Hormonal modification of microsomal oxidase activity in the housefly

Life Sciences Vol . 10, Part II, pp. 1173-1185, 1971 . Pried in Great Britain

Pergamon Press

HORMONAL MODIFICATION OF MICROSOMAL OXIDASE ACTIVITY IN THE HOUSEFLY* S . J . Yu and L . C . Terriere Department of Entomology, Oregon State University Corvallis, Oregon 97331 (Received 5 August 1971; in final form 22 September 1971) Su~ary The effect of treatments with synthetic juvenile hormone and ecdysterone on microsonal heptachlor epoxidase activity was investigated in female adults of the Isolan-B strain of the housefly . Plies were treated topically or by injection and the nicrosames prepared and assayed at intervals up to 36 hours later . Both hormones caused increased epoxidase activity, up to 65~t above controls, with the maximim for ecdysterone occurring at 6 hours and for the juvenile hormone at 18 to 24 hours after treatment . Enhancement in the case of the juvenile hormone xas preceded by an inhibitory phase at 12 to 18 hours . The increased activity of these enzymes could be prevented by simultaneous treatment with actinomycin_D . The enhancement caused by the juvenile hormone could also be blocked by treatment with cycloheximide but this inhibitor had no effect on the activity of ecdysterone, presumably because of the shorter time involved. Microsomal epoxidase assay of untreated larvae revealed a burst of activity in the late third instar about 11 hours before pupation . Attempts to induce this activity by treatment with ecdysterone were unsuccessful . In insects the growth and molting hormone, ecdysone, stimulates the synthesis of RNA and protein in various tissues, and increases mitochondria and the endoplasmic reticules in epidermal cells (1,2,3,4) :

Karlson and Sekeris (S)

have demonstrated that in Calliphora larvae ecdysone induces dope decarboxylase, one of the key enzymes in the production of .N-acetyldopamine, the sclerotizing agent.

Later they suggested that the biochemical mechanism of this hormone was

to regulate certain genes by de-repression (6) . The juvenile hormone stimulates RNA and protein synthesis in isolated fat body cell nuclei but the degree of stimulation depends on the stage of development of the larvae (Calliphora) being treated (7) .

These workers also found

*Oregón Agricultural Experiment Station, Technical Paper No. 3129 supported by USPHS grant No . 5-RO1-ES00362-13 . 1173

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H~sefly Microeomal Oxidase Activity

Vol. 10, No . 20

ecdysone to stimulate RNA and protein synthesis in the fat body cell nuclei and observed that when both hormones were added their stimulating effects were neutralized .

Since the fat body is rich in the microsomal oxidases, it seemed

likely to us that these enzymes would be among those whose production was increased as a result of hormonal action .

We now report the stimulation of

microsomal heptachlor epoxidase activity in housefly microsames after treatment of adults with ecdysterone and synthetic juvenile hormone .

Our results indicate

that this stimulation is due to induction of enzyme synthesis . Materials and Methods All of the work reported here was done with the housefly strain which we have designated Isolan-B .

These insects were used because of their very high

microsomal oxidase activity (8) making the enzyme assays more convenient . Ecdysterone (20-hydroxyecdysone,~-ecdysone or cxustecdysone) was purchased from Mann Research Laboratory . ecdysone .

We will refer to the endogenous counterpart as

Synthetic juvenile hormone (9) was obtained from Calbiochem .

Acti

nomycin D and cycloheximide were purchased from Sigma Chemical Company . Hormone treatment Groups of 50 female adults were anesthetized with C0 2 and topically treated on the abdomen with juvenile hormone or ecdysterone in 1 pl acetone . Controls were treated with 1 pl acetone . For the ecdysterone study, female adults were also injected in the thorax with 1 pl of an aqueous solution of ecdysterone .

Larvae were ligated at the

seventh segment to isolate the neuroendoerine system and 1

ul

of an aqueous

solution of ecdysterone was injected into the body cavity of the ligated posterior part by inserting the needle through the ligature loop .

Controls were

treated similarly with 1 ~1 water . In experiments with the inhibitors actinomycin D and cycloheximide, female adults were injected in the thorax with 1 ~1 aqueous solution of the inhibitor or with a mixture of the inhibitor and ecdysterone . with 1 K1 water,

Controls were injected

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Housefly Microsomal Oxidase Activity

1175

After treatment, adults were held in pint jars with food and water provided . Larvae were placed on moist filter paper in Petri dishes at 21 °C . EnzYm e-assa s The oxidase activity of the microsomes was assayed with helptachlor as substrate, the product being heptachlor epoxide . methods described previously (10) .

Microsomes were prepared by

In the case of larvae treated with ecdy

sterone, microsanes were prepared from body parts posterior to the ligature . The standard 5 ml incubation mixture was of 0 .1 M sodium phosphate buffer, pH 7 .5 a~ contained microsomes equivalent to 10 insects ; 1 .8 pmoles of NADP ; 18 pmoles of glucose 6-phosphate ; 0 .5 units of glucose 6-phosphate dehydrogenase and 100 nmoles of heptachlor in 0 .1 ml methyl cellosolve .

The incubations were

carried out with shaking at 34 ° C in an atmosphere of air for 15 min . bations were in duplicate .

All incu-

Heptachlor epoxide was measured by GLC (11) . Results

Microsomal oxidase activity in developmental stages It is well known that larval molting of insects is induced by the coordinated action of hormones liberated from the corpora allata (juvenile hormone) and the prothoracic gland (molting hormone) .

Metamorphosis occurs in mature

larvae when the juvenile hormone is lacking (12) . If the inductive action of these hormones increases the level .of the microsomal oxidases in vivo , one would expect to see this increase about the time of pupation and again as the adults near sexual maturity .

We began a careful

investigation of this point by assaying the microsomal oxidase activity of eggs, larvae, pupae and adults at frequent intervals throughout their grwth period . No measurable epoxidase activity was observed in egg miciosomes though 10002000 eggs were used for each assay, Figure 1 . larval stage was very low .

Epoxidase activity in the early

However, during the late third instar, about 15 hr

before pupation (larvae 3 .23 days old), enzyme activity increased dramatically reaching a maximum about 11 hr before pupation .

Epoxidase activity then

Housefly Mícrosomal Oxidase Activity

1178

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declined sharply and only a trace remained in pupae and phorate adults .

Activ-

ity could be detected again after emergence and climbed steadily in adults . These results differ rather sharply from those of Khan (13) who performed similar experiments on a related strain and found maximum oxidase in 1-day old larvae . vals,

In part, this discrepancy could be due to Khan's choice of assay inter especially in the critical period, as the burst of microsomal oxidase

activity lasts less than 24 hr .

Our results are in general agreement with those

of Parry and Buchner (14) who found a similar~ ..maxim~ for cytochrome P-450 in 3-day old larvae of the housefly .

Z i

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saL

0

30

20

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0 2 4 13 15 I7 19 EGGS ~ LARVAE ~TIIIAE IN ~ ADULTS PuPARlulu DEVELOPM ENTAL STAGE (DAYS) FIG . 1

Microsomal epoxidase activity in the developmental stages of Isolan-B strain of houseflies . Ne were unsuccessful in our attempts to demonstrate increased microsomal epoxidase activity in larvae injected with ecdysterone although, as will be seen

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Hóusefly Microsomal Oaidase Activity

later, we were successful in experiments with adults .

1177

One problem was that lar-

vae in early instars could not tolerate injection or ligation .

After their guts

were cleared in the third instar, ligation and injection was less of a problem . Injection of ecdysterone (0 .15 pg/larvae) beyond the ligation point in third instar larvae resulted in an acceleration of pupation in the treated portion of the body .

However, instead of the expected increase in heptachlor epoxidase

activity in microsomes prepared from the tissue posterior to the ligature, there was a 28t reduction 3 hours after treatment and a 531 decrease 8 hwrs after treatment .

Pupation began within 15 hours .

Two additional experiments were performed to see if the peak in microsomal epoxidase activity in third instar larvae was related to ecdysone titer .

Four

hours before the expected increase in microsomal oxidase activity, mature larvae were ligated at the 7th segment so as to isolate the ecdysone secreting ring glands .

They were kept in this condition for 9 hours before preparing microsomes

from tissue posterior to the ligature .

No epoxidase activity was detected in

these microsa®es, but an epoxidase activity of 2 .3 pmoles heptachlor epoxide/ segment/min was found in microsames prepared from the same body region of nonligated larvae . The second experiment was based

on an observation of Ohtaki et al .

(15)

who found that the endocrine system of Sarcrophaga is activated by exposure to dry conditions .

In the housefly this would be expected to occur when late

third instar larvae clear their guts of food materials and move to drier conditions in the upper part of the rearing jars in preparation for pupation . found that the microsomal epoxidase activity of third instar larvae old) held in dry conditions

We

(2 .5 days

(media 40~ moisture) for 12 hr at 21 °C was 19 .9

pmoles heptachlor epoxide/larvae/min and 4 .9 pmoles for those kept in wet conditions (media saturated with water) .

It thus appears that microsomal oxidase

activity in the larvae may coincide with increased levels of ecdysone, this event occurring about 15 hours prior to pupation,

Housefly Microsomal Oaidase Activity

1178

Yol . 10, No . 20

Ecdysterone and juvenile hormone treatment of adults Although ecdysone is not produced in adults (16), we were interested in determining if it would induce the microsomal oxidases in this stage of the housefly .

Three-day old females were injected with 0 .025 ug of ecdysterone

(see Methods) and examined at intervals up to 18 hours for evidence of increased microsomal epoxidase activity .

There was 40 to 54$ increase in activity at

approximately 6 hours after treatment and the activity returned to normal within 12 hours, Figure 2 .

In topically treated insects, 0 .025 pg/fly, the epoxidase

activity was increased by 10$ at 12 hours and by 65$ at 24 hours .

Longer

exposures were not included .

160

140

H

c~ Q O tY

H 2 O u

120

100

80

i

i

i

~

3

6

9

12

IS

18

TIME AFTER TREATMENT (HR) FIG . 2 Effect of ecdysterone on microsomal epoxidase activity in Isolan-B houseflies . ~, 3-day old females injected with 0 .025 ug ecdysterone per f1y ;I~, repeated experiments .

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Housefly Microsomal Oxidase Activity

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At the time of these experiments the normal epoxidase activity of 3-day old Isolan-B females was approximately 20 pmoles heptachlor epoxide/fly/min, Fig . 1 .

At this time we decided to conduct an additional selection of the strain

with the insecticide, Baygon, the original selecting agent .

This increased the

microsomal oxidase activity of the females to about 40 pmoles héptachlor epoxide/ fly/min .

When these flies were treated with ecdysterone. at 0 .025 pg per fly

(injection), there was no evidence of increased epoxidase activity during the 18 hour observation period .

However, when the dose response was re-examined in

another experiment, increased epoxidase activity was again seen, Table 1, but the optimum dose at 6 hours was 0 .1 pg ecdysterone pèr fly . TABLE 1 Effect of Ecdysterone on Microsomal Epoxidase Activity in the . Adult Housefly

Heptachlor Epoxide, ~ of Control Ecdysterone, ug/fly*

Groupa

Group b

0 .05

128

113

0 .10

138

125

0 .25

113

116

* 3-day old Isolar-B females, 6 hr treatment .

Controls injected with water .

There was no effect on microsomal epoxidase activity when ecdysterone (up to 100 pg) was added directly to the incubation mixture, indicating that the observed changes in enzyme activity were not due to hormone-microsome inter actions . This is also evidence, in agreement with Ohtaki et al . ecdysterone is not metabolized by the microsamal oxidases .

(15) that

According to them,

inactivation of à -ecdysone is oxidative in nature . This is supported by Karlson and Bode (17) who have located a soluble enzyme in the fat body of Calliphora larvae which rapidly inactivates ecdysone .

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Housefly MiCrosomal Oaidase Activity

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140

r t-

u a

120

100

O

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80

A' 60

8

IY

i 18

~ 24

i 30

v 36

TIME AFTER TREATMENT (HR) FIG . 3 Effect of juvenile hormone on microsomal epoxidase activity in the Isolan-B housefües .~-~, 1-day old females, 0 .1 pg JH/fly ;~r-~, 6-day old females, 0 .1 ug JH/fly ;- " -eand~-~ , 2-day old females, 0 .5 pg JH/fly, two separate experisents . We also studied the effect of synthetic juvenile hormone on the Isolan-B strain during periods of high and low microsomal oxidase activity .

Enhancement

of epoxidase activity was seen in 1 to 6 day old female adults, the maximum increase noted approximately 24 hours after treatment, Fig . 3 .

The effective

dose was 0 .1 ytg per fly, topically applied, and there was an initial decline to about 60t of control activity during the first 12-18 hours .

No observations

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Housefly Microsomal Oaidaee Activity

1181

were made after 24 hours in these experiments and we do not know whether the enhancement

became even greater or when the epoxidase activity returned to

norral . The effective dose for the improved Isolan-B strain was 0 .5 hg per fly, Fig . 3, and the point of Maximum activity (454 increase) occurred at 18 hours; declining to nor~al in 36 hours . shorter and less evident .

The early inhibitory phase was correspondingly

Levels of the hormone up to 25

vg

per incubation

aixture did not affect the activity of the epoxidase system . Bffect of inhibitors The stiaulatory effect of the hormones can be elisinated or reduced by inhibitors of protein synthesis, Table 2 .

Cycloheximide failed to cavpletely

block the ecdysterone enhancement although it was fully effective in this respect when the juvenile hormone was applied .

This difference .ay be due to the more

rapid onset of activation after ecdysterone treatment, Fig . 2 . a longer post-treatment period would be futile .

As can be seen,

The results agree with those

of Sekeris ind Karlson (18) who found that in Calliphora larvae inhibitors of protein and~RNA synthesis blocked the ecdysone induction of dope decarboxylase . Discussion These results indicate that both hormones are capable of stimulating sicrosomai oxidase activity in the housefly but they do not prove a direct connection between this event and the physiological and morphological changes which occur as a result of hox~one action .

That the processes are related in save way is

indicated by the burst of microsaval epoxidase activity in last instar larvae just before pupation .

This, according to Shaaya and Karlson (19.), is

the point

in blowfly larvae at which ecdysone titer begins to rise . The connection between ecdysone titer and microsomal oxidase activity in housefly larvae . has also been made by showing that ligation reduces epoxidase activity and that dry conditions in the late instar increase presumably as a result of increased hormone production .

epoxidase activity,

We were unsuccessful,

however, in our attest to demonstrate induction of the microsomal. oxidases by

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TABLE 2 . Induction of Micorsomal Heptachlor Epoxidase Activity in Houseflies Injected with Insect Hormones and Inhibitors of Protein Synthesis .

Treatment

$ of Control Activity Expt . 1

Expt . 2

Expt . 3

Eçdysterone* Ecdysterone (0 .025 Pg/fly)

145

140

Actinomycin D (0 .02 ug/fly)

110

108

96

73

Ecdysterone (0 .025 ug/fly)+ actinamycin D (0 .02 u8/fly) Cycloheximide (0 .2 pg/fly)

110

Ecdysterone (0 .025 ug/fly)+ cyclohexinide (0 .2 ug/fly)

127

154

Cycloheximide (1 wg/fly)

-

-

133

Ecdysterone (0 .025 ug/fly)+ cycloheximide (1 ug/fly)

-

-

131

131

115

Cycloheximide (1 pg/fly)

88

63

J . H. (0 .1 u8/fiY)+ cycloheximide (1 pg/fly)

77

85

Actinamycin D (0 .02 ug/fly)

-

72

J. H. (0 .1 ug/fly)+ actinomycin D (0 .02 wg/fly)

-

71

Juvenile hormone+ J . H . (0 .1 ug/fly)

* 3-day old Isolan-B females, injected with hormone and inhibitors 6 hr before micros~e preparation . + 1-day old Isolan-B females, injected with inhibitors and topically treated with juvenile hormone 24 hr before microsame preparation.

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Housefly Microsomal O~ddase Activity

1183

direct treatment of larvae with ecdysterone, the treatment actually resulting in decreased activity . of the treatment .

We believe this may have been due to improper dose or timing We plan additional work in search of the answer to this

question . Even though ecdysone is absent in adult flies (16) our demonstration of its action on the heptachlor epoxidase system should be of significance because we have no reason to believe that the microsomal oxidases differ in basic function in mature and immature stages .

This, along with our results with larvae, suggest

that stimulation of these oxidases may be one-of the functions of this hormone in initiating the molting process . In their study of the short term effects of ecdysterone injected into Calliphora larvae, Neufeld et al . ïnsect .

(1) used doses ranging from 0 .002 to 0 :2 pg per

These doses caused a depression in fat body protein synthesis at 1 hour

and an acceleration in protein synthesis at 4 hours ; réturning to normal at 6 hours .

We saw no evidence of the inhibitory phase but, since no examinations

were made less than three hours after treatment, this effect might have been missed .

The doses which were effective (0 .025 to 0 .25 pg per fly) and the time

of maximum stimulation (approximately 6 hours) do agree quite well with those observed by Neufeld et al .

(1) .

These facts suggest that among the proteins

which were stimulated in these worker's experiments with Calliphora were those of the microsomal oxidase system . Our results with the synthetic juvenile hormone agree with those of Adams and Nelson .(20) who observed that a synthetic juvenile hormone stimulated fat body development in the housefly . . The dose used by these workers was 25 pg per fly and the observations were made at 7 days after treatment, while we were able to detect changes in enzyme activity 24 hours after treatment with 0 .1 to 0 .5 pg per fly .

We did not study higher doses since preliminary experiments indicated

a plateau in enzyme activity at the 0 .05 to 0 .1 pg/fly level (the low oxidase Isolan-B population) .

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Housefly Microsomal Oaidase Activity

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We have no ready explanation of the inhibitory phase of juvenile hormone action which reduced the epoxidase activity by as much as 47~ during the first 18 hours .

One possibility is that offered by Means and Hamilton (21) in their

study of estrogen action in the rat uterus .

They suggest that the rapid initial

synthesis of RNA depletes the cell of ATP resulting in a slowdown in protein synthesis .

An attractive possibility is suggested by the results of Congote

et al . (7) who found that stimulation of RNA synthesis in the nuclei of fat body cells ( Calliphora) by ecdysone was inhibited in the presence of juvenile hormone . Thus the inhibitory phase we have observed might be part of the interaction between these two hormones . The juvenile hormone action also seems to be dose dependent in the sense that a larger dose is required when the flies are in a condition of high oxidase activity than when oxidase activity is low .

~ince our results with actinomycin D

indicate that the action of these hormones is at the DNA of the nucleus, the dose effect in this strain may be explained on the basis of repeated gene sequences as we have suggested elsewhere(8) . References 1.

G . J . Neufeld, J . A . Thamson, and D . S . H . Horn, J . Insect Physiol . (1968)

2.

T . S . Sahota and A . Mansingh, J . Insect Physiol .

3.

R . Arking and E . Shaaya, J . Insect Physiol .

4.

R . L . Patton, Introducto Insect Ph siolo and London, P . 183 1963 .

5.

P . Karlson and C . E . Sekeris, Biochem . Bioptgs .Acta .

63, 489 (1962) .

6.

P, Karlson and C . E . Sekeris, Rec . Prog . Horen . Res .

22, 473 (1966) .

7.

L . F . Congote, C . E . Sekeris and P . Karlson, Exp . Cell Res .

8.

L . C . Terriere, S . J . Yu, and R . Hoyer, Science, ~, 581 (1971) .

9.

J . H . Law, C . Yuan and C . M . Williams, Proc . Nat . Acad : Sci ., U .S .A . (1966) .

16, 1649 (1968) .

15, 287 (1969) . W . B . Saunders Co ., Philadelphia

56, 338 (1969) .

10 .

R . D . Schonbrod and L . C . Terriere, J . Econ . Entomol .

11 .

C . R . IValker and L . C . Terriere, Ent . Exp . and Appl . 13, 260 (1970) .

12 .

P . Karlson, J . Cell Physiol .

66, 69 (1965) .

14, 789

55,576 _

59, 1411 (1966) .

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Houseflq Mícrosomal O~dase Actívitq

1185

13 .

M . A. Q . Khan, Biochee . Pharmacol .

19, 903 (1970) .

14 .

A. S.Perry and A. J . Buchner, Life Sciences

15 .

T . Ohtaki, R . D. Milkman and C . M . Williams, Biol . Bull .

16 .

V . B. Wigglesworth, Insect Hormones, W . H . Freeman and Company, San Francisco, P . 12 (1970) .

17 .

P . Karlson and C . Bode, J . Insect Physiol .

18 .

C . E . Sekeris and P . Karlson, Arch . Bioches . Biophs .

19 .

E . Shaaya and P . Karlson, J . Insect Physiol .

20 .

T . S . Adams and D . R. Nelson, J . Insect Physiol .

21 .

A . R. Means and T . H . Hamilton, Proe . Nat . Acad . Sci ., U .S .A .

9, 335 (1970) 135, 322 (1968) .

15, 111 (1969) . 105, 485 (1964) .

11, 65 (1965) . 15, 1729

(1969) . 56, 686 (1966) .