Insect Biochem., Vol. 8, pp. 349 to 352 0020-1700/78/1001-0349502.00/0 Pergamon Press Ltd. 1978 Printed in Great Britain

JUVENILE HORMONE EPOXIDE HYDRASE IN HOUSE FLIES, FLESH FLIES AND BLOW FLIES*

S. J. Yu and L. C. TERRIERE

Department of Entomology. Oregon State University, Corvallis, Oregon 97331, U.S.A.

(Received 16 December 1977)

Abstract--Juvenile hormone (JH) epoxide hydrase activity of the house fly (Musca domestica L.), flesh fly (Sarcophaga bullata Parker), and black blow fly (Phormia regina (Meigen)) was measured in vitro. Juvenile hormone esterase activity, present in the same in vitro system, was eliminated by the addition of paraoxon. Most of the enzyme is located in the microsomal fractions of insect tissue homogenates. The hydrase is NADPH-independent and is not affected by carbon monoxide or other inhibitors ofmicrosomal oxidases. It is inhibited by certain JH analogs containing an epoxy group. No marked difference in hydrase activity was observed between strains of insecticide-susceptible and resistant house flies. The enzyme is induced in the house fly by feeding phenobarbital for three days.

Key Word Index: Juvenile hormone epoxide hydrase, hormone metabolism, Diptera, microsomal enzyme

I N T R O D U C T I O N

ONE Or the important primary reactions in the metabolism of the juvenile hormones (JH) in insects is the hydration of the 10,11-epoxy ring to produce the corresponding dihydro-dihydroxy compound known as JH-diol (SLADE and ZmITT, 1972; AJAMI and RIDDtFORD, 1973; WroTE, 1972). Judging from the age- dependent changes in the activity of this enzyme in the southern armyworm (Spodoptera eridania (Cramer) (SLADE et al., 1976) and in the house fly (Yc and TERRtEgE, 1977a), it may have a function in the regulation of JH levels during growth and develop- ment. Not much is known about this enzyme however, probably due in part to the difficulty of its assay.

In vitro assays of JH metabolism as well as those conducted in vivo are difficult to interpret with respect to the role o f the hydrase itself because of the competing activity of JH esterase. Not only is the parent compound attacked simultaneously by more than one enzyme, but the products of these initial reactions may become substrates for the interfering enzymes (Yu and TERRIERE, 1977a). We have found that such complications can be avoided, in studies of epoxide hydrase, by the addition of the JH esterase inhibitor, paraoxon, during the assays. The conditions required for opt imum in vitro activity of this enzyme and some of its characteristics have been studied with this technique and are reported here.

M A T E R I A L S AND M E T H O D S

Insects

The house fly (Musca domestiea L.), flesh fly (Sarcophaga bullata Parker), black blow fly (Phormia regina (Meigen)) were reared in the laboratory as described previously (TERRIERE and YU, 1976a,b). The experiments with house flies involved two insecticide-susceptible strains (CSMA and SRS) and two insecticide-resistant strains (Rutgers and Fc).

*Oregon Agricultural Experiment Station Technical Paper No. 4730.

The resistant strains, Rutgers and Fc, are resistant to diazinon and DDT, respectively. Unless otherwise stated, one week-old female adults were used throughout the experiments.

Chemicals

10-[3H]-JH ! [methyl(2E,6E,10Z)-10,1 l-epoxy-7-ethyl- 3,11-dimethvl-2,6-tridecadienoate], specific activity 13.5 Ci/mmole, was purchased from New England Nuclear Corp., Boston, MA. This compound was at least 98% pure as indicated by thin layer chromatography (TLC). Unlabelled pure JH I was obtained from Eco-Control, Inc., Cambridge, MA.

RO 7-9767 [6,7-epoxy-3,77diethyl-1 -(3,4-(methylene- dioxy) phenoxy)-2-octene], and RO 20-3600 [6,7-epoxy-3- methyl-7-ethyl- 1-(3,4-(methylenedioxy) phenoxy-2-octene],

• both as mixtures of isomers, were obtained from Hoffman- La Roche Inc., Nutley, NJ. R 20458 [l-(4'-ethylphenoxy)- 3,7-dimethyl-6,7-epoxy-trans-2-octene], 92.1% pure, was obtained from Stauffer Chemical Co., Mountain View, CA.

JH I diol was synthesized from [3H]-JH ! diluted with cold JH I by the method of SLADE and ZIBITT (1972) and its structure was confirmed by mass spectrometry.

Enzyme assays

The methods used in preparing microsomes for routine assays were as described previously (Yu and TERI~IERE, 1975). JH epoxide hydrase activity was measured as follows: the 5- ml incubation mixture contained microsomes equivalent to ten insects (abdomens in the case of adults); 0. l M sodium phosphate, pH 7.5, I0 #g of cold JH I and 5.5 ng (5.5× l0 s dis/rain) of [3H]-JH I, 10 -3 M paraoxon, and I0 mg of bovine serum albumin. The incubations were carried out in an atmosphere of air with shaking at 34°C £or 1 hr. All incubations were performed in duplicate.

The reaction was stopped by saturating the incubation mixture with granular ammonium sulphate and the mixtures were extracted three times with 2.5 ml of a 2:1 (v/v) mixture of diethyl ether-ethanol. Aliquots of the combined extracts were then analyzed by TLC followed by liquid scintillation counting as described previously (Yu and TERRIF~E, 1977b). The counting efficiency was 34--37%.

When the subcellular distribution of JH epoxide hydrase was studied, the tissues were prepared according to the metho~t described previously (Yu and TERRI~E, 1975).

The protein content of each enzyme preparation was 349

350 S.J. Yu AND L. C. TERRIERE

0 .

jH i i i i

16 12 8 4

Fig, 1. Typical thin-layer radioachromatogram of metabolite produced when house fly microsomes are incubated with [3H]-JH l in the presence of paraxon at 10 -3 M.

determined by the method of BRADFORD (1976) using bovine incubation and the enzyme was active over a broad pH serum albumin as standard, range, 6.6-8.0, with a slight peak around pH 7.2-7.5.

The specific activity of microsomes prepared from R E S U L T S AND D I S C U S S I O N house fly abdomens was higher than those prepared

from whole flies or other body segments, Table 2. Characteristics o f J H epoxide hydrase The three JH analogs, RO 7-9767, RO 20-3600 and

Microsomes prepared from house flies, flesh flies R 20458 strongly inhibited the JH epoxide hydrase, and blow flies contain two enzymes which attack the Table 3. However, other insecticidal epoxides such as juvenile hormone, the epoxide hydrase and the dieldrin and heptachlor epoxide appeared to be poor esterase(s) (Yu and TERRmRE, 1977a). With the two inhibitors. No inhibition was observed with the well- enzymes thus competing for the same substrate, it is known inhibitors of microsomal oxidases such as difficult to study certain characteristics of either piperonyl butoxide, S K F 525A, or carbon monoxide. enzyme. We were able to overcome this problem in our Styrene oxide and cyclohexene oxide did not affect the experiments with the epoxide hydrase by inhibiting the hydrase. action of the esterase with the organophosphate, The epoxide hydrase activity was increased 78°o paraoxon. As shown in Fig. 1, only one reaction (average of two experiments)when one-day-old adult product, JH I diol, was obtained when house fly female house flies (CSMA strain) were fed a diet microsomes were incubated with JH I in the presence containing l°~o sodium phenobarbital for three days. of 10- 3 M paraoxon. Under these assay conditions, This increase due to phenobarbital treatment is greater the activity of the epoxide hydrase was more than than that observed earlier (a 19~o increase) in a study double that found in similar assays without paraoxon, of JH metabolism in the house fly (Yu and TERRIERE, No significant metabolism of the JH occurred when 1977a). In that case, however, both the esterase and boiled microsomes were used as enzyme source, the hydrase were active, probably making it more

As expected from work with vertebrates, the micro- difficult to detect the additional metabolism due to the somal fraction of the tissue homogenates contained induction of the epoxide hydrase. the highest concentration of the enzyme, Table I. The The epoxide hydrase activity in various life stages of plot of enzyme activity against time of incubation was the Rutgers house fly is shown in Fig. 2. During the essentially linear during the first sixty minutes of immature stages the highest activity was found in one-

day-old larvae when based on microsomal protein or

Table 1. JH epoxide hydrase activity in subcellular fractions body weight. However, when activity was expressed of house fly, flesh fly, and blow fly homogenates

Table 2. Distribution of J H epoxide hydrase activity in body JH ! diol produced (nmole/10 segments of Rutgers house flies

abdomens/hr)* Fraction House fly (Rutgers) Flesh fly Blow fly JH l diol produced*

. . . . . . . . . . . . . . . . . . . . Tissue (pmole/mg (nmole/mg Cell debris source tissue/'hr) protein/hr)

nuclei 2.9 + 0.8 1.3 ___ 0.1 1.2 _+ 0.06 . . . . . . . . . . . . . . . Mitochondria 3.9 ± 0.9 3.4 + 0.2 1.6 ± 0.01 Whole fly 67.1 ± 2.5 4.6 + 0.1 Microsomes 13.0 + 0.1 6.3 ± 0.1 9.7 _+ 1.13 Headless fly 65.7 + 4.8 5.7 _+ 0.2 Soluble 4.2 ± 0.3 0.3 + 0.04 0.2 _+ 0.01 Thorax 79.0 ± 10.1 4.2 _+ 0.5

Abdomen 116.8 +_ 4.8 12.8 _+ 0.5 * Mean _+ range or S.E. Number of determinations: house

fly and flesh fly, 2; blow fly, 4. * Mean +_ S.E. of 4 determinations.

JH epoxide hydrase in Diptera 351

Table 3. Effect of inhibitors on JH epoxide hydrase activity Table 4. JH epoxide hydrase activity in microsomes from in Rutgers house flies female adults of house flies, flesh flies, and blow flies

Compound & Epoxide hydrase activity, concn. (M)* % of controH"

RO 7-9767 10 -4 13 + 0.2§ 10 -~ 55 _+ 6.1

RO 20-3600 1 0 - 4 8 + 0 . 4 §

10 -s 24 ± 5.6 RO 20458 10 -4 9 ± 0.7 Dieldrin 10 -4 89 + 3.4 Heptachlor epoxide 10 -4 83 ± 0.7 Styrene oxide 10 -4 102 _+ 0.5 Cyclohexene oxide l0 -4 95 ± 1.5 SKF 525A 10 -4 103 _+ 3.0 Piperonyl butoxide 10 -4 102 ± 2.7 CO~ - - 105 +_ 1.1

* See Materials and Methods for chemical names. All compounds (except CO) were added in 0.05 ml methyl cellosolve.

i'Mean + range or S.E. Number of determinations: 2 except that §, 4.

Bubbled gently for 1 min prior to incubation.

per insect, the peak activity was found in three-day-old larvae. Enzyme activity in adults increased ~ith age up to seven days after emergence but was less evident when this was based on protein content.

Comparative aspects

Table 4 shows the JH epoxide hydrase activity in house flies, flesh flies, and blow flies. There was no marked difference in enzyme activity between suscept- ible and resistant strains of the house fly. The activity in the flesh fly and blow fly was lower than the house fly when based on body weight or microsomal protein content.

Comparisons of insect epoxide hydrases attacking

JH I diol produced* (nmole/10 (pmole/mg (nmole/mg

Species abdomens/hr) body wt/hr) protein/hr)

House fly CSMA 14.9 _+ 0.9 125.4 ± 8.2 9.3 _+ 0.6 SRS 11.0 _+ 0.1 105.2 ± 1.0 10.0 _+ 0.1 Rutgers 13.6 _+ 0.1 122.8 _ 0.9 9.0 _+ 0.1 Fc 9.1 _+__ 0.4 92.1 + 4.2 7.2 ± 0.3

Flesh fly 10.6 ± 0.1 22.6 + 0.1 4.2 + 0.0 Blow fly 8.1 + 0.1 35.3 ± 0.3 5.0 ± 0.0

* Mean of 2 determinations.

JH I, R 20458 and H E O M (l,2,3,4,9,9-hexachloro- (l ,2,3,4,9,9-hexachloro-6,7-epoxy- 1,4,4a,5,6,7,8,8a- octahydro-l ,4-methanonaphthalene) reveal that al- though there are some similarities, such as enzyme distribution, there are differences in their susceptibility. to inhibitors. For example, piperonyl butoxide and SKF 525A both of which are moderate to good inhibitors of the HEOM epoxide hydrase in Spodoptera, Calliphora and Tenebrio (SLADE et al., 1975; CRAVEN et al., 1976) did not affect the house fly JH epoxide hydrase. Also, 1,1,l-trichloropropene 2,3-oxide which is a potent inhibitor of H E O M epoxide hydrase in Spodoptera, Calliphora and Tenebrio had no inhibitory effect on the house fly R 20458 epoxide hydrase (HAMMOCK et al., 1974).

On the other hand, cyclohexene oxide which inhibited certain mammalian epoxide hydrases (OEsCH et al., 1971) had no effect on the JH epoxide hydrase (Table 4) or R 20458 epoxide hydrase in house flies (HAMMOCK et al., 1974). Styrene oxide which is a substrate for mammalian epoxide hydrase (OEscH et al., 1971) did not affect these house fly epoxide

? I

I I

H

60 240

A

5C iL 200

,.? 4:

4C "-. 160

3 ( =a 120 E

20 f 80

I0 40

0 0

/i ,' t i ].

ii T . /

I I f I I I I 3 6 9 12 IS 18 2~

Larva [ Puparium I Adult

Developmental Stage (days )

2 4

C

ca

c

¢o

8~ o "o

4 H

0 2 4

Fig. 2. Microsomal JH epoxidase hydrase activity in the developmental stages O f Rutgers house flies. Microsomes were prepared from whole insects of mixed sex except for adults when female abdomens were used. Larval stage: day- 1,2nd instar; day-2, 3rd instar; day-3, 3rd instar; day-3.8, 3rd instar with clearing gut. Each point represents the mean (_+ S.E.) for 2-4 determinations. Standard errorCnot shown were less than

the size of the data points.

352 S.J. Yu AND L. C. TERRIERE

hydrases. These observations suggest that the house fly enzymes are different from the mammalian enzyme.

The strong inhibition of JH epoxide hydrase by some JH analogs is of interest. Since R 20458 is known to be metabolized by house fly epoxide hydrase (HAMMOCK et al., 1974), it is possible that these JH analogs act as alternative substrates for the JH epoxide hydrase, resulting in the inhibition which we observed (Table 3). These results are in agreement with those of SLADE and WILKINSON (1973) who found that the epoxy-type JH analogs, RO 7-9767 and N I A 16388 (10, 1 l - epoxy-N-e thy l -3 ,7 ,1 l - t r i m e t h y l - 2 , 6 - d o d e - cadienamide), inhibited JH epoxide hydrase in the southern armyworm.

Acknowledgements--This work is supported by USPHS Grant No. 5-ROI-ES-00362-18. The authors thank Stauffer Chemical Co. for supplying R 20458, and Hoffman-La Roche Inc. for RO 7-9767 and RO 20-3600. The technical assistance of Mr. DAN FARNSWORTH is also appreciated.

R E F E R E N C E S

AJAMI A. M. and R1DDIFORD L. M. (1973) Comparative metabolism of the cecropia juvenile hormone. J. Insect Physiol. 19, 635-645.

BRADFORD M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254.

CRAVEN A. C. C., BROOKS G. T. and WALKER C. H. (1976) The inhibition of HEOM epoxide hydrase in mammalian liver microsomes and insect pupal homogenates. Pestic, Biochem. Physiol. 6, 132-141.

HAMMOCK B. D., GILL S. S. and CASIDA J. E. (1974) Insect metabolism of a phenyl epoxygeranyl ether juvenoid and related compounds. Pestic Bioehem Physiol 4, 393-406.

OESCH F., KAUBISH N., Jerina D. M. and DALY J. W. (1971) Hepatic epoxide hydrase. Structure-activity relationship for substrates and inhibitors. Biochemistry t0, 4858-4866.

SLADE M., BROOKS G. T., HETNARSK1 H. K. and WILKINSON C. F. (1975) Inhibition of the enzymatic hydration of the epoxide HEOM in insects. Pestle. Biochem. Physiol. 5, 35-46.

SLADE M., HETNARSKI H. K. and WILKINSON C. F. (1976) Epoxide hydrase activity and its relationship to development in the southern armyworm, Prodenia eridania. J. Insect Physiol. 22, 619-622.

SLADE M. and WILKINSON C. F. (1973) Juvenile hormone analogs: a possible case of mistaken identity'?. Science 181, 627-674.

SLADE M. and ZmITT C. H. (1972) In Insect Juvenile Hormones (Ed. by MENN J. J. and BEROZA M.), pp. 155-176. Academic Press, New York.

TERRIERE L. C. and Yu S. J. (1976a) Interaction between microsomal enzymes of the house fly and the moulting hormones and some of their analogs. Insect Biochem. 6, 109-114.

TERRIERE L. C. and Yu S. J. (1976b) Juvenile hormone analogs: in vitro metabolism in relation to biological activity in blow flies and flesh flies. Pestic. Biochem. Physiol. 7, 161-168.

WHrrE A. F. (1972) Metabolism of the juvenile hormone analogue methyl farnesoate 10,11-epoxide in two insect species. Life Sci. 17, 619-626.

Yu S. J. and TERRIERE L. C. (1975) Microsomal metabolism of juvenile hormone analogs in the house fly, Musca domestica L. Pestic. Biochem. Physiol. 5, 418-430.

Yu S. J. and TERRIERE L. C. (1977a) Metabolism ofJH l by microsomal oxidase, esterase and epoxide hydrase of Musca domestica and some comparison with Phormia regina and Sarcophaga bullata. Pestic. Biochem. Physiol. (In press).

Yu S. J. and TERRIERE L. C. (1977b) Metabolism of ~4 C- hydroprene (ethyl 3,7,11-trimethyl-2,4-dodecadienoate) by microsomal oxidases and esterases from three species of Diptera. J. Agric. Fd Chem. 25, 1076-1080.