compounds and t h e i r metabo l i t e s . The p o t e n t i a l l y great p r e d i c t i v e value of these t e s t systems has l e d to t h e i r widespread, but o f t e n u n c r i t i c a l use. The importance of a standardised p r e p a r a t i o n c o n t a i n i n g a c t i v e microsomal mono-oxygenase i s appreciated but the r o l e of the many other enzymes i n the S-9 f r a c t i o n has been l a r g e l y ignored. 1
The widespread use and e n t h u s i a s t i c r e c e p t i o n gained by t h i s simple, quick and p o t e n t i a l l y very u s e f u l system has l e d to a r e a c t i o n from c e r t a i n quarters and a heated debate i s c u r r e n t l y being conducted. There i s c l e a r l y a need to d e f i n e the r e a c t i o n s o c c u r r i n g i n the t e s t system. The balance between a c t i v a t i o n and d e a c t i v a t i o n i s c r i t i c a l to i t s relevance to the i n v i v o s i t u a t i o n . The s t a t e of the v a r i o u s enzymes i n the S-9 f r a c t i o n and the concentrations of the various c o f a c t o r s (many of which are described above) r e q u i r e s measurement and c o n t r o l . Species v a r i a t i o n s are important. For example i t i s p o s s i b l e that 2-aminoanthracene (mutagenic i n the presence of S-9 from r a t l i v e r ) would not give a p o s i t i v e response i f dog l i v e r S-9 f r a c t i o n s were used. The f i r s t step i n i t s b i o a c t i v a t i o n ( N - a c e t y l a t i o n , see Figures 9 and 10) i s i n o p e r a t i v e . Glutathione-dependent d e a c t i v a t i o n i s l a r g e l y i n o p e r a t i v e i n standard S-9 f r a c t i o n because, although the g l u t a t h i o n e S-transferases are present, g l u t a t h i o n e i t s e l f i s l a r g e l y destroyed by catabolism, d i l u t i o n and o x i d a t i o n (145). A study of the various enzyme a c t i v i t i e s and c o f a c t o r concentrations i n human l i v e r f r a c t i o n (prepared as S-9) would a l s o prove very u s e f u l i n the i n t e r p r e t a t i o n of r e s u l t s of the Ames t e s t .
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RECEIVED
December
20, 1978.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
7 Methods for the Study of Metabolism of Xenobiotics i n Insect Cell and Organ Cultures EDWINP.MARKS Metabolism and Radiation Research Laboratory, Agricultural Research, Science and Education Administration,U.S.Department of Agriculture, Fargo,ND58102 MALCOLM J. THOMPSON and WILLIAME.ROBBINS Insect Physiology Laboratory, Agricultural Research, Science and Education Administration, U.S. Department of Agriculture, Beltsville,MD20705 Although insect organ and cell culture methodologies have been used for severa endocrinology and developmen made of these techniques in studying the metabolism of xenobiotics in insect tissues. However, as our knowledge of insect biochemistry and the necessity for environmentally compatible pesticides increase, the use of such methodologies will grow. The physiological studies that have been done with insect organ cultures so far have shown us that when experiments are carried out using appropriate techniques and carefully defined conditions, cultured tissues can be induced to behave much the same way as they do in whole insects (1). To date, studies have included such diverse processes as the biosynthesis and secretion of hormones (4,5), the production and deposition of cuticle (6,7), the biosynthesis of yolk materials (8,9), the regeneration of nerve tissues (10), and the differentiation of epidermal cells into setae (11). In all these studies, acceptable approximations of what is known to occur in vivo have also been demonstrated in vitro. Thus, it seems reasonable that cell and organ culture systems could be used to advantage for studying the metabolism of xenobiotics in insects, particularly as i t relates to mode of action. The questions, then, are what can be gained from the use of tissue culture methodology and whether the advantages justify the additional effort and expense. Organ Culture Systems Most of the physiological and biochemical studies on insect cell and organ cultures to date have been done with organ culture systems. Organ culture differs from other in vitro techniques in that an organ must be cultured for more than 24 hours under aseptic conditions and in medium that contains an adequate energy source. In such a culture, the This chapter not subject to U.S. copyright. Published 1979 American Chemical Society
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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a r c h i t e c t u r e of the organ remains i n t a c t , and some degree of organotypic a c t i v i t y can be a n t i c i p a t e d (12). Under these c o n d i t i o n s the t i s s u e s a r e o f t e n v i a b l e f o r s e v e r a l weeks or even months. For example, processes such as nerve regeneration or c u t i c l e d e p o s i t i o n may begin weeks a f t e r the t i s s u e i s explanted and may continue f o r as long as 90 days (10,11). In organ c u l t u r e s the problems encountered w i t h short-term i n c u b a t i o n s , such as s u r g i c a l trauma and m i c r o b i a l contamin a t i o n , a r e no longer a f a c t o r i n i n t e r p r e t i n g the r e s u l t s . Advantages of Organ Culture Techniques One of the main advantages of organ c u l t u r e i s the experimental f l e x i b i l i t y . The i n v e s t i g a t o r can add or remove t i s s u e s , s u b s t r a t e s , and products a t any time during the i n c u b a t i o n p e r i o d ; can s u b s t r a t e s ; and can i n c l u d the same c u l t u r e . A l s o , the medium can be sampled at i n t e r v a l s , and substrate l e v e l s can be c o n t r o l l e d and by-products removed without s e r i o u s l y d i s t u r b i n g the system. This experimental f l e x i b i l i t y i s demonstrated by the use of a hanging drop p r e p a r a t i o n to show that the r e l e a s e of peptide neurohormones synthesized by c u l t u r e d b r a i n s could be induced by adding c u l t u r e d c a r d i a c a , which, i n turn, sequestered and stored the neurohormones r e l e a s e d i n t o the medium (13). A second advantage of the organ c u l t u r e methodology i s that a l a r g e r q u a n t i t y of metabolites can be obtained from a given amount of t i s s u e because of the prolonged i n c u b a t i o n . A l s o , feedback i n h i b i t i o n can be prevented by frequent renewal of the medium. I s o l a t i o n and p u r i f i c a t i o n of metabolites are f r e q u e n t l y e a s i e r from the r e l a t i v e l y c l e a n medium than from whole body e x t r a c t s . These advantages have been discussed p r e v i o u s l y (14). A d d i t i o n a l advantages i n c l u d e the e x c l u s i o n of unwanted t i s s u e from the c u l t u r e s and the p r e s e r v a t i o n of i n t a c t c e l l membranes i n the l i v i n g c e l l s . Thus, when one i s working w i t h whole animals, i t has been v i r t u a l l y impossible to determine whether or not α-ecdysone i n i t s e l f has any a c t i v i t y s i n c e i t i s r e a d i l y converted by adjacent t i s s u e s to 20hydroxyecdysone i n q u a n t i t i e s s u f f i c i e n t to produce an endocrine response (15). With homogenates, although t i s s u e s p e c i f i c i t y i s maintained, the c e l l membranes a r e destroyed and endogenous i n h i b i t o r s and other i n t r a c e l l u l a r products that a r e normally r e t a i n e d by the c e l l s are r e l e a s e d , and these i n t u r n induce a v a r i e t y of u n c o n t r o l l e d r e a c t i o n s . Thus, organ c u l t u r e s , i n which the i n t e g r i t y of the c e l l s i s maintained, provide a biochemical s p e c i f i c i t y that i s d i f f i c u l t to o b t a i n i n other kinds of i n v i t r o systems. F i n a l l y , a most important advantage of the organ c u l t u r e approach i s that the i n v e s t i g a t o r can i n c l u d e both the m e t a b o l i z i n g and the t a r g e t t i s s u e s i n the same system. This
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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permits study of the i n t e r r e l a t i o n s h i p or i n t e r a c t i o n between the metabolism of the t e s t compound and the mode or s i t e of a c t i o n . T h i s i s i l l u s t r a t e d by our work on the metabolism of 22,25-dideoxyecdysone (16,17). Disadvantages of Organ C u l t u r e Techniques The disadvantages of organ c u l t u r e are as numerous and as apparent as the advantages. For example, i s o l a t e d t i s s u e s may l a c k substrates or c o f a c t o r s normally present i n the i n t a c t animal, which may, i n t u r n , cause minor pathways to assume a dominant r o l e , and thereby confuse the true p i c t u r e (18). Thus, i n v i t r o r e s u l t s must be compared c a r e f u l l y with what i s known from i n v i v o s t u d i e s . Some t i s s u e s do not s u r v i v w e l l i v i t r d thu cannot be used s u c c e s s f u l l example mature muscle f r e q u e n t l y y explantation Another d i f f i c u l t y l i e s i n o b t a i n i n g uncontaminated t i s s u e s f o r c u l t u r e . With holometabolous pupae or eggs, t h i s i s not a problem (19) but i n some other stages of the l i f e c y c l e , i t may be extremely d i f f i c u l t (20). The problem of contaminated t i s s u e a l s o puts a p r a c t i c a l l i m i t on the number of expiants that can be placed i n a s i n g l e c u l t u r e . For example, i f one expiant out of four i s contaminated, 75% success may be expected i n c u l t u r e s c o n t a i n i n g one expiant per c u l t u r e , l e s s than 45% success would be expected i n c u l t u r e s c o n t a i n i n g 3 expiants and there i s only a 17% chance of success i n c u l t u r e s c o n t a i n i n g s i x expiants and contamination i s a v i r t u a l c e r t a i n t y . Thus, when l a r g e amounts of t i s s u e are needed, the expiants must be c u l t u r e d s e p a r a t e l y or i n small groups, an arduous task. Even with l a r g e numbers of c u l t u r e s , the amounts of metabolites are o f t e n v a n i s h i n g l y s m a l l , so microchemical methods must be used (4). F i n a l l y , organ c u l t u r e i s expensive, both i n time and m a t e r i a l s , and should be used only when l e s s l a b o r i o u s methods are not a v a i l a b l e . Metabolism of 22,25-Dideoxyeedysone ( T r i o l ) i n Organ C u l t u r e Having discussed the use of organ c u l t u r e i n the a b s t r a c t , we would now l i k e to d i s c u s s i n some d e t a i l a study of the metabolism of the e c d y s t e r o i d 22,25-dideoxyecdysone ( t r i o l , F i g . 1 ) . In d i f f e r e n t species of i n s e c t s , or at d i f f e r e n t times or stages d u r i n g the development of the same species of i n s e c t , t h i s i n t e r e s t i n g e c d y s t e r o i d may e i t h e r enter the major pathway(s) of molting hormone metabolism and serve as a p r e c u r s o r f o r the b i o s y n t h e s i s of the i n s e c t molting hormones (21,22) or i t may be t r e a t e d as a x e n o b i o t i c and metabolized to a complex mixture of hydroxylated s t e r o i d s (22,23). The same d u a l r o l e i s r e f l e c t e d i n the b i o l o g i c a l or p h y s i o l o g i c a l a c t i v i t y and the a c t i o n of 22,25-dideoxyecdysone: In c e r t a i n i n s e c t s or b i o l o g i c a l t e s t systems, i t has molting hormone
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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Ο
22,25-Dideoxyecdysone (Triol)
Figure 2.
Schematic of leg regenerate—Rose chamber technique.
The mesothoracic legs are removed from freshly molted, late instar cockroach nymphs. After 28 days the coxal stump is removed and the leg regenerate is dissected out. The test tissues are placed under the dialysis strip and the chamber is assembled and filled with medium. The test compound(s) are placed under the dialysis strip with a microsyringe.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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a c t i v i t y (21,24,25); i n a number of other i n s e c t species i t i s a potent i n v i v o i n h i b i t o r of development, metamorphosis, and r e p r o d u c t i o n (24,25,26). Our i n i t i a l s t u d i e s w i t h the t r i o l were c a r r i e d out i n Rose multipurpose t i s s u e chambers. These chambers, which we have found to be extremely u s e f u l f o r many kinds of experiments, c o n s i s t of a s i l i c o n e gasket that i s h e l d between two c o v e r s l i p s by a p a i r of metal p l a t e s . With a s t r i p of d i a l y s i s membrane to hold the l e g regenerate t i s s u e i n p l a c e , ( F i g . 2) the Rose chamber permits experimental f l e x i b i l i t y combined w i t h a high q u a l i t y o p t i c a l image f o r v i s u a l e v a l u a t i o n of the t i s s u e w i t h a compound microscope (27). A l s o , the s o f t s i l i c o n e gaskets permit the use of a m i c r o s y r i n g e to p l a c e the experimental compound i n d i r e c t contact w i t h the t i s s u e In chambers c o n t a i n i n g a t h r e s h o l ecdysone (0.05 yg/ml), c u t i c l l e g regenerates between 10 and 14 days a f t e r the molting hormone i s added (10). When the t r i o l was t e s t e d against c u l t u r e d l e g regenerates of the cockroach Leucophaea maderae ( F . ) , i t produced c u t i c l e formation i n only 27% of the regenerates and gave e r r a t i c r e s u l t s even at high doses (28) (Table I ) . The experimental r e s u l t s obtained were not r e a d i l y understood, e s p e c i a l l y s i n c e the responses that d i d occur were not c o n c e n t r a t i o n dependent. Only when the t r i o l was looked upon as a s u b s t r a t e that could be converted to b i o l o g i c a l l y a c t i v e e c d y s t e r o i d s by p e r i p h e r a l t i s s u e s such as blood or f a t body, d i d the r e s u l t s become meaningful. When we t e s t e d t h i s hypothesis by c o - c u l t u r i n g f a t body and l e g regenerates, the i n c i d e n c e of Table I.
I n d u c t i o n of c u t i c l e formation i n cockroach l e g regenerates by c e r t a i n e c d y s t e r o i d s when tested i n the presence or absence of f a t body.
Cuticle Ecdysteroids
Control 20-Hydroxyecdysone a-Ecdysone 22,25-Dideoxyecdysone 22-Isoecdysone
a
Leg Regenerates
Formation Leg Regenerates + Fatbody
Ν
%
Ν
10 14 17 22 12
10 93 82 27 8
10
0
-
-
22 10
93 0
A l l e c d y s t e r o i d s t e s t e d at 10
\ig
per chamber.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
%
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238
To confirm these f i n d i n g s , we made a second s e r i e s of experiments i n which we s e p a r a t e ^ c u l t u r e d cockroach f a t bodies and l e g regenerates with C-labeled t r i o l f o r 6 days i n 1 ml of M20S medium i n c l e a n , s t e r i l e g l a s s s c i n t i l l a t i o n vials. Two volumes of methanol were added, and the c u l t u r e s were worked up and then analyzed f o r r a d i o l a b e l e d s t e r o i d s by t h i n - l a y e r radiochromatography. The c u l t u r e s c o n t a i n i n g only l e g regenerate t i s s u e and t r i o l produced no a d d i t i o n a l s t e r o i d a l compounds. They contained only the unmetabolized triol. However, the f a t body c u l t u r e s showed chromatographic peaks f o r t e t r a o l s , pentaols, and conjugates as w e l l as the t r i o l ( F i g . 3 ) , evidence that the metabolism of the t r i o l had proceeded v i a h y d r o x y l a t i o n and conjugation (17). Thus, our i n i t i a l hypotheses were confirmed: the t r i o l a c t s as a precursor r a t h e r than a regenerate and f a t bod capabilities. Table I I .
Compound
E f f e c t of c e r t a i n a z a s t e r o i d s and n o n s t e r o i d a l amines and an amide i n i n h i b i t i n g the a c t i v i t y of 22,25-dideoxyecdysone ( t r i o l ) i n cockroach l e g regenerate-fat body c u l t u r e s .
a
Control I II III IV V VI
All
C u t i c l e Formation
Inhibition
%
%
93 0 16 50 50 70 80
0 100 83 46 46 25 14
coumpounds t e s t e d a t 10 \xg per chamber.
From t h i s p o i n t , the work proceeded i n two d i r e c t i o n s . One t h r u s t was concerned w i t h a group of compounds c o n s i s t i n g of c e r t a i n 25-azasteroids and n o n s t e r o i d a l amines and amides ( F i g . 4) that d i s r u p t s t e r o i d metabolism and molting and metamorphosis i n i n s e c t s (29,30); these were t e s t e d f o r i n h i b i t i o n of the a c t i v i t y of the t r i o l i n inducing c u t i c l e d e p o s i t i o n i n cockroach l e g regenerate-fat body c u l t u r e s (16). The r e s u l t s obtained with s e v e r a l of these compounds i n d i c a t e d s i g n i f i c a n t i n h i b i t i o n of metabolism of the t r i o l to an a c t i v e e c d y s t e r o i d by the c u l t u r e d t i s s u e (Table I I ) . Subsequent metabolic s t u d i e s with three of the more a c t i v e
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
7.
MARKS E T A L .
Insect Cell and Organ
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TRIOL
DISTANCE
F R O M ORIGIN (CM)
Figure 3. Radiochromatograms of 4-C -ecdysteroids extracted from the medium of cockroach tissue cultures incubated with 4- C-22,25-dideoxyecdysone (triol): (A) from cultures containing leg regenerates; (B) from cultures containing leg regenerates plus fat body. 14
14
Figure 4.
Azasteroids, nonsteroidal amines, and an amide
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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i n h i b i t o r s (17) demonstrated that they have a pronounced e f f e c t on the metabolism of the t r i o l i n cockroach f a t body c u l t u r e s (Table I I I ) . Taken together, these two s t u d i e s provided the f i r s t i n v i t r o p h y s i o l o g i c a l data and f i r s t biochemical evidence that c e r t a i n of the i n h i b i t o r y a z a s t e r o i d s and n o n s t e r o i d a l amines represent a new c l a s s of i n s e c t hormonal chemicals with a novel mode of a c t i o n — t h e y i n t e r f e r e with the metabolism of the endogenous molting hormones of insects. Table I I I .
Inhibitory Compounds
The e f f e c t s o f an a z a s t e r o i d açji n o n s t e r o i d a l amines on the metabolism of 4- C-22,25-dideoxyecdysone ( t r i o l ) i n cockroach f a t body c u l t u r e s during an i n c u b a t i o n p e r i o d of 6 days
% Unmetabolized ^ 22,25-Dideoxyecdysone
% Metabolites
Conjugates Pentaols T e t r a o l s
Control II III IV
45.5 6.0 15.9 18.7
35.6 55.0 40.8 34.7
11.3 0.0 5.6 5.2
7.6 39.0 37.8 41.5
The concentrations o f the i n h i b i t o r y compounds and of 422,25-dideoxyecdysone were 10 yg each per c u l t u r e f l a s k .
C-
^ Determined by r a d i o TLC analyses.
The second t h r u s t , which i s perhaps more germane to the metabolism of x e n o b i o t i c s , i n v o l v e d over 100 c u l t u r e s of f a t body that were incubated with t r i o l . The p r i n c i p a l pathways of metabolism of t r i o l i n the f a t body c u l t u r e s turned out to be h y d r o x y l a t i o n and conjugation, as they are i n v i v o . I n t e r e s t i n g l y , enzymic h y d r o l y s i s of the conjugate f r a c t i o n showed no unmetabolized t r i o l . The major t e t r a o l metabolite was 22-deoxyecdysone, and other t e t r a o l and pentaol metabolites were hydroxylated a t the 25, 26, or 20 p o s i t i o n s ( 1 7 ) ( F i g . 5 ) . Most of the metabolites found had been p r e v i o u s l y i s o l a t e d from the f r a s s of t r i o l - f e d l a r v a e of the tobacco hornworm Manduca sexta (L.) (23). I n t h i s r e s p e c t , the metabolic pathways f o r t r i o l i n c u l t u r e d cockroach f a t body resembled the pathways that occur i n hornworm l a r v a e . A l s o , i n both cases the mechanism f o r h y d r o x y l a t i o n of the t r i o l a t C-22 appears to be l a c k i n g . When samples of the t e t r a o l and p e n t a o l f r a c t i o n s were t e s t e d against c u l t u r e d l e g regenerates,
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
7.
MARKS E T A L .
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241
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TETRADS
PENTAOLS OH
Η
OH
-Ι
OH
22,25-DFDEOXYECDYSONE (TRIOL)
OH
O H
Figure 5. Hydroxylation of 22,25-dideoxyecdysone (triol) by cockroach fat body cultures
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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242
both f r a c t i o n s induced c u t i c l e formation (Table I V ) . Since the l e g regenerates cannot m e t a b o l i c a l l y a c t i v a t e the t r i o l d i r e c t l y , they are probably unable to e f f i c i e n t l y hydroxylate the C-25 p o s i t i o n . In a d d i t i o n , assuming that 22-deoxyecdysone, l i k e α-ecdysone, i s e f f e c t i v e i n the l e g regenerate system only a f t e r metabolic conversion (31), then f u r t h e r metabolism of the t e t r a o l and pentaol metabolites by the l e g regenerates may a l s o occur. Thus, both f a t body and l e g regenerate t i s s u e may be necessary to t r a v e r s e the e n t i r e pathway from t r i o l to an e c d y s t e r o i d ( s ) with molting hormone a c t i v i t y i n the l e g regenerate system. Table IV.
E f f e c t of metabolite f r a c t i o n s i s o l a t e d from cockroach f a t bod t i s s u c u l t u r e incubated with 22,25-dideoxyecdyson by c u l t u r e d g regenerates
C u t i c l e Formation
Metabolite Fractions
Ν
Control Tetraol Pentaol
4 5 4
% 0 62 80
X days
10.8 11.3
Tested at 10 yg e q u i v a l e n t s as determined
by UV analyses.
This study i s s t i l l i n progress, and a number of questions remain unanswered. One of these concerns the b i o l o g i c a l a c t i v i t y of the v a r i o u s t e t r a o l and pentaol metabolites of the t r i o l that are produced i n f a t body c u l t u r e s . Other questions i n v o l v e the s i t e and mode of a c t i o n of c e r t a i n of the i n h i b i t o r y a z a s t e r o i d s and n o n s t e r o i d a l amines. During the course of t h i s work, we learned c e r t a i n things that may be of use to i n v e s t i g a t o r s who use s i m i l a r techniques: One of these i s that the use of a n t i b i o t i c s to c o n t r o l contamination must be approached with extreme c a u t i o n . We found that the presence of gentamicin, an a n t i b i o t i c commonly used i n t i s s u e c u l t u r e s , i n t e r f e r e d with the metabolism of the t r i o l i n f a t body c u l t u r e s . I t was a l s o observed that the amount of s u b s t r a t e and/or the length of i n c u b a t i o n produce changes i n the r a t i o s of the metabolic products formed (17). The purpose of d i s c u s s i n g the work on the metabolism of 22,25-dideoxyecdysone has been to show how organ c u l t u r e methodology has been a p p l i e d to the study of t h i s e c d y s t e r o i d that o f t e n behaves as a x e n o b i o t i c . Our r e s u l t s obtained by u s i n g organ c u l t u r e techniques so f a r agree q u i t e w e l l with
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what has been learned by using i n v i v o methods. However, the usefulness of t h i s methodology i s l i m i t e d somewhat by the expense i n both time and m a t e r i a l s compared with the use of l i v e i n s e c t s . In general, these i n v i t r o methods would appear to be more u s e f u l f o r the in-depth i n v e s t i g a t i o n of c r i t i c a l problem areas r a t h e r than f o r general usage. C e l l C u l t u r e Systems f o r Metabolic Studies The use of i n s e c t c e l l c u l t u r e systems f o r the study of the metabolism of x e n o b i o t i c s has not, to our knowledge, been attempted to date. The p e r i p h e r a l l i t e r a t u r e c o n s i s t s of only a few papers on the e f f e c t s of p e s t i c i d e s (32,33) and i n s e c t growth r e g u l a t o r s (34,35) on i n s e c t c e l l l i n e s and a s e r i e s of s t u d i e s on the e f f e c t s of v a r i o u s ecdysteroids on the morphology of c u l t u r e would l i k e to d i s c u s s b r i e f l u s i n g i n s e c t c e l l l i n e s f o r the study of the metabolism of xenobiotics. C e l l c u l t u r e s d i f f e r from organ c u l t u r e s p r i m a r i l y i n that they c o n s i s t of populations of d i v i d i n g i n d i v i d u a l c e l l s r a t h e r than h i g h l y organized t i s s u e s from a s i n g l e organism. As a r e s u l t , the l i f e of an organ c u l t u r e i s f i n i t e , while the l i f e of c e l l c u l t u r e s i s p o t e n t i a l l y i n f i n i t e (12). Primary c u l t u r e s that are derived d i r e c t l y from the donor may or may not develop i n t o s e l f r e p l i c a t i n g e s t a b l i s h e d c e l l l i n e s . Monolayer c u l t u r e s of f a t body c e l l s from pupae of l a r g e i n s e c t s such as the tobacco hornworm and embryonic c e l l s from the oothecae of grasshoppers and cockroaches have been s u c c e s s f u l l y prepared i n s u f f i c i e n t q u a n t i t i e s f o r experimental work (19) ( F i g . 6). However, because such small amounts of t i s s u e are a v a i l a b l e from most i n s e c t s and the contamination problems a r i s i n g from the use of l a r g e numbers of t i s s u e donors are so severe, the use of primary c u l t u r e s of i n s e c t c e l l s f o r experimental purposes o f f e r s few advantages over organ c u l t u r e s . The number of e s t a b l i s h e d l i n e s of i n s e c t c e l l s i s r a p i d l y i n c r e a s i n g , and many of these are a v a i l a b l e f o r experimental purposes (39). Advantages of C e l l Culture Systems The use of c e l l l i n e s f o r metabolic s t u d i e s has a number of inherent advantages and disadvantages (40). Perhaps the most obvious advantage i s that l a r g e amounts of uniform m a t e r i a l are a v a i l a b l e f o r experimentation. Large-scale r e p l i c a t i o n i s p o s s i b l e and there i s good sample u n i f o r m i t y when spinner f l a s k s (41) and r o l l e r b o t t l e s are used (42). The problems of s u r g i c a l shock and m i c r o b i o l o g i c a l contamination can be v i r t u a l l y e l i m i n a t e d ; and automatic devices f o r handling and counting c e l l s are a v a i l a b l e to keep labor at a minimum. C e l l l i n e s are a v a i l a b l e from a number of major d i p t e r a n , l e p i d o p t e r a n , and homopteran pests, although to
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(turc
Figure 6. Schematic of technique for preparing primary cell cultures from predorsal closure cockroach embryos. In preparing primary cultures of fat body cells, trypsinization is required to disperse the cells.
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date, none are a v a i l a b l e from eoleopteran or hymenopteran s p e c i e s (39). Disadvantages of C e l l C u l t u r e Systems At the present time, only a l i m i t e d number of c e l l l i n e s f o r which the t i s s u e of o r i g i n can be i d e n t i f i e d i s a v a i l a b l e . Even f o r these l i n e s , the evidence f o r i d e n t i t y i s weak (3). S k i l l f u l and determined e f f o r t s w i l l be necessary to develop new l i n e s from known t i s s u e s such as f a t body or to i d e n t i f y the t i s s u e of o r i g i n f o r present l i n e s . Another problem i s that w h i l e organ c u l t u r e s can be r e l a t e d back to i n d i v i d u a l i n s e c t s , c e l l l i n e s r e f l e c t the dynamics of l a r g e populations of i n d i v i d u a l s kept under constant s e l e c t i o n p r e s s u r e . The c e l l s must t h e r e f o r e be f r e q u e n t l y cloned and kept as f r o z e n stocks to maintain s t a b i l i t biochemical c h a r a c t e r i s t i c yet, very l i t t l e i s known about the metabolic c a p a b i l i t i e s of e x i s t i n g i n s e c t c e l l l i n e s and much remains to be done before such c e l l l i n e s can be considered u s e f u l t o o l s f o r the study of metabolism. At the present time we are working w i t h a c e l l l i n e (MRRL-CH-2) d e r i v e d from embryos of the tobacco hornworm (44). These c e l l s respond with a change i n morphology to the presence of 20-hydroxyecdysone at p h y s i o l o g i c a l c o n c e n t r a t i o n s and are capable of h y d r o x y l a t i n g the C-20 p o s i t i o n of aecdysone (43) (Table V ) . Other biochemical parameters of t h i s c e l l l i n e are c u r r e n t l y under i n v e s t i g a t i o n . Table V.
α-Ecdysone and 20-hydroxyecdysone present i n c e l l c u l t u r e s during i n c u b a t i o n of tobacco hornworm embryonic c e l l l i n e with 30 \xg of a-ecdysone.
Incubation Days
a-Ecdysone
20-Hydroxyecdysone
yg
Vtg
0 1 3 5 7
26.2 25.9 15.5 20.0 18.0
0.0 0.0 0.1 0.1 0.2
C e l l C u l t u r e s as a Source of S u b c e l l u l a r Components We have r e c e n t l y been u s i n g another tobacco hornworm embryonic c e l l l i n e (MRRL-CH-1) as a source of c e l l membrane f o r s t u d i e s of membrane t r a n s p o r t systems. As a r e s u l t of t h i s work i t has become apparent to us that c e l l l i n e s have
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some unique advantages as a source of subcellular components for various types of metabolism studies. We found that cell cultures can be subjected to bioassay and indeed to some biochemical procedures in the same medium in which they were grown (45). Thus, several of the hazards involved in tissue preparation such as osmotic shock and contamination with microorganisms are eliminated. Furthermore, large amounts of highly uniform material are available. Mammalian and plant tissue cultures are being used for such purposes (46,47), but few examples of such work with insect tissue cultures have been reported. (48). Concluding Remarks Insect organ cultures have proved to be extremely useful tools for studies of th dideoxyecdysone. The experimenta methodology and its use for the confirmation and extension of information obtained from in vivo studies make organ culture the method of choice for certain types of studies. The use of insect cell cultures for studying the metabolism of xenobiotics is s t i l l only in the early stages of development. Much work will have to be done before cell cultures can be considered useful tools for such studies. However, insect cell cultures are presently being used successfully to provide a highly uniform source of subcellular components for metabolic studies. Literature Cited 1. Marks, E. P., "Invertebrate Tissue Culture, Research Applications", (1976), Academic Press, NY, 177. 2. Agui, N., "Invertebrate Tissue Culture, Research Applications", (1976), Academic Press, NY, 133. 3. Landureau, C., "Invertebrate Tissue Culture, Applications in Medicine, Biology and Agriculture", (1976), Academic Press, NY, 101. 4. Judy, K., Schooley, D., Dunhanm L., Hall, Μ., Bergot, J., and Siddall, J., Proc. Natl. Acad. Sci. U. S. Α., (1973) 70: 1509. 5. King, D., Bollenbacher, W., Borst, D., Vedeckis, W., O'Conner, J., Ittycheriah, P., and Gilbert, L . , Proc. Natl. Acad. Sci. U. S. Α., (1974) 71: 973. 6. Marks, E. P., Biol. Bull., (1971) 73: 83. 7. Agui, Ν., Appl. Entomol. Zool., (1974) 9: 256. 8. Wyatt, G. R., Chen, T., and Couble, T., "Invertebrate Tissue Culture, Applications in Medicine, Biology and Agriculture", (1976), Academic Press, NY, 195. 9. Koeppe, J., and Offengand, J., "Invertebrate Tissue Culture, Applications in Medicine, Biology and Agriculture", (1976), Academic Press, NY, 185.
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23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
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Marks, Ε. P., Biol. Bull., (1973) 145: 171. Marks, Ε. P., Biol. Bull., (1968) 135: 520. Federoff, S., In Vitro, (1966) 2: 155. Marks, E. P., and Holman, G., J. Insect. Physiol., (1974) 20: 2087. Schooley, D., Judy, Κ., Bergot, J . , Hall, Μ., and Siddall, J . , Proc. Natl. Acad. Sci. U. S. Α., (1973) 70: 2921. King. D. S., and Marks, E. P., Life Sci., (1974) 15: 147. Marks, E. P., Robbins, W. Ε., and Thompson, M. J., Lipids, (1978) 13: 259. Thompson, M. J., Marks, E. P., Robbins, W. E., Dutky, S. R., Finegold H. and F i l i p i P. Lipids in press Müller, P., Masner Life Sci. (1974) Marks, E. P., "Tissue Culture, Methods and Applications", (1973), Academic Press, NY, 153. Judy, K. and Marks, E. P., Gen. Comp. Endocrinol., (1971) 17: 351. Kaplanis, J. Ν., Robbins, W. Ε., Thompson, M. J., and Baumhover, Α., Η., Science, (1969) 166: 1540. Kaplanis, J. Ν., Dutky, S. R., Robbins, W. Ε., and Thompson, M. J., "Invertebrate Endocrinology and Hormonal Heterophylly", (1974), Springer-Verlag, NY, 161. Kaplanis, J. Ν., Thompson, M. J., Dutky, S. R., Robbins, W. Ε., and Lindquist, E., Steroids, (1972) 20: 105. Robbins, W. Ε., Kaplanis, J. Ν., Thompson, M. J., Shortino, T. J., and Joyner, S. C., Steroids, (1970) 16: 105. Robbins, W. Ε., Kaplanis, J. Ν., Thompson, M. J., Shortino, T. J., Cohen, C. F., and Joyner, S. C., Science, (1968) 161: 1158. Earle, N., Padovani, I., Thompson, M. J., and Robbins, W. E., J. Econ. Entomol., (1970) 63: 1064. Rose, G., Pomerat, C., Shindler, T., and Trunnel, J., J. Biophys. Biochem. Cytol., (1958) 4: 761. Marks, E. P., "Invertebrate Tissue Culture, Applications in Medicine, Biology and Agriculture", (1976) Academic Press, NY, 223. Svoboda, J . Α., Thompson, M. J., and Robbins, W. Ε., Lipids, (1972) 7: 553. Robbins, W. Ε., Thompson, M. J., Svoboda, J. Α., Shortino, T. J., Cohen, C. F., Dutky, S. R., and Duncan, O., Lipids, (1975) 10: 35. Marks, E. P., Gen. Comp. Endocrinol., (1973) 21: 472. Grace, T. D. C. and Mitsuhashi, J., "Arthropod Cell Cultures and Their Application to the Study of Viruses", (1971), Springer-Verlag, NY, 108.
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33. Mitsuhashi, J., Grace, T. D. C., and Waterhouse, D., Entomol. Exp. Appl., (1970) 13: 327. 34. Cohen, E. and Gilbert, L., J. Insect Physiol. (1973) 19: 1857. 35. Wyss, C., Experientia, (1976) 32: 1272. 36. Courgeon, Α. Μ., Exp. Cell Res., (1972) 75: 327. 37. Best-Belpomme and Courgeon, A. M., C. R. Acad. Sci., (1976) 283: 155. 38. Cherbas, P., Cherbas, L . , and Williams, C. Μ., Science, (1977) 197: 275. 39. Hink, W. F., "Invertebrate Tissue Culture, Research Applications", (1976), Academic Press, NY, 319. 40. Fry, J. R. and Bridges, J. W., "Progress in Drug Metabolism", (1977) John Wiley & Sons NY 71 41. Thayer, P. S., "Tissu (1973), Academi , , 42. Whittle, W. and Kruse, P., "Tissue Culture, Methods and Applications", (1973), Academic Press, NY, 327. 43. Marks, E. P. and Holman, G. Μ., In Vitro, (1978) in press. 44. Eide, P., Caldwell, J., and Marks, E. P., In Vitro, (1975) 11: 395. 45. Marks, E. P., In Vitro, (1978) 14: 374. 46. Gregor, H. D., Protoplasma, (1977) 91: 201. 47. North, H. and Menzer, R., Pestic. Biochem. Physiol., (1972) 2: 278. 48. Davis, J. and Hartig, W., Insect Biochem., (1977) 7: 77. RECEIVED
December 20,
1978.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
8 The Use of Insect Subcellular Components for Studying the Metabolism of Xenobiotics C. F. W I L K I N S O N Department of Entomology, Cornell University, Ithaca, NY 14853
Our a b i l i t y to use to maximum advantage and i n r e l a t i v e s a f e t y the vast number of drugs, p e s t i c i d e s and other l i p o p h i l i c xenobiotics currently a extent on our a b i l i t y t organisms. Metabolic s t u d i e s i n mammals are e s s e n t i a l i n assessing the e f f i c a c y and s a f e t y of new drugs and data on the nature and t o x i c o l o g i c a l p r o p e r t i e s of p e s t i c i d e metabolites are mandatory i n e v a l u a t i n g the p o t e n t i a l hazard to man of residues of these m a t e r i a l s i n food or i n the environment. Comparative s t u d i e s with f i s h , b i r d s and other species are important i n determining the hazards posed to these animals by a large v a r i e t y of environmental p o l l u t a n t s . In a d d i t i o n to t h e i r d i r e c t importance i n safety/hazard e v a l u a t i o n metabolic s t u d i e s are b a s i c to our understanding of the mode of a c t i o n of b i o l o g i c a l l y a c t i v e m a t e r i a l s . They o f t e n y i e l d important information on enzymatic a c t i v a t i o n and d e t o x i c a t i o n processes and f r e q u e n t l y provide a mechanistic explanation of cases of s e l e c t i v e t o x i c i t y . I t i s i n the quest to o b t a i n a b e t t e r understanding of these processes and to u t i l i z e t h i s information i n the design of more e f f e c t i v e and s a f e r i n s e c t i c i d e s that has stimulated i n t e r e s t i n metabolic s t u d i e s i n i n s e c t s . As i s obvious from the p r e s e n t a t i o n s i n t h i s symposium, metabolic s t u d i e s may be c a r r i e d out i n v i v o i n the l i v i n g organism or i n v i t r o i n a v a r i e t y of preparations c o n s i s t i n g of i s o l a t e d organs, t i s s u e s , c e l l s or s u b c e l l u l a r components. In v i v o i n v e s t i g a t i o n s provide q u a n t i t a t i v e information on the overa l l r a t e of metabolism and on the nature of the t e r m i n a l metabol i t e s ; they seldom provide data on the nature of the metabolic intermediates or on the enzymatic mechanisms by which they are formed. In v i t r o experiments on the other hand permit the i d e n t i f i c a t i o n and study of i n d i v i d u a l r e a c t i o n mechanisms and products but are u s u a l l y of only l i m i t e d use i n e x p l a i n i n g the r a t e and p a t t e r n of metabolism i n the i n t a c t organism. C l e a r l y any complete metabolic study has to i n c l u d e both i n v i v o and i n v i t r o components; the s i t u a t i o n i s analogous to a puzzle where the 0-8412-0486-l/79/47-097-249$09.00/0 © 1979 A m e r i c a n C h e m i c a l Society
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i n v i v o s t u d i e s provide the pieces to the puzzle and the i n v i t r o s t u d i e s y i e l d the clues which enable the pieces to be put together i n a meaningful way. T h i s chapter w i l l address i t s e l f to the use of i n s e c t subc e l l u l a r components i n i n v i t r o s t u d i e s of the metabolism of x e n o b i o t i c s . Since most of the advantages and disadvantages of i n v i t r o s t u d i e s are of a general nature and l a r g e l y independent of the species employed, emphasis w i l l be given to d i s c u s s i n g some of the major problems encountered i n developing s u i t a b l e i n v i t r o systems and techniques with i n s e c t s p e c i e s . I t i s not the o b j e c t i v e of t h i s chapter to conduct a comprehensive survey of metabolic r e a c t i o n s with i n d i v i d u a l compounds or to d i s c u s s i n d e t a i l the c h a r a c t e r i s t i c s of the various enzymes i n v o l v e d i n x e n o b i o t i c metabolism. Those i n t e r e s t e d i n these areas are r e f e r r e d to some of th a v a i l a b l e (1-10), and th H i s t o r i c a l development The c a p a c i t y of i n s e c t s to metabolize s y n t h e t i c organic chemicals i n v i v o was f i r s t recognized during the l a t e 1940s when i t was discovered that metabolic d e h y d r o c h l o r i n a t i o n of DDT to the r e l a t i v e l y nontoxic DDE was a major causative f a c t o r i n the development of i n s e c t r e s i s t a n c e to t h i s i n s e c t i c i d e . In the years immediately f o l l o w i n g t h i s discovery i n v i v o i n v e s t i g a t i o n s with other i n s e c t i c i d e s i n c l u d i n g the c y c l o d i e n e s , the organophosphorus compounds and the carbamates revealed that almost a l l groups of compounds were s u s c e p t i b l e to metabolic attack by both i n s e c t s and a v a r i e t y of non-target organisms and that t h i s was o f t e n the dominant f a c t o r i n determining the degree and duration of t h e i r t o x i c a c t i o n . Recognition of the c r i t i c a l r o l e of metabolism i n r e l a t i o n to s e l e c t i v e t o x i c i t y and i n s e c t r e s i s t a n c e to i n s e c t i c i d e s gave f u r t h e r impetus to metabolic s t u d i e s and the intense a c t i v i t y of agro chemical i n d u s t r y during the 1950s provided a steady flow of new compounds with which to work. As a r e s u l t , i n v i v o metabolic s t u d i e s with i n s e c t s , mammals and other organisms assumed an ever important p o s i t i o n i n i n s e c t i c i d e research and were aided considerably by improved technology, p a r t i c u l a r l y the use of r a d i o a c t i v e t r a c e r s and the i n t r o d u c t i o n of more s o p h i s t i c a t e d instrumentation f o r the r e s o l u t i o n , d e t e c t i o n and i d e n t i f i c a t i o n of small amounts of metabolites. Since almost a l l of the e a r l y s t u d i e s with i n s e c t s were conducted i n v i v o only the end products of metabolism were observed. In most cases these were water s o l u b l e secondary conjugates which were d i f f i c u l t to i d e n t i f y and which provided l i t t l e or no information on the s t r u c t u r e s of the primary or intermediate metabolites from which they were derived or on the nature of the enzyme systems e f f e c t i n g the i n i t i a l a t t a c k on the parent compound. Indeed i n most of the e a r l y work these conjugates were simply c l a s s i f i e d as "water s o l u b l e s " and no f u r t h e r attempt was
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made to i d e n t i f y them. In s p i t e of these problems the importance of s e v e r a l primary metabolic r e a c t i o n s was w e l l e s t a b l i s h e d and i t was known that these could c a t a l y z e both the a c t i v a t i o n and d e t o x i c a t i o n of the parent compound. Dehydrochlorination was known to c o n s t i t u t e a major metabolic pathway f o r DDT and i t s d e r i v a t i v e s and h y d r o l y s i s and d e s u l f u r a t i o n were w e l l recognized r e a c t i o n s with a v a r i e t y of organophosphorus compounds. In a d d i t i o n the probable involvement of enzymatic o x i d a t i o n was s t r o n g l y i n d i c a t e d by the i d e n t i f i c a t i o n of metabolites such as epoxides, s u l f o x i d e s and sulfones from s e v e r a l compounds although the f u l l extent of t h i s involvement was not obvious. This then was the general s i t u a t i o n which p e r t a i n e d i n i n s e c t metabolic s t u d i e s up to and during the e a r l y 1950s. Somewhere around t h i s time there occurred a r a t h e r sudden r e a l i z a t i o n that the natur and mammals were e s s e n t i a l l r e f l e c t e d a b a s i c s i m i l a r i t y i n the enzymatic mechanisms i n v o l v e d . This tended to open up new and improved l i n e s of communication between i n d i v i d u a l s conducting metabolic s t u d i e s i n i n s e c t s and those working with mammals. The use of a v a r i e t y of i n v i t r o systems such as l i v e r s l i c e s , homogenates and s u b c e l l u l a r f r a c t i o n s had already proved u s e f u l i n mammalian s t u d i e s and i t was not long before the f i r s t attempts were made to develop s i m i l a r systems from i n s e c t s . E a r l y i n v i t r o s t u d i e s i n i n s e c t s u s u a l l y employed i n t a c t t i s s u e s or crude minces and homogenates. Although s e v e r a l organophosphorus e s t e r s were found to be hydrolyzed by the "aromatic e s t e r a s e " of the bee (Apis m e l l i f e r a ) abdomen (11) and malaoxon and acethion were shown to be degraded slowly by carboxylesterase a c t i o n i n cockroach ( P e r i p l a n e t a americana) minces and whole guts (12), homogenates of other i n s e c t s proved g e n e r a l l y i n a c t i v e towards a v a r i e t y of organophosphorus i n s e c t i c i d e s (13). Somewhat e a r l i e r than t h i s , i n t a c t i n s e c t t i s s u e s e s p e c i a l l y the gut had been found capable of a c t i v a t i n g schradan (14) and c a t a l y z i n g the o x i d a t i v e a c t i v a t i o n ( d e s u l f u r a t i o n ) of phosphorothionates such as parathion (15). Fenwick (16,17) was the f i r s t to demonstrate phosphoramidate a c t i v a t i o n i n a s u b c e l l u l a r preparation from l o c u s t ( S c h i s t o c e r c a gregaria) f a t body. He reported that 84% of the schradan o x i d i z i n g a c t i v i t y of a f a t body homogenate was i n the upper l a y e r of a heterogeneous 14,000g c e n t r i f u g a l sediment ( r e f e r r e d to somewhat questionably as the microsomal f r a c t i o n ) and that a c t i v i t y required the a d d i t i o n of e i t h e r the supernatant or exogenous NADPH. I t i s i n t e r e s t i n g that as a r e s u l t of these and r e l a t e d s t u d i e s Fenwick (17) as l a t e as 1958 found i t worthwhile to note that the l o c u s t f a t body homogenate contained " . . . (at l e a s t ) two d i s t i n c t types of p a r t i c l e s which resemble mammalian l i v e r mitochondria and microsomes." By the l a t e 1950s i t was w e l l e s t a b l i s h e d that i n mammals the primary metabolic attack on a l a r g e number of l i p o p h i l i c drugs and x e n o b i o t i c s was e f f e c t e d by a s e r i e s of o x i d a t i v e
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC METABOLISM
252
r e a c t i o n s a s s o c i a t e d with h e p a t i c microsomes (18). The f i r s t i n v i t r o demonstration of microsomal enzyme a c t i v i t y i n i n s e c t s was that of Agosin et a l . (19) who showed that NADPH-fortified microsomes prepared from whole german cockroaches ( B l a t t e l l a germanica) and other species c a t a l y z e d the h y d r o x y l a t i o n of DDT. This represented a milestone of s o r t s s i n c e i t provided the f i r s t r e a l i n d i c a t i o n that i n s e c t s contained an a c t i v e microsomal oxidase system s i m i l a r to that i n mammalian l i v e r and a l s o that i n v i t r o metabolic systems could be developed to reproduce the r e a c t i o n s o c c u r r i n g i n the l i v i n g i n s e c t . Since t h i s time i n v i t r o s t u d i e s employing s u b c e l l u l a r f r a c t i o n s derived from whole i n s e c t s or i n s e c t t i s s u e s have become almost r o u t i n e p r a c t i c e i n many l a b o r a t o r i e s . As a r e s u l t a great d e a l of information has been obtained on the types of metabolic r e a c t i o n s whic the general biochemical these r e a c t i o n s (1,6-10). I t has become c l e a r that as i n mammals, x e n o b i o t i c metabolism i n i n s e c t s i s accomplished by r e l a t i v e l y few general types of r e a c t i o n s . The importance of e s t e r cleavage by a s e r i e s of hydrolases (phosphatases, c a r b o x y l e s t e r a s e s , amidases, etc.) has been demonstrated with v a r i o u s substrates (20) and g l u t a t h i o n e t r a n s f e r a s e s are known to play important r o l e s i n primary r e a c t i o n s such as d e a l k y l a t i o n and d e a r y l a t i o n (21,22), d e c h l o r i n a t i o n (22,23,24) and thiocyanate metabolism (22,25). Epoxide hydratase a c t i v i t y i s known to be widely d i s t r i b u t e d i n i n s e c t s (20,26) and conjugating enzymes are known to c a t a l y z e numerous secondary r e a c t i o n s such as g l u c o s i d a t i o n and s u l f a t i o n (22). But perhaps most important of a l l i s c l e a r r e c o g n i t i o n of the dominance of the cytochrome P-450 mediated system i n i n s e c t s and i t s a b i l i t y to c a t a l y z e the primary metabolic a t t a c k on a l a r g e number of l i p o p h i l i c x e n o b i o t i c s through r e a c t i o n s such as epoxidation, h y d r o x y l a t i o n , N- and O-dealkyiation t h i o e t h e r o x i d a t i o n and d e s u l f u r a t i o n (1,8,9,10,27). What i s now emerging from comparative s t u d i e s i s a p i c t u r e of remarkable f u n c t i o n a l u n i t y with respect to the r e a c t i o n s of x e n o b i o t i c metabolism. There are of course some d i f f e r e n c e s between species as w i l l be discussed l a t e r i n t h i s symposium by Dr. T e r r i e r e but i n s p i t e of the dramatic v a r i a t i o n s i n s i z e , morphology, n u t r i t i o n , e c o l o g i c a l h a b i t a t and general l i f e s t y l e between say mammals and i n s e c t s , one cannot help but be impressed by the b a s i c s i m i l a r i t i e s which e x i s t at the s u b c e l l u l a r l e v e l . At the present time, however, the p i c t u r e i s s t i l l out of focus and much remains to be done, p a r t i c u l a r l y at the i n v i t r o l e v e l , before a more complete understanding can be achieved. General c o n s i d e r a t i o n s
r e l a t i n g to i n v i t r o
studies
The f i r s t major d e c i s i o n s which have to be made i n i n i t i a t i n g an i n v i t r o study are the species and l i f e stage of the i n s e c t to be employed. This w i l l be d i c t a t e d l a r g e l y by the purpose of the
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
8.
WILKINSON
Insect Subcellular
Components
253
proposed study. In v i t r o metabolic s t u d i e s are u s u a l l y d i r e c t e d towards p r o v i d i n g i n f o r m a t i o n e i t h e r on the products of a p a r t i c u l a r r e a c t i o n ( s ) or on the biochemical character and mechanism of the enzyme which c a t a l y z e s the r e a c t i o n . In the former case, the i n v i t r o system i s used as a t o o l to produce primary or i n t e r mediary metabolites of a p a r t i c u l a r x e n o b i o t i c which may be r e q u i r e d to confirm the i d e n t i t y of t r a c e metabolites or metabolic pathways suggested from corresponding i n v i v o s t u d i e s ; i n the l a t t e r case s e l e c t e d x e n o b i o t i c s are used as t o o l s to c h a r a c t e r i z e the system which can then be employed as a source of more q u a n t i t a t i v e metabolic data or i n comparative s t u d i e s . If the proposed i n v e s t i g a t i o n i s concerned with e s t a b l i s h i n g the primary metabolites of some i n s e c t i c i d e i n a s p e c i f i c i n s e c t pest then c l e a r l y the specie mined and the l i f e stage a d u l t ) are presumably those at which the i n s e c t i c i d e i s d i r e c t e d i n the f i e l d , i . e . those causing the most economic damage. The major requirement here i s to develop an i n v i t r o system that w i l l reproduce metabolites observed i n v i v o . In t h i s type of study a d e t a i l e d knowledge of the system i s not of primary importance and, indeed, i n many cases the use of i n t a c t t i s s u e s or crude homogenates may be more u s e f u l than i n d i v i d u a l s u b c e l l u l a r components. If on the other hand the o b j e c t i v e of the study i s to charact e r i z e the enzymatic system r e s p o n s i b l e f o r a c e r t a i n type of metabolic r e a c t i o n , i t i s u s u a l l y d e s i r a b l e to work with more homogeneous, p u r i f i e d f r a c t i o n s and to give greater emphasis to o b t a i n i n g q u a n t i t a t i v e i n f o r m a t i o n . For s t u d i e s of t h i s type there i s c o n s i d e r a b l y more leeway i n the s e l e c t i o n of the s p e c i e s to be employed and i n view of the estimated existence of 2-10 m i l l i o n species of i n s e c t s , the t h e o r e t i c a l p o s s i b i l i t i e s are enormous. In p r a c t i c e , however, the choice of s p e c i e s i s u s u a l l y determined by a s e r i e s of convenience f a c t o r s which r e l a t e mainly to the amount of biomass a v a i l a b l e f o r study. T h i s i s probably the major l i m i t i n g f a c t o r i n most s t u d i e s on i n s e c t biochemistry. Consequently, wherever p o s s i b l e a r e l a t i v e l y l a r g e i n s e c t species should be s e l e c t e d . In many cases the l i f e c y c l e s of l a r g e i n s e c t s are q u i t e long so that i t i s o f t e n necessary to reach a compromise between s i z e and turnover time ( i . e . length of generat i o n ) . The species s e l e c t e d f o r study should be r e a d i l y amenable to mass r e a r i n g under l a b o r a t o r y c o n d i t i o n s (the a v a i l a b i l i t y of a s a t i s f a c t o r y a r t i f i c i a l d i e t i s advantageous) and should be a v a i l a b l e on a year-round b a s i s . C l e a r l y a species with only one generation per year or one with an o b l i g a t i v e p e r i o d of diapause would not be convenient f o r continuous study. I t i s as a r e s u l t of requirements of t h i s type that to date i n v i t r o s t u d i e s have been l i m i t e d to perhaps 30-40 i n s e c t species very few of which can be considered s e r i o u s pests of a g r i c u l t u r e or p u b l i c h e a l t h .
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC METABOLISM
254 Tissue
sources
Having s e l e c t e d an appropriate i n s e c t species and l i f e stage with which to work the next step i s t o decide on the t i s s u e source to be employed as a s t a r t i n g point f o r s u b c e l l u l a r f r a c t i o n a t i o n . Because of obvious l i m i t a t i o n s on the amount of t i s s u e u s u a l l y a v a i l a b l e , most of the e a r l y metabolic s t u d i e s were conducted with homogenates or s u b c e l l u l a r f r a c t i o n s derived from whole i n s e c t s . The use of whole-insect preparations i s s t i l l common i n s e v e r a l l a b o r a t o r i e s and can f r e q u e n t l y provide u s e f u l q u a l i t a t i v e information on x e n o b i o t i c metabolism. More o f t e n than not, however, enzyme a c t i v i t y i n such p r e p a r a t i o n s , p a r t i c u l a r l y that a s s o c i a t e d with the microsomal f r a c t i o n , i s very low or non e x i s t e n t and u s u a l l y bears l i t t l e resemblance to the true enzymatic c a p a b i l i t y o i n view of the heterogeneou i t i s r e a l l y q u i t e s u r p r i z i n g that such whole-insect preparations e x h i b i t any enzyme a c t i v i t y at a l l . C l e a r l y , the homogenization of whole i n s e c t s causes a t o t a l d i s r u p t i o n of t i s s u e and c e l l u l a r o r g a n i z a t i o n and r e s u l t s i n the r e l e a s e of a l a r g e number of endogenous m a t e r i a l s with p o t e n t i a l i n h i b i t o r y e f f e c t s on the enzyme under i n v e s t i g a t i o n . Several d i f f e r e n t types of endogenous i n h i b i t o r s have been encountered and i d e n t i f i e d i n the course of s t u d i e s with i n s e c t microsomes (8,10) and there i s no doubt that they o f t e n represent a s e r i o u s p r a c t i c a l problem i n i n v i t r o i n v e s t i g a t i o n s . The i n s e c t eye pigment, xanthommatin has been e s t a b l i s h e d as an important i n h i b i t o r y f a c t o r i n preparations from whole house f l i e s (28,29), f r u i t f l i e s (Drosophila melanogaster) and honey bees (Apis m e l l i f e r a ) (30). I t causes s u b s t a n t i a l i n h i b i t i o n of house f l y epoxidase a c t i v i t y a t concentrations as low as 5 χ 10" M and i n h i b i t o r y a c t i v i t y i s accompanied by a marked increase i n NADPH o x i d a t i o n (2_8,2_9). Studies on the mode of a c t i o n of xanthommatin have shown that i t accepts e l e c t r o n s from the f l a v i n , NADPH cytochrome c_ reductase, of the microsomal e l e c t r o n t r a n s p o r t chain thereby a c t i n g as an e l e c t r o n s i n k to impede the flow of reducing e q u i v a l e n t s to cytochrome P-450 (Figure 1) (28,29). The a b i l i t y of dihydroxanthommatin to undergo a u t o x i d a t i o n and to be o x i d i z e d i n the presence of cytochrome c_ or t y r o s i n a s e suggests the r a p i d regeneration of xanthommatin and a consequent enhance ment i n i t s i n h i b i t o r y p o t e n t i a l . Since xanthommmatin i s widely d i s t r i b u t e d as an i n s e c t eye pigment t h i s type of i n h i b i t i o n i s of p o t e n t i a l importance i n almost a l l preparations from whole insects. What may be a s i m i l a r type of i n h i b i t i o n has a l s o been encountered i n attempts to measure microsomal o x i d a t i o n i n prepa r a t i o n s from whole l a s t - i n s t a r lepidopterous l a r v a e j u s t p r i o r to pupation (31,32). In t h i s case, i n h i b i t i o n i s a s s o c i a t e d with s o l u b l e products (a v a r i e t y of quinones) of the m e l a n i z a t i o n or darkening process which i n v o l v e s the tyrosinase-mediated oxidation 7
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
WILKINSON
Insect Subcellular
V
XANTHOMMATIN
Components
DIH YDROXANTHOMMATIN
Auto oxidation Cytochrome c Tyrosinase
Drug Metabolism Reviews Figure 1.
Inhibition of microsomal oxidation by xanthommatin (S)
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
256
XENOBIOTIC METABOLISM
of a v a r i e t y of ortho-dihydroxy compounds (e.g. DOPA) and t h e i r subsequent i n c o r p o r a t i o n i n t o the i n s e c t c u t i c l e . In l e p i d o p t e r ous l a r v a e , t h i s type of i n h i b i t i o n appears to be important only during l a t e l a r v a l development when the t y r o s i n a s e system i s a c t i v a t e d i n p r e p a r a t i o n f o r formation of the pupal coat. I t can be counteracted by the a d d i t i o n of l - p h e n y l - 2 - t h i o u r e a to the p r e p a r a t i o n s to i n h i b i t t y r o s i n a s e a c t i v i t y (31,32). A similar type of i n h i b i t i o n has a l s o been i m p l i c a t e d i n the i n s t a b i l i t y of mixed-function oxidase a c t i v i t y i n house f l y microsomes where the a d d i t i o n of cyanide (also a t y r o s i n a s e i n h i b i t o r ) has a marked s t a b i l i z i n g e f f e c t (33). In theory, quinones may be formed whenever c a t e c h o l s are brought i n t o contact with t y r o s i n a s e and s i n c e both are common i n i n s e c t s , workers should beware of preparations which become p r o g r e s s i v e l y darker on exposure to a i r Another major grou s e r i o u s l y impede i n v i t r those a s s o c i a t e d with the i n s e c t gut contents. Potent i n h i b i t o r s of microsomal o x i d a t i o n s have been reported i n the gut contents of several insect species including several lepidopterous larvae (34,35), a c a d d i s f l y l a r v a (Limnephilus sp.) (37), a sawfly l a r v a (Macremphytus varianus) (38) and the house c r i c k e t (Acheta domesticus) (38,39). The i n h i b i t o r y f a c t o r s i n the gut contents of the southern armyworm (Spodoptera e r i d a n i a ) (40) and the house c r i c k e t (39) have been p a r t i a l l y p u r i f i e d and c h a r a c t e r i z e d as p r o t e o l y t i c enzymes with molecular weights of 26,000 and 16,500 respectively. They are both undoubtedly n a t u r a l l y o c c u r r i n g d i g e s t i v e p r o t e i n a s e s q u i t e s i m i l a r to t r y p s i n and l i k e t r y p s i n t h e i r i n h i b i t o r y a c t i o n r e s u l t s from a d i r e c t p r o t e o l y t i c a t t a c k on the microsomal p r o t e i n (39,41). The e f f e c t of these proteases on the microsomes i s q u i t e s p e c i f i c i n that they cause the s o l u b i l i z a t i o n of the f l a v o p r o t e i n , NADPH cytochrome c_ reductase, and consequently d i s r u p t e l e c t r o n flow to cytochrome P-450 (39,41) (Figure 2 ) . There appears to be no e f f e c t on cytochrome b$ of P-450 and no e f f e c t on s u b s t r a t e b i n d i n g to the l a t t e r (41) so i t appears that the f l a v o p r o t e i n i s the major t a r g e t , p o s s i b l y due to i t s v u l n e r a b l e l o c a t i o n on the outer surface of the membrane. Although s i m i l a r i n t h e i r o v e r a l l e f f e c t on the microsomes, the armyworm and c r i c k e t gut content m a t e r i a l s e x h i b i t p r o p e r t i e s i n d i c a t i n g they are not i d e n t i c a l (Table 1). Thus i n c o n t r a s t to the armyworm m a t e r i a l which shows a s i m i l a r i n h i b i t o r y e f f e c t on i n s e c t and mammalian l i v e r microsomes the c r i c k e t m a t e r i a l i s much l e s s a c t i v e towards the l a t t e r and while both appear to be s e r i n e p r o t e i n a s e s s u s c e p t i b l e to i n h i b i t i o n by phenylmethanes u l f o n y l f l u o r i d e (PMSF) the c r i c k e t but not the armyworm m a t e r i a l i s s e n s i t i v e to soy t r y p s i n i n h i b i t o r . Bovine serum albumin (BSÀ) has a marked p r o t e c t i v e e f f e c t on microsomes i n the presence of the armyworm gut i n h i b i t o r whereas t h i s i s observed with the c r i c k e t m a t e r i a l only at high l e v e l s of BSA (39). The p r o t e c t i v e a c t i o n of BSA against at l e a s t some of these p r o t e o l y t i c enzymes undoubtedly accounts f o r the enhanced microsomal enzyme a c t i v i t y
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
WILKINSON
Figure 2.
Insect Subcellular
Components
Solubilization of NADPH-Cytochrome c reductase by gut contents inhibitor of southern armyworm (S. eridania) (41).
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
258
XENOBIOTIC METABOLISM
TABLE I Properties
of gut content i n h i b i t o r s from a/ S. e r i d a n i a and A. domesticus— Source of i n h i b i t o r
Property
S. e r i d a n i a
A.
Molecular weight
26,000
16,500
Yes
Yes
Proteolytic activity
(casein)
Mg
2+ , , Ca (mM)
Soy
t r y p s i n i n h i b i t o r (lmg/inc.)
w
2+
domesticus
x
Stimulate
effec
No e f f e c t (F )
Inhibition (F )
Inhibition
Inhibition
Armyworm midgut
0.97
0.17
Mammalian l i v e r
^2.0 (F^)
2
PMSF
X
I n h i b i t i o n epoxidase (I^Q, mg/inc.)
(mouse)
7.0 (F ) X
(rat)
Reversal of microsomal i n h i b i t i o n BSA PMSF Soy
(0.5-1.0mM) t r y p s i n i n h i b i t o r (lmg/inc.)
Yes
No ( s l i g h t )
Yes
Yes Yes
— Data from K r i e g e r and Wilkinson (40) and B r a t t s t e n and W i l k i n s o n (39). F^ i s crude f r e e z e - d r i e d gut contents s o l u b l e f r a c t i o n ; F2 i s f r e e z e - d r i e d m a t e r i a l a f t e r passage through Sephadex G-50 or G-100.
observed f o l l o w i n g a d d i t i o n of t h i s m a t e r i a l to homogenates of whole house f l i e s (42). C a s e i n o l y t i c a c t i v i t y has been reported i n whole i n s e c t homogenates of s e v e r a l species (40) and i t i s l i k e l y that p r o t e o l y t i c enzymes of t h i s type are of broad general significance i n i n v i t r o studies i n insects. I t should be obvious from t h i s d i s c u s s i o n that a v a r i e t y of endogenous i n h i b i t o r s l i b e r a t e d during homogenization make whole i n s e c t s a g e n e r a l l y poor t i s s u e source f o r i n v i t r o s t u d i e s p a r t i c u l a r l y those concerned with the biochemical c h a r a c t e r i z a t i o n
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
8.
WILKINSON
Insect Subcellular
Components
259
of the enzymes involved i n metabolism. In some eases, however, due to f a c t o r s such as very small s i z e (e.g. mosquitoes or aphids) or l i m i t e d a v a i l a b i l i t y of the species i n question the use of whole i n s e c t preparations i s unavoidable and can o f t e n be u s e f u l i n gross metabolic s t u d i e s . In t h i s case compromises must be made. The chances of encountering endogenous i n h i b i t o r s are, of course, considerably decreased when s p e c i f i c body regions of the i n s e c t are employed. Thus the use of house f l y abdomens has proved advantageous over whole house f l i e s (8,9,43) ( s i n c e i t immediately avoids the problems a s s o c i a t e d with xanthommatin) and ingenious methods have been developed f o r the mass separation of i n s e c t body segments. Further improvement may be achieved by the a d d i t i o n of BSA (39), cyanide (33) or other p r o t e c t i v e agents to the preparations although care must be take not having other e f f e c t In general, the c l e a r message i s that wherever the s i z e of the i n s e c t permits, i n d i v i d u a l t i s s u e s or organs should be employed as a t i s s u e source f o r homogenization and s u b c e l l u l a r f r a c t i o n a t i o n . Unfortunately, t i s s u e d i s s e c t i o n i s o f t e n a tedious process and there i s u s u a l l y no way to avoid the i n d i v i dual handling of l a r g e numbers of i n s e c t s . The most convenient procedures f o r t h i s vary considerably from one species to another. As a r e s u l t of considerable work i n recent years, i t has been e s t a b l i s h e d that the patterns of t i s s u e d i s t r i b u t i o n of microsomal oxidase a c t i v i t y vary considerably both between d i f f e r e n t orders of i n s e c t s and between species i n a s i n g l e order. In general, however, the t i s s u e s found to be most a c t i v e are various p o r t i o n s of the alimentary t r a c t , the f a t body or the Malpighian tubules (Figure 3) (8). In lepidopterous l a r v a e maximum oxidase a c t i v i t y i s a s s o c i a t e d with the gut t i s s u e s , p a r t i c u l a r l y those of the midgut (Figure 4) (34). This d i s t r i b u t i o n p a t t e r n has been demonstrated with approximately 40 d i f f e r e n t species (44,45) the only exception to date being the cabbage looper ( T r i c h o p l u s i a n i ) where preparat i o n s from the f a t body are more a c t i v e than those from the gut (35). High t i t e r s of microsomal oxidase a c t i v i t y have a l s o been reported i n the gut t i s s u e s of various other i n s e c t s (J3) although i n many cases t h i s i s a s s o c i a t e d with a much broader p a t t e r n of t i s s u e d i s t r i b u t i o n as i n various orthopteran s p e c i e s . In s e v e r a l species of cockroach epoxidation and h y d r o x y l a t i o n a c t i v i t y occur i n v a r i o u s t i s s u e s i n c l u d i n g the gut, f a t body and Malpighian tubules the r e l a t i v e a c t i v i t y of each v a r y i n g with the species (Figure 4B,C) (8). In the house c r i c k e t (Acheta domesticus) maximum a c t i v i t y i s found i n the Malpighian tubules (Figure 4D) (38). As i n mammals, t h e r e f o r e , microsomal oxidase a c t i v i t y i n i n s e c t s i s found i n s e v e r a l d i f f e r e n t t i s s u e s and the observed patterns may r e f l e c t a s t r a t e g i c l o c a l i z a t i o n l i n k e d with the major p o r t a l s of entry of x e n o b i o t i c s i n t o the organism (8,27).
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC METABOLISM
260
FOREGUT
"V
HEAD
"
Figure 3.
MIDGUT
ν
V
THORAX
ABDOMEN
Insect tissues involved in microsomal oxidation
FG
GC
M G H G
MT
F Β
Figure 4. Distribution of microsomal epoxidase activity in insect tissues: (A) southern armyworm (S. eridania) (34); (B) Madagascar cockroach (G. portentosa) (C) American cockroach (P. americanum) (48); (D) house cricket (A. domesticus) (38). Solid bars represent specific activity (per mg protein); striped bars represent activity per insect.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
8.
WILKINSON
Insect Subcellular
261
Components
Thus the gut t i s s u e s may c o n s t i t u t e the major s i t e f o r metabolism of x e n o b i o t i c s ingested i n the food whereas the f a t body and/or Malpighian tubules p o s s i b l y p l a y a greater r o l e i n the metabolism of m a t e r i a l s e n t e r i n g the i n s e c t by d i r e c t integumental penetrat i o n . This can be viewed as analogous to the d i s t r i b u t i o n of microsomal o x i d a t i o n i n the s k i n , lung, gut and l i v e r of mammals where they appear to be s t r a t e g i c a l l y l o c a t e d to act as the f i r s t l i n e of defense against x e n o b i o t i c s . In a s s e s s i n g the r e l a t i v e metabolic importance of a given t i s s u e to the i n t a c t i n s e c t , i t i s important to consider not only the s p e c i f i c a c t i v i t y of the enzyme i n a homogenate or s u b c e l l u l a r f r a c t i o n ( i . e . a c t i v i t y / m g p r o t e i n ) but a l s o the r e l a t i v e biomass of the v a r i o u s t i s s u e s . Consequently, a t i s s u e of l a r g e biomass but low s p e c i f i c oxidase a c t i v i t y may be more important i n i t s o v e r a l l metabolic c a p a c i t specific activity. Thi patterns of d i s t r i b u t i o n of oxidase a c t i v i t y based on measurements of s p e c i f i c a c t i v i t y with those c a l c u l a t e d on a per i n s e c t b a s i s (Figure 4). Even when working with i n d i v i d u a l t i s s u e s , i t i s important to be aware of the p o s s i b i l i t y of encountering endogenous enzyme i n h i b i t o r s . These can o f t e n be detected by combining homogenates of d i f f e r e n t t i s s u e s and examining the a c t i v i t y data f o r i n h i b i t o r y e f f e c t s . As shown i n Table 2, the t o t a l a c t i v i t i e s of combinations of homogenates of v a r i o u s armyworm t i s s u e s are equal to the sum of t h e i r i n d i v i d u a l a c t i v i t i e s and, t h e r e f o r e , can be assumed to be f r e e of i n h i b i t o r y f a c t o r s . In other cases, the
TABLE 2 Epoxidase a c t i v i t i e s of armyworm midgut homogenate alone and i n presence of homogenates of other t i s s u e s
Protein (mg/incubât ion)
Tissue
Epoxidase a c t i v i t y (nmoles/10 min) Observed Calculated
2.1
14.8
Foregut Foregut + MG
1.0 3.1
0.5 15.1
15.3
Hindgut Hindgut + MG
1.2 3.3
0.6 15.6
15.4
Fat body Fat body + MG
6.6 8.7
3.3 17.8
18.1
Malpighian tubules Malpighian tubules + MG
1.0 3.1
2.2 16.8
17.0
Midgut
(MG)
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC METABOLISM
262
a c t i v i t i e s of homogenates or s u b c e l l u l a r f r a c t i o n s of t i s s u e s can be compared with that of the i n t a c t t i s s u e . In the case of adult worker honey bees, epoxidase a c t i v i t y of i n t a c t midguts was decreased approximately 90% simply by opening the gut by l o n g i t u d i n a l i n c i s i o n and was t o t a l l y l o s t f o l l o w i n g homogenization of the t i s s u e (Table 3) (46). Subsequently a potent i n t r a c e l l u l a r i n h i b i t o r y f a c t o r was i s o l a t e d and p a r t i a l l y p u r i f i e d from the s o l u b l e f r a c t i o n of the gut (47). I n h i b i t i o n was a s s o c i a t e d with the n u c l e i c a c i d (RNA) moiety of a macromolecule ( p o s s i b l y a n u c l e o p r o t e i n ) with a molecular weight of approximately 19,000 and could be reversed by d i g e s t i o n with r i b o n u c l e a s e (RNase T^ of TABLE 3 Epoxidase a c t i v i t honey bee (adul Midgut treatment
) midgu Epoxidase a c t i v i t y pmole/min/mg wet. wt.
Intact
1.31
Opened
0.14
Homogenized
Not d e t e c t a b l e
Data from G i l b e r t and Wilkinson (46)
p a n c r e a t i c RNase) (Table 4). F o l l o w i n g t h i s f i n d i n g , i t was d i s covered that s e v e r a l commercially a v a i l a b l e n u c l e i c a c i d s i n c l u d i n g core and t r a n s f e r RNA from baker's yeast and t r a n s f e r RNA from T o r u l a yeast a l s o i n h i b i t e d i n s e c t (armyworm gut) microsomal oxidases but had l i t t l e or no e f f e c t on those from mammalian l i v e r (47). Although to date t h i s i s the only i n h i b i t o r of t h i s type r e p o r t e d , i t i s p o s s i b l e that others w i l l be found i n v a r i o u s insect preparations. Another important parameter which has to be considered i n the s e l e c t i o n of a s u i t a b l e source of t i s s u e f o r i n v i t r o i n v e s t i g a t i o n i s the age or l i f e stage o f the i n s e c t to be used. I t i s now w e l l e s t a b l i s h e d that the a c t i v i t y of the enzymes i n v o l v e d i n x e n o b i o t i c metabolism change d r a m a t i c a l l y with age and stage of development i n v a r i o u s i n s e c t s p e c i e s ( 8 ) . T h i s i s c l e a r l y i l l u s t r a t e d by the p a t t e r n s of microsomal oxidase a c t i v i t y shown i n F i g u r e 5. In g e n e r a l , microsomal oxidase a c t i v i t y i s only found i n a c t i v e l y feeding l i f e stages of the i n s e c t . Thus maximum a c t i v i t y i s u s u a l l y a s s o c i a t e d with the l a r v a l or nymphal stages of development and i n the a d u l t s where these feed; i n s e c t eggs and pupae are u s u a l l y devoid of oxidase a c t i v i t y . In southern armyworm l a r v a e , microsomal o x i d a t i o n a c t i v i t y ( s p e c i f i c a c t i v i t y ) i n
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
WILKINSON
Insect Subcelluhr
Age
(weeks)
Components
263
littar
(tine i i weeks)
Drug Metabolism Reviews Figure 5. Age-activity profiles of microsomal epoxidation in (A) southern armyworm (S. eridania) (34); (B) house cricket (A. domesticus) (38); and (C and D) Madagascar cockroach (G. portentosa) (48).
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC METABOLISM
264 TABLE 4
E f f e c t of RNase T^ and p a n c r e a t i c RNase on of the honey bee midgut
inhibitor Reversal of i n h i b i t i o n (%)
Enzyme (unit) RNase Τ -1
activity
0 24 100
(5) (260) (2800)
P a n c r e a t i c RNase
(0.072)
14
(0.360)
50
Data from G i l b e r t and Wilkinson
(47)
the gut increases about 30-fold during development from the f o u r t h to s i x t h l a r v a l i n s t a r (Figure 5A) and shows an e q u a l l y dramatic d e c l i n e as the l a r v a e terminate feeding and prepare f o r pupation (34); a s i m i l a r p a t t e r n has been observed with s e v e r a l other species (44). The increase i n a c t i v i t y does not occur i n a continuous manner, however, but shows a marked decrease during each l a r v a l molt (dotted l i n e s Figure 5A). Studies with other i n s e c t species have emphasized the remarkable patterns of oxidase a c t i v i t y which occur during i n s e c t development p a r t i c u l a r l y those o c c u r r i n g during the l a r v a l or nymphal molts. Thus a l d r i n epoxidation a c t i v i t y i n the Malpighian tubules of the house c r i c k e t i s low during the molts from 8th to 9th nymphal i n s t a r and from the 9th i n s t a r to the adult and then increases as the adult matures (Figure 5B) (38). T h i s rhythmic developmental c y c l e becomes much c l e a r e r i n the midgut-caeca p r e p a r a t i o n from the Madagascar cockroach (Figure 5C and D) where i t i s q u i t e evident that oxidase a c t i v i t y i s low during the molt and passes through a maximum about midway through each i n s t a r (48). The r e s u l t s of these and other i n v e s t i g a t i o n s s t r o n g l y suggest that microsomal oxidase a c t i v i t y i s under s t r i c t metabolic c o n t r o l and that t h i s i s c l o s e l y l i n k e d with the process of metamorphosis. From a p r a c t i c a l viewpoint these r a p i d and dramatic changes i n enzyme a c t i v i t y can provide a r e a l headache i n i n v i t r o s t u d i e s . Researchers i n t h i s f i e l d would be w e l l advised to e i t h e r e s t a b l i s h the a c t i v i t y patterns i n the i n s e c t species with which they are working or to avoid working with i n s e c t s c l o s e to the molt. In some types of i n v e s t i g a t i o n s (e.g. enzyme i n d u c t i o n s t u d i e s ) where comparisons between t r e a t e d and c o n t r o l groups are r e q u i r e d , i t i s e s s e n t i a l to use groups of i n s e c t s c l o s e l y matched
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
8.
WILKINSON
Insect Subcelluhr
Components
265
(+2h) with respect to age (49). Procedures f o r o b t a i n i n g i n s e c t s u b c e l l u l a r components As with other t i s s u e s , the p r e p a r a t i o n of s u b c e l l u l a r components from i n s e c t s i s e f f e c t e d by two successive steps: homogenization and f r a c t i o n a t i o n . In general, the methods which have been employed are based on procedures which have proved s u c c e s s f u l f o r mammalian t i s s u e s . However, due to l a r g e v a r i a t i o n s i n the nature and morphological heterogeneity of the i n s e c t t i s s u e s employed, numerous m o d i f i c a t i o n s have been made i n attempts to optimize the procedures. These have been discussed at some length i n previous reviews (8,9,10). Various procedures have been described f o r the homogenization of whole i n s e c t s , i n s e c and u s u a l l y the method o process of t r i a l and e r r o r f o r any given t i s s u e source. Homogeniz a t i o n i s u s u a l l y achieved with e i t h e r a mechanical blender, a hand-operated or motor-driven t i s s u e grinder or a mortar and p e s t l e (8,9,10). There i s a general concensus of opinion that the procedure should be as gentle as p o s s i b l e and yet s u f f i c i e n t l y d r a s t i c to s a t i s f a c t o r i l y d i s r u p t c e l l s i n heterogeneous t i s s u e s o f t e n c o n t a i n i n g s c l e r o t i z e d c u t i c l e , muscle f i b e r s , e t c . A l l operations should be c a r r i e d out at 0-2°C. Mechanical blenders (Waring blender, Omni-mixer, etc.) have been used f o r l a r g e batches of whole i n s e c t s or i n s e c t body regions (e.g. house f l i e s or house f l y abdomens) but i n general, oxidase a c t i v i t y i n microsomal f r a c t i o n s derived from such homogenates i s considerably lower than i n those from homogenates prepared with a P o t t e r Elvehjem t i s s u e grinder (43,50) or by a gentle pounding a c t i o n i n a mortar (10,51). T h i s l o s s of enzyme a c t i v i t y may r e s u l t i n part from denaturation through excessive abrasive a c t i o n of hard c h i t i n o u s p o r t i o n s of the i n s e c t or by the more e f f i c i e n t r e l e a s e of endogenous i n h i b i t o r s . V i o l e n t mechanic a l blending undoubtedly a i d s i n the r e l e a s e of the s p a r i n g l y s o l u b l e xanthominatin i n p r e p a r a t i o n s c o n t a i n i n g i n s e c t heads and there are reports that microsomes from blended whole house f l i e s contain a b-type cytochrome (probably cytochrome oxidase) which i s a s s o c i a t e d with t h o r a c i c sarcosomes or sarcosomal fragments and which i n t e r f e r e s with s p e c t r a l determination of microsomal cytochrome P-450 (10,51,52). The l a t t e r i s not observed i n house f l y homogenates prepared by the s o - c a l l e d "mortar" procedure (10,51). The degree or d u r a t i o n of homogenization of whole i n s e c t s or body regions i s a l s o important even when a t i s s u e grinder i s employed. U s u a l l y only a few passes of the plunger are required and b e t t e r r e s u l t s are achieved by use of a r e l a t i v e l y l o o s e - f i t t i n g p e s t l e of T e f l o n or smooth g l a s s . The homogenization of i n d i v i d u a l t i s s u e s u s u a l l y presents fewer problems and here a v a r i e t y of hand-operated or motord r i v e n t i s s u e g r i n d e r s provide the best r e s u l t s . The small
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
266
XENOBIOTIC METABOLISM
amounts of t i s s u e a v a i l a b l e as w e l l as the heterogenous o f t e n " s t r i n g y " nature of the t i s s u e (e.g. armyworm gut) u s u a l l y make mechanical blenders q u i t e i n e f f e c t i v e f o r homogenization purposes. Homogenization with a t i s s u e grinder should be as gentle and b r i e f as p o s s i b l e and again T e f l o n or smooth glass p e s t l e s are p r e f e r able to those of ground g l a s s . Several d i f f e r e n t types of homogenization media have been employed by v a r i o u s workers and obviously can have an e f f e c t on the f i n a l enzymatic a c t i v i t y of the preparation. Although there are reports that the i o n i c strength of the medium may be important (43) the most commonly used media are 0.15 M KC1 or 0.25 M sucrose which may or may not be b u f f e r e d at various pH l e v e l s (8,9,10). O c c a s i o n a l l y BSA (1-2% w/v) or other m a t e r i a l s such as cyanide, phenylmethanesulfonyl f l u o r i d e , e t c . may be added to at l e a s t p a r t i a l l y counteract th (8,42,43). S u b c e l l u l a r f r a c t i o n a t i o n of homogenates of whole i n s e c t s or i n s e c t t i s s u e s i s u s u a l l y achieved by the technique of d i f f e r e n t i a l c e n t r i f u g a t i o n , f i r s t pioneered by A l b e r t Claude with mammal i a n l i v e r c e l l s and subsequently widely a p p l i e d to many other t i s s u e s (53). B r i e f l y the technique i n v o l v e s c e n t r i f u g i n g the i n i t i a l homogenate at about l,000xg f o r 10 minutes to remove n u c l e i and l a r g e r c e l l d e b r i s followed by a 9,000-12,000xg c e n t r i f u g a t i o n f o r 10-30 minutes to sediment mitochondria and other intermediate s i z e d p a r t i c l e s and c e l l o r g a n e l l e s . Further c e n t r i f u g a t i o n of the postmitochondrial supernatant f o r 60 minutes at 100,000-200,OOOxg y i e l d s the s o - c a l l e d microsomal f r a c t i o n and a " s o l u b l e " supernatant. With i n s e c t t i s s u e s t h i s general procedure i s often modified and the a c t u a l c e n t r i f u g a l c o n d i t i o n s ( f o r c e , time) u s u a l l y vary somewhat between d i f f e r e n t l a b o r a t o ries. The procedure may a l s o i n c l u d e f i l t r a t i o n of the o r i g i n a l b r e i through cheese c l o t h , cotton or g l a s s wool to remove the o f t e n s u b s t a n t i a l amounts of c h i t i n o u s debris and l i p i d s r e l e a s e d during homogenization. The a p p l i c a t i o n of t h i s technique to the s u b c e l l u l a r f r a c t i o n a t i o n of i n s e c t t i s s u e homogenates i s based on the l a r g e l y unproven assumption that the s u b c e l l u l a r o r g a n e l l e s and membrane p a r t i c l e s derived from i n s e c t t i s s u e s e x h i b i t s i m i l a r sedimentat i o n c h a r a c t e r i s t i c s to those from mammalian l i v e r c e l l s . In view of the h i g h l y heterogeneous nature of most of the i n s e c t t i s s u e courses used f o r s t u d i e s of x e n o b i o t i c metabolism t h i s i s a dangerous assumption which should be only made with extreme care. Moreover the procedures found s a t i s f a c t o r y f o r one t i s s u e may not be a p p l i c a b l e to another. S u b c e l l u l a r f r a c t i o n s can be c l a s s i f i e d morphologically by means of l i g h t or e l e c t r o n microscopy as containing n u c l e i , mitochondria, microsomal p a r t i c l e s (from the membranous endoplasmic reticulum) e t c . In a d d i t i o n , the discovery that c e r t a i n types of enzymes are u s u a l l y a s s o c i a t e d with a s i n g l e c l a s s of p a r t i c l e s has l e d to the development of a s e r i e s of "marker" enzymes which
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
8.
WILKINSON
Insect Subcelluhr
Components
267
can be used to c l a s s i f y the various s u b c e l l u l a r f r a c t i o n s i n b i o chemical terms. Few attempts have yet been made to c h a r a c t e r i z e i n s e c t s u b c e l l u l a r f r a c t i o n s e i t h e r morphologically or biochemi c a l l y and most workers i n t h i s f i e l d s t i l l seem content to a s s o c i a t e t h e i r enzyme a c t i v i t i e s with s u b c e l l u l a r f r a c t i o n s defined only by values obtained from the u l t r a c e n t r i f u g e . L i t t l e a t t e n t i o n i s a l s o given to c a l c u l a t i n g a complete balance sheet of the t o t a l enzyme a c t i v i t y and p r o t e i n concentration i n each f r a c t i o n and comparing these with values f o r the o r i g i n a l homogenate. Thus i n the absence of t o t a l p r o t e i n concentration the high s p e c i f i c a c t i v i t y of an enzyme i n some s u b c e l l u l a r f r a c t i o n y i e l d s l i t t l e information with regard to i t s o v e r a l l d i s t r i b u t i o n and a meaningful d i s c u s s i o n of s u b c e l l u l a r f r a c t i o n a t i o n i s d i f f i c u l t . From the s t u d i e s which have been conducted i t i s reasonable to conclude that as i n metabolism i n i n s e c t s ar or the microsomal f r a c t i o n s of the c e l l . Although i t i s p o s s i b l e that problems could be encountered by the adsorbance of s o l u b l e enzymes onto l a r g e r c e l l o r g a n e l l e s , the preparation of s a t i s f a c tory s o l u b l e enzyme f r a c t i o n s does not appear to have been a serious problem to date. Most a t t e n t i o n has been focussed on s u b c e l l u l a r f r a c t i o n a t i o n procedures to obtain s a t i s f a c t o r y microsomal f r a c t i o n s from i n s e c t t i s s u e s . In s e v e r a l cases, microsomal f r a c t i o n s e x h i b i t i n g high l e v e l s of drug and i n s e c t i c i d e o x i d i z i n g a c t i v i t y can be prepared by a d i f f e r e n t i a l c e n t r i f u g a t i o n procedure s i m i l a r to that used f o r mammalian l i v e r . Thus i n the case of the armyworm (Spodoptera e r i d a n i a ) l a r v a l gut preparation, the microsomal f r a c t i o n (100,000xg, 60 minutes) i s c l e a r l y the major i n t r a c e l l u l a r l o c a t i o n of the enzymes c a t a l y z i n g epoxidation and N-demethylation (Figure 6A and Β r e s p e c t i v e l y ) and Figure 6B shows that the d i s t r i b u t i o n pattern f o r N-demethylase a c t i v i t y i s e s s e n t i a l l y i d e n t i c a l to that f o r NADPH-cytochrome c_ reductase a recognized microsomal marker enzyme (54) ; that t h i s f r a c t i o n i s derived mainly from the endoplasmic r e t i c u l u m has been obtained by e l e c t r o n microscopy (55). In other cases, however, the preparation of i n s e c t microsomes by d i f f e r e n t i a l c e n t r i f u g a t i o n has proved much more d i f f i c u l t . One of the most common problems which i s encountered i s that most of the x e n o b i o t i c o x i d i z i n g a c t i v i t y associated with the micro somal f r a c t i o n i s sedimented at unexpectedly low g-forces (8). T h i s has been reported f o r f a t body preparations from the American cockroach ( P e r i p l a n e t a americana) (56,57), and blowfly l a r v a ( C a l l i p h o r a erythrocephala) (58) and homogenates of c r i c k e t (Acheta domesticus) Malpighian tubules (38) and Madagascar cock roach (Gromphadorhina portentosa) gut-caeca t i s s u e s (48). Thus i n the l a t t e r example approximately 48% of the a l d r i n epoxidase a c t i v i t y was sedimented during a 15 minute s p i n at 12,000xg (Figure 6C) using the t y p i c a l d i f f e r e n t i a l c e n t r i f u g a t i o n proce dure. I f , instead the crude homogenate ( i n 0.25M sucrose
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC METABOLISM
268
Figure 6.
Subcelluhr fractionation of microsomal oxidase activity from insect tissues.
A and Β are epoxidation (34) and Ν-demethylation (54) activities, respectively, in south ern armyworm midgut tissues; dotted line in Β is NADPH-cytochrome c reductase activity. M is 10,000-12,000 g X 10-15 min pellet, Ρ is 105,000 g X 60-90 min pellet, S is supernatant. C and D are epoxidation activity from midgut caeca tissue of Mada gascar cockroach (48).
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
8.
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containing 50mM T r i s - H C l , pH 7.4) i s layered over 1.6M sucrose and c e n t r i f u g e d f o r 45 minutes at 150,000xg i n a swinging bucket r o t o r two major p a r t i c u l a t e f r a c t i o n s are obtained a dense p e l l e t ( P ) which sediments to the bottom of the tube and a l i g h t p r o t e i n Band (P-, ) at the sucrose i n t e r f a c e . The l a t t e r which represents about 28% of the t o t a l p r o t e i n contains 90.4% of the t o t a l epoxidase a c t i v i t y (Figure 6D) has been shown by e l e c t r o n microscopy t o c o n s i s t l a r g e l y of membranous v e s i c l e s from the endoplasmic reticulum. A s i m i l a r procedure i n v o l v i n g sucrose density gradient c e n t r i f u g a t i o n has been found u s e f u l i n p r e p a r i n g microsomal f r a c t i o n s from c r i c k e t Malpighian tubules (38) and honey bee l a r v a l gut t i s s u e s (46). It t h e r e f o r e appears that under c e r t a i n c o n d i t i o n s , the microsomal p a r t i c l e s i n homogenates of some i n s e c t t i s s u e s e i t h e r aggregate or become adsorbe such as mitochondria. Worker t h i s p o s s i b i l i t y and where necessary modify t h e i r procedures a c c o r d i n g l y . The suspension medium may have some e f f e c t on t h i s behavior s i n c e i t can modify the physicochemical forces i n v o l v e d . I t i s i n t e r e s t i n g to note that i n the case of microsomes from the southern armyworm midgut, the morphological appearance of the membrane v e s i c l e s prepared i n 0.25M sucrose and 0.15M KC1 a r e q u i t e d i s t i n c t ; thus although both f r a c t i o n s e x h i b i t e s s e n t i a l l y i d e n t i c a l l e v e l s of microsomal oxidase a c t i v i t y the v e s i c l e s prepared i n sucrose are r e l a t i v e l y smooth and rounded compared with the angular appearance of those prepared i n KC1. As a r e s u l t of the sometimes broad d i s t r i b u t i o n of microsomal oxidase a c t i v i t y i n s e v e r a l s u b c e l l u l a r f r a c t i o n s , the a c t i v i t y measured i n the microsomal f r a c t i o n per se may be a poor i n d i c a t o r of the t o t a l enzyme c a p a b i l i t y of a given i n s e c t t i s s u e . In comparative s t u d i e s , t h e r e f o r e , where a measure of t o t a l metabolic c a p a c i t y i s o f t e n r e q u i r e d , i t may be more appropriate to measure the a c t i v i t y i n l e s s homogeneous f r a c t i o n s such as the 10 OOOxg supernatant or even i n crude homogenates. Indeed, such preparat i o n s are probably most convenient i n many metabolic s t u d i e s s i n c e t h e i r use s i g n i f i c a n t l y decreases the time of p r e p a r a t i o n . The s t a b i l i t y of enzyme a c t i v i t y i n homogenates and s u b c e l l u l a r f r a c t i o n s from i n s e c t t i s s u e s v a r i e s considerably depending on the t i s s u e source, the method of p r e p a r a t i o n and the storage c o n d i t i o n s employed (8,9,10). As a general r u l e they should be used without delay a f t e r p r e p a r a t i o n . Oxidase a c t i v i t y i n preparations from whole house f l i e s or house f l y abdomens u s u a l l y i s r a p i d l y l o s t on storage at 0-2°C (8,10,31,43) but may be r e t a i n e d f o r s e v e r a l weeks under c e r t a i n c o n d i t i o n s (8,10,31). F r e e z i n g of these microsomes has been reported t o be d e l e t e r i o u s (8,10,51). However, microsomes from armyworm gut may be stored f o r up to a month as w e l l - d r a i n e d p e l l e t s at -15°C (59) (Table 5) and microsomal suspensions from c r i c k e t Malpighian tubules l o s t only about 15% of t h e i r i n i t i a l epoxidation a c t i v i t y a f t e r 3 weeks at the same temperature (38). 2
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC METABOLISM
270 TABLE 5 E f f e c t of f r e e z i n g on oxidase a c t i v i t y content
and cytochrome P-450
i n armyworm midgut microsomal preparations Oxidase a c t i v i t y
Storage (weeks) 0 1 2 3 4
3 (nmoles/mg protein/min) (xlO ) Epoxidation Hydroxylat i o n 92.5 92.5 88.0 94.6 98.2
Cytochrome P-450 (nmoles/mg p r o t e i n )
46.5 54.2 46.0 48.4
Data from K r i e g e r and Wilkinson (59) Microsomes were stored as w e l l - d r a i n e d p e l l e t s at
1.37 1.30 1.37 1.48
-15°C
Measurement of enzymatic a c t i v i t y Following p r e p a r a t i o n of a s u i t a b l e s u b c e l l u l a r f r a c t i o n with which to work the next step i s to measure i t s enzymatic a c t i v i t y towards an appropriate s u b s t r a t e . The i n c u b a t i o n mixture and c o n d i t i o n s to be used w i l l c l e a r l y depend on the nature of the enzyme, s u b c e l l u l a r f r a c t i o n and substrate under i n v e s t i g a t i o n and, of course, the purpose of the study. The o p t i m i z a t i o n of i n c u b a t i o n and assay c o n d i t i o n s i s extremely important and can i n i t s e l f provide a great d e a l of information on the nature and mechanism of the enzyme concerned. Unfortunately i t i s o f t e n overlooked or given minimal a t t e n t i o n where the s u b c e l l u l a r f r a c t i o n s are being used p r i m a r i l y as biochemical t o o l s to generate metabolites. The procedures employed to optimize c o n d i t i o n s f o r measuring enzyme r e a c t i o n s i n i n s e c t s u b c e l l u l a r f r a c t i o n s are e s s e n t i a l l y those p e r t a i n i n g to mammalian systems. Major parameters which should be evaluated are the type, s t r e n g t h and pH of the b u f f e r , the temperature under which the incubations are conducted and the a d d i t i o n of appropriate c o f a c t o r s or other m a t e r i a l s . Phosphate or T r i s b u f f e r s of v a r y i n g pH (7.0-8.5) and i o n i c s t r e n g t h u s u a l l y prove adequate f o r most i n v i t r o s t u d i e s on x e n o b i o t i c metabolism (8,9,10). Although most of the e a r l y i n v i t r o s t u d i e s were c a r r i e d out at 37°C i t i s now g e n e r a l l y agreed that i n s e c t enzymes seem to f u n c t i o n more s a t i s f a c t o r i l y at a temperature of about 30°C (8) and i n many cases optimum values of 20-25°C have been reported (35,36). Since enzyme a c t i v i t y i n many i n s e c t preparations i s l e s s s t a b l e than that i n corresponding mammalian f r a c t i o n s , l i n e a r i t y of the r e a c t i o n with respect to both time and p r o t e i n c o n c e n t r a t i o n should be a s c e r t a i n e d . The a d d i t i o n of c o f a c t o r s i s u s u a l l y necessary to o b t a i n maximal i n v i t r o a c t i v i t y and the nature of these c l e a r l y depends
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
8.
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on the type of enzyme under study. For the enzymes i n v o l v e d i n x e n o b i o t i c metabolism there are only a few major types of cofactors. Microsomal o x i d a t i o n a c t i v i t y i s dependent p r i m a r i l y on the a d d i t i o n of NADPH or an appropriate generating system, the l a t t e r o f t e n proving p r e f e r a b l e by extending the time l i n e a r i t y of the r e a c t i o n (8). Incubations must be conducted a e r o b i c a l l y as oxygen i s a requirement f o r oxidase a c t i v i t y . Although m a t e r i a l s such as EDTA and/or nicotinamide are o f t e n added to enhance microsomal a c t i v i t y i n mammalian microsomes where they s t a b i l i z e NADPH through b l o c k i n g l i p i d p e r o x i d a t i o n and p y r i d i n e n u c l e o t i d a s e a c t i v i t y r e s p e c t i v e l y , t h e i r i n c l u s i o n i n i n s e c t microsomes appears to have l i t t l e or no b e n e f i c i a l e f f e c t on oxidase a c t i v i t y (8). T h i s i s a l s o g e n e r a l l y true of most metal ions (8) BSA (1-2% w/v) i s sometime act the a c t i o n s of r e s i d u a taken that t h i s or other a d d i t i o n s do not have other d e l e t e r i o u s e f f e c t s on enzyme a c t i v i t y . Soluble enzymes of importance i n x e n o b i o t i c metabolism i n c l u d e a v a r i e t y of hydrolases (phosphatases, carboxyesterases, amidases and p y r e t h r o i d hydrolases) (20), s e v e r a l g l u t a t h i o n e (GSH) r e q u i r i n g enzymes ( a l k y l and a r y l t r a n s f e r a s e s , DDTdehydrochlorinase and organothiocyanate metabolizing enzymes) (21,22) and numerous other conjugating enzymes ( g l u c o s y l t r a n s f e r a s e , sulphotransferase, etc.) (22). The p r o p e r t i e s and i n v i t r o requirements, many of these enzymes have already been discussed i n t h i s symposium. Perhaps a few words should be added at t h i s point with respect to the substrate employed i n i n v i t r o s t u d i e s . Unfortu n a t e l y most of the substrates which are used i n x e n o b i o t i c s t u d i e s are of n e c e s s i t y h i g h l y l i p o p h i l i c m a t e r i a l s and t h e i r a d d i t i o n to an aqueous i n c u b a t i o n medium o f t e n presents a problem. U s u a l l y t h i s can be achieved by d i s s o l v i n g the substrate i n a small volume of ethanol, acetone or other organic solvent (care should be taken to ensure that t h i s does not i n h i b i t the enzyme) but even then i t can o f t e n be observed to p r e c i p i t a t e out of the i n c u b a t i o n medium. This r a i s e s s e r i o u s questions about the a c t u a l concentration of substrate i n the incubation mixture and how t h i s should be expressed. Studies with microsomal preparations from mammalian l i v e r and armyworm midgut have shown that i n the presence of a constant amount of microsomal p r o t e i n , the amount of a l d r i n epoxidized to d i e l d r i n i s independent of the t o t a l volume of the r e a c t i o n mixture and r e l a t e d only to the absolute amount of a l d r i n added (Table 6) (60,61). T h i s suggests that the true concentra t i o n of substrate a v a i l a b l e f o r enzyme conversion i s not that expressed by the molar concentration c a l c u l a t e d from the t o t a l incubation volume but i s probably the unknown concentration e x i s t i n g i n the l i p i d phase of the microsomes. There are, there f o r e , problems inherent i n expressing the concentrations of l i p o p h i l i c substrates i n molar terms and Κ values and other
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC METABOLISM
272 TABLE 6
E f f e c t of a l d r i n s o l u b i l i t y on epoxidation i n p i g l i v e r microsomes Incubation Medium Total vol. (ml)
1 2 3 4
Microsomal suspension (ml)
Aldrin added
0.2 0.2 0.2 0.2
Dieldrin produced
(yg)
(yg)
50 50 50 50
9.2 9.3 9.4 10.2
Aldrin Concentration (yg/ml)
(μΜ)
50 25 12.5 10
137 68.,5 45..7 34..3
Data from Lewis et^ a l . a/ — Based on t o t a l volume of i n c u b a t i o n medium k i n e t i c parameters reported i n t h i s way should be accepted with r e s e r v a t i o n . Perhaps the best way of expressing substrate concen t r a t i o n i s i n terms of the absolute amount added to the r e a c t i o n medium s i n c e the a c t u a l c o n c e n t r a t i o n i s d i r e c t l y r e l a t e d to t h i s value. P a r t i c u l a r l y i n the case of the microsomal oxidase system, the choice of s u b s t r a t e s f o r i n v i t r o s t u d i e s i s l a r g e and many e x c e l l e n t and s e n s i t i v e assays are c u r r e n t l y a v a i l a b l e . Initial s t u d i e s to optimize the c o n d i t i o n s f o r some r e a c t i o n or to charac t e r i z e a given s u b c e l l u l a r f r a c t i o n are o f t e n f a c i l i t a t e d by using a model substrate which y i e l d s a s i n g l e metabolic product. Having thus e s t a b l i s h e d the c h a r a c t e r i s t i c s of the system these can u s u a l l y be a p p l i e d d i r e c t l y to s t u d i e s with more complex drug or i n s e c t i c i d e substrates which may have s e v e r a l s i t e s at which enzyme a t t a c k can occur and which consequently produce s e v e r a l d i f f e r e n t metabolites. A p p l i c a t i o n of i n v i t r o s t u d i e s To date, the major a p p l i c a t i o n of i n v i t r o s t u d i e s with i n s e c t s u b c e l l u l a r f r a c t i o n s has been to complement i n v i v o s t u d i e s on the metabolic f a t e and pathways of i n s e c t i c i d e chemi cals. In v i v o s t u d i e s are o f t e n complicated by the f a c t that the t e r m i n a l metabolic products (conjugates) are the ones commonly observed and these o f t e n provide l i t t l e information on the nature of primary or intermediary metabolites which may e x i s t at t r a c e l e v e l s or be of a t r a n s i e n t nature. In i n s e c t s the s i t u a t i o n i s compounded by the f a c t that with h i g h l y t o x i c m a t e r i a l s only very small doses of m a t e r i a l can be a p p l i e d without k i l l i n g the i n s e c t . With i n v i t r o systems t o x i c i t y does not have to be considered and high concentrations of t o x i c a n t s can be employed. Furthermore, the use of v a r i o u s s u b c e l l u l a r f r a c t i o n s permits the i n d i v i d u a l
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
8.
WILKINSON
Insect Subcellufor
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273
study of i s o l a t e d components of the o v e r a l l metabolic machinery i n a r e l a t i v e l y concentrated form without the complications a r i s i n g from the presence of other metabolic components. Thus using a s u i t a b l y f o r t i f i e d microsomal f r a c t i o n the nature of the primary o x i d a t i v e metabolites can be studied i n the absence of the type I I conjugating systems found l a r g e l y i n the s o l u b l e f r a c t i o n . E a r l y i n v i v o metabolic s t u d i e s with the carbamates, f o r example, showed that these i n s e c t i c i d e s were r a p i d l y metabolized by i n s e c t s to water s o l u b l e products and many of which were conju gates of primary hydroxylated metabolites (5) (Figure 7). Subse quent i n v i t r o s t u d i e s u s i n g i s o l a t e d microsomal and s o l u b l e f r a c t i o n s have enabled these primary and secondary products to be more r e a d i l y i d e n t i f i e d and the appropriate enzyme systems b e t t e r characterized. Of p a r t i c u l a r s i g n i f i c a n c components i n i n v i t r o metabolis e x i s t s f o r manipulating the system by the omission or a d d i t i o n of s p e c i f i c c o f a c t o r s or i n h i b i t o r s . Thus the requirement of a c e r t a i n s u b c e l l u l a r f r a c t i o n f o r a p a r t i c u l a r c o f a c t o r to produce a c e r t a i n metabolite o f t e n provides a f a i r l y c l e a r p i c t u r e of the type of enzyme which i s i n v o l v e d i n the conversion. This type of experiment i s e s p e c i a l l y i n f o r m a t i v e where a compound may be metabolized to the same product by two d i f f e r e n t enzyme systems. The data shown i n Table 7 were obtained during the course of a study of d i a z i n o n and diazoxon metabolism i n house f l i e s (62). TABLE 7 E f f e c t of reduced g l u t a t h i o n e and NADPH on the degradation of d i a z i n o n and diazoxon by house f l y s u b c e l l u l a r f r a c t i o n s i n v i t r o Enzyme^ . source— Microsomes :
Soluble fraction:
Cofactors
Degradation Diazinon
None
0
GSH
3.2
ο (mymoles/hr/g + abdomen) Diazoxon 0 0
NADPH
122.1
23.2
NADPH + GSH
164.3
27.0
52.3
5.2
259.0
80.6
NADPH
47.0
1.5
NADPH + GSH
251.0
78.4
None GSH
Data from Yang _et a l . (62) a/ 9 — Microsomes and s o l u b l e f r a c t i o n s equivalent t o 35 and 10 + house f l y abdomens r e s p e c t i v e l y
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC METABOLISM
274
In In Microsomal
vivo vitro Soluble
fraction
?
fraction
Cofactors
N A D P H , O-
OCNHCH^HMHCH OH
CH,S
2
Glucosides Sulfates .
( C H
3
) N ^
CH , Η Ν - * ^
V
^ C H
OCH
5
4OH PRIMARY
OXIDATIVE
SECONDARY CONJUGATES
METABOLITES
Figure 7.
EtO
Metabolism
of carbamate insecticides by microsomal and soluble fractions
EtO^ ^ O
x
Ρ Εt o '
Microsomes NADPH, 0
ν
—
θγ
Ν
γθΗ(ΟΗ ) 3
kyN CH a
2
Soluble GSH Microsomes NADPH,0 2
EtQ^ S(0) Ρ #
Eto' ^ O H "
Microsomes NADPH,0 2
+ unknowns Figure 8.
Metabolism of diazinon and diazoxon (62)
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
:
8.
WILKINSON
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Components
275
These c l e a r l y show that because of the requirement f o r NADPH the microsomal degradation of d i a z i n o n and diazoxon i s p r i m a r i l y o x i d a t i v e i n nature. In c o n t r a s t the s o l u b l e f r a c t i o n contains an e f f i c i e n t GSH-dependent enzyme capable of degrading both compounds. The r e s u l t s of these s t u d i e s combined with an a n a l y s i s of the metabolites found i n the i n v i t r o incubations i n d i c a t e d that d i a z i n o n could be e i t h e r o x i d a t i v e l y a c t i v a t e d ( d e s u l f u r a t i o n ) to diazoxon or could be degraded by microsomal o x i d a t i o n or s o l u b l e GSH-transferase to d i e t h y l phosphorothioic a c i d . Diazoxon was l i k e w i s e s u s c e p t i b l e to the two degradation pathways to y i e l d d i e t h y l phosphoric a c i d (Figure 8). Several groups of compounds are known to act as i n h i b i t o r s of the enzymes i n v o l v e d i n x e n o b i o t i c metabolism and some of these have value as i n s e c t i c i d e s y n e r g i s t s which enhance the i n s e c t i c i d a l potency of v a r i o u s m a t e r i a l microsomal o x i d a t i o n i n c l u d t i v e s , a r y l - 2 - p r o p y n y l e t h e r s , 1,2,3-benzothiadiazoles and a l a r g e number of s u b s t i t u t e d imidazoles (63). Many of the s o l u b l e hydrolases such as carboxylesterase and the p y r e t h r o i d metaboliz ing esterases are e f f e c t i v e l y blocked by phosphates such as t e t r a ethylpyrophosphate and t r i - o - c r e s y l phosphate (20,64) or carbamates such as 1-naphthyl N-propylcarbamate (65) and many GSHt r a n s f e r a s e s are blocked by compounds such as p-chloromercuribenzoate (21). L i k e the v a r i o u s c o f a c t o r s p r e v i o u s l y discussed such i n h i b i t o r s can be extremely u s e f u l i n p i n p o i n t i n g the enzymes involved i n c e r t a i n r e a c t i o n s . For example i n some cases a x e n o b i o t i c may be metabolized by more than one type of enzyme system i n the same s u b c e l l u l a r fraction. In t h i s case s p e c i f i c enzyme i n h i b i t o r s can be employed to d i s c r i m i n a t e between the two enzymes. To i l l u s t r a t e how t h i s technique can be a p p l i e d i n v i t r o we w i l l use the r e s u l t s from a mammalian study. I t has been known f o r some time that many of the modern p y r e t h r o i d s are metabolized i n mammals by both esterases and mixed-function oxidases i n l i v e r microsomes (66). Recent jLn v i t r o s t u d i e s have e s t a b l i s h e d s t r u c t u r e - b i o d e g r a d a b i l i t y r e l a t i o n s h i p s with a s e r i e s of 44 p y r e t h r o i d s and model compounds and have c l e a r l y shown the comparative r o l e of esterases and oxidases i n t h e i r metabolism. Each of the t e s t compounds was incubated with microsomes alone (esterase a c t i v i t y ) with microsomes + NADPH (esterase plus oxidase a c t i v i t y ) and with microsomes + NADPH + tetraethylpyrophosphate, an esterase i n h i b i t o r (oxidase a c t i v i t y ) . The r e s u l t s shown i n Table 8 f o r the isomers of phenothrin and cyanophenothrin t y p i f y the r e s u l t s obtained with a l l compounds t e s t e d . Thus the primary a l c o h o l e s t e r s of IR, t r a n s - s u b s t i t u t e d cyclopropane c a r b o x y l i c a c i d s (e.g. phenothrin) were most r a p i d l y metabolized by both esterase and oxidase a t t a c k , the corresponding IR, c i s isomers were degraded mainly by oxidase a c t i o n and were q u i t e r e s i s t a n t to esterase a t t a c k and the α-cyano compounds were r e s i s t a n t to the degradative a c t i o n of both types of enzymes.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC METABOLISM
276 TABLE 8
E f f e c t of s t r u c t u r e on i n v i t r o metabolism of 3-phenoxybenzyl chrysanthemum e s t e r s by mouse l i v e r microsomes Structure
(IR, trans
isomer)
Percent metabolism r a t e r e l a t i v e
Compound and
isomer
Phenothrin (R = IR,
Esterase
Oxidase
+
Oxidase calc.
H)
trans
IR, çis Cyanophenothrin (R = IR,
t r a n s , a-RS
IR,
c i s , a-RS
59 + 3
27 + 2
78
+7
86
<4
37 + 5
37 + 3
37
3+1
5+1
11+2
8
<3
8+1
12+1
8
CN)
a/ — Esterase + oxidase a c t i v i t y with IR, its t j i s 3.3 + 0.8 min. 1
Esterase found
to
trans resmethrin i s 100
and
2
Data from Soderlund and
Casida
(64)
In a d d i t i o n to showing how s p e c i f i c enzyme i n h i b i t o r s can be used i n v i t r o to more c l e a r l y i d e n t i f y the metabolic r o l e of other enzymes i n the same system, the r e s u l t s of t h i s study i l l u s t r a t e s another important a p p l i c a t i o n of i n v i t r o s t u d i e s , i . e . to establ i s h r e l a t i o n s h i p s between chemical s t r u c t u r e and b i o d e g r a d a b i l i t y i n l a r g e s e r i e s of s t r u c t u r a l l y r e l a t e d compounds. This type of information i s of considerable importance i n the development of new drugs, i n s e c t i c i d e s and other b i o l o g i c a l l y a c t i v e compounds. Other examples of the use of In v i t r o systems i n s t r u c t u r e a c t i v i t y s t u d i e s are those employed i n the search f o r new and p o t e n t i a l l y more a c t i v e i n s e c t i c i d e s y n e r g i s t s . I t i s now w e l l e s t a b l i s h e d that most known s y n e r g i s t s act i n v i v o by v i r t u e of t h e i r a b i l i t y to block microsomal o x i d a t i o n (63). Consequently measurement of t h e i r a b i l i t y to i n h i b i t microsomal mixed-function o x i d a t i o n i n v i t r o provides a r a p i d and e f f e c t i v e way of a s s e s s i n g t h e i r s y n e r g i s t i c p o t e n t i a l i n v i v o and of e s t a b l i s h i n g the
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
8.
WILKINSON
Insect Subcellular
Components
277
s t r u c t u r a l requirements or physicochemical parameters on which t h e i r a c t i v i t y depends. Regression a n a l y s i s of data on the potencies of twenty 5-, 6- and 5,6-substituted 1,2,3-benzothiadiaz o l e s as i n h i b i t o r s of epoxidation i n armyworm midgut preparations revealed that i n h i b i t i o n could be s a t i s f a c t o r i l y d e s c r i b e d by the f o l l o w i n g equation i n terms of hydrophobic (π** and IT) and e l e c t r o n i c (σ) parameters (67). S i m i l a r s t r u c t u r e - a c t i v i t y s t u d i e s have been conducted with s e l e c t e d s e r i e s of s u b s t i t u t e d imidazoles. pI
5 Q
= -0.2491T
[negative l o g . I Q ( M ) ] 5
2
+ 0.761π - 0.457σ + 3.834 η = 20;
r = 0.942; s =
0.212
In the same way tha f u l q u a l i t a t i v e and q u a n t i t a t i v biologica a c t i v i t y or ease of degradation of a s e r i e s of chemicals, a s i n g l e model compound (or type of r e a c t i o n ) can be employed as an i n d i c a t o r f o r comparing enzymatic a c t i v i t y i n a s e r i e s of d i f f e r ent s t r a i n s or species of organisms. Comparative s t u d i e s of t h i s type where the emphasis i s on e s t a b l i s h i n g the biochemical s i m i l a r i t i e s or d i f f e r e n c e s between d i f f e r e n t s p e c i e s or s t r a i n s of r e s i s t a n t i n s e c t s are extremely important i n the design of compounds which e x h i b i t some degree of s e l e c t i v i t y i n t h e i r biological activity. Since the comparative aspects of t h i s subject w i l l be covered by Dr. T e r r i e r e i n the next p r e s e n t a t i o n i t w i l l not be given f u r t h e r c o n s i d e r a t i o n here. R e l a t i o n s h i p between i n v i t r o and i n v i v o s t u d i e s In the process of conducting In v i t r o s t u d i e s with s u b c e l l u l a r f r a c t i o n s we have e s s e n t i a l l y dismantled the i n s e c t i n an attempt to s i m p l i f y the system and understand more c l e a r l y the p r o p e r t i e s and f u n c t i o n s of some of i t s component p a r t s . The study of an i s o l a t e d enzyme i n v i t r o i s c l e a r l y a h i g h l y unphysiol o g i c a l s i t u a t i o n s i n c e we have t o t a l l y destroyed the morphologi c a l and f u n c t i o n a l i n t e g r i t y of the system and the complex and d e l i c a t e i n t e r r e l a t i o n s h i p s on which the i n t a c t animal depends. In i n t e r p r e t i n g the r e s u l t s of such s t u d i e s , we must be aware of the p o s s i b i l i t y of a r t i f a c t s . Nonetheless one of the requirements of any i n v i t r o study i s that i t should have some relevance to the i n t a c t animal and an attempt should always be made to evaluate this. Although i t i s q u i t e c l e a r that the i n v i v o t o x i c i t y of many compounds to i n s e c t s i s o f t e n r e l a t e d d i r e c t l y to metabolism, c o r r e l a t i o n s between the r e s u l t s of i n v i t r o metabolic s t u d i e s and i n v i v o t o x i c i t y are r a t h e r few and f a r between and to date are only of a q u a l i t a t i v e nature. We are s t i l l a long way from being able to approximate i n q u a n t i t a t i v e terms the i n v i v o metabolic c a p a b i l i t y of an i n t a c t organism from the sum of the i n d i v i d u a l
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC METABOLISM
278
metabolic c a p a c i t i e s of i t s i n d i v i d u a l organs. Indeed i n view of the l a r g e number of f a c t o r s which have to be taken i n t o c o n s i d e r a t i o n ( p e n e t r a t i o n b a r r i e r s , b i n d i n g to i n e r t t i s s u e s , etc.) i n the l i v i n g animal, i t i s u n l i k e l y that t h i s w i l l be accomplished i n the near f u t u r e . The best q u a l i t a t i v e i n v i t r o / i n v i v o c o r r e l a t i o n s can be made i n those cases where r e l a t i v e l y l a r g e changes i n metabolic a c t i v i t y occur w i t h i n a s i n g l e s p e c i e s . This s i t u a t i o n e x i s t s i n the dramatic age-related v a r i a t i o n s i n microsomal oxidase a c t i v i t y which occur i n s e v e r a l i n s e c t species and a l s o where microsomal oxidase a c t i v i t y can be enhanced by treatment of the i n s e c t s with various inducing agents. In the house c r i c k e t (Acheta domesticus) microsomal enzyme a c t i v i t y i s l o c a t e d p r i m a r i l y i n the Malpighian tubules; i t increases d r a m a t i c a l l y durin l i f e and t h e r e a f t e r decrease i n v i v o importance of the Malpighian tubules i n i n s e c t i c i d e d e t o x i c a t i o n i s s t r o n g l y suggested by the f a c t that these changes i n the t o x i c i t y of c a r b a r y l and i t s synergism by p i p e r o n y l butoxide to c r i c k e t s of d i f f e r e n t age and sex (Figure 9) (68). A good c o r r e l a t i o n has a l s o been observed i n the t o x i c i t y of c a r b a r y l to c o n t r o l and induced armyworm larvae (Table 9) (49). Thus armyworms with microsomal a c t i v i t y i n the gut t i s s u e s enhanced 3 - f o l d as a r e s u l t of a 3-day d i e t a r y exposure to pentamethylbenzene (2,000 ppm) showed a remarkable 11-fold increase i n t o l e r a n c e to o r a l l y administered c a r b a r y l ; a good c o r r e l a t i o n was a l s o observed with the l e s s e f f e c t i v e inducing agent hexamethylbenzene. TABLE 9 E f f e c t of i n d u c t i o n on i n v i t r o microsomal oxidase a c t i v i t y and v i v o t o l e r a n c e of armyworm l a r v a e to o r a l l y administered
LD Percent c o n t r o l Inducer treatment Control
in
carbaryl
5 Q
activity Carbaryl
Epoxidase
Cytochrome P-450
(yg/g)
100
100
30
225
219
67
314
299
350
He xame thylbenzene (2,000 ppm
i n diet)
Pentamethylbenzene (2,000 ppm
i n diet)
Data from B r a t t s t e n and Wilkinson
(49)
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
8.
WILKINSON
Insect Subcelluhr
i
Components
I
I
I
1
1
3 Age
of
Adult
279
1
I
5 (weeks)
J o u r n a l o f Economic Entomology
Figure 9. Retotionship between epoxidation activity in Malpighian tubules, susceptibility to carbaryl, and degree of synergism of carbaryl by piperonyl butoxide in adult crickets (A. domesticus) of different age and sex: ( ), females; ( ), males (68).
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC
280
METABOLISM
Figure 10. Activity of 1-alkylimidazoles in insects in vitro and in vivo: (Φ ·), pl aldrin epoxidase activity in armyworm gut preparation; ( Ο — Ο ), synergistic ratio with carbaryl to house flies (69). 50
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
8.
WILKINSON
Insect Subcellular Components
281
F i n a l l y , the i n v i t r o potency of many inhibitors of microsomal oxidation i n insects i s often reflected i n their a b i l i t y to synergize i n s e c t i c i d a l a c t i v i t y i n vivo. This i s evident from Figure 10 which shows that the a c t i v i t y of a series of 1-alkylimidazoles as inhibitors of microsomal a l d r i n epoxidation i n armyworm midgut preparations closely p a r a l l e l s their i n vivo a c t i v i t y as synergists for .carbaryl i n house f l i e s . Conclusion Although numerous problems are encountered i n i n v i t r o studies with insect subcellular fractions, i t i s apparent that such studies can and w i l l provide a great deal of valuable i n f o r mation concerning the metabolism of xenobiotics Substantial progress has already bee logy for the preparatio insects and i n establishing the biochemical characteristics of the many of the enzymes concerned. Indeed, within the constraints imposed by the limited a v a i l a b i l i t y of insect tissues there i s no reason to suppose that insect subcellular fractions and purified enzyme components cannot be used i n exactly similar ways to those from mammalian tissues. Abstract In v i t r o studies employing subcellular components from whole insects or insect organs can provide valuable information on the structure of primary metabolites l i k e l y to be encountered i n i n vivo studies and the use of specific cofactors or inhibitors can prove useful i n pinpointing reaction mechanisms and indicating qualitative differences i n metabolite patterns between species or strains. There are, however, several problems which require special attention i n conducting i n v i t r o studies with insect subcellular components and little can be assumed i n moving from one species to another. P a r t i c u l a r l y i n the case of microsomal oxidation, the presence of a variety of endogenous inhibitors i n homogenates from whole insects can have serious effects on enzyme a c t i v i t y and the s t a b i l i t y of the preparation and the patterns of organ l o c a l i z a t i o n change i n different species. Even i n preparations derived from specific organs, optimal preparatory procedures must be established and anomalous centrifugal sedimentation characteristics of subcellular fractions are often observed. Furthermore, i n v i t r o enzyme a c t i v i t y i n subcellular components changes dramatically with physiological factors such as age and stage of development and i s highly susceptible to the presence of inducing agents i n the diet. Data so far obtained emphasize the basic s i m i l a r i t i e s which exist i n the enzymatic composition and metabolic function of insect and mammalian subcellular components.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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XENOBIOTIC METABOLISM
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49.
Brattsten, L. B. and Wilkinson, C. F. Pestic. Biochem. Physiol. (1973), 3, 393. 50. Schonbrod, R. D. and Terriere, L. C. J. Econ. Entomol. (1966), 59, 1411. 51. Morello, Α., Bleecker, W. and Agosin, M. Biochem. J. (1971), 124, 199. 52. Philpot, R. M. and Hodgson, E. Chem.-Biol. Inter. (1972), 4, 399. 53. DeDuve, C. J. Theoret. Biol. (1964), 6, 33. 54. Crankshaw, D. L . , Hetnarski, K. and Wilkinson, C. F. J. Insect Biochem. (1978). In press. 55. Cassidy, J. D., Smith, E. and Hodgson, E. J. Insect Physiol. (1969), 15, 1573. 56. Nakatsugawa, T. and Dahm P A J Econ Entomol (1965) 58, 500. 57. Brindley, W. A. an , (1970), 63, 31. 58. Price, G. M. and Kuhr, R. J. Biochem. J. (1971), 112, 133. 59. Krieger, R. I. and Wilkinson, C. F. J. Econ. Entomol. (1970), 63, 1343. 60. Lewis, S. Ε., Wilkinson, C. F. and Ray, J. W. Biochem. Pharmac. (1967), 16, 1195. 61. Krieger, R. I. (1970). Ph.D. dissertation, Cornell University, Ithaca, N.Y. 62. Yang, R. S. Η., Hodgson, E. and Dauterman, W. C. J. Agr. Food Chem. (1971), 19, 14. 63. Wilkinson, C. F. in "Insecticides for the Future: Needs and Prospects" (eds. Metcalf, R. L. and McKelvey, J. J., Jr.) p. 195. John Wiley and Sons, Inc., N.Y., 1976. 64. Soderlund, D. M. and Casida, J. E. Pestic. Biochem. Physiol. (1977), 7, 391. 65. Jao, L. T. and Casida, J. E. Pestic. Biochem. Physiol. (1974), 4, 456. 66. Casida, J. Ε., Ueda, Κ., Gaughan, L. C., Jao, L. T. and Soderlund, D. M. Arch. Envir. Contam. Toxicol. (1975/76), 3, 491. 67. Gil, D. L. and Wilkinson, C. F. Pestic. Biochem. Physiol. (1977), 7, 183. 68. Benke, G. M. and Wilkinson, C. F. J. Econ. Entomol. (1971), 64, 1032. 69. Wilkinson, C. F., Hetnarski, Κ., Cantwe11, G. P. and DiCarlo, F. J. Biochem. Pharmac. (1974), 23, 2377. RECEIVED
December 20, 1978.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
9 The Use of In Vitro Techniques to Study the Comparative Metabolism of Xenobiotics L. C. TERRIERE Department of Entomology, Oregon State University, Corvallis, OR 97331
The u n d e r l y i n g m o t i v e of a l l b i o c h e m i c a l c o m p a r i s o n s , i n v i v o o r i n v i t r o , of d i f f e r e n t s p e c i e f plant d a n i m a l i th s e a r c h for differences be u s e f u l i n m a n y w a y s - - t h e i m p r o v e m e n t of d r u g s and p e s t i c i d e s , the p r o t e c t i o n of b e n e f i c i a l s p e c i e s and of man, the u n d e r standing of e v o l u t i o n , and the d e v e l o p m e n t of new p r i n c i p l e s . Indeed, s o m e r e f l e c t i o n about past a c c o m p l i s h m e n t s w i l l show that s p e c i e s c o m p a r i s o n s have l e d to s o m e of our m o s t i m p o r t a n t discoveries in biochemistry. G i l l e t t e (I) has d e s c r i b e d the m a n y w a y s i n w h i c h s p e c i e s m a y d i f f e r i n t h e i r m e t a b o l i c d i s p o s i t i o n of f o r e i g n c o m p o u n d s and why i t i s d i f f i c u l t to i n t e r p r e t s u c h d i f f e r e n c e s on the b a s i s of u r i n a r y e x c r e t i o n o r the p l a s m a l e v e l of t h e s e c o m p o u n d s or t h e i r m e t a b o l i t e s . Of the s e v e r a l m a j o r p a r a m e t e r s w h i c h c a n c a u s e s p e c i e s d i f f e r e n c e s , he c o n s i d e r s the quantity and q u a l i t y of the e n z y m e s and t h e i r i n t e r a c t i o n s w i t h endogenous and exogenous c o m p o u n d s to be the m o s t i m p o r t a n t . S i n c e the i n v i t r o m e t h o d i s w e l l s u i t e d to the study of the k i n e t i c s , s u b s t r a t e s p e c i f i c i t i e s , and m e t a b o l i t e s of s i n g l e as w e l l as m u l t i p l e e n z y m e s y s t e m s , i t w o u l d a p p e a r that a f u l l u n d e r s t a n d i n g of s p e c i e s d i f f e r e n c e s i n m e t a b o l i s m cannot be g a i n e d w i t h o u t t h i s tool. S o m e of the m o r e c o m m o n u s e s of the i n v i t r o m e t h o d to study s p e c i e s d i f f e r e n c e s i n c l u d e : 1) c o m p a r i s o n s of e n z y m e s and e n z y m e s y s t e m s , 2) c o r r e l a t i o n of e n z y m e a c t i v i t y w i t h t o x i c i t y o r o t h e r b i o l o g i c a l r e s p o n s e , 3) s t u d y of the s e l e c t i v i t y of d r u g s and p e s t i c i d e s , 4) e v o l u t i o n a r y c o m p a r i s o n s , i . e . a s s o c i a t i o n of d e t o x i c a t i o n a c t i v i t y w i t h h a b i t a t or feeding h a b i t s , 5) m o d e of a c t i o n s t u d i e s , 6) e n v i r o n m e n t a l s t u d i e s , i . e . the d e t o x i c a t i o n c a p a c i t y of n o n - t a r g e t s p e c i e s , and 7) d e v e l o p m e n t of the technique to r e p l a c e i n v i v o m e t h o d s . In s e a r c h of s u c h 0-8412-0486-l/79/47-097-285$09.50/0 © 1979 American Chemical Society
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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i n f o r m a t i o n b i r d s have b e e n c o m p a r e d w i t h m a m m a l s , i n s e c t s have b e e n c o m p a r e d w i t h f i s h and m a m m a l s , f i s h w i t h c r u s t a ceans, and r e p t i l e s w i t h a m p h i b i a n s , f i s h , b i r d s , m a m m a l s , and i n s e c t s , j u s t to m e n t i o n a few of the m o r e u n u s u a l c o m b i n a t i o n s . In v i t r o m e t h o d s o f f e r the s a m e advantages i n c o m p a r a t i v e b i o c h e m i s t r y a s i n other f i e l d s but, b e c a u s e of the i n h e r e n t d i f f e r e n c e s i n p h y s i o l o g y , m o r p h o l o g y , g e n e t i c s , and b e h a v i o r of s p e c i e s , t h e i r use i s m o r e u n c e r t a i n . T h e i n v e s t i g a t o r who u s e s i n v i t r o m e t h o d s i n t h i s w a y s h o u l d be a w a r e of the p o t e n t i a l f o r e r r o r i n the d e s i g n of e x p e r i m e n t s a n d i n the i n t e r p r e t a t i o n of r e s u l t s . T h e p u r p o s e of t h i s r e v i e w i s to c r i t i c a l l y e x a m i n e c u r r e n t p r a c t i c e s i n the u s e of t h e s e m e t h o d s i n s p e c i e s c o m p a r i sons, to s u g g e s t s o m e and to m e n t i o n p r o b l e m D u e to t h e i r i m p o r t a n c e i n the m e t a b o l i s m of d r u g s , p e s t i c i d e s , a n d o t h e r x e n o b i o t i c s b y plant a n d a n i m a l s p e c i e s , t h e e n z y m e s of the m i c r o s o m a l o x i d a s e s y s t e m h a v e b e e n the s u b j e c t of m o s t c o m p a r i s o n s i n v i t r o . T h i s h a s r e s u l t e d i n m o r e i n f o r m a t i o n about m e t h o d s a n d p r o b l e m s and i s the r e a s o n f o r t h e i r p r o m i n e n c e i n this r e v i e w . H o w e v e r , the i d e a s d i s c u s s e d h e r e s h o u l d a p p l y e q u a l l y w e l l to o t h e r m e t a b o l i c s y s t e m s . Historical Q u i n n et a l (2) a p p e a r to be the f i r s t to u s e the n e w l y d i s c o v e r e d m i c r o s o m a l oxidase a s s a y to explain species d i f f e r e n c e s i n r e s p o n s e t o a d r u g . T h e y o b t a i n e d a good c o r r e l a t i o n b e t w e e n h e x a b a r b i t a l s l e e p i n g t i m e a n d the i n v i t r o m e t a b o l i s m of t h i s d r u g i n the m o u s e , r a b b i t , r a t , and dog. S i m i l a r r e s u l t s w e r e r e p o r t e d b y B r o d i e e t a l . (3) who, i n a d d i t i o n , n o t e d a s e x d i f f e r e n c e i n h e x a b a r b i t a l m e t a b o l i s m i n r a t s a n d w e r e a b l e to e x p l a i n t h i s on the b a s i s of m i c r o s o m a l o x i d a s e a c t i v i t y . A n i n t e r e s t i n the e v o l u t i o n of d e t o x i c a t i o n m e c h a n i s m s l e d B r o d i e a n d M a i c k e l (4) t o c o m p a r e the m i c r o s o m a l m e t a b o l i s m of s e v e r a l d r u g s i n m a m m a l s , b i r d s , r e p t i l e s , a m p h i b i a n s , f i s h e s , a n d i n v e r t e b r a t e s ( c r a b , l o b s t e r , and c r i c k e t ) . A l t h o u g h s o m e of t h e i r c o n c l u s i o n s w e r e not s u p p o r t e d by l a t e r w o r k , they stimulated m u c h i n t e r e s t and many s i m i l a r c o m p a r i s o n s began to a p p e a r i n the l a t e 1960*s a n d h a v e c o n t i n u e d to t h i s t i m e . A p p a r e n t l y the f i r s t s p e c i e s c o m p a r i s o n , i n v i t r o , i n v o l v i n g a q u a t i c s p e c i e s , w a s that of P o t t e r and O ' B r i e n (5J who s t u d i e d the c o n v e r s i o n of the i n s e c t i c i d e , p a r a t h i o n , to .its m o r e t o x i c product, p a r a o x o n .
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
9.
TERRiERE
Comparative
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The i n v i t r o m e t h o d w a s u s e d to c o m p a r e the a z o and n i t r o r e d u c t a s e a c t i v i t y of the f i v e m a j o r c l a s s e s of v e r t e b r a t e s (6), s h o w i n g that the f i s h e s w e r e m u c h l e s s a c t i v e i n s u c h m e t a b o l i s m than the b i r d s and m a m m a l s . A n i m p o r t a n t c o n t r i b u t i o n of this w o r k w a s the d e m o n s t r a t i o n that the s p e c i e s v a r i e d i n t h e i r temperature optima f o r m a x i m u m enzyme activity, aquatic s p e c i e s r e q u i r i n g l o w e r t e m p e r a t u r e s (21-26 C) w h i l e the r e d u c t a s e s of the b i r d s w e r e m o r e a c t i v e at 40°C). I n the c a s e of the t u r t l e , n i t r o r e d u c t a s e a c t i v i t y w a s 20 t i m e s g r e a t e r a t 21°C t h a n at 37°C. E a r l y e v i d e n c e that the s u b s t r a t e s p e c i f i c i t y of the m i c r o s o m a l o x i d a s e s m i g h t v a r y w i t h the s p e c i e s w a s obtained b y C r e a v e n et a l . (7.) w h o b i p h e n y l by 11 s p e c i e s pound i n b o t h the 2 and 4 p o s i t i o n w h i l e o t h e r s f a v o r e d one but not the other of these p o s i t i o n s . T h i s w a s p r o b a b l y one of the e a r l i e r i n d i c a t i o n s that c y t o c h r o m e P-450 e x i s t s i n m o r e t h a n one f o r m . A n e a r l y a t t e m p t to use i n v i t r o t e c h n i q u e s to c o m p a r e the m e t a b o l i s m of x e n o b i o t i c s i n i n s e c t s w a s that of C h a k r a b o r t y and S m i t h (8) but t h e i r i n v i t r o t e s t s w e r e u n s u c c e s s f u l i n d e t e c t i n g the e x p e c t e d m e t a b o l i c a c t i v i t y . T h i s w a s p r o b a b l y due to the p r e s e n c e of n a t u r a l i n h i b i t o r s r e l e a s e d d u r i n g p r e p a r a t i o n of the h o m o g e n a t e s . A n o t h e r e a r l y use of the i n v i t r o m e t h o d i n the study of i n s e c t s p e c i e s w a s that of S c h o n b r o d et a l . (9). T h e m i c r o s o m a l h y d r o x y l a t i o n of naphthalene w a s s h o w n to c o r r e l a t e f a i r l y w e l l w i t h naphthalene t o x i c i t y to r e s i s t a n t and s u s c e p t i b l e house f l i e s a n d to b l o w f l i e s . The R e l i a b i l i t y of i n v i t r o M e t h o d s i n S p e c i e s C o m p a r i s o n s If i n v i t r o m e t h o d s a r e to be u s e d i n p r e d i c t i n g o r e x p l a i n i n g s p e c i e s d i f f e r e n c e s i n the m e t a b o l i s m of x e n o b i o t i c s , t h e i r r e l i a b i l i t y m u s t be e s t a b l i s h e d . T h i s i s done m o s t r e a d i l y b y c o m p a r i n g r e s u l t s f r o m a p p r o p r i a t e e x p e r i m e n t s i n v i t r o and i n v i v o o n the s a m e s p e c i e s . Not m a n y i n v e s t i g a t o r s do t h i s , howe v e r , s o i t i s n e c e s s a r y to use o t h e r c r i t e r i a s u c h as a g r e e m e n t b e t w e e n l a b o r a t o r i e s s t u d y i n g the s a m e s p e c i e s . In s u c h c a s e s a r e l i a b l e m e t h o d of e n z y m e a s s a y s h o u l d r e s u l t i n the s a m e r e l a t i v e a c t i v i t y f o r the s p e c i e s s t u d i e d . A n o t h e r t e s t of r e l i a b i l i t y i s w h e t h e r the p a t t e r n s a n d p r o d u c t s of m e t a b o l i s m r e v e a l e d b y i n v i t r o t e c h n i q u e s a g r e e w i t h those of the i n t a c t o r g a n i s m . T h i s i s , of c o u r s e , v e r y n e c e s s a r y i n the use of
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i n v i t r o m e t h o d s to r e l a t e o r e x t r a p o l a t e b e t w e e n m a n a n d experimental animals. A good e x a m p l e of s u c h c o m p a r i s o n s i s the r e p o r t of C h i p m a n et a l . (10) w h o s t u d i e d the i n v i v o and i n v i t r o m e t a b o l i s m of the d i e l d r i n a n a l o g , 1, 2, 3, 4, 9, 9 - h e x a c h l o r o - e x o - 5 , 6epoxy- 1, 4, 4a, 5, 6, 7, 8, 8 a - o c t a h y d r o - 1 , 4-methano-naphthalene ( H C E ) i n r a t s , r a b b i t s , pigeons, a n d J a p a n e s e q u a i l . T h e i r i n v i v o m e t h o d i n c l u d e d the u s e of a r e - e n t r y b i l e c a n n u l a w i t h p r o v i s i o n s f o r the r o u t i n e c o l l e c t i o n of s a m p l e s . T h e n a t u r e a n d q u a n t i t y of m e t a b o l i t e s c o l l e c t e d w i t h the b i l e , a s w e l l a s the u r i n e , w e r e c o m p a r e d w i t h t h o s e found a f t e r a 30 m i n u t e i n c u b a t i o n of H C E w i t h l i v e r m i c r o s o m e s (in v i t r o at 37°C f o r m a m m a l s , 42°C f o r b i r d s ) w h i c h w e r e l a r g e l y conjugate c o m p o u n d s p r i o r to m e a s u r e m e n t . A s s h o w n i n F i g u r e 1, H C E u n d e r g o e s t w o r o u t e s of metabol i s m , e p o x i d e h y d r a t i o n to the t r a n s - d i o l , a m i n o r p r o d u c t i n the s p e c i e s s t u d i e d , and h y d r o x y l a t i o n of the e p o x i d e to H H C , t h e major metabolites i n a l l four species. This is followed by e p i m e r i z a t i o n and f u r t h e r h y d r o x y l a t i o n to the d i h y d r o x y epoxide, DHHC. T h e r e s u l t s of C h i p m a n e t a l . a r e s u m m a r i z e d i n T a b l e I and F i g u r e 2. It w i l l be s e e n that the f a i l u r e of the pigeon to c o n v e r t the epoxide, H C E , to i t s t r a n s - d i o l w a s c o r r e c t l y p r e d i c t e d b y the m i c r o s o m a l s t u d i e s , T a b l e I. A l s o , the i n v i t r o e x p e r i m e n t s c o n f i r m e d the r e l a t i v e o r d e r of i m p o r t a n c e of the o x i d a t i v e m e t a b o l i t e s H H C , endo-HHC, a n d D H H C ( T a b l e I) i n the pigeon, q u a i l , a n d r a b b i t , a n d r e v e r s i n g o n l y the endo-HHC and D H H C i n the r a t . T h e two m e t h o d s d i s a g r e e o n the r e l a t i v e i m p o r t a n c e of e p o x i d e h y d r a t i o n i n the r a b b i t . F i g u r e 2 shows that t i m e i s a n i m p o r t a n t v a r i a b l e i n s u c h c o m p a r i s o n s . O n l y i n the s h o r t t e s t s (20 m i n u t e s i n v i t r o , 1 d a y i n v i v o ) do the two m e t h o d s a g r e e that H H C i s the m a j o r m e t a b o l i t e . A f t e r 2 days i n v i v o the o r d e r , m o s t to l e a s t , i s D H H C , HHC, a n d e n d o - H H C a n d a f t e r 80 m i n u t e s i n v i t r o it i s endo-HHC, D H H C , and H H C , a l t h o u g h the d i f f e r e n c e s h e r e a r e m u c h l e s s . T h i s d i s a g r e e m e n t i s p r o b a b l y due to the a v a i l a b i l i t y , i n v i v o , of a d d i t i o n a l e n z y m e s , the c o n j u g a s e s , w h i c h a l t e r the l e v e l s of H H C and endo-HHC. S u l l i v a n et a l . (11) have d e v e l o p e d a t i s s u e m a i n t e n a n c e technique f o r the c o m p a r a t i v e study of d r u g m e t a b o l i s m . T h e m e t h o d i n v o l v e s the i n c u b a t i o n of s m a l l p i e c e s (e.g. 2 m m cubes) of f r e s h t i s s u e f o r up to 18 h r s w i t h the r a d i o a c t i v e d r u g . The i n c u b a t i o n m e d i u m i s then e x t r a c t e d and the m e t a b o l i c
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
9. T E R R i E R E
Comparative
Xenobiotic
Metabolism
Biochemical Pharmacology Figure 1.
Metabolism of HCE (10)
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In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
g Liver represented/ incubation 1.5 0.8 1.0 1.0
α
19 4.6
14 4 5
3 0
4 0 0 8 0 13
X
Data from Chipman et a l
#
(10),
6
Others
I n d i v i d u a l metabolites as I° of t o t a l metabolites HCE endotrans HHC HHC DHHC diol Other 41 33 20 5 0 41 4 10 30 15 45 31 20 0 4 70 12 8 7 0
10 6.4
7 46 19
M e t a b o l i t e s , % of T o t a l HCE endotrans HHC DHHC diol
Only traces of r a d i o a c t i v i t y were found i n r a t urine and r a b b i t b i l e . A l l urine and b i l e collections were made during the f i r s t 2 hours f o l l o w i n g dosing (15 mg kg"' ^C-HCE). Urine samples were adjusted to 2 M HCI before i n c u b a t i n g f o r 30 min. a t 75*C t o break down conjugates. Microsomes incubated 30 min. a t 37* C o r 42 C ( b i r d s ) . F i v e m i l l i l i t e r s of incubation medium contained 40 μg HCE.
#
(b) i n v i t r o (microsomes) 7o Sub strate converted r a t (6) 60 r a b b i t (3) 75 pigeon (3) 55 Jap q u a i l (3) 25
0
68 89
i n urine* 87 95
r a b b i t (3) pigeon (4)
#
5
61 50 63
HHC
r a t (7) pigeon (4) Jap q u a i l (4)
Species
14 C, % extractable in b i l e * 89 91 57
(a) i n vivo
Table I, HCE Metabolites Found i n v i t r o and i n v i v o .
r
W Ο
>
m Η
Η
δ
ο
χ w
9.
TERRiERE
Comparative
Xenobiotic
291
Metabolism
In ¥i¥0 DHHC
Days after "C-HCE treatment
80h
Λ
· 60 ο ·> Ε
endo-HHC Έ "κ Ο ο
DHHC HHC 20
J
JL 20
40
60
L 80
Time of incubation (Minutes) Biochemical Pharmacology Figure 2. Pattern of HCE oxidative metabolism observed in the rat with respect to time. In vivo: metabolites found in bile with intermittent collections; in vitro: metabolism by hepatic microsomes reinforced with NADPH and 0 (\Q). 2
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
292
XENOBIOTIC METABOLISM
roducts r e s o l v e d by D E A E c e l l u l o s e chromatography. Using - c a r b a r y l , r i n g and N - m e t h y l l a b e l l e d , as a m o d e l , they w e r e a b l e to o b t a i n good a g r e e m e n t i n the n u m b e r and n a t u r e of m e t a b o l i t e s p r o d u c e d , w i t h those p r e v i o u s l y found i n i n v i v o s t u d i e s . The c o m p a r i s o n s w e r e m a d e w i t h l i v e r e x p i a n t s of r a t , dog, guinea pig, and man. T h e i r r e s u l t s a l s o have s o m e q u a n t i t a t i v e value, i n d i c a t i n g m a j o r and m i n o r m e t a b o l i t e s i n the f o u r s p e c i e s . S o m e t y p i c a l r e s u l t s f r o m t h e i r study a r e s h o w n i n F i g u r e s 3 and 4. In a f u r t h e r d e m o n s t r a t i o n of t h e i r method, C h i n et a l . (12) compared c a r b a r y l metabolism i n s e v e r a l human tissues. This study a l s o i l l u s t r a t e d another u s e f u l a s p e c t of the method, the a b i l i t y to c o m p a r e v a r i o u species with respect t It i s c l e a r f r o m the r e s u l t s of S u l l i v a n et a l . and of C h i n et a l . that the t i s s u e e x p i a n t s e x h i b i t m o s t , i f not a l l , of the p r i m a r y and s e c o n d a r y m e t a b o l i c p r o c e s s e s e x p e c t e d of the o r g a n i n v i v o . In a d d i t i o n to the o x i d a s e and c o n j u g a s e s y s t e m s o b s e r v e d i n the m e t a b o l i s m of c a r b a r y l , ( N - d e m e t h y l a t i o n , e p o x i d a t i o n , r i n g h y d r o x y l a t i o n , epoxide h y d r a t i o n , g l u c u r o n i d a tion, and s u l f a t i o n ) , the a u t h o r s c l a i m to have o b s e r v e d n i t r o r e d u c t i o n and s u l f o x i d a t i o n . A l l t h i s was a t t a i n e d w i t h o u t added co-factors. In s e e k i n g an e x p l a n a t i o n f o r the 1000-fold g r e a t e r o r a l t o x i c i t y of the i n s e c t i c i d e c h l o r f e n v i n p h o s to r a t s than to dogs, H u t s o n and H a t h w a y (13) found s e v e r a l d i f f e r e n c e s i n the two s p e c i e s . R a t b r a i n a c e t y l c h o l i n e s t e r a s e w a s about 10 t i m e s m o r e s e n s i t i v e to i n h i b i t i o n by c h l o r f e n v i n p h o s , r a t e r y t h r o c y t e s a b s o r b e d the t o x i c a n t m o r e r e a d i l y f r o m p l a s m a ( d i f f e r e n c e about 3-fold) and t h e r e was a g r e a t e r uptake of c h l o r f e n v i n p h o s by r a t b r a i n . T h e r e was about 1 5 - f o l d l e s s t o x i c a n t i n the b l o o d of the dogs, p r o b a b l y due to a c o m b i n a t i o n of a b s o r p t i o n and metabolic differences. The m e t a b o l i s m of c h l o r f e n v i n p h o s by l i v e r s l i c e s f r o m the r a t , m o u s e , r a b b i t , and dog was s t u d i e d i n a f u r t h e r i n v e s t i g a t i o n of s p e c i e s d i f f e r e n c e s i n the t o x i c i t y of t h i s i n s e c t i c i d e (14). The l i v e r s l i c e s w e r e i n c u b a t e d w i t h the -labelled compound f o r 1-2 h r s at 37°C. The m e t a b o l i s m was s h o w n to be due to a m i c r o s o m a l m o n o - o x y g e n a s e , r e s u l t i n g i n the e l i m i n a t i o n of one of the e t h y l g r o u p s . T h i s had a l r e a d y b e e n s h o w n to be the m a j o r i n v i v o r e a c t i o n i n both r a t s and dogs. I n i t i a l r e a c t i o n r a t e s f o r the d e - e t h y l a t i o n , c a l c u l a t e d f r o m the c u r v e s s h o w n i n F i g u r e 5, p l a c e the f o u r s p e c i e s i n the o r d e r 1, 8, 24, 88 (rat, m o u s e , r a b b i t , dog). I n c o m p a r i s o n , the acute, o r a l L D ^ Q 'S (mg/kg) of
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
9. T E R R i E R E
Comparative
Xenobiotic
Metabolism
293
Toxicology and Applied Pharmacology Figure 3. DEAE-cedlulose chromatogram of in vivo and in vitro rat metabolites of carbaryl-naphthyl- C. Gradient elution program: (I) 0.01M Tris · HCl buffer, pH 7.5 to 0.05M Tris · HCl buffer, pH 7.5; (II) 0.05M Tris · HCl buffer, pH 7.5 to 0.1M Tris · HCl buffer, pH 7.5; (III) 0.1 M Tris · HCl buffer, pH 7.5 to 0.5M Tris · HCl buffer, pH 7.5 (11). 14
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
294
XENOBIOTIC METABOLISM
4
8
12
16
20
24
28
32
36
40
44
Fraction Number * 10
1
π
+
+
m
Toxicology and Applied Pharmacology Figure 4. DEAE-cellulose chromât ο gram of in vivo and in vitro guinea pig metabolites of carbaryl-naphthyl- C. Gradient elation program as in Figure 3 (11). 14
T i m e (h) Biochemical Journal Figure 5.
Relative rates of dealkyhtion of chlorfenvinphos in liver slices: (Φ), rat; (A)> mouse; ( » , rabbit; (O), dog (14).
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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Xenobiotic
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c h l o f e n v i n p h o s i n these s p e c i e s i s 10 (rat), 100 (mouse), 500 (rabbit), a n d > 1200 (dog). T h u s the i n v i t r o e x p e r i m e n t s w o u l d have c o r r e c t l y p r e d i c t e d t h e o r d e r of acute t o x i c i t y i n the f o u r species. O n l y two i n v i t r o s t u d i e s c o u l d be found i n w h i c h the s a m e s p e c i e s a n d the s a m e d r u g s w e r e c o m p a r e d . T h e s e w e r e t h e r e p o r t s of C h h a b r a et a l . (15) w h o c o m p a r e d the m i c r o s o m a l o x i d a s e a c t i v i t i e s of l i v e r a n d i n t e s t i n e a g a i n s t f o u r c o m p o u n d s in f i v e s p e c i e s ( h a m s t e r , g u i n e a pig, r a t , m o u s e , r a b b i t ) a n d L i t t e r s t e t a l . (16) w h o u s e d s o m e of the s a m e d r u g s t o c o m p a r e m i c r o s o m a l m e t a b o l i s m i n lung, l i v e r , and k i d n e y of the s a m e s p e c i e s . T a b l e II shows that they found v e r y l i t t l e d i f f e r e n c e i n c y t o c h r o m e P-450 content h a v i n g h i g h e r P-450 l e v e l s t u d i e s a g r e e i n r a n k i n g t h e r a t as the l o w e s t of the f i v e s p e c i e s and i n the n a r r o w range, l e s s than 2 - f o l d , of c y t o c h r o m e P-450 concentration. T a b l e II.
A C o m p a r i s o n of H e p a t i c C y t o c h r o m e P-450 i n F i v e Species
L i t t e r s t et a i . (16) A A 490-450/mg Species protein /ml
C h h a b r a et a l . (15) n m o l e s /mg protein Species
rabbit hamster guinea p i g mouse rat
guinea pig hamster rabbit mouse rat
0. 177 0.140 0. 125 0. 108 0.098
+ Ο.Οβδ^ + 0.023 + 0.038 + 0.022 +0.025
1.45 Ι.26 1.1 1.1 0.84
b/ + 0. Ιό+ 0.07 +0.32 +0.07 + 0.07
- ± S. D. ~ ± S. E . T h e r e w a s r e a s o n a b l e a g r e e m e n t i n the r a n k i n g of the f i v e s p e c i e s a c c o r d i n g to t h e i r m i c r o s o m a l a n a l i n e h y d r o x y l a s e a c t i v i t i e s , T a b l e ΙΠ, the h a m s t e r a n d m o u s e b e i n g h i g h e s t i n b o t h s t u d i e s , a n d b o t h s h o w i n g the r a b b i t and g u i n e a pig a s the l o w e s t . T h e a c t i v i t i e s d i f f e r e d about 4 - f o l d i n the L i t t e r s t et a l . study. In the c a s e of b i p h e n y l h y d r o x y l a s e , T a b l e IV, the t w o s t u d i e s a g r e e o n the r a t a s l o w e s t i n l i v e r h y d r o x y l a s e a c t i v i t y but t h e r e i s l i t t l e a g r e e m e n t i n the r a n k i n g of the other s p e c i e s . The m e a n a c t i v i t i e s f o r the b i phenyl h y d r o x y l a s e v a r i e d about 3-fold i n both studies. F r o m this l i m i t e d r e v i e w of r e s u l t s f r o m two l a b o r a t o r i e s , it a p p e a r s that the technique i s r e a s o n a b l y r e l i a b l e i n the r a n k i n g
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC METABOLISM
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T a b l e III. A C o m p a r i s o n of H e p a t i c A n a l i n e H y d r o x y l a s e i n F i v e Species L i t t e r s t et a l . (16) Activity Species
n m o l e s /mg protein/min
hamster mouse rat g u i n e a pig rabbit
2.7 + 0 . 9 ^ 1.5+0.3 0.8 + 0. 0.8 + 0. 0.6 + 0.4
C h h a b r a et a l . (15) Activity Species
n m o l e s /mg protein/15 m i n
mouse hamster
24.1 + 1 . 9 ^ 19.1 + 1.3
rabbit
9.8 + 2.3
- ± S. D.
T a b l e I V . A C o m p a r i s o n of H e p a t i c B i p h e n y l H y d r o x y l a s e i n F i v e Species L i t t e r st e t a l . (16) Activity Species guinea p i g hamster mouse rabbit rat
- ± S. D.
n m o l e s /mg protein/min 4.1 3.4 2.8 1.7 1.6
+ ± ± ± ±
3. 11.3 0.6 0.5 0.4
C h h a b r a et a l . (15) Activity Species mouse guinea pig rabbit hamster rat
n m o l e s /mg protein/15 m i n 95.3 + 1 0 . 0 ^ 7 7 . 9 + 1.8 5 8 . 1 + 1.6 4 7 . 4 + 3.0 34.4± 1.4
^ ± S . E .
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
9.
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Xenobiotic
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297
of m i c r o s o m a l o x i d a s e a c t i v i t y i f the d i f f e r e n c e s b e t w e e n s p e c i e s a r e at l e a s t 2 - f o l d . In c o m p a r i n g the e n z y m e a c t i v i t i e s of T a b l e III and I V w i t h the c y t o c h r o m e P-450 c o n c e n t r a t i o n s s h o w n i n T a b l e II, i t w i l l be s e e n that t h e r e i s often l i t t l e c o r r e l a t i o n b e t w e e n the two p a r a m e t e r s e v e n though c y t o c h r o m e P-450 i s k n o w n to be the k e y e n z y m e i n these o x i d a s e s y s t e m s . T h i s h a s a l s o b e e n o b s e r v e d i n s t u d i e s of s i n g l e s p e c i e s . Indeed, i n v i e w of o u r p r e s e n t u n d e r s t a n d i n g of this i m p o r t a n t h e m o p r o t e i n , a d i r e c t r e l a t i o n s h i p o n the b a s i s of c y t o c h r o m e P-450 content a l o n e w o u l d b e s u r p r i s i n g s i n c e t h i s e n z y m e i s k n o w n to e x i s t i n s e v e r a l f o r m s with different substrate specificities. A c a r e f u l study o i n v o l v e d i n the a c t i v a t i o s p e c i e s f a i l e d to p r o v i d e a n e x p l a n a t i o n f o r the d i f f e r i n g t o x i c i t i e s of p a r a t h i o n (17). T h i s i n s p i t e of the f a c t that t h e r e w e r e l a r g e d i f f e r e n c e s , 2 0 - f o l d i n p a r a o x o n f o r m a t i o n and 8 - f o l d i n p a r a o x o n d e g r a d a t i o n , a m o n g the s p e c i e s s t u d i e d . T h e a u t h o r s c o n c l u d e d that o r g a n s o r t i s s u e s other than the l i v e r m u s t b e i n v o l v e d i n p r o t e c t i n g the a n i m a l a g a i n s t s u c h t o x i c a n t s . O f c o u r s e i t i s a l s o p o s s i b l e that the t a r g e t of the t o x i c a n t , the a c e t y l c h o l i n e s t e r a s e s y s t e m , a l s o v a r i e d i n s e n s i t i v i t y among the nine species. T h e a g r e e m e n t b e t w e e n i n v i t r o m e t a b o l i s m and t o x i c i t y i n f i v e m a m m a l i a n and t h r e e a v i a n s p e c i e s w a s a l s o u n s a t i s f a c t o r y i n a study of the i n s e c t i c i d e , d i a z i n o n (18). O n l y w i t h the sheep, w h i c h c a n t o l e r a t e 2-50 t i m e s m o r e d i a z i n o n t h a n pigs, g u i n e a pigs, cows, r a t s , t u r k e y s , c h i c k e n s , and d u c k s , w a s the t o x i c i t y relatively w e l l c o r r e l a t e d w i t h i n vitro m e t a b o l i s m . The authors c o n c l u d e d that e x t r a h e p a t i c m e t a b o l i s m w a s m o r e i m p o r t a n t than l i v e r m e t a b o l i s m . F a c t o r s A f f e c t i n g C o m p a r i s o n s - - S o u r c e s of E r r o r T h e r e a r e s e v e r a l b i o l o g i c a l , experimental, and even e n v i r o n m e n t a l f a c t o r s w h i c h c a n a f f e c t the p e r f o r m a n c e of i n v i t r o s y s t e m s . S o m e e x a m p l e s w h i c h a r e to b e found i n the c u r rent l i t e r a t u r e include: 1. S e x and age e f f e c t s : D e W a i d e (19) found that the v a r i a t i o n i n a m i n o p y r i n e N - d e m e t h y l a s e and a n i l i n e h y d r o x y l a s e a c t i v i t y b e t w e e n i n d i v i d u a l s i n four s p e c i e s of f i s h o b s c u r e d a n y p o s s i b l e s e x d i f f e r e n c e s i n the a c t i v i t i e s of t h e s e e n z y m e s . S e x d i f f e r e n c e s i n m i c r o s o m a l o x i d a s e a c t i v i t y a r e w e l l known, h o w e v e r . F o r e x a m p l e , the r a t and m o u s e e x h i b i t s e x
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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298
d i f f e r e n c e s i n k i n e t i c c o n s t a n t s ( K and V ) but the g u i n e a pig, r a b b i t , and m o n k e y do not (20). A n o t h e r e x a m p l e i s s e e n i n the r e p o r t of W h i t e h o u s e et a l . (17) who found a s e x d i f f e r e n c e i n the d e s u l f u r a t i o n of p a r a t h i o n i n two of nine s p e c i e s t e s t e d , guinea pig and r a t , and i n i t s h y d r o l y s i s i n o n l y one, the r a t . D i f f e r e n c e s i n the r a t e of e n z y m e d e v e l o p m e n t a r e w e l l k n o w n f r o m s t u d i e s of the c o m m o n l a b o r a t o r y a n i m a l s , h e n c e s i m i l a r c h a r a c t e r i s t i c s s h o u l d be e x p e c t e d i n other s p e c i e s . Some i n v e s t i g a t o r s f a i l to take t h i s into account i n c o m p a r i s o n s of i n v e r t e b r a t e s o r of w i l d v e r t e b r a t e s , h o w e v e r , p e r h a p s b e c a u s e they have no c h o i c e o r b e c a u s e they a r e u n a w a r e of the p o s s i b l e effects of age d i f f e r e n c e s . E x t r e m e v a r i a t i o n s i n m i c r o s o m a l oxidase activit stages of d e v e l o p m e n t a ( F i g u r e s 6, 7, 8) show. S i m i l a r o b s e r v a t i o n s have b e e n m a d e w i t h the e s t e r a s e s w h i c h m e t a b o l i z e j u v e n i l e h o r m o n e a n a l o g s (21, 22) and w i t h e p o x i d e h y d r a s e w h i c h m e t a b o l i z e s the j u v e n i l e h o r m o n e (23). A g e dependent f l u c t u a t i o n s i n m i c r o s o m a l o x i d a s e a c t i v i t y have a l s o b e e n s e e n i n the h o u s e c r i c k e t (24) and the c o c k r o a c h (25). S p e c i e s d i f f e r e n c e s i n the r a t e of d e v e l o p m e n t have b e e n o b s e r v e d w i t h the g l u c u r o n i d a t i o n s y s t e m (26). C h i c k e m b r y o s w e r e a l m o s t as a c t i v e i n the g l u c u r o n i d a t i o n of O - a m i n o p h e n o l at 12 d a y s as i n 6 w e e k - o l d c o c k r e l s w h e r e a s the l i v e r of the 16 d a y - o l d m o u s e fetus was n e g a t i v e and e v e n the 10-day i n f a n t l i v e r c o n t a i n e d o n l y 5 0 % of the a d u l t a c t i v i t y . 2. S o u r c e of e n z y m e s ; D e W a i d e (19) m e a s u r e d the a p p a r e n t V and K f o r the _p_-hydroxylation of a n i l i n e of e i g h t t i s sues, l i v e r , kidney, h e a r t , lung, gut, m u s c l e , blood, and s p l e e n i n pigeon, r a t , trout, r o a c h , and c r a b ( s u b s t i t u t i n g hepatopanc r e a s f o r l i v e r i n the l a t t e r ) . In the c r a b , t r o u t , and r o a c h , he a l s o i n c l u d e d the g i l l i n t h e s e m e a s u r e m e n t s . The l i v e r was the m o s t a c t i v e s o u r c e of e n z y m e i n the f o u r v e r t e b r a t e s , but the c r a b g i l l was c o n s i d e r a b l y m o r e a c t i v e t h a n i t s h e p a t o p a n c r e a s . The k i d n e y was n e a r l y as a c t i v e as the l i v e r i n the c a s e of the t r o u t and b o t h the k i d n e y and lung w e r e i m p o r t a n t s o u r c e s of the e n z y m e i n the pigeon. P o h l et a l . (27) a l s o found d i f f e r e n c e s i n the d i s t r i b u t i o n of e n z y m e a c t i v i t y a m o n g the o r g a n s of d i f f e r e n t s p e c i e s . In the l i t t l e s k a t e , f o r e x a m p l e , the l i v e r and k i d n e y had a p p r o x i m a t e l y the s a m e a c t i v i t y , per mg of m i c r o s o m a l p r o t e i n , w h i l e i n the w i n t e r f l o u n d e r the l i v e r w a s a p p r o x i m a t e l y 20 t i m e s as a c t i v e as the k i d n e y . m
m
a
x
m
a
x
m
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
9.
TERRiERE
Comparative
"Ό
Egg I
Xenobiotic
Larva
Metabolism
| Puparium |
299
Adult
"5
DAYS Figure 6.
Microsomal aldrin epoxidases activity in the developmental stages of Isohn-B and SR house flies
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
300
XENOBIOTIC METABOLISM
DEVELOPMENTAL STAGS (DAIS)
Pesticide Biochemistry and Physiology Figure 7.
O
Microsomal aldrin epoxidase activity in developmental stages of the blow fly (39)
U LARVA
1 8
1216 PUPARIUM DEVELOPMENTAL STAGE
2k
20 I
28
32
ADULT
(DAYS)
Pesticide Biochemistry and Physiology Figure 8.
Microsomal aldrin epoxidase activity in developmental stages of the fleshfly(39)
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
9.
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Comparative
Xenobiotic
Metabolism
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T h e r e i s c o n s i d e r a b l e u n c e r t a i n t y about the s i t e of m f o a c t i v i t y i n i n v e r t e b r a t e a n i m a l s . P o h l et a l . (27) w e r e u n a b l e to d e t e c t h y d r o x y l a s e o r d e a l k y l a s e a c t i v i t y i n the h e p a t o p a n c r e a s of the l o b s t e r o r c r a b w h i l e B u r n s (28) d i d f i n d s o m e d r u g m e t a b o l i z i n g a c t i v i t y i n t h i s t i s s u e i n the c r a b but found m o r e i n g i l l and i n c l a w m u s c l e t i s s u e . D e W a i d e (19) a l s o found m o r e a c t i v i t y i n the g i l l t i s s u e of the c r a b t h a n i n the h e p a t o p a n c r e a s . E l m a m l o u k and G e s s n e r (29) w e r e a l s o u n a b l e to d e t e c t h y d r o x y l a s e o r d e m e t h y l a s e a c t i v i t y i n the m i c r o s o m a l f r a c t i o n of the h e p a t o p a n c r e a s of the l o b s t e r but t h e y d i d f i n d s o m e a n i l i n e h y d r o x y l a s e and n i t r o r e d u c t a s e a c t i v i t y i n the s o l u b l e fraction. A n interesting resul activity i n various tissue ity of c l a w m u s c l e . In t e r m s of s p e c i f i c a c t i v i t y , the g i l l m i c r o s o m e s w e r e c o n s i d e r a b l y m o r e a c t i v e than t h o s e of hepatopanc r e a s o r c l a w m u s c l e but the t o t a l c a p a c i t y of the m u s c l e s y s t e m was h i g h e r than that of g i l l and h e p a t o p a n c r e a s c o m b i n e d , 3.6 n m o l e s per h o u r c o m p a r e d to 1.3 a n d 1.2 n m o l e s per h o u r f o r g i l l and h e p a t o p a n c r e a s . 3. C e l l f r a c t i o n ; I n c o m p a r i n g the u s u a l c e n t r i f u g a l f r a c tions of l i v e r h o m o g e n a t e s of the r a t and the t r o u t , D e W a i d e (19) found that t r o u t m i c r o s o m e s c o n t a i n e d o n l y 4 7 % of the N_d e m e t h y l a s e a c t i v i t y of the homogenate w h e r e a s the r a t l i v e r m i c r o s o m e s c o n t a i n e d 7 9 % of the a c t i v i t y . E v e n the p r o c e s s of c e n t r i f u g i n g the 9000 x G f r a c t i o n (containing m i c r o s o m e s a n d s o l u b l e f r a c t i o n ) a t 100, 000 x G f o r 1 hour, then r e m i x i n g w i t h a t i s s u e g r i n d e r , r e s u l t e d i n a 3 1 % l o s s of a c t i v i t y b y the t r o u t s y s t e m c o m p a r e d to a n e g l i g i b l e l o s s b y the r a t s y s t e m . T h e s e r e s u l t s l e d D e W a i d e to use the 9000 x G f r a c t i o n i n h i s s p e c i e s c o m p a r i s o n s . A s he points out, t h i s not o n l y r e d u c e s e r r o r s due to the d i f f e r e n t i a l l o s s of a c t i v i t y i n t e s t s i n v o l v i n g d i f f e r e n t s p e c i e s and t i s s u e s , but i t s i m p l i f i e s the p r o c e d u r e s b y s h o r t e n ing the t i m e i n v o l v e d and e l i m i n a t i n g o t h e r o p p o r t u n i t i e s f o r l o s s of a c t i v i t y . O t h e r o b s e r v a t i o n s s u p p o r t the use of the m o r e i n c l u s i v e i n t e r m e d i a t e f r a c t i o n of t i s s u e h o m o g e n a t e s (e.g. the 9000 x G f r a c t i o n , e t c . ) . B u h l e r and R a s m u s s e n (30) found that the n i t r o r e d u c t a s e a c t i v i t y of s e v e r a l s p e c i e s of f i s h was a l m o s t e n t i r e l y i n the s o l u b l e f r a c t i o n w h i l e i n m a m m a l i a n s p e c i e s it i s e q u a l l y d i s t r i b u t e d b e t w e e n the m i c r o s o m a l and s o l u b l e f r a c t i o n (31 ). O t h e r e x a m p l e s of t h i s k i n d a r e the r e p o r t by E l m a m l o u k and G e s s n e r (29) w h o o b s e r v e d that the a n i l i n e h y d r o x y l a s e a c t i v i t y
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
302
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of l o b s t e r h e p a t o p a n c r e a s h o m o g e n a t e s w a s i n the s o l u b l e f r a c t i o n w h i l e n i t r o r e d u c t a s e a c t i v i t y was i n b o t h m i c r o s o m a l and soluble fraction. E i g h t s p e c i e s w e r e s t u d i e d i n a d i r e c t c o m p a r i s o n of m i c r o s o m a l and w h o l e h o m o g e n a t e f r a c t i o n s of the l i v e r as the e n z y m e s o u r c e f o r the d e s u l f u r a t i o n of p a r a t h i o n (17). The two s y s t e m s a g r e e d i n the r e l a t i v e p o s i t i o n s of the f i r s t f i v e s p e c i e s but d i s a g r e e d i n the p o s i t i o n s of the l a s t t h r e e . The d i f f e r e n c e s a m o n g these t h r e e w e r e o n l y about 2 - f o l d , h o w e v e r . T h e i n s e c t e s t e r a s e s w h i c h h y d r o l y z e the j u v e n i l e h o r m o n e a n a l o g s a r e d i s t r i b u t e d b e t w e e n s o l u b l e f r a c t i o n (20%) and m i c r o s o m a l f r a c t i o n (40%) i n the h o u s e f l y (21) and f l e s h f l y (22) w h i l e the d i s t r i b u t i o n i n the c a s (60%) and m i c r o s o m a l f r a c t i o 4. E n z y m e s t a b i l i t y ; In c o m p a r i s o n s b e t w e e n the r a t and the r o a c h D e W a i d e (19) s u b j e c t e d the 9000 x G s u p e r n a t a n t f r a c t i o n to v a r i o u s t r e a t m e n t s p r i o r to i n c u b a t i o n w i t h the s u b s t r a t e . W h e n the e n z y m e s w e r e a l l o w e d to s t a n d f o r 5 h r s at r o o m t e m p e r a t u r e p r i o r to a s s a y the N - d e m e t h y l a t i n g s y s t e m of the r o a c h l o s t 4 2 % of i t s a c t i v i t y w h i l e that of the r a t l o s t 1 4 % . S i m i l a r t r e a t m e n t of the w h o l e h o m o g e n a t e (used i n the a s s a y of j D - n i t r o p h e n o l g l u c u r o n i d a s e ) r e s u l t e d i n a 6 2 % i n c r e a s e i n the a c t i v i t y of the r o a c h and a 1 6 % i n c r e a s e i n t h a t of the r a t . O t h e r t r e a t m e n t s ( s t o r a g e of the e n z y m e s at -15°C f o r 15 h r s , r e p e a t e d f r e e z i n g and t h a w i n g of the e n z y m e s , and u l t r a s o n i c a tion) a f f e c t e d the N - d e m e t h y l a s e and h y d r o x y l a s e s y s t e m s of the two s p e c i e s to a p p r o x i m a t e l y the s a m e extent. A d i f f e r e n c e i n e n z y m e s t a b i l i t y was a l s o o b s e r v e d i n a study of the e f f e c t of t e m p e r a t u r e on m i c r o s o m a l a l d r i n e p o x i d a s e i n e i g h t s t r a i n s of the house f l y (32). M i c r o s o m a l o x i d a s e s of the W o r l d H e a l t h O r g a n i z a t i o n S t a n d a r d R e f e r e n c e S t r a i n of f l i e s c o u l d be i n c u b a t e d at 47. 5°C f o r 5 m i n and s t i l l r e t a i n 8 1 % of t h e i r m a x i m u m e p o x i d a s e a c t i v i t y w h e r e a s s e v e n o t h e r s t r a i n s l o s t 6 2 - 9 0 % of t h e i r m a x i m u m a c t i v i t y a f t e r s u c h t r e a t m e n t . 5. T e m p e r a t u r e of i n c u b a t i o n ; The e r r o r w h i c h c a n o c c u r by the use of the w r o n g t e m p e r a t u r e i n i n c u b a t i o n s of m i c r o s o m a l o x i d a s e e n z y m e s c a n be a p p r e c i a t e d by a study of DeWaide's r e s u l t s i n w h i c h the t e m p e r a t u r e - a c t i v i t y c u r v e s w e r e d e t e r m i n e d f o r eight s p e c i e s , F i g u r e 9. In a l l c a s e s the r e g i o n of m a x i m u m a c t i v i t y w a s n a r r o w , about 5°C, d r o p p i n g off s h a r p l y at t e m p e r a t u r e s o u t s i d e t h i s r a n g e . D e W a i d e c o n c l u d e d , i n a g r e e m e n t w i t h e a r l i e r s t u d i e s b y A d a m s o n (6) and o t h e r s , that the l i v e r d e m e t h y l a s e of b i r d s (based on s t u d i e s w i t h the pigeon) s h o u l d be i n c u b a t e d at 42 C., t h o s e of m a m m a l s at 37 C and
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
9.
TERRiERE
Comparative
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activity
e
temperature ( C )
temparaturef
C)
J. H. DeWaide (thesis) Figure 9. Ν-demethylation of aminopyrine by 9000 g supernatants prepared from liver homogenates of various animal species. Activity was measured for 10 min at different temperatures. The values of each species were expressed in relation to the maximal value (numerically 100) (19).
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC
304
METABOLISM
t h o s e of f i s h at 25°C. T h e s e t e m p e r a t u r e s w o u l d not be o p t i m u m i n e v e r y c a s e , h o w e v e r , as was s h o w n by f u r t h e r c o m p a r i s o n of e n z y m e a c t i v i t i e s at t h e s e and at the s p e c i f i c t e m p e r a t u r e o p t i m a for e a c h s p e c i e s . W i t h s o m e s p e c i e s the N - d e m e t h y l a s e a c t i v i t y was as l i t t l e as 4 6 % that o b s e r v e d at the s p e c i f i c o p t i m u m . The d i f f e r e n c e s w e r e l e s s i n the J D - h y d r o x y l a t i o n of a n i l i n e w h e r e the o p e r a t i v e t e m p e r a t u r e r e s u l t e d i n a c t i v i t i e s a t l e a s t 6 4 % of t h o s e o b t a i n e d i n the o p t i m u m r a n g e . P r e s u m a b l y i t was not p r a c t i c a l to c o n d u c t the i n c u b a t i o n s f o r e a c h s p e c i e s a t its o p t i m u m l e v e l . A n o t h e r e x a m p l e of a t e m p e r a t u r e effect d u r i n g e n z y m e a s s a y i s m e n t i o n e d b y B e n d (33). T h e l i v e r O - d e a l k y l a s e s y s t e m of the l i t t l e s k a t e was m u c h l e s s a c t i v e at b o t h 41 C and 12 C t h a n a t 30 °C. Recent reports indicat s u c h s t u d i e s i s s t i l l open. P o h l et a l . (27) i n c u b a t e d m i c r o s o m e s p r e p a r e d f r o m the l i v e r s of m a r i n e s p e c i e s o r the h e p a t o p a n c r e a s of c r a b and l o b s t e r at 30 C., w h i l e B u r n s (28) u s e d t e m p e r a t u r e s of 14.5 C and 20 C f o r m i c r o s o m e s f r o m the f i d d l e r c r a b and E l m a m l o u k and G e s s n e r (29, 34) i n c u b a t e d l o b s t e r he pato panc r e a s m i c r o s o m e s at 20°C. None of t h e s e a u t h o r s p r o v i d e d a t a i n s u p p o r t of t h e i r u s e of t h e s e t e m p e r a t u r e s . D e W a i d e (19) a l s o found a t e m p e r a t u r e d i f f e r e n t i a l i n the s t a b i l i t y of the N - d e m e t h y l a s e s y s t e m of r a t and t r o u t , the t r o u t s y s t e m r e m a i n i n g l i n e a r f o r a l o n g e r p e r i o d (20-30 m i n ) at i n c u b a t i o n t e m p e r a t u r e s n e a r i t s o p t i m u m , than that of the r a t (5-10 m i n ) w h e n i n c u b a t e d at i t s o p t i m u m . 6. K i n e t i c s ; A s i d e f r o m its v a l u e i n c h a r a c t e r i z i n g e n z y m e s , the k i n e t i c c o n s t a n t K i s u s e f u l i n the d e t e r m i n a t i o n of the o p t i m u m l e v e l of s u b s t r a t e to be u s e d i n m e a s u r i n g e n z y m e activity. A s a general practice substrate concentrations should be at l e a s t t w i c e the K to a c h i e v e m a x i m u m a c t i v i t y of the enzyme being measured. F r e q u e n t l y investigators comparing a c t i v i t y of a n e n z y m e i n d i f f e r e n t s p e c i e s use the s a m e s u b s t r a t e c o n c e n t r a t i o n , a p p a r e n t l y a s s u m i n g that the K f o r the e n z y m e i s the s a m e i n a l l s p e c i e s o r that the s u b s t r a t e c o n c e n t r a t i o n c h o s e n w i l l e x c e e d e v e n the h i g h e s t K . DeWaide's study ( T a b l e V) shows that t h i s a s s u m p t i o n i s not a l w a y s j u s t i f i e d . The K v a l u e s f o r N - d e m e t h y l a s e v a r i e d 2 4 - f o l d a m o n g the 16 s p e c i e s i n v e s t i g a t e d ( f r o m 0.42 m M f o r the p i g e o n to 10 m M f o r the pike) and 2 0 0 - f o l d f o r the h y d r o x y l a s e s y s t e m (0.05 m M f o r the r a t to 10 m M f o r the w h i t e b r e a m ) . The f o r the g l u c u r o n i d a s e s y s t e m , c o m p a r e d i n 11 s p e c i e s , v a r i e d m u c h l e s s , f r o m 0.32 m M f o r the m o u s e and l i z a r d to 1. 3 m M f o r the e e l . m
m
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In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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— K - v a l u e s i n mM; m e a n s of 2 o r m o r e d e t e r m i n a t i o n s ; f o r a n i m a l s of w h i c h the a p p a r e n t K - v a l u e s a r e d e t e r m i n e d by 4-10 d e t e r m i n a t i o n s t h e ^ t a n d a r d e r r o r of .the m e a n i s g i v e n . — A s s a y e d w i t h 9000 g —/Range of a m i n o p y r i n e c o n c e n t r a t i o n used: 1.5-35 m M . s u p e r n a t a n t of g i l l ^ R a n g e of a n i l i n e c o n c e n t r a t i o n used: 0. 1-22 m M . homogenate. — R a n g e of p_-nitrophenol c o n c e n t r a t i o n used: 0. 14-1.4 m M . D a t a f r o m D e W a i d e (19)*
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XENOBIOTIC METABOLISM
306
K n o w l e d g e of a p p a r e n t K m and V x values is important i n a n o t h e r way, the i n t e r p r e t a t i o n of i n v i t r o d a t a i n t e r m s of the i n v i v o c o n d i t i o n s . T h i s was d i s c u s s e d by C a s t r o and G i l l e t t e (20) i n t h e i r s t u d i e s of the N - d e m e t h y l a t i o n of e t h y l m o r p h i n e b y m i c r o s o m e s of r a t , m o u s e , r a b b i t , g u i n e a pig, and m o n k e y . A s t h e y state, the d i f f e r e n c e s i n s u b s t r a t e c o n c e n t r a t i o n s b e t w e e n the i n v i t r o and i n v i v o s i t u a t i o n c o u l d o b s c u r e a d i f f e r e n c e i n e n z y m e a c t i v i t y i n one c a s e o r the o t h e r i f the K i s not t a k e n into a c c o u n t . 7. N a t u r a l i n h i b i t o r s of e n z y m e a c t i v i t y : S e v e r a l of the e a r l y f a i l u r e s to d e t e c t e n z y m e a c t i v i t y i n the t i s s u e s of i n s e c t s w e r e l a t e r found to be due to the p r e s e n c e of n a t u r a l i n h i b i t o r s w h i c h totally or p a r t i a l l s u r p r i s i n g s i n c e the s m a l of s p e c i f i c o r g a n s o r t i s s u e s . T h u s the h o m o g e n i z i n g of w h o l e i n s e c t s o r m a j o r body s e g m e n t s m i g h t be e x p e c t e d to i n t r o d u c e c a t a b o l i c e n z y m e s into the s u b c e l l u l a r f r a c t i o n b e i n g a s s a y e d . T h i s was the r e a s o n f o r low m f o a c t i v i t y i n l e p i d o p t e r o u s l a r v a e (35), the h o u s e c r i c k e t (36), and the honey bee (37). The i n h i b i t i o n of m f o a c t i v i t y i n m i c r o s o m e s p r e p a r e d f r o m w h o l e house f l i e s was t r a c e d to eye pigments (38) a n d was c o r r e c t e d by r e m o v i n g the heads b e f o r e h o m o g e n i z a t i o n . S u c h i n h i b i t o r s a r e a l s o p r e s e n t i n the f l e s h f l y and b l o w f l y (39). Two n a t u r a l i n h i b i t o r s of h o u s e f l y m i c r o s o m a l o x i d a s e s have b e e n c h a r a c t e r i z e d by J o r d a n and S m i t h (40), one of t h e s e p r o b a b l y the eye p i g m e n t i d e n t i f i e d by S c h o n b r o d and T e r r i e r e (38). A n o t h e r p o t e n t i a l s o u r c e of i n t e r f e r e n c e w i t h m i c r o s o m a l o x i d a s e a c t i v i t y i n s p e c i e s c o m p a r i s o n s i s the l i p i d p e r o x i d a s e s y s t e m . A 1 5 - f o l d d i f f e r e n c e i n the a c t i v i t y of t h i s s y s t e m b e t w e e n r a t l i v e r and r a b b i t l i v e r m i c r o s o m e s has b e e n r e p o r t e d (41). T h i s was s u f f i c i e n t to change the r e l a t i v e a c t i v i t y of the r a t d e m e t h y l a s e c o m p a r e d to that of the r a b b i t f r o m 2.2 t i m e s (rat m o r e than r a b b i t ) to 3.4 t i m e s w h e n the p e r o x i d a t i o n was p r e v e n t e d by the a d d i t i o n of E D T A . P o h l et a l . (27) c o u l d not d e t e c t m f o a c t i v i t y i n the hepatop a n c r e a s of l o b s t e r o r c r a b , a t t r i b u t i n g t h i s i n p a r t to the p r e s e n c e of d i g e s t i v e j u i c e s r e l e a s e d d u r i n g h o m o g e n i z a t i o n . T h e s e j u i c e s w e r e found to be i n h i b i t o r y of the h y d r o x y l a t i o n of a n i l i n e and the N_-demethylation of b e n z p h e t a m i n e . However, the i n h i b i t o r had o n l y a s l i g h t effect on the d e - e t h y l a t i o n of 7 - e t h o x y c ο uma r i η. T h i s o b s e r v a t i o n c o u l d e x p l a i n the f a i l u r e of other w o r k e r s (28, 29) to d e t e c t m i c r o s o m a l o x i d a s e a c t i v i t y i n the he pato p a n c r e a s of the c r a b and l o b s t e r . m
a
m
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
9.
TERRiERE
Comparative
Xenobiotic
Metabolism
307
8. E n v i r o n m e n t a l e f f e c t s : D u r i n g h i s study of the d r u g m e t a b o l i z i n g a c t i v i t y of l i v e r m i c r o s o m e s f r o m f i s h s p e c i e s o b t a i n e d f r o m the R h i n e R i v e r a n d i t s t r i b u t a r i e s , D e W a i d e (19, 42) o b s e r v e d a s e a s o n a l v a r i a t i o n i n e n z y m e a c t i v i t y . F u r t h e r i n v e s t i g a t i o n r e v e a l e d that the N- de m e thy l a s e a c t i v i t y of the r o a c h ( F i g u r e 10) a n d the r u d d ( T a b l e V I ) c o l l e c t e d d u r i n g the s u m m e r m o n t h s w a s m o r e than t w i c e that of s p e c i m e n s c o l l e c t e d i n the w i n t e r . T h e d i f f e r e n c e w a s not e x p l a i n e d on the b a s i s of l i v e r w e i g h t o r p r o t e i n content or o n d i f f e r e n c e s i n w a t e r t e m p e r a t u r e . It w a s c o n c l u d e d that the p r e s e n c e of e n z y m e i n d u c i n g c h e m i c a l s i n the f i s h e s e n v i r o n m e n t d u r i n g the s u m m e r m o n t h s was the m a i n c a u s e of t h e s e d i f f e r e n c e s i n e n z y m e a c t i v i t y . T h e r e i s now c o n s i d e r a b l species are induced b P C B ' s (43, 44, 45). I n c r e a s e s i n a r y l h y d r o c a r b o n h y d r o x y l a s e a c t i v i t i e s (benzo-(a )-pyrene as s u b s t r a t e ) up to 4 - f o l d have b e e n noted (46). H o w e v e r , not a l l s p e c i e s a r e a f f e c t e d nor a r e the e f f e c t s on the x e n o b i o t i c m e t a b o l i z i n g e n z y m e s the s a m e as noted by P a y n e (47). F o r e x a m p l e , the a r y l h y d r o c a r b o n h y d r o x y l a s e was i n d u c e d i n the t r o u t but N - d e m e t h y l a s e w a s not a f f e c t e d . N e i t h e r e n z y m e w a s i n d u c e d i n the c r a b a n d l o b s t e r . Y a w e t z (48) a l s o r a i s e d t h i s q u e s t i o n o n f i n d i n g b o t h D D E and P C B ' s i n the t i s s u e s of s i x s p e c i e s of b i r d s i n the i n v i t r o study of a l d r i n e p o x i d a t i o n . R e s i d u e c o n c e n t r a t i o n i n h e a r t t i s s u e ( l i v e r w a s not a n a l y z e d ) w e r e 0. 1-1.2 p p m f o r the t w o inducers. T h e r e m a y a l s o be a n e f f e c t o n e n z y m e a c t i v i t y b y the e n v i r o n m e n t a l t e m p e r a t u r e of the s p e c i e s under study. D e W a i d e (49) found that t r o u t and r o a c h h e l d a t 5 C f o r 2 w e e k s had l i v e r h y d r o x y l a s e and Ν - de m e thy las e a c t i v i t i e s about t w i c e those of f i s h h e l d at 18 C. In s i m i l a r e x p e r i m e n t s w i t h the h a m s t e r a n d the r a t ( t e m p e r a t u r e s 5 C a n d 23 C ) the d i f f e r e n c e s w e r e about 1. 5 - f o l d o r l e s s . 9* B a s i s of c o m p a r i s o n ; N e a r l y a l l i n v e s t i g a t o r s of x e n o b i o t i c m e t a b o l i s m r e p o r t m e t a b o l i c a c t i v i t y i n t e r m s of the p r o t e i n content of the f r a c t i o n b e i n g a s s a y e d . T h i s i s b a s e d o n the a s s u m p t i o n that the p r e p a r a t i v e m e t h o d s u s e d w i l l r e s u l t i n c o n stant and r e p r o d u c i b l e a m o u n t s of e n z y m e p r o t e i n . In c o m p a r a t i v e s t u d i e s a s e c o n d a s s u m p t i o n i s r e q u i r e d - - t h a t the t i s s u e s o r o r g a n s of the d i f f e r e n t s p e c i e s w i l l y i e l d the s a m e p r o p o r t i o n s of a c t i v e p r o t e i n i f the m e t h o d s u s e d a r e the s a m e . T h e r e a r e good r e a s o n s to doubt t h i s , h o w e v e r , c o n s i d e r i n g the p o s s i b i l i t i e s f o r variation in tissue constituents, especially non-specific protein and i n p h y s i c a l t e x t u r e (thus a l t e r i n g g r i n d i n g c o n d i t i o n s ) . T h e s e 1
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC
308
METABOLISM
Ν-demethylation of amwopyrinc (/Amok* formaldehyde produced /$ liver, h) 12
τ
I—ι—r—ι—r—ι—ι—ι—ι—ι—ι—ι—ι—ι July Jan. 196Ô 1969
ι
ι ι July
ι—ι—ι
ι
ι—ι—ι—ι—\—• Jan. 1970
t • July
•
J. H. DeWaide (thesis) Figure 10. Seasonal variation in hepatic drug-metabolizing capacity in the wild roach. Measurements were performed with 9000 g supernatants derived from liver homogenates. Curves are drawn through the median values of the samples (19).
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
9.
TERRiERE
Table VI.
Comparative
Xenobiotic
Metabolism
309
H e p a t i c d r a g o x i d a t i o n i n the r u d d at d i f f e r e n t t i m e s of the y e a r D a t e of a s s a y M a r c h 12, 1970 June 29, 1970 (n = 25) (n = 11)
N - d e m e t h y l a t i o n of amino pyrine activity per— g fresh liver mg l i v e r protein mg l i v e r D N A 100 g b o d y - w e i g h t
/
1.37 0.0119 0.63 2.40
+ + + +
0.52~ 0.0042 0.28 0.86
3.60 0.0379 1.87 10.8
+1.83 + 0.0197 + 0.89 + 6.3
0.41 0.0035 0. 19 0.72
+ + + +
0. 17 0.0015 0.09 0.28
0.87 0.0093 0.47 2.76
+ 0.45 + 0.0046 + 0.25 + 1.67
ρ - H y d r o x y l a t i o n of aniline activity per— g fresh liver mg liver protein mg l i v e r D N A 100 g b o d y - w e i g h t
μΐηοΐββ f o r m a l d e h y d e p r o d u c e d p e r h o u r . b M e a n s w i t h s t a n d a r d d e v i a t i o n . T h e c o r r e s p o n d i n g v a l u e s of the two dates a r e s i g n i f i c a n t l y d i f f e r e n t a t Ρ < 0 . 0 1 ( W i l c o x o n twosided two-sample test). c μΐϊΐοΐββ jD-aminophenol p r o d u c e d p e r h o u r . D a t a f r o m D e W a i d e (19)·
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
XENOBIOTIC METABOLISM
310
f a c t o r s c o u l d l e a d to d i f f e r e n c e s i n the a m o u n t s of s p e c i f i c and n o n - s p e c i f i c p r o t e i n i n the f r a c t i o n f i n a l l y a s s a y e d . I n a s m u c h as the m e t h o d s u s e d f o r p r o t e i n d e t e r m i n a t i o n do not d i s t i n g u i s h b e t w e e n a c t i v e and i n a c t i v e p r o t e i n , the c h a n c e s f o r e r r o r a r e obvious. T h e p o s s i b l e extent of the e r r o r c a n be s e e n i n data f r o m P o h l et a l . (27) who found a 6 - f o l d v a r i a t i o n i n the y i e l d of m i c r o s o m a l p r o t e i n p e r g r a m of l i v e r i n 12 v e r t e b r a t e s p e c i e s . A v a r i a t i o n i n m i c r o s o m a l p r o t e i n content i s r e p o r t e d i n a study of s i x s p e c i e s of w i l d b i r d s (7.0 + 1.3 to 12. 6 _+ 2. 5 mg p r o t e i n per g l i v e r ) (48) and f o r the r a t , r a b b i t , m o u s e , h a m s t e r , and g u i n e a pig ( 2 4 . 0 + 3 . 9 to 35.9 + 10.3 mg p r o t e i n per g l i v e r ) (16). D e W a i d e (19) c o m p a r e and g l u c u r o n i d a t i o n a c t i v i t b a s i s of l i v e r weight, l i v e r p r o t e i n , l i v e r DNA, and body w e i g h t . A s m i g h t be e x p e c t e d , the d r u g m e t a b o l i z i n g a c t i v i t i e s of the l i v e r of the v a r i o u s s p e c i e s r a n k e d d i f f e r e n t l y w h e n e x p r e s s e d i n d i f f e r e n t p a r a m e t e r s . In the N - d e m e t h y l a t i o n of a m i n o p y r i n e , the r a t (average of 30 a n i m a l s ) was 14 t i m e s m o r e a c t i v e than the t r o u t ( a v e r a g e of 30 a n i m a l s ) w h e n c o m p a r e d on the b a s i s of l i v e r weight, 10 t i m e s m o r e a c t i v e on the b a s i s of l i v e r p r o t e i n , 12 t i m e s m o r e a c t i v e on the b a s i s of l i v e r DNA, and 35 t i m e s m o r e a c t i v e on the b a s i s of b o d y w e i g h t . In the h y d r o x y l a t i o n of a n i l i n e , the m u l t i p l e s w e r e 4, 3, 3, and 9 f o r c o m p a r i s o n s on the b a s i s of l i v e r weight, l i v e r p r o t e i n , l i v e r DNA, and b o d y weight. T h i s p r o b l e m has b e e n d i s c u s s e d b y H a r p e r et a l . (50). T h e y p r e f e r to c o m p a r e a c t i v i t i e s b e t w e e n s p e c i e s on the b a s i s of V r a t h e r than s p e c i f i c a c t i v i t i e s and they a l s o show c o m p a r i s o n s on the b a s i s of c y t o c h r o m e P-450 content ( c a t a l y t i c constant, k ^ - ) . T h e b e n z e n e m e t a b o l i s m of l i v e r and lung e n z y m e s f r o m r a t , r a b b i t , and h a m s t e r i s r a n k e d a c c o r d i n g to t h e s e p a r a m e t e r s i n T a b l e VII, c o m p i l e d f r o m t h e i r d a t a . T h e r e i s no a g r e e m e n t b e t w e e n r e l a t i v e a c t i v i t i e s b a s e d o n c y t o c h r o m e P-450 content and those b a s e d on p r o t e i n content o r V . The r e a s o n f o r t h i s i s now u n d e r s t o o d as m e n t i o n e d e a r l i e r i n t h i s symposium. m
a
x
ca
m
a
x
Discussion T h r e e g e n e r a l c o n c l u s i o n s e m e r g e f r o m the f o r e g o i n g r e v i e w of the use of i n v i t r o m e t h o d o l o g y i n s p e c i e s c o m p a r i s o n s : 1) m o s t of the p r o b l e m s o c c u r i n s t u d i e s w i t h a q u a n t i t a t i v e r a t h e r than a q u a l i t a t i v e a s p e c t ; 2) the p r o b l e m s i n c r e a s e as the
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
9.
TERRiERE
Comparative
Xenobiotic
Metabolism
311
T a b l e V I I . M i c r o s o m a l B e n z e n e H y d r o x y l a s e A c t i v i t y of R a t , R a b b i t , and H a m s t e r C o m p a r e d i n T h r e e W a y s
Microsomes from
V
5/
B a s i s of C o m p a r i s o n Specific Activity-^
cat
r a t lung
1. 13 + 0 . 2 9
0.49 + 0 . 1 8
-
h a m s t e r lung
3.16 + 0.35
2.41 + 0 . 3 5
27
10.36 + 1.14
4.65+0.44
18
rat l i v e r
1.35 + 0 . 5 5
1.09 ± 0 . 2 7
1.8
hamster liver
9.29+4.03
4.26+0.84
2.2
rabbit liver
3.86 + 0.20
2.08 + 0 . 2 9
1.8
r a b b i t lung
— nmol/min/mg protein ^ n m o l / m i n / m g protein c/ — n m o l /nmol cytochrome P-450/min D a t a f r o m H a r p e r et a l . (50)
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
312
XENOBIOTIC METABOLISM
s y s t e m s b e c o m e m o r e h i g h l y defined; a n d 3) the p r o b l e m s a r e m o r e l i k e l y to o c c u r i n s t u d i e s of i n v e r t e b r a t e s and w i l d v e r t e b r a t e s . It i s a l s o a p p a r e n t that, i n s p i t e of the d i f f i c u l t i e s w h i c h have b e e n i d e n t i f i e d , the m e t h o d has p r o d u c e d m u c h u s e f u l k n o w l e d g e not a v a i l a b l e by o t h e r m e a n s , about s p e c i e s d i f f e r ences. M u c h of the w o r k w h i c h has b e e n done s o f a r i n t h i s a r e a , e s p e c i a l l y that i n v o l v i n g n o n - l a b o r a t o r y a n i m a l s , w o u l d have b e e n i m p r o v e d by m o r e a t t e n t i o n to the u s e of o p t i m u m c o n d i t i o n s . A g e n e r a l r e c o m m e n d a t i o n f o r the f u t u r e u s e of i n v i t r o m e t h o d s i n c o m p a r a t i v e s t u d i e s i s that none of the e x p e r i m e n t a l c o n d i t i o n s o r b i o l o g i c a l f a c t o r s be t a k e n f o r g r a n t e d . T h i s i n c l u d e s the pH a n d t e m p e r a t u r sub-cell fractions, substrat p r e p a r a t i o n , use, s t o r a g e , a n d e n z y m e s o u r c e , the p o s s i b i l i t y of endogenous i n h i b i t o r s , a n d the e f f e c t of a g e a n d s e x . S o m e s u g g e s t i o n s f o r d e a l i n g w i t h s o m e of these p r o b l e m s a r e as follows: 1. C h o i c e of i n v i t r o s y s t e m : T h e c h o i c e of i n v i t r o s y s t e m to b e u s e d i n the c o m p a r i s o n s w i l l depend on the o b j e c t i v e s of the e x p e r i m e n t . W h e n m e t a b o l i c a c t i v i t i e s a r e to be c o m p a r e d q u a n t i t a t i v e l y , the m o r e c o m p l e t e s y s t e m s s u c h a s t i s s u e e x p i a n t s and s l i c e s s h o u l d be u s e d . W i t h these t h e r e i s l e s s danger of l o s i n g i m p o r t a n t e n z y m e s a n d c o - f a c t o r s a n d the c e l l u l a r o r g a n i z a t i o n of the c o n s t i t u e n t s i s p r e s e r v e d . A new a p p r o a c h to s u c h s y s t e m s i s the i n t a c t o r w h o l e c e l l , u s u a l l y p r e p a r e d f r o m the l i v e r a n d thus known a s the h e p a t o c y t e (51). T h i s t e c h n i q u e a p p e a r s to o f f e r s e v e r a l advantages o v e r the l i v e r s l i c e o r e x p i a n t method, m a i n t a i n i n g the o r g a n i z a t i o n a l i n t e g r i t y of the o r i g i n a l t i s s u e w i t h o u t the a r t i f a c t s i n t r o d u c e d b y m e c h a n i c a l i n j u r y to the c e l l s and b y a b n o r m a l i t i e s i n the d i f f u s i o n of s u b s t r a t e o r oxygen. Some r e c e n t u s e s of this t e c h n i q u e a r e d e s c r i b e d below. It i s not p o s s i b l e to p r e p a r e s u c h f r a c t i o n s f r o m s m a l l a n i m a l s , e i t h e r b e c a u s e m e t h o d s a r e not known o r b e c a u s e of the s i z e of the t i s s u e o r o r g a n . In t h e s e c a s e s t h e r e s e e m s no c h o i c e but to u s e a h o m o g e n a t e of the e n t i r e a n i m a l o r a m a j o r body s e g m e n t f o l l o w e d by m i l d c e n t r i f u g a t i o n . A l t h o u g h p a r t i a l l y d i s o r g a n i z e d , this s y s t e m s h o u l d c o n t a i n a l l of the n a t u r a l c o n s t i t u e n t s r e q u i r e d f o r the r e a c t i o n s under study. H o w e v e r , the i n v e s t i g a t o r s s h o u l d c h e c k the r e q u i r e m e n t f o r s u p p l e m e n t a l c o - f a c t o r s s u c h as N A D P H . M o r e h i g h l y d e f i n e d f r a c t i o n s s u c h as m i c r o s o m e s o r m i c r o s o m a l s u p e r n a t a n t a n d s u b f r a c t i o n s of these a r e a p p r o p r i a t e
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w h e n the o b j e c t i v e of the e x p e r i m e n t s i s m o r e q u a l i t a t i v e i n n a t u r e , s u c h as the c o m p a r i s o n of e n z y m e s p e c i f i c i t i e s , s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s , e f f e c t of i n h i b i t o r s a n d a c t i v a t o r s , k i n e t i c s t u d i e s , etc* In t h e s e c a s e s the i n v e s t i g a t o r i s w i l l i n g to s a c r i f i c e o r g a n i z a t i o n and c o m p l e t e n e s s f o r b e t t e r c o n t r o l of e x p e r i m e n t a l c o n d i t i o n s . 2. T e m p e r a t u r e of i n c u b a t i o n ; T h e t e m p e r a t u r e f o r o p t i m u m m e t a b o l i c a c t i v i t y s h o u l d be d e t e r m i n e d f o r e a c h of the s p e c i e s b e i n g c o m p a r e d . W h e n t h i s i s not f e a s i b l e , i t i s proba b l y b e s t to i n c u b a t e e n z y m e s f r o m p o i k i l o t h e r m i c s p e c i e s at o r i n the t e m p e r a t u r e r a n g e of t h e i r n a t u r a l h a b i t a t . 3. E n z y m e k i n e t i c s : W h e n the c o m p a r i s o n s a r e b e i n g m a d e with specific sub-cellula soluble fraction, it is importan s u b s t r a t e b e i n g s t u d i e d i n o r d e r that s a t u r a t i n g c o n c e n t r a t i o n s c a n be u s e d i n e a c h i n c u b a t i o n . W h e n t i s s u e s l i c e s , t i s s u e e x p i a n t s , h e p a t o c y t e s , e t c . , a r e used, K determinations w i l l have l i t t l e v a l u e , s i n c e , p r e s u m a b l y , the s u b s t r a t e conc e n t r a t i o n w i t h i n the c e l l w i l l be d e t e r m i n e d b y p e r m e a b i l i t y a n d d i f f u s i o n p r o p e r t i e s c h a r a c t e r i s t i c of the t i s s u e s b e i n g s t u d i e d . In t h e s e c a s e s , i t i s i m p o r t a n t to m a i n t a i n the s u b s t r a t e l e v e l of the m e d i u m at s a t u r a t i n g c o n d i t i o n s . 4. A g e , sex, a n d s o u r c e of a n i m a l s ; W h e n e v e r p o s s i b l e i n v i t r o c o m p a r i s o n s of m e t a b o l i c a c t i v i t y s h o u l d b e m a d e o n l y a f t e r the r e l a t i o n s h i p b e t w e e n sex, age, a n d d e v e l o p m e n t a l stage and e n z y m e a c t i v i t y have b e e n d e t e r m i n e d . T h e e x p e r i m e n t s s h o u l d then be d e s i g n e d to a c c o m m o d a t e t h e s e v a r i a b l e s . I n the c a s e of s p e c i e s o b t a i n e d f r o m the f i e l d , a t t e n t i o n s h o u l d be g i v e n to e n v i r o n m e n t a l b a c k g r o u n d , e s p e c i a l l y to s u c h f a c t o r s as t e m p e r a t u r e r a n g e of the h a b i t a t and the p o s s i b i l i t y of i n d u c t i o n of e n z y m e s by e n v i r o n m e n t a l p o l l u t a n t s . 5. E n d o g e n o u s i n h i b i t o r s : T h e p r e s e n c e of i n h i b i t o r s w h i c h prevent o r reduce metabolic activity during i n v i t r o assays s h o u l d be a s s u m e d i n l i e u of e v i d e n c e t o the c o n t r a r y w h e n e v e r new s p e c i e s o r new o r g a n s a n d t i s s u e s a r e b e i n g e x a m i n e d . T h i s i s p a r t i c u l a r l y i m p o r t a n t i n s t u d i e s of s m a l l i n v e r t e b r a t e s w h e r e i t i s n e c e s s a r y to h o m o g e n i z e a l l o r p a r t of the a n i m a l i n o r d e r to p e r f o r m the a s s a y . O n e m e t h o d of d e t e c t i n g i n h i b i t i o n i s to i n c u b a t e the s u s p e c t e d f r a c t i o n w i t h one of k n o w n a c t i v i t y f o r e v i d e n c e of i n h i b i t i o n of the l a t t e r . 6. B a s i s of c o m p a r i s o n ; T h e c o m m o n p r a c t i c e of b a s i n g m e t a b o l i c a c t i v i t y o n the p r o t e i n content of the f r a c t i o n b e i n g a s s a y e d i s p r o b a b l y the b e s t w h i c h c a n be d e v i s e d at p r e s e n t . H o w e v e r , s u f f i c i e n t d a t a s h o u l d b e p r o v i d e d to enable o t h e r s to m
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m a k e t h e i r own e v a l u a t i o n of the a c t i v i t y of the t i s s u e s and o r g a n s c h o s e n f o r study. T h i s i n c l u d e s the y i e l d of p r o t e i n per u n i t w e i g h t of t i s s u e , the t o t a l w e i g h t of o r g a n , t i s s u e , o r a n i m a l , and the p r o t e i n m e t h o d u s e d . W h e n the s p e c i e s b e i n g c o m p a r e d a r e f r o m d i f f e r e n t t a x o n o m i c g r o u p s a t l e a s t one a d d i t i o n a l m e t h o d of e x p r e s s i n g a c t i v i t i e s s h o u l d be p r o v i d e d f o r better c r o s s - r e f e r e n c i n g between l a b o r a t o r i e s . W i t h v e r t e b r a t e s and l a r g e i n v e r t e b r a t e s , a c t i v i t i e s s h o u l d be r e l a t e d to the f r e s h w e i g h t of the t i s s u e s b e i n g s t u d i e d and f o r s m a l l i n v e r t e b r a t e s , to the body w e i g h t . It i s r e g r e t t a b l e that few i n v e s t i g a t o r s of the c o m p a r a t i v e b i o c h e m i s t r y of x e n o b i o t i c s use the s a m e s u b s t r a t e s i n t h e i r s t u d i e s . Often, of c o u r s e w o u l d not p e r m i t t h i s , bu " s t a n d a r d x e n o b i o t i c s c o u l d be u s e d . E x a m p l e s i n c l u d e e f f e c t of i n d u c e r s and i n h i b i t o r s , s e x and age dependency of e n z y m e a c t i v i t y , r e l a t i v e e n z y m e a c t i v i t y , and the o p t i m i z a t i o n of e x p e r i m e n t a l c o n d i t i o n s . T h e u s e of s u c h s t a n d a r d s u b s t r a t e s w o u l d g r e a t l y i m p r o v e c o m m u n i c a t i o n b e t w e e n l a b o r a t o r i e s and thus c o n t r i b u t e to the v a l u e of the i n v i t r o a p p r o a c h . 11
U s e f u l n e s s of the i n v i t r o m e t h o d i n s p e c i e s c o m p a r i s o n s . A l l of the m e r i t s of i n v i t r o m e t h o d s w h i c h have b e e n d i s c u s s e d i n the p r e v i o u s c h a p t e r s a p p l y as w e l l to t h e i r u s e i n species comparisons. These include improved control over e x p e r i m e n t a l c o n d i t i o n s , e l i m i n a t i o n of v a r i a b l e s , i s o l a t i o n and study of s p e c i f i c s y s t e m s , g r e a t e r p r e c i s i o n i n m e a s u r e m e n t s , m o r e f l e x i b i l i t y i n d e s i g n of e x p e r i m e n t s , and e c o n o m i e s of t i m e and l a b o r . I n a d d i t i o n , and i n s p i t e of the g r e a t e r s u s c e p t i b i l i t y to e x p e r i m e n t a l e r r o r m e n t i o n e d e a r l i e r i n t h i s c h a p t e r , the m e t h o d has s o m e s p e c i a l advantages i n s p e c i e s c o m p a r i s o n s . Some examples follow. 1. Studies of m e t a b o l i s m i n w i l d s p e c i e s . I n v i t r o m e t h o d s have a l r e a d y b e e n of v a l u e i n d r a w i n g a t t e n t i o n t o the l o w e r c a p a c i t y of f i s h , r e p t i l e s , a m p h i b i a n s , and o t h e r w i l d s p e c i e s f o r m e t a b o l i z i n g f o r e i g n c o m p o u n d s (3, 6_, 19j 27, 30, 33, 43). It i s not l i k e l y that this i n f o r m a t i o n c o u l d h a v e b e e n g a t h e r e d s o q u i c k l y and o n s u c h a l a r g e s c a l e b y any other m e a n s . T h e d i f f i c u l t i e s of c o l l e c t i n g o r r e a r i n g w i l d a n i m a l s p e c i e s i n s u f f i c i e n t n u m b e r s f o r i n v i v o s t u d i e s and of t r e a t i n g s u c h species w i t h c h e m i c a l s without introducing various s t r e s s e s , a l m o s t e l i m i n a t e s the u s e of s u c h m e t h o d s of e x p e r i m e n t a t i o n . In a d d i t i o n , w i t h a q u a t i c s p e c i e s , t h e r e m a y b e p r o b l e m s w i t h
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collecting excreted metabolites for identification. Furthermore, these m e t h o d s p e r m i t m o r e u s e of the s a m e a n i m a l s s i n c e s e v e r a l m e t a b o l i c s y s t e m s c o u l d be s t u d i e d w i t h the s a m e o r different organs. 2. M e t a b o l i s m of x e n o b i o t i c s b y h u m a n s ; T h e i n v i t r o m e t h o d has obvious advantages i n s t u d i e s of the m e t a b o l i s m of f o r e i g n c h e m i c a l s b y h u m a n s . W i t h o u t the d i r e c t e x p o s u r e of h u m a n s u b j e c t s , t h e s e m e t h o d s c o u l d p r o v i d e u r g e n t l y needed i n f o r m a t i o n about m e t a b o l i c pathways, i d e n t i t y of m e t a b o l i t e s , e f f e c t of i n h i b i t o r s , d e t e c t i o n of i n t e r a c t i o n s b e t w e e n c h e m i c a l s , l o c a t i o n of s i t e s of m e t a b o l i s m , and the d e t e c t i o n of age and s e x r e l a t i o n s h i p s . S u c h i n f o r m a t i o n c o u l d be o b t a i n e d o n l y w i t h d i f f i c u l t y by other m e a n s of l a b o r a t o r y a n i m a l s , e x p e r i m e n t s into d e c i s i o n s about h u m a n s a f e t y c o u l d b e done w i t h g r e a t e r p r e c i s i o n than at p r e s e n t . 3. U s e i n s c r e e n i n g p r o g r a m s : T h e a u t h o r i s u n a w a r e of the extent to w h i c h i n v i t r o m e t h o d s a r e b e i n g u s e d at p r e s e n t b y the d r u g and p e s t i c i d e i n d u s t r i e s . T h e t e c h n i q u e s s h o u l d be h e l p f u l i n b r i d g i n g gaps b e t w e e n s y n t h e s i s of new c o m p o u n d s a n d their testing for efficacy w i t h l a b o r a t o r y animals, insects, o r p l a n t s . It s h o u l d a l s o be u s e f u l i n g a t h e r i n g i n f o r m a t i o n o n e n v i r o n m e n t a l s a f e t y . In v i t r o s t u d i e s of m e t a b o l i s m b y the e x p e r i m e n t a l o r g a n i s m s m i g h t be h e l p f u l i n d e t e r m i n i n g the r e a s o n s f o r f a i l u r e s i n t o x i c i t y , i n d i c a t i n g w h e t h e r t h i s i s due to inadequate uptake, l a c k of t r a n s p o r t , o r too r a p i d m e t a b o l i s m . The m e t h o d i s a l s o u s e f u l i n d e t e c t i n g u n e x p e c t e d m e t a b o l i t e s or i n p r o d u c i n g m e t a b o l i t e s f r e e of i n t e r f e r i n g c o m p o u n d s . R e s e a r c h needs. The g r e a t e s t o b s t a c l e to the expanded use of i n v i t r o m e t h o d s is the l a c k of e v i d e n c e of t h e i r r e l i a b i l i t y i n e x p l a i n i n g and p r e d i c t i n g i n v i v o e v e n t s . T h i s o b s t a c l e c a n be r e m o v e d o n l y b y a d d i t i o n a l s t u d i e s i n w h i c h i n v i v o and i n v i t r o m e t h o d s a r e c o m p a r e d a s i n the e x p e r i m e n t s r e p o r t e d b y C h i p m a n et a l . (10) and by S u l l i v a n et a l . (11, 12). T h e g r e a t e r use of c o m m o n subs t r a t e s and t o x i c a n t s ( i . e . s t a n d a r d c h e m i c a l s ) b y d i f f e r e n t laboratories would help i n achieving this goal. M o r e w o r k i s needed o n e n z y m e s y s t e m s other than the m i c r o s o m a l o x i d a s e s w h i c h a r e i m p o r t a n t i n the m e t a b o l i s m of x e n o b i o t i c s . T h i s i n c l u d e s the c a r b o x y e s t e r a s e s w h i c h , along w i t h the m i c r o s o m a l o x i d a s e s , a c c o u n t f o r m o s t of the p r i m a r y m e t a b o l i s m of d r u g s and p e s t i c i d e s . M o r e i n f o r m a t i o n i s a l s o
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needed on the glutathione-S transferases, epoxide hydras e, and other enzymes which are important in the secondary (Type 2) metabolism of xenobiotics. With knowledge of the distribution and properties of these enzymes in various species, the metabolism of xenobiotics can be conducted on a broader scale and thus improve the predictive value of the method. Except for the common laboratory animals, there is a serious deficiency in our knowledge about the rate of development of xenobiotic metabolizing enzymes. It is quite likely that age and stage of development are important factors in these metabolic processes and, until the facts are known, it will be difficult to plan good experiments. Immediate attentio should b give t th f th intact cell technique in comparativ results using rat liver cells have begun to appear and a recent report describes a system using both liver and kidney cells to reconstruct the entire metabolism of a drug from its oxidation and conjugation with GSH to the production of the corresponding mercapturic acid (52). These cells have been shown to resemble the microsomal oxidase system in enzyme activity and substrate specificity. They also appear to contain the conjugation systems of the liver (53-58), and to demonstrate the effect of inducers (52, 57, 59)* Hepatocytes have also been prepared successfully from pig and human liver (60). Methods for the preparation of hepatocytes are described by Mouldes et al. (51). Abstract In addition to the usual doubts about the use of in vitro methods to replace or support those conducted in vivo, their use in comparative biochemistry encounters other uncertainties. These arise from the genetic, behavioral, morphological, and physiological differences among species. There is a need for evidence that, in spite of these special difficulties, in vitro methods can be reliable in detecting metabolic differences between species. The best method of establishing the reliability of data obtained in vitro is to compare the results with those from in vivo experiments performed on the same species. Another test of reliability is to determine how different laboratories rank the same species in terms of their relative enzyme activity. Up to the present time there are only a few reports which permit the use of either of these methods of evaluation. A critical examination of such data indicates that reliable comparisons of xenobiotic metabolism can be made in vitro providing a number of precautions are taken. In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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In vitro studies of species differences are of two types, those in search of quantitative relationships and those seeking qualitative information. There will be fewer problems in studies of the first type if the investigator uses the more complete in vitro systems such as the tissue explant or slice or, in the case of small animals, the low speed centrifugal fraction of the tissue homogenate. When the experiments are qualitative in nature, the more highly defined but less complete sub-cellular fractions are recommended. Special problems arise in the study of wild species. These include: lack of information regarding the relationship between enzyme activity and ag bolic activity; species difference enzyme activity; the presence of endogenous inhibitors of metabolic enzymes; and unpredictable environmental effects on metabolic activity. Another problem encountered in all comparative work but most acute in studies of wild species is the lack of a suitable basis of reference for comparing metabolic activity. The practice of comparing activities on the basis of protein content appears to be the best that can be devised at present, but its limitations should be understood. It is recommended that an additional system of reference be used in most species comparisons. Carefully planned in vitro experiments can be very useful in comparative studies, especially those involving wildlife species which are difficult to rear or manage. The methods should also be useful in screening programs for new drugs and pesticides and in studies of drug metabolism by humans. Research needed to expand the use of these methods includes studies of the use of hepatocytes and other intact cells as substitutes for tissue explants and slices and for sub-cell fractions. Additional information on the age dependency of metabolic enzymes and on conditions affecting in vitro assays for other enzymes such as the carboxyesterases, epoxide hydrase, and the conjugating enzyme systems is also needed.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
318
XENOBIOTIC METABOLISM
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Comparative Xenobiotic Metabolism
Terriere, L . C. and Yu, S. J., Pestic. Biochem. Physiol. (1977) 7, l6l. Yu, S. J. and Terriere, L. C., Pestic. Biochem. Physiol. (1975) 5, 418. Yu, S. J. and Terriere, L. C., Insect Biochem. (1978) ( in press ). Benke, G. M. and Wilkinson, C. F., J. Econ. Entomol. (1971) 64, 1032. Benke, G. M. and Wilkinson, C. F., J. Econ. Entomol. (1972) 65, 1221. Fyffe, J. and Dutton, G. J., Biochim. Biophys. Acta (1975) 411, 41. Pohl, R. J., Bend J. R., Drug Metab Disp (1974) Burns, Κ. Α., Marine Biol. (1976) 36, 5. Elmamlouk, T. H. and Gessner, T., Comp. Biochem. Physiol. (1976) 53C, 57. Buhler, D. R. and Rasmusson, Μ. Ε., Arch. Biochem. Biophys. (1968) 103, 582. Fouts, J. R. and Brodie, Β. Β., J. Pharmacol. Exp. Therap. (1957) 119, 197. Terriere, L . C., Schonbrod, R. D., and Yu, S. J., Bull. W.H.O. (1975) 52, 101. Bend, J. R., James, M. O., and Dansette, P. Μ., Ann. Ν. Y. Acad. Sci. (1977) 298, 505. Elmamlouk, T. H. and Gessner, T., Comp. Biochem. Physiol. (1976) 53C, 19. Krieger, R. I. and Wilkinson, C. F., Biochem. J. (1970) 116, 781. Brattsten, L . B. and Wilkinson, C. F., Comp. Biochem. Physiol. (1973) 45B, 59. Gilbert, M. D. and Wilkinson, C. F., Comp. Biochem. Physiol. (1975) 50B, 613. Schonbrod, R. D. and Terriere, L. C., Pestic. Biochem. Physiol. (1972) 1, 409. Terriere, L . C. and Yu, S. J., Pestic. Biochem. Physiol. (1976) 6, 223. Jordan, T. W. and Smith, J. Ν., Int. J. Biochem. (1970) 139. Kamataki, T. and Kitagawa, Η., Biochem. Pharmacol. (1973) 23, 1915. DeWaide, J. H. and Henderson, P. T., Comp. Biochem. Physiol. (1970) 32, 489.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
319
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XENOBIOTIC METABOLISM
Malins, D. C., Ann. Ν. Y. Acad. Sci. (1977) 298, 482. Gruger, E. H., Wekeli, M. M., Numoto, P. T., and Craddock, D. R., Bull. Environ. Contam. Toxicol. (1977) 17, 512. Yarbrough, J. D. and Chambers, J. E., Life Sci. (1977) 21, 1095. Payne, J. F. and Penrose, W. R., Bull. Environ. Contam. Toxicol. (1975) 14, 112. Payne, J. F., Marine Poll. Bull. (1977) 8, 112. Yawetz, Α., Agosin, M . , and Perry, A. S., Pestic. Biochem. Physiol. (1978) 8, 44. DeWaide, J. H., Comp. Gen. Pharmacol. (1970) 1, 375. Harper, C., Drew Metab. Disp. (1973 Moldeus, P., Hogberg, J., and Orrenius, S., Methods in Enzymology (1978) 52, 60. Moldeus, P., Jones, D. P., Ormstad, Κ., and Orrenius, S., Biochem. Biophys. Res. Comm. (1978) (in press). Inaba, T., Umeda, T., and Mahon, W. Α., Life Sci. (1975) 16, 1227. Vadi, Η., Moldeus, P., Capdevila, J., and Orrenius, S., Cancer Res. (1975) 2083. Grundin, R., Moldeus, P., Vadi, Η., and Orrenius, S., in "Cytochromes P-450 and b " (Cooper, D. Υ., Rosenthal, O., Snyder, R., and Witmer, C., Eds.) Plenum Pub. Corp., New York, N.Y., 1974. Erickson, R. R., and Holtzman, J. L . , Biochem. Pharmacol. (1976) 25, 1501. Wiebkin, P., Ery, J. R., Jones, C. Α., Lowing, R., and Bridges, J. W., Xenobiotica (1976) 6, 725. Burke, M. D., Vadi, Η., Jernstrom, Β., and Orrenius, S., J. Biol. Chem. (1977) 252, 6424. Moldeus, P., Grundin, R., von Bahr, C., and Orrenius, S., Biochem. Biophys. Res. Comm. (1973) 55, 937. Belfrage, P., Borjesson, Β., Hagerstrand, I., Nilsson, Α., Olsson, Α., Wiebe, T., and Akesson, Β., Life Sci. (1975) 17, 1219. 5
56. 57. 58. 59. 60.
RECEIVED
December 20, 1978.
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
INDEX
Absorbance, Type I Absorption of C herbicides in excised leaves root and leaf C Acetyl-JV-acetyltransferase Acetyl-coenzyme A 2-Acetylaminofluorene Acetylation α-1-Acetylmethadol N-Acetyltransferase 14
14
Acheta domesticus
156 4,11/ 8t,9i 9-17 7 203 203 20 203-20 142 203,210
256, 259, 260/,
263/, 267, 278, Activity-structure studies Acyl anilide S-Adenosyl-L- [ methyl- H ] methionine S-Adenosylmethionine Aflatoxin Bi Aglycons 3
Agrostemma githago
165 142
Apis mellifera
251,254
Apoplastic transport Apple
5 41*, 99
Arachis hypogaea
7
Arbutin Arene oxides
116 142 208
Aryl acylamidases Aryl hydroxylation Aryl-2-propynyl ethers Atrazine
279/ 276 Avena fatua 80 Avenu sativa 206 Azasteroids 206 Azobenzenes 143 51/ 42*
Alachor , 22 Alanine 212 Albumin, bovine serum 256 Aldrin 43*, 67/, 87,134, 272* Alkyl alcohols 114 Alkyl thiocyanates 217 1- Alkylimidazoles 280/, 281 Allium canadense
Anion, superoxide Antipyrine
25
94-98 18 275 10,17,21,22,105 ..
7 8
238*, 239/, 240 80 Β
Barban Barley Bathochromic shift Bean Benomyl Benzene hydroxylase activity Benzo(a)pyrene Benzoic acid 1,2,3-Benzothiadiazoles Benzoyl-CoA Benzoylprop-ethyl
Ames test 223 Amiben 8 Amines, azasteroids and nonsteroidal 238* Amino acid conjugates 50 Amino acids, conjugation with 208-214 Beta vulgaris 2- Amino-anthracene 223 Bifenox 4-Aminobenzoic acid 203 Bile cannula, reentry 4-Aminobiphenyl 204 Bioassays 2-Aminofluorene 204 Biphenyl 2-Aminonaphthalene 204 hydroxylase, hepatic l-Aminopropan-2-ol 203 Birds Aminopyrine, N-demethylation of 304/ Blattetta germanica Amitrole 10,17 Blowfly larva Analine hydroxylase, hepatic 287* Boston ivy Analines 80 Botran Bromacil Animal subcellular fractions 149-174,181-233 Buffer(s) Anthranilic acid 116 Butamoxane Anti-12-hydroxy- C-endrin 189 Butylate 14
321
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
108 7,8 160 42-43*, 114 29 312* 142,143 143 275, 277 208 101 42*
101 288 66-68 90,143 297* 192 252
267 41* 83 25 ...188,270 142 22
322
XENOBIOTIC
C D A A (N,N-diallylchloroaeetamide) 113 C - S lyase Ill Caffeic acid 116 Calliphora erythrocephah 267 Callue 95 Canavalie ensiformis 40* Carbamates 250 insecticides 80 Carbanion form of substrates 169 Carbaryl 22,42-43*, 134,135,138,192 metabolism of 65/ (1-naphthyl methylcarbamate) . . . . . 62 toxicity of 278 Carbene form of substrates 170 Carbon monoxide 159/, 160 Carboxylesterase 27 Carcinogens 22 Carrot 7,39-43* Cat 192 Catechol O-methyl transferase 206 Catharanthus roseus 68 Cell culture 137-143 primary 244/ as source of subcellular components 245-246 systems 243-245 techniques 35-71 fraction 302 intact 317 methods, isolated plant part and .... 7-29 techniques, in vitro tissue, organ and isolated 1-30 Centrifugation, differential 182-183 Centrifugation, sucrose density gradient 269 Chicken 141 Chloral hydrate 135 Chloramben 101 Chlorbromuron 22,29 Chlorcyclizine 134,135 Chlorfenvinphos, dealkylation of 295/ 3- Chloro-4-hydroxyaniline 113 Chloro-N-methylaniline 90 p-Chloro-N-methylaniline 87,90 4- Chloro-o-toluidine 139 4-Chlorobiphenyl 134 Chlorofenprop-methyl 99 p-Chloromercuribenzoate 275 Chloropropham 136 Chlorotoluron 139 Chlorphenamidine 139 Chlorpropham 19,22, 111 Chromatogram, DEAE-cellulose 294/, 295/ Chromatography, gel filtration 183-184 Cinnamic acid 89
METABOLISM
Cisanilide 9,39-41*, 45,47/, 48*, 111 Citrus sinensis 41* Clover 42-43* Cockroach 259,267 embryos 244/ leg, cuticle formation in 237* leg, 22,25-dideoxyecdysone in .238*, 242* Coconut milk 36 Collagenase/hyaluronidase 141 Condensations 78 Conjugates 139 Conjugating agent 181 Conjugation 77,103-118,238,240 with amino acids 208-214 with glutathione 142,214-223 with sulfate 142,197-203 Convolvulus arvenis 40* 50 Cotton 7,9,21,25,39-41* p-Coumaric acid 116 Cows 212 tri-o-Cresyl phosphate 275 Cricket/ 259,278 Cucumber 99 Cuticle 4 formation in cockroach leg 237* Cyanophenothrin 275 Cyclizine 135 Cyclodienes 250 Cysteine conjugates Ill Cysteine S-transferase activity 104* Cytochrome P-450 87,143,149-151 Class Β type of 165 clusters 170 cyclic function of 154 cyclic reaction of 173/ ferric 157/ hepatic 296* multiple types of 171-172 reductase, N A D P H 158/ reduction of 158-159 Cytosol 182 Cytotoxicity 137
2,4-D 40-41* D D E , nontoxic 250 DDT 43*, 66,251,252 Dandelion 95,96-97* Dansylamide 142 Daucus carota 7, 39*, 40*, 42* Dealkylation 252 of chlorfenvinphos 295/ N-Dealkylation 80,87 N- and O-Dealkylation thioether 252
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
323
INDEX
O-Dealkylation 87 Dearylation 252 Decarboxylations 78 Dechlorination 252 Dehalogenation 94 Dehydrochlorination 251 N-Demethylase 90 Demthylations 78,142 N-Demethylation 21 of aminopyrine 304/ of monuron 18 Desulfuration 87,252 Detoxication 99 mechanisms 286 in plants 23 N,N-Diallyl-2,2-diehloroacetamide .85,111 Diazinon 272,274/ Diazoxon 273,274 2,6-Dichloro-4-nitrophenol 20 2,4-Dichloroacetophenone 220 Dichlorodiphenylacetic acid ( D D A ) 136 1,2-Dichloronitrobenzene 108 2,4-Dichlorophenacyl chloride 220 2,4-Dichlorophenoxyacetic acid 20,36 2,4-Dichlorophenylethanol 220 3,4-Dichloropropionanilide 44 Diclofop-methyl 9-21 22,25-Dideoxyecdysone in cockroach leg 238* hydroxylation of 241/ metabolism of 235 Dieldrin 67/, 134,193 Differential centrifugation 182-183 Digestion, enzymatic 28 Digestive juices inhibitory 307 5,6-Dihydro-5,6-dihydroxycarbaryl .... 139 l,2-Dihydro-l,2-dihydroxynaphthalene 142 Dihydrodiol 193 Dimefox 136 Dimethoate 101,139 N,N-Dimethyl-2,2-diphenylacetamide 44 cis-2,5-Dimethyl- 1-py rrolidinecarboxanilide 45 Ν,Ν-Dimethylaniline 90 Dimethylvinphos 220 Dinoben ( 2,5-dichloro-3-nitrobenzoic acid) 94 Dinoseb 25 Diphenamid 39-41*, 44,45/, 46*, 116 Diphenylacetic acid 212 Diphenylether 105 Dipropetryn 17 Dismutase, superoxide 165 Dissociation of superoxide 166/ Diuron 20 Dogs Drosophih mehnogaster
204,212 254
Drugs metabolism and toxicity of metabolizing capacity oxidation, hepatic Duochromator Dyfonate
249 181 309/ 310* 153/ 22,206
E P T C (S-Ethyl dipropylthiocarbamate ) 108 α-Ecdysone 245 Ecdysteroid 235,237* Electron the second 166-169 transfer components, microsomal .. 159* transport complexes 172 Enzymatic activity Enzymatic digestion Enzyme activity development induction proteolytic source of stability studies, in vitro substrates for hepatic Epoxidase activities microsomal aldrin Epoxidation microsomal aldrin Epoxide hydratase Ester cleavage Esterases Ether-solubles Ethinimate Ethoxycoumarin Ethylenechlorohydrin Ethylenethiourea Ethylmorphine Excised leaves E y e pigments
270-272 28 269,307 299 162 256 299 303 77-121 306* 261*, 262* 300/, 301/ 87,279/ 263/ 281 142,193,252 252 99-102 52/ 142 143 114 18 142 6 307
F Felidae Fenuron Ferret Ferulic acid Field bindweed Fish Flamprop-isopropyl Flavoprotein Fluometuron Fluoride, phenylmethanesulfonyl
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
192 97 212 116 40-41* 192 101 150/, 256 29,139 256
324
XENOBIOTIC
Fluorodifen
8-10,16,17,38, 40-41*, 94,105 metabolism 26, 27*, 44/ Flurenol butyl ester 87 Foetus 211 Fractionation 265-266 subcellular 182 of microsomal oxidase activity . . . 268/ Freezing, effect on oxidase activity .... 270* Fungicides 1 G G S H conjugates, formation of 106* G S H S-transferase activity 104*, 106* Galium verum 42* Gel filtration chromatography 183-18 Gentiobioside 11 Geranium 41 Gibberellin A 92 Glucose conjugation 114—118 Glucosides 114 Glucosyltransferase 114 Glucuronic acid conjugation 187-197 Glucuronidation 182,192 of methylumbelliferone 193* N-Glucuronide 139 Glucuronyl transferase 142,181,188 Glucose ester 101 N-Glucoside 101 Glutamic acid 212 Glutamine 208,212 γ-Glutamyltransferase 214 Glutathione conjugation 18,23,214-223 S-transferases 107*, 110* X-transferase 103-113 S Glutathione 217 Glycine 208,211,212 N-acylase 208 Glycine max 7,39—43*, 50 Gossypium hirsutum 7, 39* Gromphadorhina portentosa 260/, 263/, 267 Guinea pigs 201, 204,295/ J
35
H Hamster Hank's buffer Harmalol Harmol Helianthus annus Hemeprotein Hens Hepatocyte isolated
204,312* 141 199 199 40* 154 192 313 131,141-143
METABOLISM
Herbicides phenylurea by plant tissue culture, metabolism of Hexamethylbenzene Hippuric acid Homogenization Honey bee Hordeum vulgare Hormones of insects Hornworm larvae House flies Hydratropic acid Hydrogen peroxide Hydrolysis Hydrolytic reactions Hydroperoxidase
1,38-62 139 39-41* 278 208,212 265-266 264*, 269 7 240 240 273 212 166,168/ 77,78 94 85
N-Hydroxyacetanilide 202 N-Hydroxy-2-acetylaminofluorene 198 N-Hydroxy-2-acetyl-aminonaphthalene 202 N-Hydroxy-N-arylaeetamides 202* Hydroxyatrazine 18 Hydroxy benzoic acids 116 4-Hydroxybiphenyl 143 4- Hydroxycarbaryl 135,139 5- Hydroxycarbaryl 139 N-Hydroxy-4-chloroacetanilide 202 7-Hydroxychlorpromazine 189 st/n-12-Hydroxy dieldrin 193 20-Hydroxyecdysone 245 Hydroxylase, hepatic 297* Hydroxylation 78, 87,142, 238, 240, 252 aryl 18 of D D T 252 of 22,25-dideoxyecdysone 241/ N-Hydroxyphenacetin 193,202 Hypsochromic effect 156 Hypsochromic spectral shift 160
I Imidazoles Imipramine Incubation, temperature of Indoleactic acid Induction Inhibition Inhibitors endogenous natural of enzyme activity Inhibitory factors intracellular
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
275 134,135 303 58 99 99 254 287 307 256 262
325
INDEX
Insect age or life stage of the 262 preparation, whole 254, 259 subcellular components 249-281 Insecticides 1,62-70 carbamate 80,274/ by plant tissue culture, metabolism of 42-43* synergists 275-276 In vitro enzyme studies 77-121 methods in species comparisons .287-298 studies, application of 272-277 techniques 285-319 In vivo studies 277-281 Indole 85 Intestines 136 Iodide 84* Ioxynil 8 Iron, ferric 154 Iron—sulfur protein 158 Isonazid 203 Isosuccinimide 0-glucoside 118 Isovanillic acid 116 Isozymes 78,99
Lipophilic drugs 251 Lipophilic zenobiotics 249 Liver 132,136,141 expiants 293 microsomal Cytochrome P-450 .162/, 164/ microsomes, preparation of 152 perfusion apparatus 133/ slices 293,295/ Lubrol 188 Lung 132,136,138 Lycopersicon esculentum
18
Lymphoma cells
138 M
Macremphytus varianus
Malpighia
256
,
Manduca sexta
240
Manometry Matacil Melanization
136 80 254
Meliotus alba
42*
Membrane diagram of the microsomal 170/ fluidity 171 J role of the 170-171 Jackbean 40-41* structure 159 Johnsongrass 105 transport systems 245 Metabolism of 4- C-22,25-dideoxyecdysone 240* Κ of cisanilide 47/ Kelthane 43*, 66 of 22,25-dideoxyecdysone 235 Kidneys 136 of diphenamid 45/ sulphotransferase, bovine 198* fluorodifen 26,27*, 44/ Kinetin 6, 29 of H C E 289/ of herbicides by plant tissue culture 39-41* L in higher plants, pesticide 77-121 in higher plants, xenobiotic 35-71 Lactuca satim 42* of insecticides by plant tissue Leaf culture 42-43* cells, separated 23-28 in leaf discs or sections 21-22 discs 6,19-23 esticide 36 excised y plants, 2,4-D 57/ absorption and translocation in .. 9-17 in rats, hexabarbital 286 radioautographs of soybean 12/ in separated cells 25-28 and roots 7—19 thiocyanage 252 zenobiotic metabolism in 17 of triol 238,240 Lepidopterous larvae 259 in wild species 315 Lettuce 42-43* of xenobiotics 4,7,249-281,285-319 Leucophaea maderae 237 by humans 316 Ligand, soft 160 in insect cell and organ Light retardation of senescence 6 cultures 233-246 Lignification 80 by plant tissue culture 38 Limnephilus 256 Type I 149-174 Lindane 42^43*, 63 Type II 181-223 Lipid peroxidase 307 14
E
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
326
XENOBIOTIC
Metabolites C-carbaryl 64* in cells and medium 46* 2,4-D 51*, 59-61/ 2,4-D-l- C ...54-56* HCE 290* isolation and characterization of .... 23 Metabolizing tissues 234 Metamorphosis, process of 264 Methadone 134,135 8-Methoxybutamoxane 142 N-Methylaniline 90 Methylation 206-207 Methylenedioxyphenyl derivatives .... 275 Methylparathion 217 Methyltransferases 206 4-Methylumbelliferone 143,201 Metobromuron 13 Metribuzin 40-41* Mice 212 Microdissection 28 Microsomal fraction of liver 151 membrane 170 pigments, spectrophotometric analysis of 152-153 Microsomes 188 hepatic 252 Mirex 134 Monkeys 192,204 Monophenyl phosphate 203 Monuron 18-25 Mouse 138,204 Multienzyme complexes 159 Mutagens 222 14
14
Ν N A D P H Cytochrome c reductase 254,256,257/ N A D P H oxidation 254 Naphthalene 142,193 Naphthalene-l,4-diol 139 Naphthalene-l,5-diol 139 Naphthaleneacetic acid 36,116 α-Naphthol 85 1-Naphthol 135,142,192 1- Naphthylacetic acid 212 2- Naphthylacetic acid 212 2-Naphthylamine 203 1-Naphthyl methylcarbamate-iVglucuronide 139 1-Naphthyl ZV-propylcarbamate 275 N I H shift 90 Nicotiana glauca 42* glutinosa 42* sylvestris 42* tabacum 24,40*, 42*
METABOLISM
p-Nitoanisole p-Nitrophenol 4-Nitrophenyl sulphate Nitroreductases Nonsteriodal amines
87,90 134,142,187 199 94 239/
Ο Oat shoots, radioautographs of excised Organ culture systems culture techniques perfusion systems, perfused Organophosphorothioates Organophosphorus compounds
14/ 233 235 132-135 131 89 250
Oxene complex of Cytochrome P-450 169 Oxene type intermediate 169/ Oxenoid. species of oxygen 169 Oxidase activity, effect of freezing on 270* activity, microsomal 259 -like activity 93* mixed function 87-93 Oxidation 77,87,252 inhibition of microsomal 255/ metabolism, H C E 292/ microsomal 260/ NADPH 254 reactions 77-93 Oxycytochrome P-450 163,164/ reduction of 168/ Oxygen, activated 154 Oxygenase, mixed function 142 Oxygenation reactions 166
Ρ P C N B (pentachloronitrobenzene) ... 107 Panax ginseng 69 Papaver somniferum 89 Paraoxon 80,95,134,135 Paraquat 29 Parathion 80,95,134,135 Parenchyma cells 37 Parsley 42-43* Parthenocissus tricuspidata 41*, 62 Pea 8 Peanut 9,26 Pelagonium hortorum 41* Pentachloronitrobenzene 94 Pentamethylbenzene 278 Pentobarbital 134-135 Peptidase 214 Perfluidone 8,18 Perhydroxyl radical 165
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
327
INDEX
Periphneta americana 251, 260/, 267 Peroxidases 62,78-87 Peroxycytochrome P-450 166,168/ Pesticides 249 metabolism 36,77-121,181 Petioles 7 Petroselinum hortense 42-43* Phaseolus radmtus 85 Phaseolus vulgaris .17,43*, 85 Phenols 78, 85,114 Phenothrin 275 3-Phenoxybenzoic acid 212-214 3-Phenoxybenzoyltaurine 214 3-Phenoxybenzoyl chrysanthemum esters 276* Phenylacetic acid 212 Phenylcarbamates 80 Phenylmethanesulfonyl fluoride Phenylurea 80 herbicides 139 Phorate 89 3'-Phosphoadenosine-5'-phosphosulphate 197 Phosphorylation 203 Photophosphorylation, cyclic 6 Photosynthesis 20 inhibition of 106* Phytotoxicity 49,66-68 Picea glauca 62 Pig 192,204 Pisum sativum 8 Placenta 192 Plants discotyledonous 7 enzymes 77,91* monocotyledonous 7 part and cell methods, isolated 7-29 pesticide metabolism in higher 77-121 protoplasts 28-29 radioautographs of oat 15/ radioautographs of soybean 13/ tissue culture 36-38 metabolism of herbicides by 39-41* metabolism of insecticides by .. .42-43* metabolism of xenobiotics by ...... 38 xenobiotic metabolism in higher . 3 5 - 7 1 Phntago major 21 Plantain 21 Plasmalemma 4 Poikilothermic species 314 Polyacrylamide gel electrophoresis 171,172/ Polychlorinated biphenyls 134 Polymorphism 203 Potato 42-43* Precipitation 183 Propachlor 9,108,113 Propanil 22, 39-41*, 44,95,97
Propham Propoxyphene 6-Propyl-2-thiouracil Protein, iron-sulfur Protein synthesis Protoplasts, plant Pseudomonas putida
97,136 142 206 158 6 28-29 151,155/, 160/, 161,163,164/, 165 Purple cockle 42-43* Putidaredoxin 167 Pyrethroid metabolizing esterases 275 Pyridine 206 nucleotide, reduced 150/ Pyroloxygenase 92 Pyrolysis 83
Quinin Quinones
192,254 R
Rabbit 203,204,210-212,312* Radioactivity of 2,4-D 57* Radiochromatograms of 4 - C ecdysteroids 239/ Rat 141,192,201,204,210,212,312* metabolites 294/ Reduction 77 of Cytochrome P-450 158-159 reactions 94-102 Resonance, paramagnetic 155/ Respiration 20 Rice 7,39-41*, 95,96-99* Roach, wild 309/ Roots, excised leaves and 7-19 Ruta graveohns 68 14
Saccharum officianarum Schistocera gregaria Screening programs Selectivity Senescence Sepharose 2B Serine Sex difference Shamouti orange Shrew Simazine Sinapic acid Solarium tuberosum Soret region Sorghum Sorghum vulgare
7 251 316 99 4, 6-7 183 212 286,298 41* 204 8 116 42-43* 162 7,23,105,107* 7
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
328 Soybean 7,16*, 25, 39-43* Spectral shift, hypsochromic 160 Spectrophotometry, difference absorbance 156 Spectroscopy, E P R 156 Spectrum, photochemical action 161/ Spodoptera eridania 256,257/, 260/, 263/, 267 Structure-activity studies 276 Sundangrass 105 Sunflower 40-41* Sugarcane 7,105 Sulfate, conjugation with 197-203 Sulfate and glucuronic acid conjugation 142 Sulfobromophthalein 108 Sulfones 89 Sulfoxide 89,9 Sulfur oxidation 8 Sulphotransferase enzymes 197 Sumithion 95 Superoxide 165/ anion 165 dismutase 165 dissociation of .. 166/ Suspension cultures 49 Symplastic transport 5 Synergistic effect 169 of N A D H 168 Syringic acid 116 Τ Target tissues 234 Taurine 208,211,212 Temperature of incubation 303 Temperature optima 287 Teratogens 222 Tetraethylpyrophosphate 275 Tetraphenylboron 141 Thiocarbamate sulphoxides 218 Thiocyanate metabolism 252 Thione transferases 214 Tissue culture, plant 36-38 culture techniques 35-71 expiants 293,313 maintenance technique 288 metabolizing 234 organ and isolated cell techniques, in vitro 1-30 slices .....136-137 sources 254-264 target 234 Tobacco 36,40-45 hornworm 245 Toruh 262 Toxicity of carbaryl 278
XENOBIOTIC
METABOLISM
Transferases, thione Transferases, G S H Translocation in excised leaves of C herbicides Transpiration stream Transport, apoplastic Transport, symplastic Tree shrew Triazines 5-Triazines 5-Triazinyl mercapturic acid Trichloroacetic acid Trichloroethanol glucuronide Trichloroethylen
214 275 4-7,11/ 9-17 8*, 9* 7 17 5 5 192 9 108 218 135 135 135 134,135
1 4
Triol, metabolic pathways for Triol, metabolism of Triticum aestivum Triticum monococcum Trypan blue exclusion test Trypsin Tulip Tyrosinase system
240 238,240 7 40* 142 141 95,96,97* 256
U Uridine-5'-diphospho-a-D-glucuronic acid
188
V Vanillic acid
116
W Wheat W i l d oat W i l d onion
7,40-41*, 114 7,16* 25
X Xanthommatin 254,255/ Xenobiotic(s) 188 in insect cell and organ cultures, metabolism of 233-246 metabolism of ... 1,4-7,249-281,285-319 in excised leaves 17 in plants 1-30, 35-71 Type I 149-174 Type II 181-223
Ζ Zea mays Zextran Zinia
In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
,
7,40* 80 26
