Resistance Mechanisms to Carbamate and Organophosphate

Chapter 3

Resistance Mechanisms to Carbamate and Organophosphate Insecticides

Downloaded by MICHIGAN STATE UNIV on June 11, 2013 | http://pubs.acs.org Publication Date: February 23, 1990 | doi: 10.1021/bk-1990-0421.ch003

L. B. Brattsten Department of Entomology, Rutgers University, J. B. Smith Hall, New Brunswick, NJ 08903-0231

Organophosphates (OPs), introduced i n 1944, and carbamates, i n t r o d u c e d i n 1956, remain w i d e l y used and effective insecticides although not f r e e from r e s i s t a n c e p r o b l e m s . Metabolic r e s i s t a n c e t o OPs was reported 14 years a f t e r t h e i r i n t r o d u c t i o n , compared t o only 7 years f o r DDT and 5 f o r the carbamates. The complex metabolic f a t e o f t h e OPs, i n c l u ding attack by cytochrome P-450 l e a d i n g e i t h e r t o a c t i v a t i o n o r d e t o x i f i c a t i o n , as w e l l as by g l u t a t h i o n e t r a n s f e r a s e s and esterases, may p l a y a r o l e in this delay. Carbamates a r e not b i o a c t i v a t e d ; they are d e t o x i f i e d by cytochrome P-450. In the presence o f a continued s e l e c t i o n pressure, metabolic r e s i s t a n c e may facilitate the e v o l u t i o n o f other defenses such as t a r g e t site r e s i s t a n c e , r e p o r t e d f o r t h e OPs and carbamates 6 and 10 y e a r s a f t e r m e t a b o l i c r e s i s t a n c e . Target s i t e r e s i s t a n c e t o OPs and carbamates r e s i d e s i n modified forms o f acetyl-cholinesterases (AChEs) with reduced affinity for the insecticides. AChE-based t a r g e t s i t e r e s i s t a n c e does not n e c e s s a r i l y confer cross r e s i s t a n c e t o all other OPs and carbamates and may be unstable i n the absence of a s e l e c t i o n pressure. Historical

Perspective

The organophosphorous (OP) i n s e c t i c i d e s were among t h e f i r s t s y n t h e t i c i n s e c t i c i d e s t o be used on a l a r g e s c a l e ; they a r e s t i l l widely used today. The f i r s t organophos0097-6156/90/0421-0042$06.00/0 © 1990 American Chemical Society

In Managing Resistance to Agrochemicals; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

3. BRATTSTEN

Resistance Mechanisms to Carbamates & Organophosphates


phate t o be used as an i n s e c t i c i d e was TEPP ( t e t r a e t h y l pyrophosphate), introduced i n 1944. T h i s compound was soon discontinued, however, because of i t s very high mammalian t o x i c i t y . Parathion was the f i r s t s u c c e s s f u l OP but a l s o s u f f e r e d from very high mammalian t o x i c i t y . Parathion was introduced i n 1947 and q u i c k l y followed by a l a r g e number of other OPs, some of which have improved s e l e c t i v i t y between i n s e c t s and mammals because of small changes i n the molecule. For instance, methyl parathion, introduced i n 1949, i s l e s s t o x i c t o mammals because of the phosphoester methyl s u b s t i t u e n t s , and f e n i t r o t h i o n i s a f u r t h e r improvement thanks t o the methyl s u b s t i t u e n t on the p h e n o l i c r i n g (Table 1) (1-3). Table 1. Comparison of acute t o x i c i t i e s ( L D , mg/kg) of OPs and carbamates t o mammals ( r a t , o r a l ) and i n s e c t s (house f l y , t o p i c a l ) . The data are compiled from s e v e r a l sources (1-3) 50

Insecticide Parathion Methyl parathion Fenitrothion Malathion Carbaryl Propoxur Carbofuran

Mammal

Insect

3.6 24 250 885 500 95 8

0.9 1.2 2.3 26.5 100 26 4.6

Because of t h e i r combination of high acute mammalian t o x i c i t y and very high e f f i c i e n c y i n c o n t r o l l i n g i n s e c t s , the OPs are c r e d i t e d with having given a strong impetus f o r n e u r o p h y s i o l o g i c a l and neurobiochemical studies i n both mammalian and i n s e c t systems, prompting the development of what are now h i g h l y s o p h i s t i c a t e d areas i n s c i e n ce. The i n t r o d u c t i o n of malathion i n 1950 increased the acceptance of t h i s type of compounds f o r commercial f i e l d use, as m a l a t h i o n was t h e f i r s t OP i n s e c t i c i d e w i t h remarkable mammalian s a f e t y (Table 1). The development of the N-methy1carbamate i n s e c t i c i d e s and t h e s u c c e s s f u l i n t r o d u c t i o n of c a r b a r y l i n 1956 seemed to s o l v e the problems with the high mammalian toxi c i t y of the OPs, but many subsequently developed carbamates are a l s o h i g h l y t o x i c t o v e r t e b r a t e s (Table 1). In f a c t , only two new c l a s s e s of i n s e c t i c i d e s have been developed f o r commercial use i n the l a s t 30 years. Both the s y n t h e t i c p y r e t h r o i d s and the avermectins have e x c e l l e n t mammalian s a f t e t y . However, both are encumbered by p r e v i o u s l y evolved t a r g e t s i t e r e s i s t a n c e , s e l e c t e d by over-use of DDT and cyclodiene i n s e c t i c i d e s , r e s p e c t i v e l y . Thus, the remaining importance of the OPs and the carbamates i s obvious.



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Structures As shown i n F i g u r e 1, the b a s i c s t r u c t u r a l s k e l e t o n of both OPs and carbamates allows a very l a r g e v a r i e t y of s u b s t i t u e n t s t o be attached without l o s s of t o x i c i t y . With t h e e x c e p t i o n o f t h e oxime carbamates, e. g. a l d i c a r b and methomyl, the s u c c e s s f u l carbamates have aromatic or heteroaromatic s u b s t i t u e n t s . There are three c a t e g o r i e s of OPs: the a l i p h a t i c OPs, e x e m p l i f i e d by malathion; the aromatic OPs, e x e m p l i f i e d by methyl parat h i o n ; and the heteroaromatic OPs, e x e m p l i f i e d by c h l o r p y r i f o s . The l a s t category, c o n t a i n i n g the l e a s t biodegradable compounds of the three (4) , i s the one most w i d e l y used i n a g r i c u l t u r a l a p p l i c a t i o n s . Recently however, a l i p h a t i c OPs are used i n c r e a s i n g l y as systemic a g r i c u l t u r a l i n s e c t i c i d e s as replacements f o r a l d i c a r b , which has caused ground water contamination problems. The b a s i c s t r u c t u r e s a l s o show t h a t , depending on the nature of the s u b s t i t u e n t s (R) , the OP and carbamate molecules are s u s c e p t i b l e t o enzymatic a t t a c k i n not j u s t one, but s e v e r a l p l a c e s . The microsomal cytochrome P-450 dependent p o l y s u b s t r a t e monooxygenases (EC 1.14.14.1) (PSMOs), c a r b o x y l e s t e r a s e s (EC 3.1.1.1 or 3.1.1.2) with phosphatase a c t i v i t y , and g l u t a t h i o n e t r a n s f e r a s e s (EC 2.5.1.18) a l l metabolize the OPs, whereas the carbamates are d e t o x i f i e d with s u f f i c i e n t speed only by cytochrome P-450. Resistance Resistance t o the OPs was not reported u n t i l 14 years a f t e r t h e i r i n t r o d u c t i o n . T h i s i s twice the time i t took i n s e c t s t o evolve r e s i s t a n c e t o DDT. Resistance t o the carbamates appeared more q u i c k l y , 5 years a f t e r t h e i r i n t r o d u c t i o n , and was probably c o n d i t i o n e d by previous use of c h l o r i n a t e d hydrocarbon and OP i n s e c t i c i d e s , both of which acted as s e l e c t i o n agents of the major d e t o x i f i c a t i o n mechanism, cytochrome P-450, f o r the carbamates. Metabolic r e s i s t a n c e mechanisms are s t i l l the most widely encountered causes f o r OP and carbamate r e s i s tance. Yet, a f t e r the f i r s t few cases of t a r g e t s i t e i n s e n s i t i v i t y were reported i n the e a r l y 1970's i n mites and t i c k s , many such cases have been found a l s o i n i n s e c t s . Resistance can a l s o be enhanced by a decreased r a t e of p e n e t r a t i o n through the integument. T h i s r e s i s tance mechanism by i t s e l f i s of minor importance, but provides an increased opportunity f o r d e t o x i f i c a t i o n . A 5 0 - f o l d i n c r e a s e i n r e s i s t a n c e to c a r b a r y l was seen i n a house f l y s t r a i n , i n which a gene f o r reduced p e n e t r a t i o n had been combined (by s e l e c t i v e breeding) with a gene f o r increased d e t o x i f i c a t i o n (5). Target s i t e r e s i s t a n c e i s probably always combined with some other form of r e s i s t a n c e ; even a r e l a t i v e l y i n s e n s i t i v e t a r g e t s i t e would e v e n t u a l l y be overwhelmed unless there were a l s o an e f f e c t i v e way t o e l i m i n a t e the i n s e c t i c i d e . Combinations of r e s i s t a n c e mechanisms are becoming i n c r e a s i n g l y frequent i n f i e l d populations of


3. BRATTSTEN


i n s e c t s under p e r s i s t e n t s e l e c t i o n pressure. In i n s e c t populations t h a t are s p e c i a l i z e d t o feed on t o x i c p l a n t s , the occurrence of s e v e r a l or many r e s i s t a n c e mechanisms appears t o be a r u l e with extremely few exceptions ( 6 ) .


Metabolic Resistance t o Carbamates Oxidation. Any and a l l biotransformations o f carbamate i n s e c t i c i d e s r e s u l t i n t h e i r l o s s o f anti-AChE a c t i v i t y . Oxidation i s the most important metabolic f a t e o f the carbamates and cytochrome P-450 i s t h e most important c a t a l y s t . One o r more isoenzymes o f cytochrome P-450 can o x i d i z e a carbamate molecule i n many d i f f e r e n t p l a c e s , depending on i t s s t r u c t u r e . In Figure 2, arrows i n d i c a t e p l a c e s s u s c e p t i b l e t o o x i d a t i o n i n s e l e c t e d carbamate molecules. Cytochrome P-450 removes the N-methyl group by d i r e c t h y d r o x y l a t i o n of the carbon atom adjacent t o the n i t r o gen; the aromatic s u b s t i t u e n t can be hydroxylated a t s p e c i f i c carbons or undergo epoxidation a t e l e c t r o n dense s i t e s , followed by conversion t o the d i h y d r o d i o l e i t h e r spontaneously o r c a t a l y z e d by epoxide hydrolases (7) . E a r l y experiments with synergized carbamates l e d t o the r e a l i z a t i o n t h a t i n s e c t s are w e l l endowed with "drugmetabolizing enzymes" and t h a t these, i n p a r t i c u l a r c y t o chrome P-450, are i n v o l v e d i n i n s e c t i c i d e d e t o x i f i c a t i o n . T h e r a p e u t i c a l l y used drug extenders, such as SKF 525A and L i l l y 18947, were shown t o be s y n e r g i s t s f o r carbamates by v i r t u e o f i n h i b i t i n g d e t o x i f i c a t i o n (8-10). As i n v i t r o methods f o r studying cytochrome P-450 i n i n s e c t s became a v a i l a b l e (11-13), i t soon became c l e a r t h a t i n s e c t s with high cytochrome P-450 a c t i v i t i e s were r e s i s t a n t t o carbamates and most other i n s e c t i c i d e s . T h i s phenomenon i s termed m e t a b o l i c c r o s s r e s i s t a n c e and d e r i v e s from the c h a r a c t e r i s t i c of cytochrome P-450 of accepting a very wide range of molecular s t r u c t u r e s as s u b s t r a t e s ; t h e cytochrome b i n d s t h e s u b s t r a t e v e r y l o o s e l y by a l i p o p h i l i c i n t e r a c t i o n and r a p i d l y o x i d i z e s i t by an oxygen f r e e radical-mediated r e a c t i o n , a very powerful combination. Moreover, the cytochrome occurs i n s e v e r a l o r many d i f f e r e n t isoenzymic forms with broadly overlapping substrate preferences. A normally i n f r e q u e n t form may be s e l e c t i v e l y induced by a l l e l o c h e m i c a l s i n the crop p l a n t s (14) , and i f the induced form has s u r v i v a l value i n the presence of an i n s e c t i c i d e , i t could be sel e c t e d t o dominate i n the exposed p o p u l a t i o n (15). S e l e c t i o n o f l a b o r a t o r y s t r a i n s of i n s e c t s with i n s e c t i c i d e s has r e p e a t e d l y r e s u l t e d i n p o p u l a t i o n s with c o n s i d e r a b l y higher cytochrome P-450 a c t i v i t i e s than the o r i g i n a l ones (16, 17) . The r o l e of cytochrome P-450 i n the d e t o x i f i c a t i o n and metabolism of the carbamates has been s t u d i e d e x t e n s i v e l y (18.) . High cytochrome P-450 a c t i v i t i e s are a s s o c i a t e d with v i r t u a l l y a l l cases of carbamate r e s i s t a n c e .


MANAGING

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A m e r i c a n C h e m i c a l Society Library 1155 15th St., N.W.

In ManagingWashington, Resistance to Agrochemicals; D.C 20036Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

48

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and p o s s i b l y harder t o overcome, r e s i s t a n c e mechanisms. Despite r e s u l t s of l a b o r a t o r y s e l e c t i o n experiments with mixtures, i t i s p r e d i c t a b l e , that mixtures with few components w i l l be n e a r l y as e f f e c t i v e s e l e c t i n g agents as s i n g l e compounds. To slow r e s i s t a n c e e v o l u t i o n t o a pace comparable t o t h a t i n n a t u r a l i n s e c t - p l a n t a s s o c i a t i o n s , w i l l r e q u i r e multi-component m i x t u r e s such as those employed by p l a n t s . A l u p i n e i n Colorado e f f e c t i v e l y p r o t e c t s i t s i n f l o r e s c e n c e s a g a i n s t an o t h e r w i s e specialized c a t e r p i l l a r by a mixture of a l k a l o i d s cont a i n i n g up t o 18 d i f f e r e n t compounds (26).


Metabolic

Resistance

to

OPs

Oxidation. OPs are attacked by no l e s s than three enzyme systems, the PSMOs, carboxylesterases with phosphatase a c t i v i t y , and g l u t a t h i o n e t r a n s f e r a s e s . The l a t t e r two i n v a r i a b l y d e t o x i f y the OPs by s p l i t t i n g o f f a l k y l or other s u b s t i t u e n t s . However, the a c t i o n of cytochrome P450 can r e s u l t i n d e t o x i f i c a t i o n i f the carbon of one of the small a l k y l s u b s t i t u e n t s i s o x i d i z e d , or i n a c t i v a t i o n of phosphorothioates i f the P=S i s converted t o P=0 by o x i d a t i v e d e s u l f u r a t i o n (Figure 3). Except i n a very few c a s e s , the t h i o p h o s p h a t e (P=S) i s not an AChE i n h i b i t o r . I t i s u s u a l l y the phosphate (P=0) form of the OPs t h a t i n h i b i t s AChE, because the P=S bond does not p o l a r i z e the phosphorous atom s u f f i c i e n t l y f o r strong b i n d i n g t o the e s t e r a t i c s u b s i t e . The o x i d a t i v e d e s u l f u r a t i o n r e a c t i o n i s unusual f o r cytochrome P-450, which t y p i c a l l y o x i d i z e s carbon atoms. I t i s thought to occur i n analogy with the epoxidation r e a c t i o n of aromatic carbons, as o u t l i n e d by Nakatsugawa and M o r e l l i (27) (Figure 4). H y d r o l y s i s . Carboxylesterases are f r e q u e n t l y one of the major f a c t o r s i n OP r e s i s t a n c e . In some i n s e c t s , f o r i n stance the house f l y (28), there are h i g h l y substrate s p e c i f i c esterases which attack only one or a very few molecules. "Malathionase", the prominent esterase respons i b l e f o r many cases of malathion r e s i s t a n c e , i s h i g h l y s p e c i f i c f o r malathion. I t cleaves one or both of the e t h y l e s t e r groups l e a v i n g malathion mono- or d i a c i d (29). T h i s enzyme i s a t r u e s e r i n e carboxylesterase t h a t i s i n h i b i t e d by malaoxon (28) and does not hydrolyze any of the phosphoester bonds. In Anopheles Stephensi from Pakistan, the malathion r e s i s t a n c e decreased with a d u l t age, but there was no concommittant decrease i n general esterase a c t i v i t y as measured with 1- and 2-naphthylacet a t e as model substrates (30). Other mosquitoes have a carboxylesterase with broad substrate s p e c i f i c i t y t h a t i s a s s o c i a t e d with r e s i s t a n c e (31-33). As mentioned above, the green peach aphid has a carboxylesterase, E4, with broad s u b s t r a t e s p e c i f i c i t y t h a t sequesters t o x i c a n t s (24).


3.

BRATTSTEN



Since OPs i n h i b i t s e r i n e hydrolases such as AChE by phosphorylating them, i t f o l l o w s t h a t OPs are p o t e n t i a l i n h i b i t o r s of a l l other s e r i n e hydrolases. T h i s means t h a t OPs can be used as s y n e r g i s t s , notably f o r the p y r e t h r o i d s t h a t are d e t o x i f i e d by e s t e r h y d r o l y s i s , and OP-containing mixture have proven s u c c e s s f u l i n s e v e r a l c a s e s (14.) . The u s e f u l n e s s o f t h e s e m i x t u r e s can be prolonged by using them i n r o t a t i o n s with other mixtures and/or i n s e c t i c i d e s . Glutathione conjugation. The involvement of g l u t a t h i o n e t r a n s f e r a s e s i n OP metabolism was r e a l i z e d i n the e a r l y 1960's f35. 36). I t was d i f f i c u l t t o e s t a b l i s h t h i s f a c t because of s i m i l a r i t i e s between g l u t a t h i o n e t r a n s f e r a s e and carboxylesterase-produced metabolites. Induction of 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 i n the f a l l armyworm caused a 2- t o 3 - f o l d decrease i n the t o x i c i t y of d i a z i non, methamidophos, and methyl parathion (17). T h i s shows i n d i r e c t l y t h e importance o f 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 i n the d e t o x i f i c a t i o n of these OPs. Glutathione t r a n s f e r a s e s c a t a l y z e a s u b s t i t u t i o n react i o n at e l e c t r o p h i l i c centers of molecules. They are a l s o b i n d i n g p r o t e i n s i n analogy with the E4 e s t e r a s e ; a mammalian form, c a l l e d l i g a n d i n , binds with high a f f i n i t y to a broad spectrum of compounds but does not c a t a l y z e the subsequent s u b s t i t u t i o n r e a c t i o n (38.) . The r o l e of the t r a n s f e r a s e s i n the c a t a l y t i c r e a c t i o n i s thought t o be t o provide c l o s e proximity between the x e n o b i o t i c and the reduced g l u t a t h i o n e anion, GS . There are, as seems t o be the r u l e with enzymes i n v o l ved i n the metabolism of x e n o b i o t i c s , m u l t i p l e isoenzymes with 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 . T h i s f a c t combined with t h e i r low substrate s p e c i f i c i t y i s considered to account f o r the v a r i e t y of OP metabolites produced as shown i n Figure 5 (19). There are at l e a s t three d i f f e r e n t isoenzymes i n the house f l y (4J)) . One of the house f l y glutathione t r a n s f e r a s e s i s i d e n t i c a l t o the enzyme r e s p o n s i b l e f o r DDT d e h y d r o c h l o r i n a t i o n (41)• T h i s form i s r a t h e r substrate s p e c i f i c , and i s not known t o confer metabolic c r o s s r e s i s t a n c e between DDT and any OP. There are few h i g h l y s p e c i f i c i n h i b i t o r s of the g l u t a thione t r a n s f e r a s e s , but organisms and t i s s u e s can be depleted of endogenous g l u t a t h i o n e by diethylmaleate and other compounds, with which i t r e a c t s spontaneously. The herbicide s y n e r g i s t t r i d i p h a n e a l s o synergized d i a z i n o n t o x i c i t y towards house f l i e s by a c t i n g as an a l t e r n a t i v e substrate (42). The house f l y g l u t a t h i o n e t r a n s f e r a s e conjugated t r i d i p h a n e 22 times f a s t e r than d i a z i n o n . E v o l u t i o n of metabolic r e s i s t a n c e t o the OPs. The f o l l o wing i s an attempt t o understand why i t took such an unusually long time f o r i n s e c t s t o evolve metabolic r e s i s t a n c e t o the OPs: C l e a r l y , the s e l e c t i o n pressure i s d i l u t e d by having t o a c t on three d i f f e r e n t , g e n e t i c a l l y u n r e l a t e d enzyme systems, a l l of which d e t o x i f y the com-


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Resistance Mechanisms to Carbamate and Organophosphate

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