Induction of the polysubstrate monooxygenase system of the native budworm Heliothis punctiger (Wallengren) (Lepidoptera: Noctuidae)

Insect Btochem Vol 15 No 4, pp 551 555, 1985 Pnnted m Great Britain All rights reserved

0020-1790/85 $300+000 Copyright ,( 1985Pergamon Press Ltd

INDUCTION OF THE POLYSUBSTRATE MONOOXYGENASE SYSTEM OF THE NATIVE BUDWORM HELIOTHIS PUNCTIGER (WALLENGREN)

(LEPIDOPTERA. NOCTUIDAE) P J COLLINS* Department of Entomology, Umverslty of Queensland, St Lucre Q 4067, Australia (Received 24 Aprd 1984, revised 25 September 1984) Abstract--The effects of dietary inducers on properUes of the polysubstrate monooxygenase system were investigated in the mldgut, fat body and mtegument of Hehothts puncttger larvae Diets tested were an artificial &et (AD), AD + ~-pmene, AD + phenobarbital, and the whole plant diets of tobacco and lucerne The tobacco and ~-pmene diets reduced forms of P-450 in mldguts with a comparatively low catalytic capaoty for aldrm and a high proportion of hemoprotem in the high spin form In contrast, lucerne and phenobarbital promoted P-450 speoes with a high affimty for aldnn and hemoprotem predominantly m the low spin state P-450 specific activity and n-octylamme binding stu&es revealed that in the fat body phenobarbital pretreatment induced different forms of cytochrome P-450 compared to that predominating in larvae fed on diets high in secondary plant substances All diets promoted predominantly low spin state P-450 m both the fat body and integument Key Word Index Hehothts puncttger, Induction, mixed-function oxldase, cytochrome P-450, polysubstrate monooxygenase

INTRODUCTION The polysubstrate monooxygenase (PSMO) or mixed-function oxldase system of insects plays a central role in the primary degradation of xenoblotics, both natural and synthetic (Brattsten, 1979, Wilkinson, 1983) Two features of this system that make it a particularly effective detoxlficaUon mechamsm are its broad substrate specificity and the ease with which it is induced by a variety of chemicals Although broad substrate specificity and high affinity are inversely related, the latter is achieved m the PSMO system by employing multiple enzymes which are responsive to chemical stress (Mannering, 1980) Xenob~otlcs, therefore, induce the enzyme system that ~s most effective in catalyzing their own metabohsm The central component of the PSMO system, cytochrome P-450, m particular, is stimulated by various inducers Evidence from studies of the mammahan PSMO system has shown that each inducer seems to activate either a specific form of P-450 or a profile of P-450 forms that distinctly increases or decreases and also, that each inducer varies in its inducing capacity (Nebert, 1979) Induction of the insectan PSMO system by xenobiotics has been investigated from two aspects Firstly, paralleling mammalian studies, the effects of synthetic chemicals were studied to gain an understanding of the mechanism of induction In particular, the PSMO systems of housefly Musca domesttca ( L ) (Capdevdla et al, 1973a, 1973b, Yu and Ternere, 1973) and the southern armyworm Spodoptera err*Present address Entomology Branch, Department of Primary Industries, Melers Road, Indooroopflly Q 4068, Austraha 551

dama (Cramer) (Brattsten et al, 1980, Chang et al, 1983) were the model for these studies Secondly, the idea that a major function of the PSMO system was to protect organisms from naturally occurring toxins (secondary plant substances) In their diet was explored (Yu et al, 1979, Berry et al, 1980, Farnsworth et al, 1981, Yu, 1982) These studies have been based on a small number of lepidopteran species The responses of the PSMO system of Insects to Induction suggest that, as in mammals, a number of cytochrome P-450 species are present There has been no systematic study, however, to characterize the various effects of man-made and naturally occurring inducers in one species In addition, although the PSMO system is well known from the mldguts of lepidopteran larvae and housefly abdomens, there have been few studies of the reduction of this system in other tissues (Brattsten et al, 1980, Farnsworth et al, 1981, Tate et al, 1982, Moldenke et al 1983) In this investigation I aimed to assess the influence of synthetic and naturally occurnng inducers on the quantity and quality of mlcrosomal PSMO activities in the mldgut, fat body and integument of the native budworm, Hehothis puncttger MATERIALS AND METHODS

Chemwals Cytochrome c, NADPH, NADP, glucose-6-phosphate, phenobarbital, and bovine serum albumin (BSA) were purchased from either Sigma Chemical Co Inc, U S A or Boehnnger-Mannhelm Austraha Pty Lid, Sydney, Austraha n-Octylamme (99~) was bought from Fluka AG, Buchs, Switzerland and HHDN (aldnn) and HEOD (dieldrin), both 99 0% pure, were gifts from Shell Research Limited, Slttmgbourne, Kent, U K (+) ct-Plnene was purchased from ICN Pharmaceuticals lnc, Plalnvlew, New York,

552

P J COLLINS

Table 1 Effect of various diets on parameters of the polysubstrate monoox'rgenase ~,stem ol tissues lrom last InM,lr [tl'~dt.. t)[ H plltlt[lk'tt Tissue diet

Aldrm epoxldase a~.tl',ltV4;

Cvtochrome P - 4 5 0 t.ontent++

(ytochrome P 4gll ~pet.lhc a~tl~lt~

N ADPH cxto~.hrome lLdu~,i~L

ttAl',Ilx ¢

Mtdgut Arhficlat diet lAD} AD + phenobarbital AD + ~-pmene Tobacco Lucerne

0 424 _+0 057b 0 634 ,+ 0 074a 0 389 ,+ 0 064b 0 636 _+0 061a 0 178 + 0 041 c

0 258 ,+ 0 006t 0 "t22 _+0 (108b 0 271 _+0 010~. 0 346 ,+ 0 028a 0 064 -+ 0 01 Id

1 645 _+() 216 ~ I 975 + () 308 t I 4:~5 + 0 ~4t)a I 838 ± 0 2b~a 2 7"~6+ 136 q4b

411 It) ~ I ~2 t ~" s(I + 22 70* 1~6 II) - 21 40* NI) ' NIl

Fat bod) Artaficml d~et (AD) AD +phenobarbttal AD + x-plnene Tobacco Lucerne

0 104_+0015cd 0 143 ,+ 0 032b 0 119,+ 0011bc 0 203 _+0 027a 0 069 + 0 043d

0 121 ,+0013b 0 129 ,+ 0 014b 0 119,+0 015b 0 276 ,+ 0 051a 0 126 _~0 015b

086:~ ,+(I 10~ab 1 112 + () 24,~ t 0990-+0 ll)4a 0 7~7 + 0 100be 0 549 + I) 286t

~ ~,4 ~_ 0 II) 48 40 ~ 12 70* 41 90 ~ 400" ND ND

Integument Artificial &et (AD) AD + phenobarNtal AD + e-pmene Tobacco Lucerne

0 066 + 0013b 0 079 _+0 007a 0 059 _+0 005b 0 060 _+0 005b 0 077 + 0 003a

0057,+0011bt. 0 066 + 0 006ab 0 073 _4-0 014a 0 048 _+0 004c 0 050 _+0 01 lc

I 141 ±(I 199a 1 189 _+0 187a 0 663 _+0 037b 1 283 + 0 160a I 540 + 0 121~

15 9~, : 1 4(1 17 20 ~- 6 90 24 10 _+7 3(1" ND ND

Values are mean _+SD, n = 4 or more Means not followed by a common letter are slgmficantl~ (P < 0 05t different *Slgmficantlv&fferent (P < 0 05) from artlficml &et (Students t-test) tnmol &eldrm produced/mm per mg protein ~nmol cytochrome P-450/mg protein §nmol dieldrin produced/ram per nmol P 450 ¶nmol cytochrome c reduced/mm per mg protein IlNot done U S A All other chemicals and solvents used were of analytical reagent grade Insects

Larvae were mamtalned on an artlfiCml dtet based on navy beans and wheat germ plus vitamins and preservatives (Shorey and Hale, 19653 until the third lnstar At that time they were placed either on a whole plant diet or an artificml diet containing a chemical inducer The inducers, phenobarNtal and ~-plnene, were blended into the artlficml diet at a rate of 0 25 and 0 2% w/dry weight, respectively Larvae fed on tobacco leaves were placed onto tobacco plants growing In pots under glasshouse condmons Lucerne-fed larvae were kept in plastic boxes (about 10 larvae/box) at 30°C and ambient RH and supphed with freshly cut lucerne each day Enzyme sources and assa3 s

Last instar larvae, 24-36 hr since moulting, were used as the source of mldgut and fat body tissues and newly moulted, 0-6hr, last lnstar larvae were the source of integuments Homogenates and mtcrosomes were obtamed from these ttssues as previously described (Colhns and Hooper, 1984) The enzyme source for each replicate was dertved from a sample of 15 20 insects Aldrin epoxldase assays, N A D P H cytochrome ~ reductase assays and measurement of the n-octylamme (type II) binding spectra were performed exactly as already described (Colhns and Hooper, 1984) n-Octylamlne type II &fference spectra were used to calculate percentage htgh and low spin form P-450 using the method of Jefcoate et al (1969, 1970) Cytochrome P-450 was determined according to the method of Omura and Sato (1964) Homogenate protem was assayed using a bluret method (Layne, 1957) and mlcrosomal protein accordmg to the method of Bradford (1976) BSA was used as the standard for both assays Statzsttcal analvsts

Aldrln epoxldase assays were rephcated four times and data are reported as the mean +standard devlatmn (SD) Cytochrome P-450 measurements and N A D P H cytochrome c reductase assays were rephcated ~ 6 times with fresh mlcrosomes and data are reported as the mean + SD n-Octylamlne spectral binding studies were each rephcated

three times Analys~s of variance was used to test lor treatment &fferences Palrw|se comparisons were made using the least sIgmficant d~fference test Students t-test was also used where appropriate RESULTS I n d u c t i o n of P S M O

a~ttlttte~

P a r a m e t e r s o f the P S M O s y s t e m o f the v a r i o u s tissues were slgmficantly influenced by diet ( T a b l e 1) M l d g u t s s h o w e d a difference o f m o r e t h a n five-fold in P - 4 5 0 tltre a n d 3 6-fold in a l d r m e p o x l d a s e a c t w ity, w h e r e a s these differences were respectwely, 2 3 a n d 2 9 for fat b o d y a n d 1 5 a n d 1 3 for i n t e g u m e n t s T h e m a x i m u m o f the C O r e d u c e d c y t o c h r o m e P - 4 5 0 s p e c t r u m w a s at 4 5 0 n m for all tissues v~th all treatments T h e spectfic activity o f c y t o c h r o m e P - 4 5 0 relative to the m e t a b o h s m o f a l d r m (Table 1) w a s calculated as a m e a s u r e o f the catalytic capaclt~ o f P - 4 5 0 (Berry et a l , 1980) m each o f the tissues C h a n g e s m this catalytic c a p a c i t y reflect q u a h t a t l ~ e c h a n g e s in the p a t t e r n s o f P - 4 5 0 c y t o c h r o m e s m the m l c r o s o m e s (Schwen and Mannering 19823 T h e catalytic c a p a c i t y o f m t d g u t P - 4 5 0 f r o m larvae fed o n lucerne h a d slgmficantly ( P < 0 05) g r e a t e r activity t o w a r d s a l d r m t h a n a n y o f the o t h e r t r e a t m e n t s Th~s result s u g g e s t s t h a t one or m o r e f o r m s o f P - 4 5 0 w~th a l o w e r activity t o w a r d s aldrin were induc,'d to v a r ) lng extents, by all the dtets except lucerne o r t h a t s e c o n d a r y p l a n t s u b s t a n c e s in lucerne inhibited P S M O activity A l t h o u g h fat b o d y f r o m larvae fed o n t o b a c c o h a d high P - 4 5 0 c o n t e n t a n d a l d r m e p o x l d a s e activity w h e n c o m p a r e d to fat b o d y f r o m larvae fed o n lucerne, there w a s n o c o n c o m i t a n t l y large increase m catalytic c a p a c t l t y ( T a b l e 1) T h i s s u g g e s t s that a q u a n t l t a t w e increase r a t h e r t h a n a q u a h t a t t v e c h a n g e m c y t o c h r o m e P - 4 5 0 w a s p r o m o t e d by the t o b a c c o &et I n c o n t r a s t , the A D + p h e n o b a r b i t a l t r e a t m e n t a l t h o u g h p r o d u c i n g n o increase in P - 4 5 0 c o n t e n t

553

P o l y s u b s t r a t e m o n o o x y g e n a s e s y s t e m o f the n a t i v e b u d w o r m Table 2 n-Octylamme difference spectra of oxidized mlcrosomes from tissues of last mstar larvae of H punctlger fed various &ets A,max

Amm

AA (410-500 nm) AA (392-500 rim)

°/o High spin P-450"

Type II bmdmg~

Midgut Artificial diet (AD) AD + phenobarbital AD + apmene Tobacco Lucerne

432 432/1 430 432 432

392 410 392 392 410

094+006 095-t-005 0 13+002 073__.0 11 300+050

17 17 >50 23 <10

0032+0011 0053_+0005 0070+0007 0050+0005 0093+0015

Fat hod) Artlficml diet (AD) A D + phenobarbital AD + ct-pmene Tobacco Lucerne

430 430 430 432 430

410 410 412 412 410

300+050 275-t-050 300_+055 290+085 183+023

<10 <10 <10 <10 <10

0078+0012 0 108_+0015 0042_+0008 0036+0007 0063+0016

Integument Artificial diet (AD) AD + phenobarbital AD + :t-pmene Tobacco Lucerne

432 430 430 430 430

415 415 415 415 410

325_+1 06 250_+050 350_+070 280_+009 275+_003

<10 <10 <10 <10 <10

0070_0005 0061 _+0015 0055+_0013 0 104_+0021 0060_+0020

Tissue diet

Values are mean + S D , n = 3 or more separate experiments *Calculated from the standard curve of Jeffcoate et al (1970) ?Type I1 binding = A absorbance n-octylamme spectrum/nmol P-450

apparently caused a two-fold increase m the specific actw~ty of P-450 compared to that occurrmg m larvae fed on lucerne This suggests a quahtatwe change m the cytochrome P-450 species present Cytochrome P-450 specific actwlty relatwe to aldrm epoxldatlon (Table 1) was slgmficantly (P < 0 0 5 ) lower m integuments from AD + ~t-pmene treated larvae than that calculated for larvae fed A D + phenobarbital or tobacco and these were less actwe than mlcrosomes from larvae fed on lucerne These results suggest that ~-pmene and the secondary plant substances in tobacco may have reduced a quahtatwely different form of P-450 an integument mlcrosomes The N A D P H cytochrome c reductase actwlty of rmdguts was reduced more than three-fold by the addmon of either ct-plnene or phenobarbital to the artificial &et (Table 1) Each of these mducers also produced a seven-fold increase m the reductase activity of fat body mlcrosomes Although phenobarbital had no slgmficant effect, ~t-pmene pretreatment mcreased reductase actwlty of the integument by approx 62~ compared to the artlficml diet

n-Octylamme spectra The n-octylamme spectrum of mldgut mlcrosomes from larvae fed on either the artificial diet, AD + phenobarbital or tobacco had a double trough with minima at 392 and 410nm In all cases the 392 nm minimum was more marked than that at 410 nm In contrast, n-octylamme ehclted a spectrum with a single trough at 410 nm from mldgut mlcrosomes of lucerne fed larvae while m~crosomes from mldguts of larvae pretreated w~th AD + ~-pmene showed an n-octylamlne spectrum with a smgle trough at 392 nm The n-octylamme spectra formed with oxl&zed fat body and integument mlcrosomes showed similar patterns with all treatments (Table 2) Both fat body and integument mlcrosomes contained predominantly low spin cytochrome P-450 with all treatments However, with mldgut m~crosomes, only larvae fed on lucerne did not have measurable

amounts of h~gh spm P-450 Comparison of the results with mldguts in Tables 1 and 2 reveal an reverse relationship between the proportion of high spm state hemoproteln and the specific actwlty of cytochrome P-450 relatwe to the metabohsm of aldrm If the artificial &et is regarded as a control, then phenobarbital pretreatment favoured low spin P-450 and a relatively high level of aldnn epoxldase activity In contrast, ct-plnene mduct~on promoted a high proportion of high spin state hemoprotem m the mlcrosomes accompanied by a comparatwely low epoxtdase actw~ty A s~mdar pattern of reduction occurs when the effects of the two whole plant &ets are compared Larvae fed lucerne had a mlcrosomal P-450 species with a high rate of epoxldatlon but predominantly low spin hemoprotem whde tobacco reduced P-450 species with a lower aldrln epoxldase actwlty but a comparatxvely high amount of high spin state hemoprotem The extent of type II binding (Table 2) with fat body and integument cytochrome P-450 was as high as that of the hemoprotem from mldgut mlcrosomes Further, although there was no obwous relationship between binding capacity and other parameters of the mldgut PSMO system a &rect relatlonshxp between fat body type II bmdmg and fat body cytochrome P-450 specific actwlty (Tables 1 and 2) was apparent Tobacco and ct-plnene reduced, m fat body, P-450 species with a comparatively low affimty for noctylamme while phenobarbital treated larvae had about 40~ higher bmdmg capacity than the control mlcrosomes Slmdarly, fat body mlcrosomes from tobacco fed larvae had about half the blndmg capacity of larvae fed on lucerne Integument mlcrosomes from larvae fed on tobacco had about 40~o higher binding capacity than those from larvae fed the other &ets (Table 2) DISCUSSION

The response to xenoblotlc reduction of mldgut and fat body aldrln epox~dase activity and cyto-

554

P J COLLINS

c h r o m e P-450 c o n t e n t were in the range reported for other lepldopteran species (Brattsten a n d Wilkinson, 1973, Yu et al 1979, Brattsten et a l , 1980, Farnsw o r t h et al, 1981) W i t h the mldgut, however, the relative increase in epoxldase activity was not as high as that in P - 4 5 0 c o n t e n t This result contrasts with some o t h e r reports of investigations of lepldopterous larvae O t h e r a u t h o r s (Berr3 et al, 1980, F a r n s w o r t h et al., 1981) f o u n d t h a t induction caused a greater increase in aldrln epoxldase actlv~t5 t h a n in P-450 c o n t e n t It m a y be that not all the cytochromes P-450 m e a s u r e d from m~dguts of H pumttger were involved in the epoxldat~on of aldrln In c o n t r a s t to epoxidation actwlty, the N A D P H c y t o c h r o m e ~ reductase system of H pun~ttget was m u c h more responsive to mduct~on than t h a t of other lepldopteran larvae investigated (Brattst.'n et al 1980 Tate et a l , 1982) There are no reports ot induction of int e g u m e n t P S M O activities for c o m p a r i s o n Each o f the tissues investigated displayed a distinctive response to induction b~ xenoblotlcs With the m l d g u t P S M O system, the u-plnene a n d tobacco d~ets seemed to induce forms of ~ytochrome P-450 with a comparatively low affinlt~ for aldrln a n d h e m o p r o t e i n p r e d o m i n a n t l y in the high spin state while lucerne a n d p h e n o b a r b i t a l diets induced P-450 species with a high affinity for aldrln a n d hemoprotein p r e d o m i n a n t l y in the low spin state There is a situation s o m e w h a t analogous to this in m a m m a l s A l t h o u g h m a n y c o m p o u n d s induce the mlcrosomal P S M O system of m a m m a h a n livers to varying degrees showing d~fferent properties, two distinctive classes o f inducer, one typlhed by p h e n o b a r b i t a l (PB) a n d the other by 3 - m e t h y l c h o l a n t h r e n e (3-MC), each induce one of two m a j o r groups ot c y t o c h r o m e P-450 species ( M a n n e r l n g , 1980) These two groups o f P-450 are characterized by spectral properties and b,~ differences in substrate speclhc~ty Properties ot these two groups of cytochromes P-450 In m a m m a l s relev a n t to this investigation are (l) that 3-MC induces c y t o c h r o m e P-450 species with a ~ery low rate o f aldrln e p o x l d a t l o n while PB p r o m o t e s species with a high rate of e p o x l d a t l o n (Wolff" et al 1980) and (ll) that 3-MC induces a p r e d o m i n a n c e of high spin state P - 4 5 0 in mlcrosomes while PB type inducers p r o m o t e the f o r m a t i o n of b o t h low a n d high spin forms of h e m o p r o t e i n a l t h o u g h favouring 1o~ spin (Jefcoate et al, 1969) Therefore, the :~-plnene a n d tobacco diets (high in certain secondary plant substances) may induce forms of c y t o c h r o m e P-450 in H p u r e tiger similar m character to those induced by 3-MC m m a m m a l s and that other types o1 diet (phenobarbital a n d lucerne) induce P-450 specle~ in H p u m ttge~ s~mllar to those induced by PB m m a m m a l i a n liver mlcrosomes Unlike the situation in m a m m a l s , there was no a p p a r e n t shift in the cytoc h r o m e P-450 m a x i m u m with induction in mldgut mlcrosomes from H pumttger This may indicate t h a t the induced c y t o c h r o m e P-450 a b s o r b s maximally at the same wavelength ,is the non-Induced P-450 Alternatively, it is possible that the spectrum of an induced P-450 may be hidden b~ the larger spectrum of the n o n - i n d u c e d h e m o p r o t e l n There was also evidence for quahtatl~e changes in the P - 4 5 0 species occurring in fat body mlcrosomes Larvae fed on u-plnene and tobacco diets had fat

body P-450 with a comparatlvel) low affinlt) lol n - o c t y l a m i n e while p h e n o b a r b i t a l induced hemoprotein with a high type l l binding capacity (Table 2) Further, differences between the P-450 speclhc activity o f fat body from larvae fed on lucerne and larvae fed on diets indicate the presence of two or more forms of P-450 in fat bod~ mlcrosomes C y t o c h r o m e P-450 ~peclfiC actlVlt) relative to aldrln e p o x l d a t l o n v~as significantly ( P < 0 05) lower in integuments from c~-plnene treated larvae t h a n that calculated for larvae fed A D + p h e n o b a r b i t a l or tobacco leaves and these were less active than microsomes from larvae fed on lucerne These results suggest t h a t certain secondary plant substances ma~ have induced a qualitatively different form of P-450 in integument mlcrosomes

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