Cytochrome P-450 Involvement in the Interactions Between Plant

10 Cytochrome P-450 Involvement in the Interactions Between Plant Terpenes and Insect Herbivores LENA

B.

BRATTSTEN

Downloaded by MONASH UNIV on March 12, 2013 | http://pubs.acs.org Publication Date: January 20, 1983 | doi: 10.1021/bk-1983-0208.ch010

University of Tennessee, Department of Biochemistry and Graduate Program in Ecology, Knoxville, TN 37996

The cytochrome P-450-dependent microsomal mono-oxygenase system i s important i n several ways i n insect herbivores that feed on terpene-containing plants. Cytochrome P-450 metabolises many terpenes to polar products that can be excreted, often after further conjugation reactions, or that may be more toxic to the insect. Many terpenes induce insect cytochrome P-450 to higher a c t i v i t y . Changes i n cytochrome P-450 a c t i v i t y may influence hormone balance or pheromone production i n the insect, implicating the plant allelochemicals as factors i n the regulation of reproductive success of insect populations. Cytochrome P-450 i s therefore an important factor i n insect host-plant specialisations. Efforts at improving plant resistance to insect herbivory must take this enzyme system into consideration.

One or more o f the l a r g e v a r i e t y of terpenes, b i o s y n t h e t i c a l l y r e l a t e d t o each other as o u t l i n e d i n Figure 1, are present i n almost a l l higher p l a n t s . The terpenes are a l l f a i r l y to h i g h l y l i p o p h i l i c compounds depending upon t h e i r s t a t e o f o x i d a t i o n and g l y c o s y l a t i o n . They t h e r e f o r e have a high p o t e n t i a l f o r t o x i c i n t e r f e r e n c e w i t h the b a s i c biochemical and p h y s i o l o g i c a l funct i o n s of i n s e c t h e r b i v o r e s . Although the r o l e of the terpenes i n the p l a n t s that produce them i s s t i l l a matter of debate, evidence of an a n t i - h e r b i v o r e f u n c t i o n f o r many of them i s accumulating. Modern DNA technology, gene s p l i c i n g , and c l o n i n g techniques w i l l undoubtedly make i t p o s s i b l e to i n c o r p o r a t e s u i t a b l e defensive a l l e l o c h e m i c a l s i n t o s e l e c t e d crop p l a n t s i n order to minimise crop d e v a s t a t i o n by i n sect h e r b i v o r e s . However, i t i s necessary to understand the fund0097-6156/83/0208-0173$07.00/0 © 1983 American Chemical Society

In Plant Resistance to Insects; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

174

PLANT RESISTANCE TO INSECTS

Pheromones


Geranyl-PP

(CIO)

> Monoterpenes

•> Pyrethrum

Gossypol

Farnesyl-PP

(CIS)

> Sesquiterpenes

Steroids (C27)

> Pheromones

Triterpenes

A

*

o d

«

r o c n t

'

n

Cucurbitacins

Geranylgeranyl-PP (C20)

>

Oiterpenes

(C20)

Graya no toxin y Carotenoids Figure 1.

(C40) Outline of the biosynthetic relationships of plant terpenes.



10.

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P-450

Involvement

175

amental mechanisms by which p l a n t populations and i n s e c t populat i o n s c o e x i s t i n p r i s t i n e ecosystems i n order t o take f u l l advantage of t h i s p o t e n t i a l . I t i s c l e a r that the i n s e c t herbivores have developed many e f f e c t i v e and h i g h l y s p e c i a l i s e d adaptations f o r d e a l i n g w i t h the unavoidable presence o f these p o t e n t i a l l y t o x i c chemicals i n t h e i r food supply. Due t o the great d i v e r s i t y of s p e c i f i c s o l u t i o n s f o r s u r v i v a l that e x i s t i n nature, meaningf u l e f f o r t s a t mathematical modeling t o e s t a b l i s h p r e d i c t i v e models cannot be based simply on the gross behavior of experimenta l l y manipulated microcosms. Such models w i l l a t best be v a l i d only f o r the p a r t i c u l a r s i t u a t i o n on which they are based. Modeli n g techniques a r e p r e s e n t l y f a r ahead o f the a v a i l a b l e b a s i c b i o l o g i c a l and e c o l o g i c a l i n f o r m a t i o n upon which t r u l y p r e d i c t i v e models should be based, i n p a r t i c u l a r when t o x i c a n t s o f any k i n d are i n v o l v e d . I n undisturbed ecosystems, p l a n t and herbivorous i n s e c t popu l a t i o n s c o e x i s t i n a steady s t a t e c o n d i t i o n d i c t a t e d by e x t e r n a l b i o l o g i c a l and p h y s i c a l f a c t o r s ( p a r a s i t e s , predators, p r e c i p i t a t i o n , temperature, s o i l q u a l i t y , e t c . ) . But t h i s balance i s a l s o regulated by myriads of very fundamental i n t e r a c t i o n s , many o r even most of which are yet unknown, between the p l a n t a l l e l o c h e m i c a l s and the b i o c h e m i c a l , p h y s i o l o g i c a l , and b e h a v i o r a l f u n c t i o n s of the i n s e c t herbivores ( 1 ) . The complex i n t e r a c t i o n s of terpenes w i t h i n s e c t growth and reproduction and w i t h the i n s e c t s ' a b i l i t y to metabolise p o t e n t i a l l y t o x i c , l i p o p h i l i c f o r e i g n compounds w i l l be discussed b r i e f l y i n the f o l l o w i n g . T o x i c i t y of terpenes As i s the case w i t h p l a n t a l l e l o c h e m i c a l s i n g e n e r a l , the terpenes are not u s u a l l y a c u t e l y t o x i c . Table I provides a few examples o f terpene t o x i c i t i e s to mammals and shows that some are, indeed, h i g h l y t o x i c , e.g. grayanotoxin and e r g o s t e r o l . However, the v a s t m a j o r i t y of the terpenes show acute mammalian t o x i c i t i e s only i n the order o f s e v e r a l g/kg, i . e . they a r e not a c u t e l y t o x i c More data are a v a i l a b l e on p h y t o t o x i n t o x i c i t i e s to mammals than to i n s e c t s . I t i s not n e c e s s a r i l y true that a compound which i s t o x i c to a mammal has a s i m i l a r t o x i c e f f e c t i n an i n s e c t . I t i s , f o r i n s t a n c e , w e l l known that the p y r e t h r i n s a r e h i g h l y t o x i c t o i n s e c t s and are used w i t h s a f e t y as s e l e c t i v e i n s e c t i c i d e s . Limonene, although not t o x i c t o mammals, i s a c u t e l y t o x i c t o Dendroctonus pine b e e t l e s (2, 3). On the other hand, chemicals that a r e h i g h l y t o x i c t o mammals may not be t o x i c t o i n s e c t s . An example i s e r g o s t e r o l which i s h i g h l y t o x i c t o s e v e r a l mammals i n a d d i t i o n t o the dog, but which l a c k s acute t o x i c i t y t o l a r v a e of the southern armyworm, Spodoptera e r i d a n i a ( B r a t t s t e n , unpubl i s h e d ) . This compound i s probably used as a precursor i n the s y n t h e s i s o f c h o l e s t e r o l by a t l e a s t some i n s e c t s (4).


176

PLANT RESISTANCE TO INSECTS Table I


Mammalian acute t o x i c i t i e s of p l a n t terpenes Compound

^50

Animal, r o u t e

Grayanotoxin Ergosterol Gossypol Pyrethrum Pulegone Hymenovin a-pinene Limonene Carvone Borneol Cineole Citral Eugenol Geraniol Menthol Terpineol Caryophyllene Nepetalactone

1.2 mg/kg 4 mg/kg 550 mg/kg 200 mg/kg 120 mg/kg 150 mg/kg s e v e r a l g/kg s e v e r a l g/kg s e v e r a l g/kg s e v e r a l g/kg s e v e r a l g/kg s e v e r a l g/kg s e v e r a l g/kg s e v e r a l g/kg s e v e r a l g/kg s e v e r a l g/kg s e v e r a l g/kg s e v e r a l g/kg

mouse, i p dog, o r a l pig, oral rat, oral rat, ip mouse, o r a l rat, oral rat, oral rat, oral rabbit, oral rat, oral rat, oral mouse, o r a l rat, oral rat, oral rat, oral rat, oral rat, oral

A l l i n f o r m a t i o n from (46) except f o r hymenovin ( 4 7 ) . Terpenes as i n s e c t a t t r a c t a n t s and deterrents Numerous terpenes are a t t r a c t a n t s f o r i n s e c t s . Table I I shows some examples. The compounds a r e very o f t e n feeding a t t r a c t a n t s . They can a l s o be o v i p o s i t i o n s t i m u l i a s , f o r i n s t a n c e , a-pinene f o r the eastern spruce budworm or methyl i s o eugenol f o r the c a r r o t r u s t f l y . The e v o l u t i o n a r y d e t a i l s of some of these r e l a t i o n s , o f t e n r e v e a l f a s c i n a t i n g cases of i n s e c t h o s t - p l a n t s p e c i a l i s a t i o n s . A chemical that i s a deterrent to most i n s e c t s can become an o b l i g a t o r y feeding cue f o r a s p e c i a l i s t ^ e.g. the spotted cucumber b e e t l e ' s dependency on c u c u r b i t a c i n s f o r food r e c o g n i t i o n . Even more i n t r i c a t e r e l a t i o n s h i p s e x i s t as w i t h Ips and Dendroctonus bark b e e t l e s using the host t r e e (+)'-a-pinene as precursor f o r t h e i r own aggregation pheromone component, c i s verbenol (_5, _6) . A study w i t h h o u s e f l i e s (7) shows c l e a r l y that very s m a l l d i f f e r e n c e s i n the molecular s t r u c t u r e can r e s u l t i n d r a s t i c a l l y d i f f e r e n t b i o l o g i c a l e f f e c t s as e x e m p l i f i e d i n Table I I I by the o p t i c a l isomers (-)-limonene, a f l y a t t r a c t a n t , and (+)-limonene, a f l y d e t e r r e n t . A d i f f e r e n c e i n o x i d a t i o n s t a t e i n the funct i o n a l group as i n c i t r o n e l l o l , a f l y a t t r a c t a n t , and c i t r o n e l l a l , a f l y d e t e r r e n t , a l s o causes d i f f e r e n t responses. A d i f f e r e n c e i n the length of the carbon chain as i n f a r n e s o l (C15), a f l y a t t r a c t a n t , and g e r a n i o l (C10), a f l y d e t e r r e n t , a l s o confers d i f f e r e n t


10.

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Cytochrome

P-450

111

Involvement


Table I I P l a n t terpenes as a t t r a c t a n t s f o r i n s e c t s Insect species

Compound

Eastern spruce budworm Ips bark b e e t l e s S c o l y t i d bark b e e t l e s

a-pinene a-pinene a-pinene g-pinene limonene camphene geraniol a-terpineol geraniol geraniol citronellal a-terpineol eugenol a-pinene anethole citronellal methyleugenol mehyl isoeugenol a-pinene (3-pinene limonene caryophyllene iridodiol cucurbitacins salicin t e r p i n y l acetate linalool hexenol

Honeybee Japanese b e e t l e Pine b e e t l e Pales w e e v i l

Oriental f r u i t f l y Carrot r u s t f l y B o l l weevil

Lace wing Spotted cucumber b e e t l e Willow b e e t l e S i l k worm

Referer 48 49 50

51 52 53 54

55 56 57

58 59 60 61



178


b i o l o g i c a l p r o p e r t i e s . Table I I I a l s o shows an example of the c o n c e n t r a t i o n e f f e c t . In t h i s case a low c o n c e n t r a t i o n of carvone i s an a t t r a c t a n t , whereas a high c o n c e n t r a t i o n i s a d e t e r r e n t . This i s a very w i d e l y occuring phenomenon, and w e l l known a l s o i n human s o c i e t y , e.g. i n the contexts of s p i c e s and perfumes. Table IV gives a few examples of terpenes shown to be i n s e c t d e t e r r e n t s . As w i t h the previous examples of i n s e c t a t t r a c t a n t s i n Tables I I and I I I , the s t r u c t u r a l d i v e r s i t y of the d e t e r r e n t compounds i s remarkable. There are no c l e a r and l o g i c a l 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 among the compounds w i t h these behavi o r a l e f f e c t s . S p e c i a l i s e d and unique e f f e c t s and b e h a v i o r a l adaptations are thus the r u l e i n i n t e r a c t i o n s between species r a t h e r than the exception. The Vernonia sesquiterpene l a c t o n e * g l a u c o l i d e A, i s a feeding d e t e r r e n t f o r the southern armyworm, a broadly g e n e r a l i s t feeder. This compound i s one of the few that the southern armyworm l a r v a e r e j e c t . This i n s e c t and i t s c l o s e r e l a t i v e the f a l l armyworm, J3. f r u g l p e r d a , are capable of feeding on a l a r g e v a r i e t y of p l a n t s ( 8 ) . Both can a l s o metabolize l i p o p h i l i c f o r e i g n compounds, i n c l u d i n g p l a n t a l l e l o c h e m i c a l s and s y n t h e t i c p e s t i c i d e s , to e x c r e t a b l e , p o l a r metabolites by cytochrome P-450-dependent oxidations. Southern and f a l l armyworm growth on pulegone-laced d i e t s The mint monoterpene pulegone i s not only a feeding d e t e r r e n t f o r the f a l l armyworm but i s a l s o t o x i c to t h i s species ( 9 ) . I t i s , however, n e i t h e r a feeding d e t e r r e n t nor a c u t e l y t o x i c to the southern armyworm at s i m i l a r c o n c e n t r a t i o n s . F i g u r e 2a shows that a c o n c e n t r a t i o n of 0.1% pulegone i n the d i e t (10) i s t o x i c and k i l l s the f a l l armyworm l a r v a e (11). In c o n t r a s t , the southern armyworm l a r v a e feed f r e e l y on d i e t s (12) c o n t a i n i n g e i t h e r 0.01% or 0.1% pulegone. Their weight increases and there i s no delay i n t h e i r development (Figure 2b). However, f u r t h e r experiments w i t h the southern armyworm showed that pulegone can be a very important f a c t o r i n the s u c c e s s f u l coexistence of p l a n t s and herb i v o r e s (13). Pulegone e f f e c t s on the southern armyworm The data i n Table V (13) show that the southern armyworm l a r v a e a t t a i n higher maximal f r e s h body weights when pulegone i s present i n t h e i r d i e t up to 0.1%. But the percentage of nonwater body c o n s t i t u e n t s i s reduced as i s most obvious i n l a r v a e feeding on a 0.2% pulegone d i e t . The l a t t e r d i e t a l s o prolongs the time the l a r v a e spend i n the s i x t h i n s t a r and they undergo the pupal molt 8-10 days l a t e r than l a r v a e fed c o n t r o l d i e t s or d i e t s w i t h lower pulegone c o n c e n t r a t i o n s . The 0.2% pulegone d i e t a l s o reduces the pupation success to 57% as shown i n Table VI (13) , but a d u l t emergence from these pupaeoccursat almost normal r a t e (85%).


10.

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Cytochrome

P-450

179

Involvement


Table I I I Terpenes as housefly a t t r a c t a n t s and r e p e l l e n t s ( 7 ) . Attractants

Deterrents

(-)-limonene Citronellol Eugenol Farnesol Carvone (low)

(+)-limonene Citronellal Citral Geraniol (high) Carvone Camphene Cineol 6 -phellaudrene Carvacrol Linalool

Table IV P l a n t terpenes as d e t e r r e n t s f o r i n s e c t s Insect species

Compound

Western pine b e e t l e

Myrcene Limonene Farnesol Geranial Nerolidol Nepetalactone Azadirachtin Gossypol Terpineol Geranylacetate Geraniol Warburganal Xylomollin Caryophyllene Pulegone Glaucolide A Glaucolide A

Gypsymoth l a r v a e

Many species

Silkworm

A f r i c a n armyworm Beet armyworm F a l l armyworm Southern Armyworm

References


62 63

64 65 66 61

67 68 69 9 70 70

180


S. fruoiperdo

V Downloaded by MONASH UNIV on March 12, 2013 | http://pubs.acs.org Publication Date: January 20, 1983 | doi: 10.1021/bk-1983-0208.ch010

„4 ~«

1

1

5

1

1

1

1

1

1

1

1

1

1

1

1

1

1

10 15 Days since molting to 4th instar

5

1—

20

10 15 Days since molting to 4th instar

Figure 2. Growth from fourth instar to pupation on control or pulegone-containing diet of fall armyworm larvae (11) (top) and southern armyworm larvae (13) (bottorn). Key: control; A, 0.01% pulegone; and A, 0.1% pulegone.


10.

BRATTSTEN

Cytochrome

P-450

181

Involvement

Table V E f f e c t of d i e t a r y pulegone on body weight and accumulation of nonwater body c o n s t i t u e n t s i n southern armyworm l a r v a e and pupae (13) Diet


Control 0.01% 0.1 % 0.2 %

Larvae mg/fresh l a r v a l %dry mass 622.4 111 672.1 24* 784.8 96* 687.5 88

12 10 8* 8*

Pupae %dry mass mg/fresh pupa 282.8 278.1 323.0 296.2

22 20 53* 36

21 20 20 15*

* S i g n i f i c a n t l y d i f f e r e n t from c o n t r o l a t P< 0.002 ( T - t e s t ) Data are mean ± S.D. Larvae ate the 0.2% pulegone d i e t during the s i x t h i n s t a r o n l y ; they ate the other d i e t s from the f o u r t h i n s t a r through t o pupation.

However, a l l l e v e l s o f d i e t a r y pulegone a f f e c t the egg p r o d u c t i o n o f the moths. The data i n Table V I i n d i c a t e a reduced o v i p o s i t i o n r a t e even a t the lowest (0.01%) pulegone c o n c e n t r a t i o n i n the l a r v a l d i e t . The h i g h e s t c o n c e n t r a t i o n (0.2%) reduces the egg p r o d u c t i o n to 10% of that i n the c o n t r o l moths and hatching i s a l s o reduced t o 10% (13). This case i l l u s t r a t e s a p o s s i b l e , n a t u r a l l y occuring mechanism whereby even a minute amount o f a b i o a c t i v e p l a n t a l l e l o c h e m i c a l may reduce the r e p r o d u c t i v e c a p a c i t y of an i n s e c t herb i v o r e p o p u l a t i o n not d r a s t i c a l l y , but conceivably enough so that continued co-existence i s p o s s i b l e . The i n s e c t p o p u l a t i o n may be reduced only t o the p o i n t where the p l a n t s can s t i l l grow and r e produce s u c c e s s f u l l y , thereby i n s u r i n g a continued food supply f o r the i n s e c t h e r b i v o r e . An e n t i r e l y d i f f e r e n t , i n f a c t o p p o s i t e , e f f e c t on i n s e c t r e p r o d u c t i o n by terpenes occurs w i t h the desert l o c u s t . I n t h i s case the monoterpenes a-pinene, $-pinene, limonene, and eugen o l evaporating from desert shrubs about to bloom, p r e c i p i t a t e s synchronised sexual maturation and mating a c t i v i t y i n the l o c u s t s (14). The spruce budworm i s a l s o s t i m u l a t e d to increased f e r t i l i t y l e v e l s by host t r e e monoterpenes (R.G. Cates, personal*communi c a t i o n ) . I t i s p o s s i b l e that even opposite e f f e c t s on reproduct i o n i n i n s e c t s could occur depending on the s p e c i a l i s a t i o n o f the i n s e c t species t o i t s environment, the d i v e r s i t y o f the b i o l o g i c a l a c t i v i t i e s o f the compounds, and the high l e v e l o f complexity o f the r e p r o d u c t i v e processes.


182


Table VI E f f e c t s of l a r v a l d i e t a r y pulegone on development and o v i p o s i t i o n i n the southern armyworm Diet


Control 0.01% 0.1 % 0.2 %

%Pupation

96 92 82 57

Days to emergence 12 12 12 15

3 3 3 3

%Emergence

Days to oviposition

96 93 93 85

3.5 3.5 3.0 2.5

Eggs per female 1900-2000 1500-1800 1000-1200 100-200

Larvae ate the 0.2% pulegone d i e t d u r i n g the s i x t h i n s t a r o n l y ; they ate the other d i e t s from f o u r t h i n s t a r through to pupation. The "Days to emergence ' data i n d i c a t e that moths emerge d u r i n g a 6-day period w i t h a peak on day 12 or 15 a f t e r pupation. 1

Terpene involvement i n i n s e c t r e p r o d u c t i o n Several d i f f e r e n t molecular mechanisms could be i n v o l v e d i n the r e p r o d u c t i v e i n h i b i t i o n observed i n the southern armyworm. For i n s t a n c e , many terpene d e r i v a t i v e s mimic i n s e c t hormone action. Juvabione (15) i s the c l a s s i c a l example of a j u v e n i l e hormone (JH) mimic that prevents egg maturation i n P y r r h o c o r i s bugs. Aromatic terpene ethers (16), methylene dioxyphenyl terpene ethers (17), and other f a r n e s y l d e r i v a t i v e s a l s o have JH a c t i v i t y and the l a t t e r ones (18) a l s o cause s t e r i l i t y i n P y r r h o c o r i s . For the most p a r t JH a c t i v e terpenes are among the sesquiterpenes but s e v e r a l monoterpenes a l s o have i n s e c t s t e r i l i z i n g e f f e c t s (19, 20). The a c y c l i c monoterpene c i t r a l reduces the f e r t i l i t y of r a t s by causi n g f o l l i c u l a r degeneration (21). The precocenes, another c l a s s of terpene d e r i v a t i v e s w i t h JH a n t a g o n i s t i c e f f e c t s , i n a c t i v a t e JH s y n t h e s i s by s p e c i f i c i n h i b i t i o n of corpora a l l a t a (CA) microsomal cytochrome P-450-dependent mixed-function oxidases (22). These enzymes, of primary importance i n l i p o p h i l i c f o r e i g n compound metabolism, are e s s e n t i a l i n the b i o s y n t h e s i s of JH as o u t l i n e d i n F i g u r e 3 (23). They may a l s o c o n t r i b u t e to the i n a c t i v a t i o n of JH as the metabolic scheme i n F i g u r e 4 i n d i c a t e s (_24, ^ 5 , 26) although epoxide hydrase and esterase a c t i v i t i e s dominate here (26, 27). The presence of JH i n the female a d u l t i s necessary f o r v i t e l l o g e n i n s y n t h e s i s (28, 29) i n many i n s e c t s . Therefore, s i n c e the cytochrome P-450 oxidase system i s e s s e n t i a l f o r the maintenance of balanced JH t i t e r s , even s l i g h t changes i n the P-450 system i n response to e x t e r n a l inducers and i n h i b i t o r s could have profound e f f e c t s on the dynamic c h a r a c t e r i s t i c s of i n s e c t p o p u l a t i o n s .


BRATTSTEN

Cytochrome


Farnesyl

P-450

Involvement

pyrophosphate oxidation(s)

Farnesoic acid ^ Methyltransferase, SAM

OCH, Methylfarnesoate CA cytochrome P-450 NADPH + H +, 0

2

OCH.

Figure 3.

Outline of juvenile hormone biosynthesis based on Ref. 23.



Figure 4.

Outline of juvenile hormone inactivation based on Refs. 25 and 26.


09

H

w ο

1

Ο

H

Ο W

>

H

ce 53

Η

>

00

10.

BRATTSTEN

Cytochrome

P-450 Involvement


I n t e r a c t i o n of terpenes and cytochrome P-450.

185

Metabolism

The major metabolic f a t e o f higher p l a n t terpenes i n mammals i s o x i d a t i o n followed by conjugation u s u a l l y to g l u c u r o n i c a c i d (30). However, s p e c i f i c data on even some of the most common compounds are not r e a d i l y a v a i l a b l e . Limonene metabolism seems to have been s t u d i e d unusually i n t e n s i v e l y , maybe due to i t s therapeutic use t o d i s s o l v e p o s t - o p e r a t i v e l y r e t a i n e d g a l l s t o n e s . Limonene i s t y p i c a l l y converted to t r a n s d i o l s v i a cytochrome P-450-mediated epoxidation of e i t h e r one of the two double bonds, followed by epoxide h y d r a t i o n (31). I t i s a l s o converted t o seve r a l a l c o h o l s (32, 33) which may undergo f u r t h e r o x i d a t i o n by dehydrogenases as i n d i c a t e d i n Figure 5. A l l these products are non-toxic and are excreted, i n some cases a f t e r g l u c u r o n i d a t i o n . The cases where terpene metabolism has been studied i n i n sects are very few indeed. C e r t a i n Ips and Dendroctonus bark b e e t l e s convert monoterpenes such as a-pinene, 8-pinene and myrcene to o x i d a t i o n products, some of which have pheromonal a c t i v i t i e s (_5, ^, J34, 35). A Dendroc tonus bark b e e t l e ' s cytochrome P-450 converts a-pinene to s e v e r a l o x i d i z e d products a f t e r induct i o n by a-pinene, and to a t l e a s t one o x i d i z e d product without p r i o r i n d u c t i o n (36). Rat l i v e r cytochrome P-450 a l s o converts a-pinene t o o x i d a t i o n products (36) and t h i s a c t i v i t y i s induced by phenobarbitol and 3-naphthoflavone. There i s a l s o the i n t e r e s t i n g p o s s i b i l i t y that the b a c t e r i a l f l o r a i n the bark b e e t l e s may c o n t r i b u t e to the o x i d a t i o n of a-pinene to t r a n s - and c i s verbenol. A bacterium, B a c c i l l u s cereus, i s o l a t e d from the hindgut of Ips paraconfusus c a t a l y s e s these o x i d a t i o n s (37). The microsomal cytochrome P-450 system i n the midguts of southern armyworm l a r v a e o x i d i s e s pulegone in v i t r o . The two major products, 9-hydroxypulegone and 10-hydroxypulegone are formed by microsomes from l a r v a e induced w i t h e i t h e r pentamethylbenzene o r a-pinene. Microsomes from c o n t r o l (un-induced) l a r v a e only o x i d i s e trace amounts o f the compound. The 9-hydroxypulegone rearranges spontaneously to menthofuran (38). In both these cases where an i n s e c t cytochrome P-450 system has been shown to be r e s p o n s i b l e f o r the o x i d a t i o n of a-pinene and pulegone, the enzyme had to be induced to higher a c t i v i t y to e f f e c t i v e l y c a t a l y s e the r e a c t i o n . This leads to the question of whether i n s e c t P-450-dependent o x i d a t i o n s are s u f f i c i e n t l y a c t i v e i n n a t u r a l s i t u a t i o n s to produce a s i g n i f i c a n t amount o f the metabolites. Due to the importance of cytochrome P-450 o x i d a t i o n s i n p e s t i c i d e metabolism, there are, f o r t u n a t e l y , s e v e r a l s t u d i e s which show that the i n s e c t oxidase system i s e a s i l y and r a p i d l y induced i n response to a l a r g e v a r i e t y o f non-nutrient chemicals i n the food.




186


10.

BRATTSTEN

Cytochrome

P-450 Involvement

187


I n t e r a c t i o n s of terpenes and cytochrome P-450. Induction The c l a s s i c a l examples o f cytochrome P-450 inducers among the terpenes are the hormonal s t e r o i d s . Among the p h y t o s t e r o l s , s i t o s t e r o l , s t i g m a s t e r o l (39), and e r g o s t e r o l (Table V I I ) are i n ducers of the southern armyworm microsomal oxidases. The i n s e c t m o l t i n g hormones a-ecdysone and ecdysterone, w i d e l y occuring i n p l a n t s , a r e very potent inducers of housefly microsomal a l d r i n epoxidase a c t i v i t y (40). Some of the most a c t i v e inducers of i n sect cytochrome P-450 a r e among the monoterpenes, e.g. myrcene, camphene (39) and other shown i n Table V I I . Menthol, menthone, a-pinene, and 8-pinene induce a l d r i n epoxidase a c t i v i t y i n microsomes from the v a r i e g a t e d cutworm larvae (41). However, i n t h i s case, limonene was i n h i b i t o r y (41) whereas both (+)- and (-)limonene are good inducers of the southern armyworm oxidase system (_39 and Table V I I ) . Table V I I Terpene i n d u c t i o n of midgut microsomal cytochrome P-450 and pyrethrum-dependent NADPH o x i d a t i o n i n southern armyworm l a r v a e (42) Cytochrome P-450 (nmole/mg p r o t e i n )

Diet control stigmasterol ergosterol a-pinene 8-pinene (+)-limonene (-)-limonene a-terpinene Y-terpinene

0.350 0.396 0.371 0.881* 0.741* 0.683 0.967* 0.876* 1.028*

X-max (nm) 450.1 450.3 449.8 449.9 449.5 449.8 449.8 450.0 449.9

NADPH o x i d a t i o n (nmole/min,mg p r o t e i n ) 10.96 12.13 16.57 33.27* 33.89* 32.55* 31.20* 38.12* 41.34*

• S i g n i f i c a n t l y d i f f e r e n t from c o n t r o l a t P < 0.001 ( T - t e s t ) . S i x t h i n s t a r l a r v a e were fed ad l i b i t u m f o r 3 days on d i e t s cont a i n i n g 0.2% o f the terpene. Table V I I shows the increase i n cytochrome P-450 content i n microsomes from southern armyworm l a r v a l midguts r e s u l t i n g from d i e t a r y exposure t o s e v e r a l c y c l i c monoterpenes (42). I t a l s o shows a c l o s e l y corresponding i n c r e a s e i n the r a t e of NADPH o x i d a t i o n when pyrethrum i s the s u b s t r a t e (R) being o x i d i s e d . The microsomal cytochrome P-450 system i s arranged as o u t l i n e d i n F i g u r e 6, c o n s i s t i n g of a t e r m i n a l heme-iron p r o t e i n that i n the o x i d i s e d (Fe3+) s t a t e binds the s u b s t r a t e (R). The complex undergoes two reductions d u r i n g which bound molecular oxygen i s converted t o f r e e r a d i c a l s p e c i e s , one of which i s i n s e r t e d i n the s u b s t r a t e molecule, and the other one forms water. The reductions



188


Figure 6.

Outline of the microsomal cytochrome P-450 system.



10.

BRATTSTEN

Cytochrome

P-450

Involvement

189

are s p e c i f i c a l l y dependent upon NADPH and a f l a v o p r o t e i n which t r a n s p o r t s the reducing e q u i v a l e n t s to the cytochrome. The e n t i r e system i s deeply embedded i n the endoplasmic r e t i c u l u m membranes and depends on the membrane p h o s p h o l i p i d f r a c t i o n t o a s s i s t i n the b i n d i n g o f the l i p o p h i l i c s u b s t r a t e molecule. The NADPH-dependence o f f e r s a convenient spectrophotometric method f o r measuring the r a t e o f oxygenation o f s u b s t r a t e s i n cases where there i s no simple means of a n a l y s i n g the r a t e o f product f o r m a t i o n , o r when s e v e r a l d i f f e r e n t m e t a b o l i t e s a r e formed as i n the cases o f pyrethrum and pulegone. The r a t e s o f pulegone-dependent NADPH o x i d a t i o n i n southern armyworm microsomes are shown i n Table V I I I (38). I n t h i s case microsomes from c o n t r o l d i e t - f e d l a r v a e show only t r a c e a c t i v i t y i n agreement w i t h r e s u l t s o f the gas chromatographic m e t a b o l i t e analyses. The southern armyworm cytochrome P-450 system apparentl y more e a s i l y oxygenates pyrethrum (Table V I I ) than pulegone. I n Table V I I I the more potent inducer used i s pentamethylbenzene and, i n accordance, the r a t e of pulegone metabolism i s twice as h i g h w i t h t h i s inducer as when a-pinene i s the inducer. A l l the p l a n t monoterpenes appear t o induce a molecular form of the cytochrome that i s i d e n t i c a l t o the c o n s t i t u t i v e form as i n d i c a t e d by the p o s i t i o n of the a b s o r p t i o n maximum o f the cytochrome-CO d i f f e r e n c e spectrum. Pentamethylbenzene, on the other hand, appears t o i n duce a molecular form w i t h some minute s t r u c t u r a l d i f f e r e n c e ( s ) from the c o n t r o l form as i n d i c a t e d by a s h i f t i n the a b s o r p t i o n maximum t o 449.0 nm. I n the case o f pulegone o x i d a t i o n , the M i c h a e l i s constants (Km) i n d i c a t e t h a t both the pentamethylbenzene and a-pinene-induced cytochrome have s i m i l a r a f f i n i t y f o r the compound. However, the pentamethylbenzene-induced form h y d r o x y l a t e s pulegone i n the CIO p o s i t i o n three times more o f t e n than the a-pinene-induced form (38). The inducing e f f e c t s on the southern armyworm cytochrome P-450-mediated metabolism shown i n Tables V I I and V I I I r e s u l t e d from 3 days f e e d i n g on d i e t s c o n t a i n i n g 0.2% o f the terpene. This c o n c e n t r a t i o n may occur i n many p l a n t s p e c i e s . However, a much s m a l l e r dose of the inducer a l s o e f f e c t s a measureable d i f f e r e n c e i n the oxygenation r a t e s as shown w i t h a-pinene and s i n i g r i n i n d u c t i o n o f southern armyworm P-450 (39). The data i n Table IX show that a s i n g l e dose o f 100 yg/g o f l a r v a l body weight r e s u l t s i n a c t i v i t i e s h i g h e r than those i n c o n t r o l l a r v a e . Cons i d e r i n g the feeding behavior o f the southern armyworm l a r v a e , c o n s i s t i n g of feeding bouts o f 10-20 minutes f o l l o w e d by e q u a l l y long r e s t i n g p e r i o d s ( 4 3 ) , i t seems reasonable t o assume that the metabolism of some b i o a c t i v e l e a f component may be increased enough i n a n a t u r a l s i t u a t i o n t o c o n t r i b u t e s i g n i f i c a n t l y t o the r e v o l u t i o n a r y s t a t u s o f the i n s e c t w i t h i t s host p l a n t ( 3 9 ) . This would be a v a l i d assumption whether a d e t o x i f i e d o r an a c t i v a t e d p o l a r product i s formed. 1



0.815

1.120

a-pinene

PMB

449.0

449.9

450.0

X max (nm)

4.88

80.11 ± 11.52

39.86 ±

Trace

m a x

m

K (uM)

11.64 ±

10.28 ±

NADPH o x i d a t i o n V (nmole/mi^mg p r o t e i n )

Larvae fed ad l i b i t u m f o r 3 days on c o n t r o l d i e t o r d i e t s c o n t a i n i n g 0.2% o f the inducer.

0.346

control

Diet

Cytochrome P-450 concentration (nmole/mg p r o t e i n )

Table V I I I Pulegone-dependent NADPH o x i d a t i o n i n r e l a t i o n t o cytochrome P-450 content i n midgut microsomes form southern armyworm l a r v a e ( 3 8 ) .


2.88

1.94


1.66 2.22 1.92 2.07 1.95

±0.1 ±0.2 ±0.2 ±0.2 ±0.2 (7) (4) (4) (4) (4)

Specific activity (nmole/mi^mg p r o t e i n ) 100 134 115 125 117

Percent of c o n t r o l

P P>0.001 0.002>P>0.001 0.01>P > 0.005

Significance (2-tailed T-test)

The data a r e mean ±. S.D. of (N) experiments.

Larvae were i n d i v i d u a l l y f e d a l i m a bean l e a f d i s c loaded w i t h a terpene dose of 100 ± 2 \xg per gram of t h e i r body weight. They were k i l l e d one hour a f t e r f e e d i n g . A c t i v i t y of p - c h l o r o N - m e t h y l a n i l i n e N-demethylase was measured i n p o s t - m i t o c h o d r i a l supernatants as d e s c r i b e d e a r l i e r ( 7 1 ) ,

control d-carvone 1-carvone caryophyllene carotol

Inducer

Rapid i n d u c t i o n of microsomal oxidase a c t i v i t y By terpenes i n southern armyworm l a r v a e

Table IX



192


Conclusions I t would be c h a r i t a b l e to say that the cytochrome P-450 system i n i n s e c t h e r b i v o r e s does not e n t i r e l y d i c t a t e t h e i r i n t e r a c t i o n s w i t h the host p l a n t s . There i s a very a c t i v e cytochrome P-450 system i n the o l f a c t o r y receptor region of the dog (44), i n d i c a t i n g the t a n t a l i s i n g p o s s i b i l i t y that such an enzyme system, i f s u i t a b l y l o c a t e d i n the i n s e c t , i s a l s o c o n t r i b u t i n g to the e f f e c t s of p l a n t a l l e l o c h e m i c a l s on i n s e c t feeding beh a v i o r . The cytochrome P-450 may a l s o be i n v o l v e d i n pheromone b i o s y n t h e s i s i n more species of i n s e c t s (45) than the bark b e e t l e s . Cytochrome P-450 i s d i r e c t l y i n v o l v e d i n i n s e c t JH b i o s y n t h e s i s , of e s s e n t i a l importance i n both development and reproduction. The microsomal cytochrome P-450 system i s of c e n t r a l and c r u c i a l importance i n the metabolsim of l i p o p h i l i c f o r e i g n compounds of a l l k i n d s , and may have derived i t s major b i o l o g i c a l s i g n i f i c a n c e i n i n s e c t herbivores from exposure to the p l a n t a l l e l o c h e m i c a l s . I t i s beyond doubt that the cytochrome P-450 system s t r o n g l y c o n t r i b u t e s to the great v a r i e t y of unique s o l u t i o n s f o r s u r v i v a l that e x i s t between i n s e c t h e r b i v o r e s and plants. Acknowledgement I thank C.K. Evans, C.A. Gunderson, and J.T. Fleming f o r e x c e l l e n t a s s i s t a n c e . P r e v i o u s l y unpublished work presented here was supported by U.S. Department of A g r i c u l t u r e , (SEA-GAMO) Competitive Research Grant Program grant No. 59-1471-1-1-695-0 and NSF grant PCM 81 00081. Literature cited 1.

2. 3. 4. 5. 6. 7. 8.

9.

C l a y t o n , R.B., i n "Aspects of Terpenoid Chemistry and B i o chemistry" (T.W. Goodwin, ed.) Academic P r e s s , New York, 1971, p. 1-27. Smith, R.H. J . Econ. Entomol. 1965, 58, 509-510. Coyne, J.F., L o t t , L.H. Georgia Entomol. Soc. 1976, 11, 297301. Thompson, M.J., Svoboda, J.A., K a p l a n i s , J.N., Robbins, W.E. Proc. Roy. Soc. Lond. 1972, B180, 203-221. Renwick, J.A.A., Hughes, P.R., Ty, T.D. J . Insect P h y s i o l . 1973, 19, 1735-1740. Vité, J.P., Bakke, A., Renwick, J.A.A. Can. Entomol. 1972, 104, 1967-1975. Sharma, R.N., Saxena, K.N. J . Med. Entomol. 1974, 11, 617621. T i e t z , H.M.: "An Index to the described L i f e Histories, e a r l y Stages and Hosts of the Macrolepidoptera of the C o n t i n e n t a l United States." A.C. A l l y n P u b l . , Sarasota, 1972. Zalkow, L.H., Gordon, M.M. L a n i r , N. J. Econ. Entomol. 1979, 72, 812-815.


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P-450

Involvement

193

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Involvement

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RECEIVED September 27, 1982


Cytochrome P-450 Involvement in the Interactions Between Plant

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