Xenobiotic Metabolism: In Vitro Methods - ACS Publications

8 The Use of Insect Subcellular Components for Studying the Metabolism of Xenobiotics C. F. W I L K I N S O N

Downloaded by UNIV LAVAL on July 14, 2016 | http://pubs.acs.org Publication Date: April 5, 1979 | doi: 10.1021/bk-1979-0097.ch008

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 x e n o b i o t i c s c u r r e n t l y at our d i s p o s a l i s dependent to a large extent on our a b i l i t y to e s t a b l i s h t h e i r metabolic f a t e i n l i v i n g 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

Paulson et al.; Xenobiotic Metabolism: In Vitro Methods ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

XENOBIOTIC


250

METABOLISM

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 the recent books or review a r t i c l e s which are a v a i l a b l e (1-10), and the many references contained t h e r e i n . 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



8.

WILKINSON

Insect Subcellular

Components

251

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 nature of the metabolites produced by i n s e c t s and mammals were e s s e n t i a l l y s i m i l a r and that presumably t h i s 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


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 which take p l a c e i n i n s e c t t i s s u e s and on the general biochemical c h a r a c t e r i s t i c s of the enzymes c a t a l y z i n g 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



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 species to be employed i s already d e t e r mined and the l i f e stages of most i n t e r e s t (egg, l a r v a , nymph or 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 .



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 of the i n s e c t under i n v e s t i g a t i o n . Indeed, i n view of the heterogeneous mixture of m a t e r i a l s they contain 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



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)



256


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 group of endogenous i n h i b i t o r s which can s e r i o u s l y impede i n v i t r o s t u d i e s i n whole-insect p r e p a r a t i o n s are 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



WILKINSON

Figure 2.

Insect Subcellular

Components

Solubilization of NADPH-Cytochrome c reductase by gut contents inhibitor of southern armyworm (S. eridania) (41).


258


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

Stimulates (F )

No e f f e c t


Proteolytic activity w

2+

Mg

(casein)

2+ , , Ca (mM)

domesticus

x

No e f f e c t (F )

Xenobiotic Metabolism: In Vitro Methods - ACS Publications

Recommend Documents