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Ecdysteroid receptor binding activity and ecdysteroid agonist activity at the level of gene expression are correlated with the activity of dibenzoyl hydrazines in larvae of Bombyx mori
Vol. 42, No. 10, pp. 937-941, 1996 Copyright 0 1996 Elsevier Science Ltd
J. Insect Physiol.
Pergamon
Printed in Great Britain. All rights resewed
PII: SOO22-1910(96)00056-X
0022-1910/96$15.00 + 0.00
Ecdysteroid Receptor Binding Activity and Ecdysteroid Agonist Activity at the Level of Gene Expression are Correlated with the Activity of Dibenzoyl Hydrazines in Larvae of Bombyx mori KENICHI MIKITANI* Received 1 November 199.5; revised and accepted 26 March 1996
Ecdysteroid activities of two dibenzoyl hydrazines (RH 5849 and RH 5992) were examined using the Kc cell line. Gene expression activity was determined in the transient assay utilizing the ecdysteroid responsive Drosophila melanogaster hsp27 promoter gene. Ecdysteroid receptor binding (E& = 3.0 x lo-’ M), induction of ecdysteroid responsive gene expression (EC5,, = 3.0 x 10m6 M) and induction of cell morphological change (Et& = 3.0 x lo-’ M) were 13fold, 7-fold and 13-fold higher in RH 5992 than in RH 5849, respectively. The addition of 10 ppm RH 5849 to the diet induced head capsule slippage in Bombyx mori larvae and most of the larvae died. RH 5992 caused the same response at 1 ppm. The higher insecticidal activity of RH 5992 compared to RH 5849 is likely caused by its higher ecdysteroid agonist activity at the molecular level. Copyright 0 1996 Elsevier Science Ltd Ecdysteroid
Dibenzoyl hydrazines Gene expression Bombyx mori
INTRODUCTION
Kc cell
supports the validity of this model. Another nuclear hormone receptor (Usp) is indispensable in forming the functional ecdysteroid receptor complex (Yao et al., 1992, 1993; Thomas et al., 1993). Thus far, there is little information concerning gene expression activities of the non-steroidal ecdysteroid agonists dibenzoyl hydrazines (Retnakaran et al., 1995). RH 5992 showed apparently higher (lo- to 1OO-fold) insecticidal activities against various Lepidopteran insect species than RH 5849 (Smagghe and Degheele, 1994). In the present study, the activity of two dibenzoyl hydrazines (RH 5849 and RI-I 5992, Fig. 1) are evaluated for receptor binding, gene expression and the induction of cell morphological changes. Also, in vivo activities against larvae of Bombyx mori (silkworm) are investigated.
1, 2-Dibenzoyl- 1-tert-butylhydrazine (RH 5849) was the first non-steroidal chemical that showed ecdysteroid agonist activities at the cellular (Wing, 1988) and organismal (Wing et al., 1988) levels. Dibenzoyl hydrazines are effective chemical tools for insect hormone research (Oberlander et al., 1995). They show ecdysteroid receptor binding as well as the ability to induce acetylcholinesterase and morphological changes in Kc cells. The uptake of N-acetyl-glucosamine by a Plodia interpunctella (Indian meal moth) cell line of wing disk origin was also observed in response to both RI-I 5849 and 20-hydroxyecdysone (Silhacek et al., 1990). The mode of action of ecdysteroids is believed to be via gene expression of ecdysteroid responsive genes (Ashbumer, 1972, 1990; Ashbumer et al., 1974; Cherbas, 1993). The recent cloning of the ecdysone receptor (EcR) gene from Drosophila melanogaster (Koelle et al., 1991)
MATERIALS AND METHODS Assay for ecdysteroid
receptor
binding
The ecdysteroid receptor binding assay was performed by the automated 96-well microplate based method
*Agricultural Chemicals Research Laboratory, Sumitomo Chemical Co., Ltd, 4-2-1, Takatsukasa, Takarazuka, Hyogo 665, Japan. 937
938
K. MIKITANI
Compound RH 5849 RH 5992
Rl
R2
R3
H EH,CH~
CHs
cti3
H
FIGURE 1. Chemical structures of dibenzoyl hydrazines used in this study.
(Mikitani, 1995a, 1996). D. melanogaster Kc cells cultured in spinner flasks were collected and cell cytosols were made by sonication. After ultracentrifugation (lOO,OOOg,4°C 60 min), supematants were frozen and stored as aliquots in liquid nitrogen until use. 3H-ponasterone A (final concentration of 1.0 x lop9 M), thawed Kc cell cytosol and different concentrations of RH 5849 and RH 5992 dissolved in dimethyl sulfoxide were transferred and mixed in each well of the 96-well microplate. After a 60 min incubation at 25°C the ice-cold dextrancoated charcoal suspension was added to the well. After vigorous mixing at 4°C the 3H-ponasterone A that was not bound to receptor was absorbed to the charcoal and precipitated by centrifugation. The supematant was transferred to a solid scintillator for the measurement of radioactivity. Non-specific binding was defined as dpm bound in the presence of 2.0 x lop5 M 20-hydroxyecdysone, and each data point was corrected by subtracting the dpm value of non-specific binding from that of each total binding. Assay for ecdysteroid
responsive
gene expression
activity
Gene expression activity for each compound was assayed as reported (Mikitani, 1995a,b). The reporter plasmid pHSP27-LUC was constructed by ligation of the D. melanogaster 669 bp hsp27 gene promoter region (which contains the ecdysteroid responsive element and a TATA box sequence) with the firefly luciferase cDNA. The highly purified pHSP27-LUC plasmid was transiently introduced into Kc cells by electroporation (0.5 kV, 3 PF). The cells, diluted 20-fold with the medium in 2.5 ml aliquots, were incubated with 2.5 ~1 of dimethyl sulfoxide containing the chemicals (RH 5849 and RH 5992) for 24h in a 6 cm diameter tissue culture plastic Petri dish at 25°C under 5% CO,. After 24h, cells were lysed and the luciferase activity was measured. Protein concentration was determined by the BCA method for correction.
Assay for induction
of cell morphological
changes
Nearly confluent Kc cells were diluted with M3 (BF) medium supplemented with 2% fetal bovine serum to a density of 1.0 x lo6 cells/ml. The cells in 2.5 ml aliquots were incubated with 2.5 ~1 of dimethyl sulfoxide containing the chemicals (RH 5849, RH 5992 and 20hydroxyecdysone) for 48h in a 6 cm diameter tissue culture plastic Petri dish at 25°C under 5% COZ. Then, the cells were observed and photographed by phase-contrast microscopy. Ecdysteroid responding cells were scored as having more than twice the length of process elaboration compared to the shorter width of the cell. Activity
test against
B. mori larvae
B. mori (Kinsyu x Shugetsu) larvae were fed on an artificial diet and reared at 25°C 60% RH and 16L:8D photoperiod. The compounds (RH 5849 and RH 5992) were dissolved in dimethyl sulfoxide and diluted to the different concentrations with distilled water. A solution of 2.0 ml was poured on the artificial diet (13 g) in a plastic cup. After absorption of the solution into the diet, 10 newly ecdysed 3rd instar larvae were transferred to each cup. All experiments were duplicated and the average was taken. Cultures were checked for head capsule slippage at 24h intervals, and mortality was assessed 96h after the larvae were transferred to the cups. Chemicals RH 5849 and RH 5992 were synthesized from N-benzoyl-Ar-tert-butyl-hydrazine and N-4-ethyl-benzoyl-hrtert-butyl-hydrazine, respectively, and 99% purity products were used (Hsu, 1991; Oikawa, 1994). 20Hydroxyecdysone (98% purity) was purchased from Sigma.
939
ECDYSTEROID ACTIVITIES OF DIBENZOYL HYDRAZINES RESULTS
Ecdysteroid hydrazines
receptor
binding
activities
of dibenzoyl 40
Displacement kinetics of 3H-ponasterone A binding to the Kc cell cytosol by dibenzoyl hydrazines are shown in Fig. 2. RH 5992 at 4.0 x 1O-6 M displaced 95% of 3H-ponasterone A binding, while 4.0 x lo-’ M of RH 5849 achieved 98% displacement. For RH 5992, the EC,,, (effective concentration, 50%) was 3.0 x lo-’ M; for RH 5849, the E& was 4.0 x lop6 M. The ECso for RH 5849 was approximately the same as the value previously reported in a similar binding assay (Wing, 1988). For 20hydroxyecdysone, the E& was 8.0 x lo-* M, as was previously shown in the same binding assay system (Mikitani, 1995a, 1996). Ecdysteroid responsive dibenzoyl hydrazines
gene expression
activities
c
0 ‘c;
2
z ._
30
0
9
20
10
0 lo-’
10”
of
The luciferase reporter plasmid (pHSP27-LUC), which contains the 5’-upstream 0.67 kb sequence of the D. melanogaster hsp27 gene as the promoter gene, was transiently transfected into Kc cells by electroporation (Mikitani, 1995a,b). The luciferase induction activities of dibenzoyl hydrazines in the pHSP27-LUC plasmid-introduced cells were determined (Fig. 3). For RH 5849, the ECso was 2.0 x lop5 M; for RH 5992, the EC& was 3.0 x lop6 M. Maximal inductions by RH 5849 and RH 5992 were 43-fold and 45-fold, respectively.
10”
los
lo4
10;
Concentration (id) FIGURE 3. Dose-response curves for luciferase induction activities of RH 5849 (0) and RH 5992 (e) against pHSP27-LUC introduced Kc cells. The pHSP27-LUC plasmid DNA was transfected into Kc cells by electroporation methods and cultured with and without the chemicals. After 24h, cell extracts were prepared for the measurement of luciferase activities and protein concentrations (for correction). Each data point is the average of duplicate measurements.
Induction of morphological changes in Kc cells by dibenzoyl hydrazines Like 20-hydroxyecdysone, RH 5849 and RH 5992 also induced process protrusion in Kc cells (Fig. 4). The E&
100 90
Em g
70
8
60
$
50
lo-lo
10"
10-l
Concentration
10"
1o-5
lo4
10"
(M)
FIGURE 4. Induction of Kc cell process protrusion by RH 5849 (0), RH 5992 (0) and 20-hydroxyecdysone (0). Nearly confluent cells were cultured with the chemicals for 48h. Ecdysteroid responding cells were scored as having more than twice the length of process elaboration compared to the shorter width of the cell. Each data point represents the mean f SD (n = 3).
$Ia 8
10'
30
20 10 0 lo-lo
10-g
109
1o-7
lo6
1o-5
lo4
lo9
Concentration (M) FIGURE 2. Displacement of ‘H-ponasterone A bound to the Kc cell cytosol in the presence of RH 5849 (0) and RH 5992 (0). Specifically, bound ‘H-ponasterone A was determined as described in Materials and Methods and is plotted as percentage displacement of 3H-ponasterone A binding vs test concentration of chemicals. Each data point represents the mean k SD (n = 3).
for RH 5849 was 4.0 x lop6 M; the ECXo for RH 5992 was 3.0 x IO-’ M; the EC,,, for 20-hydroxyecdysone was 1.0 x lop8 M. The EC&, for RH 5849 was approximately the same as the value previously reported (Wing, 1988). Activities of dibenzoyl hydrazines against B. mori larvae Dibenzoyl hydrazines applied to the artificial diet in B. mori 3rd instar day 0 larvae induced head capsule
940
K. MIKITANI
and biochemical and cellular studies using D. melunoguster Kc cells (Wing, 1988), RH 5849 was reported to be an ecdysteroid agonist. In addition to the above mentioned study of MHR3 mRNA induction by RH 5992 (Retnakaran et al., 1995), a number of studies have focused on the mode of action of dibenzoyl hydrazines, both in cell lines of Diptera (Clement et al., 1993; Quak et al., 1995; Spindler-Barth et al., 1991) and in cell lines or tissues of Lepidoptera (Silhacek et al., 1990; Sobek et al., 1993; Sohi et al., 1995; Smagghe and Degheele, 1995). The effective concentrations of RH 5849 found in DISCUSSION these previous studies ranged from 10e4 M to 10m6 M. The results of the transient gene expression assay in The results of the gene expression assay (ECSo = 2.0 x this paper clearly show that dibenzoyl hydrazines induce lo-’ M) and the cell morphology assay (EC,, = 4.0 x significantly high levels of ecdysteroid responsive gene lop6 M) in the present study are within that range. In expression. Induction activity of RI-I 5992 was 7-fold some of the previous studies, RH 5992 was compared higher than that of RH 5849. In addition, the results in with RH 5849 for ecdysteroid receptor binding and biothis paper show that RH 5992 has more than a lo-fold logical activities (Quak et al., 1995; Smagghe and stronger affinity for the ecdysteroid receptor than RH Degheele, 1995). In those studies the potencies of the 5849. The gene expression activity reflects this difference two chemicals in ecdysteroid receptor binding (RH of receptor affinity, and the cellular response measured 5992> RI-I 5849) corresponded to their biological effects. as cell process protrusion also showed the same range of However, the edysteroid responsive gene expression difference (7-fold) between the two compounds. activities of the two chemicals were not investigated. The The Manduca se&a hormone receptor 3 (MHR3) present studies clearly show that RH 5992 induces apparmRNA induction activity of RH 5992 was reported ently higher levels of expression of the ecdysteroid recently (Retnakaran et al., 1995). MHR3 is a homolog responsive gene, compared with RH 5849. Differences of DHR3, which in turn is an ecdysteroid-inducible in the relative potencies of RI-I 5992 compared to RH nuclear hormone receptor in D. melanogaster (Koelle et 5849 (about lo- to lOO-fold) among the different assays al., 1992; Palli et al., 1992). Although the ecdysteroid may have been caused by the use of different materials responsive element of MHR3 was not identified as the and assay methods. hsp 27 gene, their result provides further evidence for RH 5992 also had more than a lo-fold higher potential the ecdysteroid responsive gene expression activity of for larval mortality in different orders of insects, but difRH 5992. ferences between RH 5849 and RH 5992 for metabolic or Also, the pDAChE-LUC plasmid, which contains the uptake rates were not observed (Smagghe and Degheele, ecdysteroid inducible D. melanogaster 5’-upstream 1.62 1994). Taken together with the results from this study, kb promoter sequence of the acetylcholinesterase gene, the evidence strongly suggests that the relative in vivo was introduced into Kc cells and showed luciferase potencies of these two dibenzoyl hydrazines reflect their induction by dibenzoyl hydrazines (Mikitani, 1995a). RI-I relative potencies at the molecular and the cellular levels. 5992 again possessed higher luciferase induction activity Head capsule slippage was induced within 24h in B. than RH 5849. mori larvae fed on a diet containing dibenzoyl hydraBased on in viva tests of M. sexta (Wing et al., 1988) zines, while control larvae required an additional 48h for head capsule slippage. RH 5992 showed an approximately lo-fold higher activity for head capsule slippage TABLE 1. Head capsule slippage and mortality in 3rd instar larvae of than RH-5849. Most of the larvae that displayed head Bombyx mori in response to dibenzoyl hydrazines capsule slippage died 96h after treatment, thus the toxicity of dibenzoyl hydrazines appears to be expressed % Mortality Concentration % Head capsule slippage through ecdysteroid agonist activity. Five different diben(ppm) RH5992 RH5992 RH5849 RH5849 zoyl hydrazine derivatives (including RH 5849 and RH 5992) show gene expression activity corresponding to 0 0 0 0 0.01 against lepidopteran activities their insecticidal 0 0 0 0 0.1 (Spodoptera Zituru) larvae fed on artificial diets 80 0 90 0 1.0 (unpublished). The ecdysone receptors of two other lepi100 100 70 100 10.0 100 100 100 100 100.0 dopteran insects, B. mori (Swevers et al., 1995) and IV. sexta (Fujiwara et al., 1995), have been cloned recently. *Each concentration of the chemical (2.0 ml) was soaked into the Their ligand binding domains possess high sequence artificial diet (13 g) in a plastic cup, and 10 newly ecdysed B. mori identity (approximately 70%) with that of D. melunogaslarvae were reared. Head capsule slippage was observed 24h after ter. The similarities in the ligand binding domain of the the treatment. Mortality was observed 96h after the treatment. All receptors of Lepidoptera and Diptera suggests that RH experiments were duplicated and the average was calculated. slippage 24h after application (Table 1). RI-I 5992 at 1.0 ppm induced head capsule slippage in 90% of the larvae, while the same dose of RI-I 5849 did not induce head capsule slippage. RH 5849 at 10.0 ppm was required to induce head capsule slippage in 100% of the larvae. Mortality of the larvae was observed 96h after application (Table 1). RH 5992 killed 80% of the larvae at 1.0 ppm, while the same dose of RH 5849 killed no larvae and 10.0 ppm was required for 70% mortality.
ECDYSTEROID
ACTIVITIES
5849 and RI-I 5992 would show similar agonist activities in both orders. In summary, the two dibenzoyl hydrazines showed ecdysteroid receptor binding activity, ecdysteroid responsive gene expression activity, and induction of morphological changes in Drosophila Kc cells corresponding to their in vivo potencies against B.mori larvae. REFERENCES Ashburner M. (1972) Patterns of puffing activity in the salivary gland chromosomes of Drosophila. IV. Induction by ecdysone in salivary gland of Drosophila melanogaster cultured in vitro. Chromosoma 38, 255-281. Ashbumer M., Chihara C., Meltzer P. and Richards G. (1974) Temporal control of puffing activity in polytene chromosomes. Cold Spring Harbor Symp. Quant. Biol. 38, 655-662. Ashbumer M. (1990) Puffs, genes, and hormones revisited (minireview). Cell 61, 1-3. Cherbas P. (1993) The IVth Karlson lecture: ecdysone-responsive genes. Insect Biochem. Mol. Biol. 23, 3311. Clement C. Y., Bradbrook D. A., Lafont R. and Dinan L. (1993) Assessment of a microplate-based bioassay for the detection of ecdysteroid-like or antiecdysteroid activities. Insect Biochem. Mol. Biol. 23, 187-193. Fujiwara H., Jindra M., Newitt R., Palli S. R., Hiruma K. and Riddiford L. M. (1995) Cloning of an ecdysteroid receptor homolog from Munduca sexta and developmental profile of its mRNA in wings. Insect Biochem. Mol. Biol. 25, 845-856. Hsu A. C.-T. (1991) 1,2-Diacyl-1-alkylhydrazines. In Synthesis and chemistry of agrochemicalsII (Eds Baker D. R. et al.), pp. 478490. American Chemical Society, Washington, DC. Koelle M. R., Talbot W. S., Segraves W. A., Bender M. T., Cherbas P. and Hogness D. S. (1991) The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell 61, 59-77. Koelle M. R., Segraves W. A. and Hogness D. S. (1992) DHR3: A Drosophila steroid receptor homolog. Proc. Natl. Acad. Sci. U.S.A. 89, 61676171. Mikitani K. (1995a) The molecular biological study of insect molting hormone: ecdysteroid. Ph.D. thesis, University of Tokyo, Tokyo, Japan. Mikitani K. (1995b) Sensitive, rapid and simple method for evaluation of ecdysteroid agonist activity based on the mode of action of the hormone. J. Seric. Sci. Jpn. 64, 534-539. Mikitani K. (1996) An automated ecdysteroid receptor binding assay using a 96-well microplate. J. Seric. Sci. Jpn. 65, 141-144. Oberlander H., Silhacek D. L. and Porcheron P. (1995) Non-steroidal ecdysone agonists: tools for the study of hormonal action. Arch. Insect. Biochem. Physiol. 28, 209-223. Oikawa N. (1994) Quantitative structure-activity studies of molting hormone mimics. Ph.D. thesis, Kyoto University, Kyoto, Japan. Palli S. R., Hiruma K. and Riddiford L. M. (1992) An ecdysteroidinducible Manduca gene similar to the Drosophila DHR3 gene, a member of the steroid hormone receptor superfamily. Dev. Biol. 150, 3063 18. Quak S., Fretz A., Spindler-Barth M. and Spindler K.-D. (1995) Recep-
OF DIBENZOYL
HYDRAZINES
941
tor affinities and biological responses of nonsteroidal agonists on the epithelial cell line from Chironomus tenruns (Diptera: Chironomidae). Eur. J. Entomol. 92, 341-347. Retnakaran A., Hiruma K., Palli S. R. and Riddiford L. M. (1995) Molecular analysis of the mode of action of RH-5992, a lepidopteran-specific, non-steroidal edysteroid agonist, Insect Biochem. Mol.Biol. 25, 1099117. Silhacek D. L., Oberlander H. and Patrick P. (1990) Action of RH5849, a non-steroidal ecdysteroid mimic, on Plodia interpunctellu (Htibner) in vivo and in vitro. Arch. Insect Biochem. Physiol. 15, 201-212. Smagghe G. and Degheele D. (1994) Action of a novel nonsteroidal ecdysteroid mimic, tebufenozide @H-5992), on insect of different orders. Pestic. Sci. 42, 85592. Smagghe G. and Degheele D. (1995) Biological activity and receptorbinding activity of ecdysteroids and the ecdysteroid agonists RH5849 and RH-5992 in imaginal discs of Spodoptera exigua (Lepidoptera: Noctudiae). Eur. J. Entomol. 92, 3333340. Sobek L., Bohm G.-A. and Penzlin H. (1993) Ecdysteroid receptor in last instar larvae of the wax moth Galleria mellonella L. Insect Biochem. Molec. Biol. 23, 125-129. Sohi S. S., Palli S. R., Cook B. J. and Retnakaran A. (1995) Forest insect cell lines responsive to 20-hydroxyecdysone and two nonsteroidal ecdysone agonists, RH-5849 and RH-5992. J. Insect Physiol. 41, 457464. Spindler-Barth M., Turberg A. and Spindler K.-D. (1991) On the action of RH5849, a nonsteroidal ecdysteroid agonist, on a cell line from Chironomus tentans. Arch. Insect Biochem. Physiol. 16, 1 l-18. Swevers L., Drevet J. R., Lunke M. D. and Iatrou K. (1995) The silkmoth homolog of the Drosophila ecdysone receptor (Bl isoform): cloning and analysis of expression during follicular ceil differentiation. Insect Biochem. Mol. Biol. 25, 857-866. Thomas H. E., Stunnenberg H. G. and Stewart A. F. (1993) Heterodimerization of the Drosophila ecdysone receptor with retinoid X receptor and ultraspiracle. Nature 362, 471475. Wing K. D. (1988) RH-5849, a nonsteroidal ecdysone agonist: Effects on a Drosophila cell line. Science 241, 467469. Wing K. D., Slawecki R. A. and Carlson G. R. (1988) RH-5849, a nonsteroidal ecdysone agonist: Effects on larval Lepidoptera. Science 241, 470472. Yao T.-P., Segraves W. A., Oro A. E., McKeown M. and Evans R. M. (1992) Drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation. Cell 71, 63-72. Yao T.-P., Forman B. M., Jiang Z., Cherbas L., Chen J. -D., McKeown M., Cherbas P. and Evans R. (1993) Functional ecdysone receptor is the product of EcR and Ultraspiracle genes. Nature 366, 476 479.
Acknowledgements-I thank Professor Masahiko Kobayashi, Associate Professor Masao Nagata and Associate Professor Toru Shimada of the University of Tokyo for useful and kind advice. I am grateful to Associate Professor Haruhiko Fujiwara of the University of Tokyo for information on ecdysteroid receptor genes of lepidopteran insects. I also express thanks to Professor Reio Shimura and his colleagues at Kyoto University for providing the Kc cell line. I thank Mr Kouichi Sugihara, Mr Keiichi Izumi and Mr Touhei Takagaki of Sumitomo Chemical Co., Ltd for their help and encouragement during this work.
J. Insect Physiol.
Pergamon
Printed in Great Britain. All rights resewed
PII: SOO22-1910(96)00056-X
0022-1910/96$15.00 + 0.00
Ecdysteroid Receptor Binding Activity and Ecdysteroid Agonist Activity at the Level of Gene Expression are Correlated with the Activity of Dibenzoyl Hydrazines in Larvae of Bombyx mori KENICHI MIKITANI* Received 1 November 199.5; revised and accepted 26 March 1996
Ecdysteroid activities of two dibenzoyl hydrazines (RH 5849 and RH 5992) were examined using the Kc cell line. Gene expression activity was determined in the transient assay utilizing the ecdysteroid responsive Drosophila melanogaster hsp27 promoter gene. Ecdysteroid receptor binding (E& = 3.0 x lo-’ M), induction of ecdysteroid responsive gene expression (EC5,, = 3.0 x 10m6 M) and induction of cell morphological change (Et& = 3.0 x lo-’ M) were 13fold, 7-fold and 13-fold higher in RH 5992 than in RH 5849, respectively. The addition of 10 ppm RH 5849 to the diet induced head capsule slippage in Bombyx mori larvae and most of the larvae died. RH 5992 caused the same response at 1 ppm. The higher insecticidal activity of RH 5992 compared to RH 5849 is likely caused by its higher ecdysteroid agonist activity at the molecular level. Copyright 0 1996 Elsevier Science Ltd Ecdysteroid
Dibenzoyl hydrazines Gene expression Bombyx mori
INTRODUCTION
Kc cell
supports the validity of this model. Another nuclear hormone receptor (Usp) is indispensable in forming the functional ecdysteroid receptor complex (Yao et al., 1992, 1993; Thomas et al., 1993). Thus far, there is little information concerning gene expression activities of the non-steroidal ecdysteroid agonists dibenzoyl hydrazines (Retnakaran et al., 1995). RH 5992 showed apparently higher (lo- to 1OO-fold) insecticidal activities against various Lepidopteran insect species than RH 5849 (Smagghe and Degheele, 1994). In the present study, the activity of two dibenzoyl hydrazines (RH 5849 and RI-I 5992, Fig. 1) are evaluated for receptor binding, gene expression and the induction of cell morphological changes. Also, in vivo activities against larvae of Bombyx mori (silkworm) are investigated.
1, 2-Dibenzoyl- 1-tert-butylhydrazine (RH 5849) was the first non-steroidal chemical that showed ecdysteroid agonist activities at the cellular (Wing, 1988) and organismal (Wing et al., 1988) levels. Dibenzoyl hydrazines are effective chemical tools for insect hormone research (Oberlander et al., 1995). They show ecdysteroid receptor binding as well as the ability to induce acetylcholinesterase and morphological changes in Kc cells. The uptake of N-acetyl-glucosamine by a Plodia interpunctella (Indian meal moth) cell line of wing disk origin was also observed in response to both RI-I 5849 and 20-hydroxyecdysone (Silhacek et al., 1990). The mode of action of ecdysteroids is believed to be via gene expression of ecdysteroid responsive genes (Ashbumer, 1972, 1990; Ashbumer et al., 1974; Cherbas, 1993). The recent cloning of the ecdysone receptor (EcR) gene from Drosophila melanogaster (Koelle et al., 1991)
MATERIALS AND METHODS Assay for ecdysteroid
receptor
binding
The ecdysteroid receptor binding assay was performed by the automated 96-well microplate based method
*Agricultural Chemicals Research Laboratory, Sumitomo Chemical Co., Ltd, 4-2-1, Takatsukasa, Takarazuka, Hyogo 665, Japan. 937
938
K. MIKITANI
Compound RH 5849 RH 5992
Rl
R2
R3
H EH,CH~
CHs
cti3
H
FIGURE 1. Chemical structures of dibenzoyl hydrazines used in this study.
(Mikitani, 1995a, 1996). D. melanogaster Kc cells cultured in spinner flasks were collected and cell cytosols were made by sonication. After ultracentrifugation (lOO,OOOg,4°C 60 min), supematants were frozen and stored as aliquots in liquid nitrogen until use. 3H-ponasterone A (final concentration of 1.0 x lop9 M), thawed Kc cell cytosol and different concentrations of RH 5849 and RH 5992 dissolved in dimethyl sulfoxide were transferred and mixed in each well of the 96-well microplate. After a 60 min incubation at 25°C the ice-cold dextrancoated charcoal suspension was added to the well. After vigorous mixing at 4°C the 3H-ponasterone A that was not bound to receptor was absorbed to the charcoal and precipitated by centrifugation. The supematant was transferred to a solid scintillator for the measurement of radioactivity. Non-specific binding was defined as dpm bound in the presence of 2.0 x lop5 M 20-hydroxyecdysone, and each data point was corrected by subtracting the dpm value of non-specific binding from that of each total binding. Assay for ecdysteroid
responsive
gene expression
activity
Gene expression activity for each compound was assayed as reported (Mikitani, 1995a,b). The reporter plasmid pHSP27-LUC was constructed by ligation of the D. melanogaster 669 bp hsp27 gene promoter region (which contains the ecdysteroid responsive element and a TATA box sequence) with the firefly luciferase cDNA. The highly purified pHSP27-LUC plasmid was transiently introduced into Kc cells by electroporation (0.5 kV, 3 PF). The cells, diluted 20-fold with the medium in 2.5 ml aliquots, were incubated with 2.5 ~1 of dimethyl sulfoxide containing the chemicals (RH 5849 and RH 5992) for 24h in a 6 cm diameter tissue culture plastic Petri dish at 25°C under 5% CO,. After 24h, cells were lysed and the luciferase activity was measured. Protein concentration was determined by the BCA method for correction.
Assay for induction
of cell morphological
changes
Nearly confluent Kc cells were diluted with M3 (BF) medium supplemented with 2% fetal bovine serum to a density of 1.0 x lo6 cells/ml. The cells in 2.5 ml aliquots were incubated with 2.5 ~1 of dimethyl sulfoxide containing the chemicals (RH 5849, RH 5992 and 20hydroxyecdysone) for 48h in a 6 cm diameter tissue culture plastic Petri dish at 25°C under 5% COZ. Then, the cells were observed and photographed by phase-contrast microscopy. Ecdysteroid responding cells were scored as having more than twice the length of process elaboration compared to the shorter width of the cell. Activity
test against
B. mori larvae
B. mori (Kinsyu x Shugetsu) larvae were fed on an artificial diet and reared at 25°C 60% RH and 16L:8D photoperiod. The compounds (RH 5849 and RH 5992) were dissolved in dimethyl sulfoxide and diluted to the different concentrations with distilled water. A solution of 2.0 ml was poured on the artificial diet (13 g) in a plastic cup. After absorption of the solution into the diet, 10 newly ecdysed 3rd instar larvae were transferred to each cup. All experiments were duplicated and the average was taken. Cultures were checked for head capsule slippage at 24h intervals, and mortality was assessed 96h after the larvae were transferred to the cups. Chemicals RH 5849 and RH 5992 were synthesized from N-benzoyl-Ar-tert-butyl-hydrazine and N-4-ethyl-benzoyl-hrtert-butyl-hydrazine, respectively, and 99% purity products were used (Hsu, 1991; Oikawa, 1994). 20Hydroxyecdysone (98% purity) was purchased from Sigma.
939
ECDYSTEROID ACTIVITIES OF DIBENZOYL HYDRAZINES RESULTS
Ecdysteroid hydrazines
receptor
binding
activities
of dibenzoyl 40
Displacement kinetics of 3H-ponasterone A binding to the Kc cell cytosol by dibenzoyl hydrazines are shown in Fig. 2. RH 5992 at 4.0 x 1O-6 M displaced 95% of 3H-ponasterone A binding, while 4.0 x lo-’ M of RH 5849 achieved 98% displacement. For RH 5992, the EC,,, (effective concentration, 50%) was 3.0 x lo-’ M; for RH 5849, the E& was 4.0 x lop6 M. The ECso for RH 5849 was approximately the same as the value previously reported in a similar binding assay (Wing, 1988). For 20hydroxyecdysone, the E& was 8.0 x lo-* M, as was previously shown in the same binding assay system (Mikitani, 1995a, 1996). Ecdysteroid responsive dibenzoyl hydrazines
gene expression
activities
c
0 ‘c;
2
z ._
30
0
9
20
10
0 lo-’
10”
of
The luciferase reporter plasmid (pHSP27-LUC), which contains the 5’-upstream 0.67 kb sequence of the D. melanogaster hsp27 gene as the promoter gene, was transiently transfected into Kc cells by electroporation (Mikitani, 1995a,b). The luciferase induction activities of dibenzoyl hydrazines in the pHSP27-LUC plasmid-introduced cells were determined (Fig. 3). For RH 5849, the ECso was 2.0 x lop5 M; for RH 5992, the EC& was 3.0 x lop6 M. Maximal inductions by RH 5849 and RH 5992 were 43-fold and 45-fold, respectively.
10”
los
lo4
10;
Concentration (id) FIGURE 3. Dose-response curves for luciferase induction activities of RH 5849 (0) and RH 5992 (e) against pHSP27-LUC introduced Kc cells. The pHSP27-LUC plasmid DNA was transfected into Kc cells by electroporation methods and cultured with and without the chemicals. After 24h, cell extracts were prepared for the measurement of luciferase activities and protein concentrations (for correction). Each data point is the average of duplicate measurements.
Induction of morphological changes in Kc cells by dibenzoyl hydrazines Like 20-hydroxyecdysone, RH 5849 and RH 5992 also induced process protrusion in Kc cells (Fig. 4). The E&
100 90
Em g
70
8
60
$
50
lo-lo
10"
10-l
Concentration
10"
1o-5
lo4
10"
(M)
FIGURE 4. Induction of Kc cell process protrusion by RH 5849 (0), RH 5992 (0) and 20-hydroxyecdysone (0). Nearly confluent cells were cultured with the chemicals for 48h. Ecdysteroid responding cells were scored as having more than twice the length of process elaboration compared to the shorter width of the cell. Each data point represents the mean f SD (n = 3).
$Ia 8
10'
30
20 10 0 lo-lo
10-g
109
1o-7
lo6
1o-5
lo4
lo9
Concentration (M) FIGURE 2. Displacement of ‘H-ponasterone A bound to the Kc cell cytosol in the presence of RH 5849 (0) and RH 5992 (0). Specifically, bound ‘H-ponasterone A was determined as described in Materials and Methods and is plotted as percentage displacement of 3H-ponasterone A binding vs test concentration of chemicals. Each data point represents the mean k SD (n = 3).
for RH 5849 was 4.0 x lop6 M; the ECXo for RH 5992 was 3.0 x IO-’ M; the EC,,, for 20-hydroxyecdysone was 1.0 x lop8 M. The EC&, for RH 5849 was approximately the same as the value previously reported (Wing, 1988). Activities of dibenzoyl hydrazines against B. mori larvae Dibenzoyl hydrazines applied to the artificial diet in B. mori 3rd instar day 0 larvae induced head capsule
940
K. MIKITANI
and biochemical and cellular studies using D. melunoguster Kc cells (Wing, 1988), RH 5849 was reported to be an ecdysteroid agonist. In addition to the above mentioned study of MHR3 mRNA induction by RH 5992 (Retnakaran et al., 1995), a number of studies have focused on the mode of action of dibenzoyl hydrazines, both in cell lines of Diptera (Clement et al., 1993; Quak et al., 1995; Spindler-Barth et al., 1991) and in cell lines or tissues of Lepidoptera (Silhacek et al., 1990; Sobek et al., 1993; Sohi et al., 1995; Smagghe and Degheele, 1995). The effective concentrations of RH 5849 found in DISCUSSION these previous studies ranged from 10e4 M to 10m6 M. The results of the transient gene expression assay in The results of the gene expression assay (ECSo = 2.0 x this paper clearly show that dibenzoyl hydrazines induce lo-’ M) and the cell morphology assay (EC,, = 4.0 x significantly high levels of ecdysteroid responsive gene lop6 M) in the present study are within that range. In expression. Induction activity of RI-I 5992 was 7-fold some of the previous studies, RH 5992 was compared higher than that of RH 5849. In addition, the results in with RH 5849 for ecdysteroid receptor binding and biothis paper show that RH 5992 has more than a lo-fold logical activities (Quak et al., 1995; Smagghe and stronger affinity for the ecdysteroid receptor than RH Degheele, 1995). In those studies the potencies of the 5849. The gene expression activity reflects this difference two chemicals in ecdysteroid receptor binding (RH of receptor affinity, and the cellular response measured 5992> RI-I 5849) corresponded to their biological effects. as cell process protrusion also showed the same range of However, the edysteroid responsive gene expression difference (7-fold) between the two compounds. activities of the two chemicals were not investigated. The The Manduca se&a hormone receptor 3 (MHR3) present studies clearly show that RH 5992 induces apparmRNA induction activity of RH 5992 was reported ently higher levels of expression of the ecdysteroid recently (Retnakaran et al., 1995). MHR3 is a homolog responsive gene, compared with RH 5849. Differences of DHR3, which in turn is an ecdysteroid-inducible in the relative potencies of RI-I 5992 compared to RH nuclear hormone receptor in D. melanogaster (Koelle et 5849 (about lo- to lOO-fold) among the different assays al., 1992; Palli et al., 1992). Although the ecdysteroid may have been caused by the use of different materials responsive element of MHR3 was not identified as the and assay methods. hsp 27 gene, their result provides further evidence for RH 5992 also had more than a lo-fold higher potential the ecdysteroid responsive gene expression activity of for larval mortality in different orders of insects, but difRH 5992. ferences between RH 5849 and RH 5992 for metabolic or Also, the pDAChE-LUC plasmid, which contains the uptake rates were not observed (Smagghe and Degheele, ecdysteroid inducible D. melanogaster 5’-upstream 1.62 1994). Taken together with the results from this study, kb promoter sequence of the acetylcholinesterase gene, the evidence strongly suggests that the relative in vivo was introduced into Kc cells and showed luciferase potencies of these two dibenzoyl hydrazines reflect their induction by dibenzoyl hydrazines (Mikitani, 1995a). RI-I relative potencies at the molecular and the cellular levels. 5992 again possessed higher luciferase induction activity Head capsule slippage was induced within 24h in B. than RH 5849. mori larvae fed on a diet containing dibenzoyl hydraBased on in viva tests of M. sexta (Wing et al., 1988) zines, while control larvae required an additional 48h for head capsule slippage. RH 5992 showed an approximately lo-fold higher activity for head capsule slippage TABLE 1. Head capsule slippage and mortality in 3rd instar larvae of than RH-5849. Most of the larvae that displayed head Bombyx mori in response to dibenzoyl hydrazines capsule slippage died 96h after treatment, thus the toxicity of dibenzoyl hydrazines appears to be expressed % Mortality Concentration % Head capsule slippage through ecdysteroid agonist activity. Five different diben(ppm) RH5992 RH5992 RH5849 RH5849 zoyl hydrazine derivatives (including RH 5849 and RH 5992) show gene expression activity corresponding to 0 0 0 0 0.01 against lepidopteran activities their insecticidal 0 0 0 0 0.1 (Spodoptera Zituru) larvae fed on artificial diets 80 0 90 0 1.0 (unpublished). The ecdysone receptors of two other lepi100 100 70 100 10.0 100 100 100 100 100.0 dopteran insects, B. mori (Swevers et al., 1995) and IV. sexta (Fujiwara et al., 1995), have been cloned recently. *Each concentration of the chemical (2.0 ml) was soaked into the Their ligand binding domains possess high sequence artificial diet (13 g) in a plastic cup, and 10 newly ecdysed B. mori identity (approximately 70%) with that of D. melunogaslarvae were reared. Head capsule slippage was observed 24h after ter. The similarities in the ligand binding domain of the the treatment. Mortality was observed 96h after the treatment. All receptors of Lepidoptera and Diptera suggests that RH experiments were duplicated and the average was calculated. slippage 24h after application (Table 1). RI-I 5992 at 1.0 ppm induced head capsule slippage in 90% of the larvae, while the same dose of RI-I 5849 did not induce head capsule slippage. RH 5849 at 10.0 ppm was required to induce head capsule slippage in 100% of the larvae. Mortality of the larvae was observed 96h after application (Table 1). RH 5992 killed 80% of the larvae at 1.0 ppm, while the same dose of RH 5849 killed no larvae and 10.0 ppm was required for 70% mortality.
ECDYSTEROID
ACTIVITIES
5849 and RI-I 5992 would show similar agonist activities in both orders. In summary, the two dibenzoyl hydrazines showed ecdysteroid receptor binding activity, ecdysteroid responsive gene expression activity, and induction of morphological changes in Drosophila Kc cells corresponding to their in vivo potencies against B.mori larvae. REFERENCES Ashburner M. (1972) Patterns of puffing activity in the salivary gland chromosomes of Drosophila. IV. Induction by ecdysone in salivary gland of Drosophila melanogaster cultured in vitro. Chromosoma 38, 255-281. Ashbumer M., Chihara C., Meltzer P. and Richards G. (1974) Temporal control of puffing activity in polytene chromosomes. Cold Spring Harbor Symp. Quant. Biol. 38, 655-662. Ashbumer M. (1990) Puffs, genes, and hormones revisited (minireview). Cell 61, 1-3. Cherbas P. (1993) The IVth Karlson lecture: ecdysone-responsive genes. Insect Biochem. Mol. Biol. 23, 3311. Clement C. Y., Bradbrook D. A., Lafont R. and Dinan L. (1993) Assessment of a microplate-based bioassay for the detection of ecdysteroid-like or antiecdysteroid activities. Insect Biochem. Mol. Biol. 23, 187-193. Fujiwara H., Jindra M., Newitt R., Palli S. R., Hiruma K. and Riddiford L. M. (1995) Cloning of an ecdysteroid receptor homolog from Munduca sexta and developmental profile of its mRNA in wings. Insect Biochem. Mol. Biol. 25, 845-856. Hsu A. C.-T. (1991) 1,2-Diacyl-1-alkylhydrazines. In Synthesis and chemistry of agrochemicalsII (Eds Baker D. R. et al.), pp. 478490. American Chemical Society, Washington, DC. Koelle M. R., Talbot W. S., Segraves W. A., Bender M. T., Cherbas P. and Hogness D. S. (1991) The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell 61, 59-77. Koelle M. R., Segraves W. A. and Hogness D. S. (1992) DHR3: A Drosophila steroid receptor homolog. Proc. Natl. Acad. Sci. U.S.A. 89, 61676171. Mikitani K. (1995a) The molecular biological study of insect molting hormone: ecdysteroid. Ph.D. thesis, University of Tokyo, Tokyo, Japan. Mikitani K. (1995b) Sensitive, rapid and simple method for evaluation of ecdysteroid agonist activity based on the mode of action of the hormone. J. Seric. Sci. Jpn. 64, 534-539. Mikitani K. (1996) An automated ecdysteroid receptor binding assay using a 96-well microplate. J. Seric. Sci. Jpn. 65, 141-144. Oberlander H., Silhacek D. L. and Porcheron P. (1995) Non-steroidal ecdysone agonists: tools for the study of hormonal action. Arch. Insect. Biochem. Physiol. 28, 209-223. Oikawa N. (1994) Quantitative structure-activity studies of molting hormone mimics. Ph.D. thesis, Kyoto University, Kyoto, Japan. Palli S. R., Hiruma K. and Riddiford L. M. (1992) An ecdysteroidinducible Manduca gene similar to the Drosophila DHR3 gene, a member of the steroid hormone receptor superfamily. Dev. Biol. 150, 3063 18. Quak S., Fretz A., Spindler-Barth M. and Spindler K.-D. (1995) Recep-
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tor affinities and biological responses of nonsteroidal agonists on the epithelial cell line from Chironomus tenruns (Diptera: Chironomidae). Eur. J. Entomol. 92, 341-347. Retnakaran A., Hiruma K., Palli S. R. and Riddiford L. M. (1995) Molecular analysis of the mode of action of RH-5992, a lepidopteran-specific, non-steroidal edysteroid agonist, Insect Biochem. Mol.Biol. 25, 1099117. Silhacek D. L., Oberlander H. and Patrick P. (1990) Action of RH5849, a non-steroidal ecdysteroid mimic, on Plodia interpunctellu (Htibner) in vivo and in vitro. Arch. Insect Biochem. Physiol. 15, 201-212. Smagghe G. and Degheele D. (1994) Action of a novel nonsteroidal ecdysteroid mimic, tebufenozide @H-5992), on insect of different orders. Pestic. Sci. 42, 85592. Smagghe G. and Degheele D. (1995) Biological activity and receptorbinding activity of ecdysteroids and the ecdysteroid agonists RH5849 and RH-5992 in imaginal discs of Spodoptera exigua (Lepidoptera: Noctudiae). Eur. J. Entomol. 92, 3333340. Sobek L., Bohm G.-A. and Penzlin H. (1993) Ecdysteroid receptor in last instar larvae of the wax moth Galleria mellonella L. Insect Biochem. Molec. Biol. 23, 125-129. Sohi S. S., Palli S. R., Cook B. J. and Retnakaran A. (1995) Forest insect cell lines responsive to 20-hydroxyecdysone and two nonsteroidal ecdysone agonists, RH-5849 and RH-5992. J. Insect Physiol. 41, 457464. Spindler-Barth M., Turberg A. and Spindler K.-D. (1991) On the action of RH5849, a nonsteroidal ecdysteroid agonist, on a cell line from Chironomus tentans. Arch. Insect Biochem. Physiol. 16, 1 l-18. Swevers L., Drevet J. R., Lunke M. D. and Iatrou K. (1995) The silkmoth homolog of the Drosophila ecdysone receptor (Bl isoform): cloning and analysis of expression during follicular ceil differentiation. Insect Biochem. Mol. Biol. 25, 857-866. Thomas H. E., Stunnenberg H. G. and Stewart A. F. (1993) Heterodimerization of the Drosophila ecdysone receptor with retinoid X receptor and ultraspiracle. Nature 362, 471475. Wing K. D. (1988) RH-5849, a nonsteroidal ecdysone agonist: Effects on a Drosophila cell line. Science 241, 467469. Wing K. D., Slawecki R. A. and Carlson G. R. (1988) RH-5849, a nonsteroidal ecdysone agonist: Effects on larval Lepidoptera. Science 241, 470472. Yao T.-P., Segraves W. A., Oro A. E., McKeown M. and Evans R. M. (1992) Drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation. Cell 71, 63-72. Yao T.-P., Forman B. M., Jiang Z., Cherbas L., Chen J. -D., McKeown M., Cherbas P. and Evans R. (1993) Functional ecdysone receptor is the product of EcR and Ultraspiracle genes. Nature 366, 476 479.
Acknowledgements-I thank Professor Masahiko Kobayashi, Associate Professor Masao Nagata and Associate Professor Toru Shimada of the University of Tokyo for useful and kind advice. I am grateful to Associate Professor Haruhiko Fujiwara of the University of Tokyo for information on ecdysteroid receptor genes of lepidopteran insects. I also express thanks to Professor Reio Shimura and his colleagues at Kyoto University for providing the Kc cell line. I thank Mr Kouichi Sugihara, Mr Keiichi Izumi and Mr Touhei Takagaki of Sumitomo Chemical Co., Ltd for their help and encouragement during this work.