ecological significance of mixed-function oxidations

DRUG METABOLISM REVIEWS, 10(1), 35-58 (1979)

Ecological Significance of Mixed-Function Oxidations* LENA B. BRATTSTEN Department of Biochemistry and Ecology P r o g r a m University of Tennessee Knoxville, Tennessee 37916

I.

II.

m. IV.

V.

VI.

VII . VIII ,

IX.

INTRODUCTION.. .................................. MFO METABOLISM O F PLANT ALLELOCHEMICALS..

FOREIGN COMPOUND METABOLIZING ENZYMES .... MFO STUDIES IN THE SOUTHERN ARMYWORM LARVA .......................................... MFO ACTIVITY LEVELS AND HERBIVORE FEEDING HABITS ................................. INSECT MFO INDUCTION BY PLANTS AND PLANT CHEMICALS .............................. ADVANTAGE O F MFO INDUCTION .................. INDUCTION OF VERTEBRATE MFO ACTIVITY ....... MFO ENZYMES AND HORMONES ...................

36

36

37

39

40

41

48

50

51

*Presented at the AAAS Symposium on the Nature and Functional Role of Cytochrome P-450 Mediated Systems, AAAS Annual Meeting, Houston, Texas, January 4, 1979.

35

Copyright 0 1980 hy Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may he reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying. microfilming. and recording, or by any information storage and retrieval system, without permission in writing from the publisher.

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36 BRATTSTEN

X. MFO ENZYMES AND PHEROMONES ................. 54

Acknowledgments .................................. 56

References ....................................... 56

I. INTRODUCTION

Very recently in human history have people learnt to fabricate and utilize complicated chemicals in the hope of improving life for our- selves. We found by practical, sometimes undoubtedly fatal experi- ence, the first biologically active, potentially toxic molecules in plants and have learnt to improve t6e plants' biochemistry in our synthetic chemistry laboratories. We a r e still looking to the plants for compounds to expand our chemical arsenal. Or we should. Once we got into the business of using powerful molecules, we quickly reached a stage where at least one synthetic chemical is more o r less essential to our every activity, and it is with drugs and pesti- cides that our chemical ability is presently in its most diverse, daring, and desperate state.

alkaloids, phenolics, and quinones, have varied functions in the plants. It is generally accepted that they also play an important ecological role in the protection of plants from excessive utiliza- tion by herbivores. The most damaging herbivores a r e clearly some of the phytophagous insects, not only because of their tre- mendous numbers both in terms of species and in terms of individ- uals, but also because they were on the scene by the time the plants started to evolve their defensive chemicals, They had the oppor- tunity to develop counteracting mechanisms and in many cases even to take advantage of the plants' devices for purposes of their own. A s a result, it is today an extremely r a r e plant that does not have an insect herbivore or two associated with it.

Many of the plant allelochemicals, e.g., terpenoids, steroids,

II. MFO METABOLISM OF PLANT ALLELOCHEMICALS

All tobacco growers know that tobacco is one of the most insecticide-dependent crops grown in the United States. It seems suscep- tible to almost every insect under the sun, and this is despite the sometimes very high concentrations of the highly toxic alkaloid nicotine [ 11. The mechanisms that insect herbivores use to avoid

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MIXED-FUNC "LON OXIDATIONS 37

b

FIG. 1. Mixed-function oxidase catalyzed metabolic conversions of nicotine.

nicotine poisoning range from behavioral, in the green peach aphid, to biochemical, in several lepidopterous larvae. The most important mechanism is the rapid enzymatic conversion of nicotine to one o r more of several nontoxic metabolites. All the conversions in Fig. 1 a r e catalyzed by microsomal mixed-function oxidases (MFOs). Southern armyworm (Spodoptera eridania Cramer) larvae feed with impunity on tobacco plants, and the activity level of their MFO system is directly proportional to their tolerance toward nicotine.

It is well known that MFOs a r e involved in the metabolic breakdown of numerous plant allelochemicals in addition to nicotine. Ro- tenone, the pyrethroids, and the opium alkaloids (Fig. 2) are a few examples. The metabolism of plant chemicals may be a clue to a more original function for the MFO enzymes than drug and pesticide degradation, although the MFOs a r e extremely important in the metabolism of synthetic foreign compounds.

111. FOREIGN COMPOUND METABOLIZING ENZYMES

Figure 3 shows schematically the metabolism of foreign compounds. It is conveniently categorized into primary and secondary metabolism. The purpose and basic function of foreign compound metabolism is to convert lipophilic foreign chemicals to polar metabolites that the organism can easily rid itself of by means of its excretory system. Excretory systems a re water-based and thus

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3t3 BRATTSTEN

cH30@

0 $ 8 CH,

hH3 Rotenone Thebainr

Pyrethrin I

FIG. 2. Rotenone, pyrethrin I , and thebaine.

the metabolite needs to be made hydrophilic in the metabolic process. Lipophilic foreign compounds a r e the potentially most toxic ones at low concentrations due to the difficulty in their excretion and their tendency to accumulate in membranous and other lipid-rich tissues, The MFOs comprise the most important system in the primary degradation of lipophilic foreign compounds. The products of the MFO- catalyzed conversions a re often conjugated to a highly water-soluble endogenous substance, e.g., a sugar, amino acid, sulfate, phosphate, o r short peptide such a s glutathione. The conjugation process renders the MFO metabolites highly water soluble and excretable. The MFOs thus occupy a key position and act in concert with a ple- thora of conjugating enzymes. There is also a veritable battery of esterases, reductases, group transfer enzymes, and epoxide hy- drases that a r e also involved in the primary metabolism together with and/or complementing the MFOs. This arrangement virtually insures an enzymatic attack on any and all foreign compound molecules that manage to reach the interior of the organism.

MFO enzymes occur in all organisms except anaerobic bacteria. In vertebrates they are primarily located in the liver. In a rat the liver cells have about 10 times higher specific MFO activities than other tissues and also more diversified activities [2]. MFO activity is also present in other tissues, e, g., the small intestine,

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MIXED-FUNCTION OXTDATIONS 39

LlPOPHlLlC P HYDROPHILIC

(1$ PRIMARY *o products - SECONDARY 0 products

foreign compound Oxidation Conjugation

with sugara, amino acids,

sulfate, phosphate, otc.

Rsduction

Hydrolysis

Group tra nsfcr

EXCRETION

FIG. 3. Schematic view of foreign compound metabolism,

the lungs, the kidneys, placenta, and skin. In insects there is no tissue with such predominance in MFO activity as the vertebrate liver. A s with vertebrates, MFO activities in insects a r e found in several different tissues. Beside the gut, MFOs a r e found also in fat body and Malpighian tubules (see Table 8).

anism of unavoidable foreign compounds, such as those incorporated into a vegetable diet, by virtue of its extremely diversified ability to catalyze a large number of reactions. MFOs catalyze CH-hydroxylations including N- and 0-dealkylations; n-bond oxygenations such a s aromatic hydroxylations, epoxidations, and thiophosphate oxidations; and also thioether and nitrogen oxidations [ 31. This catalytic diver- sity enables the MFO system to attack a large variety of molecular structures with only a suitable lipophilicity as a common denomina- tor. Another crucial feature of the MFOs is their sensitivity to en- vironmental factors, mainly inducing and inhibitory chemicals.

The MFO system is particularly well suited a s a detoxication mech-

IV. MFO STUDIES IN THE SOUTHERN ARMYWORM LARVA

Krieger and Wilkinson [ 4 ] showed that the midgut tissue of the broadly polyphagous herbivore, the southern armyworm larva, contains

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40 BRATTSTE N

Whole k a d Fat Gut Molp. Remains larvae body tubules

FIG. 4. Relative aldrin epoxidase activity in crude homogenates of isolated tissues of the southern armyworm larva [ 43,

a highly active MFO system (Fig. 4). We have four model substrates for measuring MFO activity, namely aldrin [4], p-chloro N-methylaniline [5], aniline [ 61, and methoqresorufin [52], in the armyworm gut tissue.

We also measure microsomal cytochrome P-450 as the reduced carbon monoxide complex [7 ] and the reduction of cytochrome c by the NADPH-dependent cytochrome P-450 reductase [ 81.

The MFO system present in the armyworm gut microsomes have been instrumental in establishing a relatively firm correlation between these enzymes and the feeding habits of herbivores.

V. MFO ACTIVITY LEVELS AND HERBIVORE FEEDING HABITS

The apparent higher tolerance to synthetic insecticides in polyphagous insect herbivores compared to insects with a more restricted range of acceptable food plants has often been observed. Swingle [ 91

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MIXED-FUNCTION OXIDATIONS 41

TABLE 1

Mixed-Function Oxidase Activity Levels in 35 Species of Lepidopterous Larvae in Relation to their Feeding Habits [ 113

Feeding range Aldrin epoxidase (no. of plant families) No. of species @mol/min/mg protein)

Monophagous (1) a 20.42 9.1

Oligophagous (2-10) 15 90.0 2 33.6

Polyphagous (11 or more) 12 297.4 L 65.9

suggested in 1939, long before the insecticide metabolizing enzymes were known and before the advent of synthetic organic insecticides, that the feeding antecedents of a lepidopterous larva were correlated to its insecticide tolerance. Gordon [lo] in 1961 pointed out the possibility that polyphagous herbivores have been forced to develop the ability to detoxify a wide variety of potential plant toxicants and will therefore be better equipped also to detoxify synthetic insecticides by enzymatic degradation. More recently, Krieger et al. [ll] concluded that generalist feeders in a sample of 35 species of lepidopterous larvae indeed have significantly higher levels of specific MFO activity in their guts than do restricted feeders (Table 1). These specific activity levels may reflect adapted and/or genetically determined detoxication capacities at the particular point in their life stages that the Lar- vae were assayed. Even more important, though, in the feeding strat- egies of herbivores, in particular those of polyphagous herbivores, is undoubtedly the insects' ability to adapt quickly and temporarily to exposure to varying levels and kinds of plant chemicals. The insect herbivore needs an MFO system that will acquire increased specific activities with enough speed and in the presence of low levels of potentially toxic plant chemicals to provide temporary protection from poisoning. In other words, they need an MFO system that can be induced easily.

VI. INSECT MFO INDUCTION BY PLANTS AND PLANT CHEMICALS

By using crude gut homogenates for measurements of aldrin epoxidation and p-chloro N-methylaniline N-demethylation, we found that

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42 BRATTSTEN

TABLE 2

Mixed-Function Oxidase Activities in Crude Midgut Homogenates of Southern Armyworm Larvae after Feeding for 24 Hours on

Foliage of the Plants Listeda

Specitic activity (nmol/mg protein, min)

N-Demethyl- Body weight Food plant ation Epoxidation @/worm)

Lima bean 0.29 2 0.03 (4) 0.26 2 0.02 (4) 0.39 2 0.03 (6)

Carrot 2.73 2 0.55 (4) 1.08 L 0.14 (4) 0.32 L 0.04 (5)

Spananthe paniculata 1.92 f 0.20 (3) 0.65 L 0.04 (3) 0.17 2 0.02 (4)

Parsley 1 . 2 4 f 0 . 1 6 (3) O.lOLO.02 (4) 0 .17L0.02 (4)

Coriander 0.99 f 0.11 (4) 0.09 2 0.01 (3) 0.19 2 0.02 (5)

Basil 0.76 0.11 (3) 0.40 2 0.06 (4) 0.34 f 0.02 (4)

Tomato 0.46 2 0.06 (3) 0.20 L 0.02 (3) 0.34 f 0.02 (3)

aMean L S.E., numbers of replicates in parentheses.

MFO levels in the armyworm gut a re totally dependent on what plant the larvae fed on during the preceeding 24 hours (Table 2). Larvae feeding on the leaves of the lima bean, Phaseolus lunatus, have the high- est body weight and lowest specific activities. This may somehow be related to the lima bean plant being the favored food plant of armyworm larvae. They feed freely and thrive on at least 40 species of plants [ 121. Of the other plants, the carrot, parsley, coriander, and Spananthe a r e umbellifers characterized by their content of coumarins, flavonoids, acetylenic compounds, and benzodioxole de- rivatives [13]. Al l of the plants shown in Table 3 stimulate the armyworm gut N-demethylase, sometimes very much (e.g., carrot). A few plants have an inhibitory effect on the epoxidase activity a t the same time that they stimulate N-demethylation. We are now studying the effects of some of the chemicals in these plants, mainly the carrot, to explore the interactions of individual chemicals on a greater variety of MFO parameters in greater detail. In another study, commercially

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TABLE 3

Effect of Diets Containing Purified Plant Chemicals on N-Demethylase Activity Levels in Crude Midgut Homogenates of Southern

Armyworm Larvae [ 141

Treatment (24 hr) % in diet Specific activity % of control

Control

trans -2-Hexenal

(+)-a-Pinene

0-Pinene

(+)- Limonene

Camphene

Myr c ene

0 -Carotene

C adin en e

Stigmast erol

Sitosterol

Quinoline

Quinazoline

Sinigrin

-

0.20

0.26

0.14

0.20

0.20

0.20

0.20

0.10

0.20

0.20

0.10

0.20

0.20

0.70 0.06

1.02 L 0.05

2.18 L 0.08

1.51 2 0.12

1.30 f 0.12

1.48 L 0.17

2.70 L 0.20

1.13 L 0.07

1.19 L 0.08

1.00 k 0.10

1 . 9 4 f 0 . 0 9

1 . 8 9 2 0 . 1 5

1 . 0 9 2 0.12

1 .39L0.06

~~

100

145

312

216

186

211

386

161

170

143

277

270

156

198

available plant chemicals were incorporated in an artificial diet based on agar and fed to larvae for 24 hours [ 143. The crude gut N-demethylase activities were in most cases substantially induced, Table 3, indicating the likelihood of the relation between MFO activities and plant chemicals in the data in Table 2. Figure 5 also illustrates the amazing flexibility of armyworm gut MFO activities. In this experi- ment armyworm larvae were fed for 24 hours on carrot foliage, lima bean foliage, o r seeds of the showy crotalaria, C. spectabilis. The microsomal N-demethylase activities and cytochrome P-450 levels in the armyworm gut a r e shown and also those in the gut tissues of two species that normally feed on these plants: larvae of the black

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44 BRATTSTEN

NCH3

nmole/min,mg protein

IS

10

5

P-450

nmolelmg protein

ST +AW- uo + c a r r o t 1 Bean I- Crotalaria-l

FIG. 5. Microsomal cytochrome P-450 levels (right ordinate) and p-chloro N-methylaniline N-demethylase activities (left ordinate) in midguts of black swallowtail (ST) larvae fed on carrot foliage; of southern armyworm (AW) larvae fed on carrot leaves, lima bean foliage, o r showy crotalaria seeds; and of bella moth (UO) larvae fed on crotalaria seeds. Incubation conditions and measuring meth- ods were a s described in Ref. 5.

swallowtail butterfly, Papilio polyxenes, on the carrot, and larvae of the bella moth, Utetheisa ornatrix, on the seeds of the showy crotalaria. The latter plant contains high levels of monocrotaline in the seeds (Brattsten and Van de r Meer, unpublished), The armyworm MFO activities adjust to levels very similar to those of the larvae that normally feed on the plant, They reach very high levels in carrot-fed larvae whereas they a re virtually completely suppressed in

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MIXED- FUNC TlON OXIDATIONS 45

control -+- I l l 1 I I

2 4 6 9 12 24

Hours on diet

FIG. 6. Increase in p-chloro N-methylaniline N-demethylase activities in crude midgut homogenates of last instar southern armyworm larvae feeding on diets containing 0.04% (+)+-pinene ( a), 0.1% sinigrin (0), 0.2% trans-2-hexenal (A), o r a control diet (0) without added chemicals. Activities are shown as a function of time of feeding. Vertical bars indicate the range in the activity data with the points indicating the mean values and N = 2 o r 3 [ 143.

the crotalaria-fed larvae. By using chemicals extracted and purified from these two plants, we a r e now establishing more firmly the con- nection between the larval MFO activities and the chemical content of the plant. Both swallowtail larvae and bella larvae can be reared on artificial diets devoid of the dominating plant chemicals. Preliminary results with bella larvae reared on a pinto bean diet [15] indicate that these larvae, conversely, a r e capable of higher MFO levels in the ab- sence of monocrotaline. The data presented so far thus illustrate that the gut MFOs of a generalist insect herbivore a r e highly influenced by a wide variety of plants, probably by the plant chemicals, after 24 hours of exposure.

It is ecologically very important that the increase in activity occurs rapidly enough to provide protection during continued feeding. Fig- u re 6 shows that induction by a-pinene and sinigrin (ally1 glucosinolate) is indeed very rapid, The induction effected by trans-2-hexenal fol- lows the course of that effected by the other inducers after a short initial depression of the activity. Whereas a-pinene and sinigrin

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46 BRATTSTEN

TABLE 4

Effect of Single Oral Dose of a-Pinene o r Sinigrin on N-Demethylase Activity Levels in Crude Gut Homogenates of

Southern Armyworm Larvae [ 141

Time after Specific activity (nmol/mg protein/min), consumption (mean S.E.)

(min) Control (+)-a-Pinene Sinigrin

2 0.886 2 0.036 0.958 L 0.078 0.866 f. 0.080

30 0.826 f 0,045 1,000 k 0. 063a 1.032 +. 0. Olga

60 0.798 & 0.017 0.953 2 0. 04ga 0.988 2 0. 105a

a P < 0.1 relative to corresponding control (Student's t-test).

have no in vitro effect on the N-demethylase activity, hexenal is slightly inhibitory, an effect that probably explains the initial depression as an artifact. The armyworm gut MFO system, in fact, responded so quickly that within 30 minutes after one oral dose of a-pinene o r sinigrin a significant (P c 0.1) increase in activity could be measured (Table 4 [14]). Further evidence of the flexibility of the armyworm gut MFO system is shown in Fig. 7 [16]. In this experi- ment larvae were fed diets containing the synthetic chemicals pentamethylbenzene o r diethyl 1,4-dihydr0-2,4,6-trimethylpyridine-3,5- dicarboxylate (DDC) for 24 hours and subsequently continued on a chemical-free control diet, The data show that with pentamethylbenzene, which undergoes rather rapid degradation, the MFO system responded very quickly not only to the introduction of the substance but also to its removal from the diet. In the case of the metabolically more stable DDC, the elevated activity level per- sisted even after its removal from the diet. Figure 8 further shows that the MFO system in the armyworm gut can be triggered to increased activities a t any point in time during the larval instar [Sl .

The question of whether the armyworm gut MFO system responds to a low enough dose of an inducing plant chemical for the induction to be of ecological significance i s answered in Fig. 9. A dose of either a-pinene, sinigrin, o r hexenal that is low enough to be

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20

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0

-er-

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n-=

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40

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Hours

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FIG. 7.

Cha

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in

p-ch

loro

N

-met

hyla

nili

ne N

-dem

ethy

lase

act

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ies

in c

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gut

hom

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B)

for

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0.2%

die

thyl

1,

4-di

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(C)

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[l6

].

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48 BRATTSTEN

I I I 1 I 10 40 60 80 100

Hours from molt

FIG. 8. Changes in p-chloro N-methylaniline N-demethylase activities in a mitochondria1 supernatant of midgut tissues of southern armyworm larvae feeding on a control diet (A), a diet containing 0.2% pentamethylbenzene for 24 h r only followed by control diet at 24 hr after the molt (B) o r a t 48 h r after the molt (C) 151.

encountered in leaf tissues stimulates the enzymes to higher activity. The figure also shows that there seems to be an upper limit to the activity. This probably results from increasing distastefulness of the diet a t high concentrations with accompanying reduction in food intake and reduced growth rates, In the case of sinigrin, a toxic metabolite is produced in the gut that causes reduced vigor of the larvae at high concentrations [ 141.

VII. ADVANTAGE OF MFO INDUCTION

The next question is whether the increased MFO levels a r e of sur- vival value to the insect herbivore in terms of increased tolerance to potential toxicants, Table 5 shows that armyworm larvae with twofold

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c

Sinigrin (+)-a- pinene Tmm-2-hexenal

FIG. 9. Dose-dependent variations in p-chloro N-methylaniline N-demethylase activity in crude midgut homogenates of southern armyworm larvae feeding for 24 h r on diets containing sinigrin, (+)- cr-pinene, o r trans-2-hexenal at the concentrations indicated (o/o of wet weight of diets). Larvae were given free access to the diets r141.

TABLE 5

Acute Toxicity of Nicotine to Southern Armyworm Larvae in Relation to their Gut N-Demethylase Activity Levels [14]

Pretreatment N-CH, LD,, L S . E. (diet, 24 hr) (% of control) Treatment (oral) (mg/kg)

Control 100 Nicotine 2670 2 430

0.10% (+)-a-pinene 227 Nicotine 4600 2 210

Control 100 Nicotine and piperonyl- 770 f 120 butoxide

0.10% (+)-a-pinene 220 Nicotine and piperonyl- 530 L 40 butoxide

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50 BRATTSTEN

95

90

80 70 60 50 40 30 20

10

5

FIG. 10. Log dosage vs probit mortality plot showing the acute, oral 24 hr toxicity of carbaryl to southern armyworm larvae that had been feeding a control diet (A), a diet containing 13.5 +mol/g of diet of hexamethylbenzene (B), o r a diet containing 13.6 pmol/g of diet (0.2%) of pentamethylbenzene [5].

increased gut MFO levels due to ingestion of a-pinene containing diet a r e twice a s tolerant to nicotine as a r e (the already tolerant) larvae with control level MFO activities, Also shown is the reduced tolerance in the presence of the MFO inhibitor piperonylbutoxide, a commercially used insecticide synergist [ 143.

The increased tolerance i s not restricted to the situation with this pair of chemicals, a-pinene and nicotine, but was also observed with a pair of synthetic chemicals, Figure 10 [51 shows the effect of MFO induction by pentamethylbenzene on armyworm tolerance to the widely used carbamate insecticide carbaryl, known to undergo MFO-catalyzed degradation [ 3 1,

VIII. INDUCTION O F VERTEBRATE MFO ACTIVITY

Most studies of vertebrate MFO induction have been done with the classical inducers phenobarbital, 3-methylcholanthrene, o r benzo(a)- pyrene. A very large body of information on the effects of these compounds on MFO systems in the vertebrate liver has been accumulated.

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Vertebrate liver MFOs a r e also induced by naturally occurring sub- stances such a s plant allelochemicals [17, 183. A s reflected by the information collected in Table 6, most work has concerned itself with effects on rat liver after some llun-naturalll route of administration. In most cases the liver induction seems to take a fairly long time and a fairly high dosage. Very few studies have dealt with vertebrate intestine, due to problems with contaminating digestive enzymes. Only in one case was a time-dependent effect of a synthetic inducer, 3- methylcholanthrene, on intestinal MFO activities studied. In this study it was shown that there was a significant increase within 90 minutes of the dosing (intragastrically). Few if any studies with rat liver microsomes have addressed the question of the time dependency of the increase in MFO activities. Therefore, no conclusions can be drawn concerning the ability of vertebrate MFOs to respond with ecologically significant speed to the presence of potential inducers o r to realistically low doses. The possibility of a l lfasterl l MFO system in alimentary tracts than in liver tissues of small herbivorous vertebrates is very interesting from an ecological standpoint. Experiments with insect tissues to study this possibility a re in progress in our laboratory. Few studies other than with the southern armyworm demonstrate the rapidity of the induction in insect tissues. Yu and Terr iere [ 533 showed a significant increase in microsomal epoxidase activity within 6 hours after injection of 0-ecdysone into adult houseflies.

tributed among insect tissues than in the vertebrate body. Table 7 shows a few examples of tissue distribution of MFOs in various insect species. In all insects listed in Table 7, MFO activity i s found in more than one tissue, Induction studies that include time-course measurements have been restricted to the use of the most active tissue, e.g., armyworm midgut.

A s mentioned earlier, MFO activities a r e often more evenly dis-

IX. MFO ENZYMES AND HORMONES

Cytochrome P-450-dependent MFOs located both in liver microsomes and in mitochondria of adrenal cortex and other steroid-hormone producing vertebrate tissues are involved in the metabolism of steroid hormones [19, 201. The mitochondria1 MFOs not only differ in their composition from the microsomal ones in having a nonheme iron-sulfur protein incorporated a s an essential part of the system, they a r e also less influenced by external chemical inducers.

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TA

BL

E 6

Indu

ctio

n of

Ver

tebr

ate

Mix

ed-F

unct

ion

Oxi

dase

s by

Pla

nt A

llel

oche

mic

als

and

3-M

ethy

lcho

lant

hren

e (3

-MC

)

Indu

cer

dose

, ro

utea

T

ime

for

MFO

sou

rce

effe

ct

Ref

s.

Gos

sypo

l, 1

00 m

g/kg

, o

ral

Pyr

ethr

um,

200

mg/

kg,

oral

a-T

erpi

neol

mix

, in

hala

tion

Isos

afro

l,

50 m

g/kg

, i.p

.

aLP

inen

e, 2

20 m

g/kg

, s.

t.

Euc

alyp

tol,

500

mg/

kg,

s. c.

Spi

rono

lact

one,

100

mg/

kg,

i.p.

Caf

fein

e, 7

5 m

g/kg

, i.

p.

Cau

lifl

ower

, o

ral

Tea

sol

ids,

ora

l

3-M

C,

100

mg/

kg,

i.p.

20 m

g/kg

, i.g

.

P-N

apht

hofl

avon

e,

80 m

g/kg

, i.

p.

Rat

liv

er

Rat

liv

er

Rat

liv

er

Rat

liv

er

Rat

liv

er

Rat

liv

er

Mou

se l

iver

Rat

liv

er

Rat

liv

er

Rab

bit

live

r

Mou

se l

iver

Rat

sm

all

inte

stin

e

Rat

col

on

4 da

ys

13 d

ays

4-7 days

3 da

ys

4 da

ys

4 da

ys

4 da

ys

3 da

ys

3-4

wee

ks

13 w

eeks

24

hou

rs

1-1/

2 ho

ur

4 da

ys

Abo

u-D

onia

and

Die

cker

t [32

]

Spr

ingf

ield

et

al.

[ 333

Cin

ti e

t al.

[34]

Vai

nio

and

Par

kki [

351

Pap

and

Sza

rvas

[36

1

Jori

et

al.

[37]

Fel

ler

and

Ger

ald

1381

Lom

broz

o an

d M

itom

a [ 3

93

Bab

ish

and

Stoe

wsa

nd [

401

Bab

ish

and

Stoe

wsa

nd [

41)

Gel

boin

et a

l. [ 4

23

Sto

hs e

t al

. [4

3]

Fan

g an

d S

trob

el [ 44

3

a- 1.p.

=

int

rape

rito

neal

ly;

s.t.

=

sto

mac

h tu

be;

s.~

. = s

ubcu

tane

ousl

y; i

.g.

= in

trag

astr

ical

ly.

W !a

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MIXED- FUNCTION OXIDATIONS 53

TABLE 7

MFO Activities in Some Insect Tissues

Order of

specific Species Stage Tissuea activity Refs,

Heliothis vi r esc ens

Larva Gut Fat body

xxxx xx

Bull and Whitten C 451

Agrotis ypsilon Larva Gut Fat body M. t.

Thongsinthusak and Krieger [46]

xxxx xx xxx

Kuhr [47] Trichoplusia ni Larva Gut Fat body M. t.

xxx xxxx

X

Krieger et al. [48] Antherea pernyi Larva Gut Fat body M.t.

xxxx X

xxx

Utetheisa ornatrix Larva Gut Fat body

X X

Spodoptera e r i dan ia

Larva Gut Fat body

xxxx xx

Periplaneta americana

Adult Gut Fat body

Turnquist and Brindley [ 491

xxx xxxx

Acheta domesticus Adult Gut Fat body Met.

Benke and Wilkin- son [50]

xx X

xxxx

Benke et al. [51] Gromphadorhina portentosa

Adult Gut Fat body M.t.

xxxx xx xxx

aM,t. = Malpighian tubules.

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54 BRATTSTEN

I

OH

FIG. 11. Structure of the gypsy moth sex attractant, disparlure, and inactive precursor (olefin) and breakdown product (transdiol).

Microsomal MFOs in the specialized steroidogenic tissues also appear more substrate specific than the liver microsomal MFOs, and conse- quently metabolize foreign compounds to a lesser extent than the liver M F O ~ r z i i .

So far, only one mitochondrial cytochrome P-450-dependent MFO system has been found in an insect. Bollenbacher et at. [ 223 dem- onstrated the conversion of a-ecdysone to the active insect molting hormone, P-ecdysone, by a mitochondrial MFO system located in the fat body of the tobacco hornworm larva, It is unknown to what extent this insect MFO system parallels the vertebrate mitochondrial MFOs. Microsomal MFOs have also been implied in the juvenile hormone metabolism in adult houseflies [23] and in Blaberus giganteus [ 241. The possibility obviously exists that insects also have several species of MFOs with differing and sometimes strictly internal functions,

X. MFO ENZYMES AND PHEROMONES

It seems rather certain that insect MFOs will one day be found responsible for the synthesis of sex pheromones, The top molecule in Fig, 11 was found in the pheromone gland of the female gypsy moth (Lymantria dispar) in 10-fold excess of the middle molecule which is the active pheromone, disparlure [25]. The bottom molecule is inactive as a sex attractant. It has been shown that the moth converts the olefin4top) to the epoxide (middle) in vivo [26]. crosomal MFO preparation from the armyworm midgut also catalyzes

An active mi-

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FIG. 12. Structure of monocrotaline and sex attractants isolated from danaid butterflies.

the epoxidation of the olefin (L. B. Brattsten, C. F. Wilkinson, and T. Taylor, unpublished). It seems likely that a microsomal epoxide hydrase could be responsible for the inactivation of the pheromone to the trans-diol (bottom) once it is no longer needed. This possibly quite specialized MFO system awaits experimental investigakion.

Another intriguing possible involvement of MFO enzymes in the production of sex attractants is the conversion of the toxic pyrro- lizidine alkaloids to pheromone molecules. Figure 12 shows monocrotaline, a rather common allelochemical in leguminous, composite, and boraginaceous plants. The three bottom molecules a r e sex attractants found in danaid butterflies [ 27, 281 and in arctiid moths [ 29; T. Eisner, personal communication]. Male queen butterflies that were deprived a s larvae of a source of the alkaloids suffer a com- plete mating failure and are devoid of the pheromone molecules [30, 311.

How did the MFOs evolve into their highly distinctive endocrine, exocrine, and ecological roles ? One possibility i s that the evolution of sterols in plant tissues reinforced an originally endocrine biochemical mechanism to expand into a system with the ability to metabolize a t first related, and then a wider variety of dietary, nonnutrient chemicals to excretablc products. Once the broad catalytic potential was established, the MFOs could be exploited again for more specific

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56 BRATTSTEN

tasks in the conversion of molecules at least sometimes derived from the diet into the biologically extremely important sex attractants.

Acknowledgments

Data not previously published were obtained in projects supported in part by NIH grant ES-00400 awarded to Dr. C. F. Wilkinson and NIH grant AI-02908 and NSF grant BMS75-15084 awarded to Dr. T. Eisner and by NIH grant BRSG-RR-07088 to Dr. Brattsten. The skilled technical assistance of Brenda Gordon and Carla A. Gunder- son i s gratefully acknowledged, May Berenbaum kindly supplied the swallowtail larvae and the Spananthe plants,

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c 23

r 31

c51

[ 61

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--

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ecological significance of mixed-function oxidations

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