Selection for resistance to methoxyfenozide and 20-hydroxyecdysone in cells of the beet armyworm Spodoptera exigua.код для вставкиСкачать
36 Mosallanejad et al. Archives of Insect Biochemistry and Physiology 67:36–49 (2008) Selection for Resistance to Methoxyfenozide and 20-Hydroxyecdysone in Cells of the Beet Armyworm, Spodoptera exigua Hadi Mosallanejad, Thomas Soin, and Guy Smagghe* In this report with an ecdysteroid-responsive cell line of the beet armyworm, Spodoptera exigua (Se4) selection for resistance against methoxyfenozide and the insect moulting hormone (20-hydroxyecdysone, 20E) was carried out to analyze the resulting resistant cells in order to elucidate possible mechanisms of resistance towards these compounds. From these cultures, five methoxyfenozide- and four 20E-resistant subclones were selected starting from 0.1 nM methoxyfenozide up to 100 µM and from 10 nM 20E up to 100 µM, respectively. To date, the selected cells kept their loss of susceptibility for 100 µM. Here we evaluated two processes known to be important in insecticide resistance, namely metabolism and pharmacokinetics, in the selected methoxyfenozide- and 20E-resistant subclones. Synergism experiments with piperonyl butoxide, S,S,S-tributyl phosphorotrithioate, and diethyl maleate, which are respective inhibitors of monooxygenases, esterases, and gluthation-Stransferases, did not affect the level of the resistance. To check the possible existence of active transport in the resistant cells, we used ouabain, an inhibitor of active membrane transport. In parallel, the absorption profile was studied in resistant and susceptible cells with use of 14C-methoxyfenozide. Interestingly, resistant subclones showed cross-resistance towards methoxyfenozide and 20E. The resistance was irreversible even after the compounds were removed from the medium. Arch. Insect Biochem. Physiol. 67:36–49, 2008. © 2007 Wiley-Liss, Inc. KEYWORDS: resistance mechanism; ecdysteroid agonist; methoxyfenozide; 20-hydroxyecdysone; Spodoptera exigua; insect cell line INTRODUCTION Pest management strategies have evolved over the years from broad-spectrum to target specific narrow-spectrum pesticides (Retnakaran et al., 2003). Non-steroidal ecdysteroid agonists such as methoxyfenozide (RH-2485), tebufenozide (RH5992), halofenozide (RH-0345), and chromafenozide (ANS-118), are a group of insect growth regulator insecticides (IGRs), also called moulting accelerating compounds (MACs) that provoke a precocious and lethal larval moulting in susceptible insects (Wing et al., 1988; Dhadialla et al., 1998). These compounds are harmless to vertebrates (Carlson et al., 2001) with little or no adverse effects on beneficial insects (Smagghe and Degheele, 1995; Dhadialla et al., 1998; Retnakaran et al., 2003). So, their narrow spectrum of activity makes them an excellent tool in integrated pest management (IPM) programs. Even though these compounds are non-steroidal, they manifest their activity via interaction with the functional ecdysteroid receptor complex, like the steroidal insect moulting hormone, 20-hydroxyecdysone (20E). Although 20E is the insect moulting hormone, selective toxicity is observed among different insect Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium Contract grant sponsor: Ministry of Science, Research and Technology (SRT); Contract grant sponsor: Agricultural Research Organization of Iran (Plant Pest and Disease Research Institute). *Correspondence to: G. Smagghe, Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium. E-mail: firstname.lastname@example.org Received 27 April 2007; Accepted 26 July 2007 © 2007 Wiley-Liss, Inc. DOI: 10.1002/arch.20220 Published online in Wiley InterScience (www.interscience.wiley.com) Archives of Insect Biochemistry and Physiology January 2008 doi: 10.1002/arch. Resistance to Methoxyfenozide and 20E in S. exigua orders for these dibenzoylhydrazine-type compounds (Smagghe and Degheele, 1994; Dhadialla et al., 1998; Sundaram et al., 1998). Methoxyfenozide and tebufenozide are the most effective of selective dibenzoylhydrazine insecticides now commercialized for the control of lepidopteran larvae. While these compounds hold promise as excellent pest control agents, some laboratory and field surveys have shown that resistance and crossresistance can evolve in response to this chemical group (Sauphanor and Bouvier, 1995; Smagghe et al., 1998, 2003; Waldstein et al., 1999; Moulton et al., 2002; Gore and Adamczyk, 2004; Cao and Han, 2006). In toxicology, established insect cell lines are tools for screening purposes and identifying the mode of action of compounds, while providing homogeneous material (Spindler et al., 1993; Smagghe, 2007). The easy handling and the fast response of these systems also allows their use as a suitable predictive method to forecast the development of insecticide resistance. Several in vivo and in vitro studies have tried to explain the differential toxicity of the non-steroidal ecdysteroid agonists to susceptible, nonsusceptible, and resistant insects and cells (Spindler-Barth et al., 1991; Smagghe and Degheele, 1993, 1994; Sundaram et al., 1998; Retnakaran et al., 2001; Hu et al., 2001; Zhang et al., 2006). The objectives of this study are to better understand the potency of resistance development to ecdysteroid agonists at the cellular level and the mechanism(s) underlying this phenomenon. Here we investigated the significance of two key processes involved in resistance development, namely metabolism and pharmacokinetics. In this project with an ecdysteroid-responsive cell line of the beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), we assessed the potency of four ecdysteroid agonists. The beet armyworm is a polyphagous noctuid species of world economic importance. Such caterpillars cause high levels of damage in >75 crop species; also many populations developed high levels of resistance towards most insecticide groups (Alford, 2000). In continuation, we challenged these cells with one of the most active compounds, methoxyArchives of Insect Biochemistry and Physiology January 2008 doi: 10.1002/arch. 37 fenozide, to select for resistance. In parallel, we selected the cells for resistance towards the natural insect moulting hormone 20E. This is the first report of a cell line selected for resistance against ecdysteroid and agonist, derived from an important target pest insect. With the resulting resistant cells, we then investigated the resistance stability, cross-resistance, and two possible resistance mechanisms towards these two compounds. MATERIALS AND METHODS Chemicals A technical grade of ecdysteroid agonists, halofenozide (RH-0345, >90% pure), methoxyfenozide (RH-2485, unlabeled >95% pure and 14C-tert-butyllabeled with a specific activity of 23.06 mCi/g), tebufenozide (RH-5992, >95% pure), and RH-5849 (>99% pure) were a kind gift of Rohm and Haas Co. (Spring House, PA). 20E (>95% pure) was purchased from Sigma Co. (Bornem, Belgium). Serial dilutions of the test compounds were prepared in ethanol. Cell Line and Culture Conditions The Se4 cell line (BCIRL/AMCY-SeE-CLG4, a kind gift from Dr. C. Goodman, USDA-ARS, BCIRL, Columbia, MO), which originated from embryos of the beet armyworm, S. exigua (Goodman et al., 2001), was cultured at 27°C in EX-CELL™ 401 and in EX-CELL™ 420 medium (JRH Biosciences, Hampshire, UK), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma Co., Bornem, Belgium). Because of the unavailability of the old version (EX-CELL™ 401) we had to use the new version (EX-CELL™ 420) of this medium. Schneider 2 (S2) cells (S2-Mt-Dl, derived from Drosophila melanogaster embryo) were cultured in HyQ SFXInsect™ Medium (Perbio Science, Erembodegem, Belgium). Comparative Potency of Ecdysteroid Agonists To determine the relative potencies of four ecdysteroid agonists (methoxyfenozide, tebufen- 38 Mosallanejad et al. ozide, halofenozide, and RH-5849) and 20E on cell proliferation, the cells were treated for 5 days with the test compounds at final concentrations ranging from 100 µM to 1 pM. A cell solution with a density of 100,000 cells/ml was prepared. After loading the required wells of a 96-well microtiter plate (Greiner labortechnik, Frickenhausen, Germany) with 100 µl of the cell solution, 1 µl of the compound was added with a microsyringe equipped with an applicator (Hamilton, Bonaduz, Switzerland). Then the plates were sealed with parafilm and incubated for 5 days at 27°C. For controls, cells were treated with 1 µl of ethanol in 100 µl cell suspension. For every concentration, 6 replicates were done and each experiment was repeated 2 or 3 times. After incubation, the cell numbers were counted with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) technique according to Decombel et al. (2005). Briefly, 100 µl of cell solution was transferred to a microtube (Eppendorf) and 100 µl of 1 mg/ml MTT solution was added. After 3 h incubation at 27°C, the formazan crystals were collected by centrifugation for 7 min at 20,000g at 4°C. Then, the formazan crystals were dissolved in isopropanol. Then, for the next 30 min, the microtubes were rotated using a test tube rotator (Labinco, Breda, The Netherlands). After centrifugation of the resulting solution again for 7 min at 20,000g, the absorbance was measured at 560 nm in a microtiter plate reader (PowerWare X340, Bio-Tek Instruments Inc., Winooski, VT). The results were transformed to relative data as the percentage of active cells in comparison to the control batch and the percentage effect was calculated. EC50’s, median effective concentration values on cell proliferation were calculated with Prism version 4® (GraphPad Software Inc., San Diego, CA). nM 20E up to 100 µM, and from 0.1 nM methoxyfenozide up to 100 µM. These starting concentrations corresponded closely with respective EC10s (data not shown). Gradually increasing amounts of 20E and methoxyfenozide (until the highest concentration, 100µM) was continued for over 15 months, which corresponds to 70 and 60 passages for 20E- and methoxyfenozide-resistant subclones, respectively. The evaluation of the observed resistance was done between passages 34 and 38 for the methoxyfenozide-resistant subclones, and between passages 18 and 21 for the 20E-resistant subclones, using the MTT assay as described above. A survey of selected concentrations is presented in Figure 1. Five and four subclones were independently selected for resistance to methoxyfenozide (Se4-RH2485-R1-5) and 20E (Se4-20E-R1-4), respectively. After reaching the highest concentration, we continued subculturing all of the resistant subclones under this pressure until the present (February 2007). Effect of Metabolic Synergists: PBO, DEF, and DEM The enzyme synergists tested were piperonyl butoxide (PBO, technical grade, Fluka, Bornem, Selection for Resistance Cell proliferation cessation by 20E and methoxyfenozide was used to select resistant subclones of the cells. Subclones were selected by exposure of cells to increasing concentrations of 20E and methoxyfenozide, starting in 2005–2006 from 10 Fig. 1. Development of resistance towards methoxyfenozide and 20E for successive selection with increasing concentrations over different passages for the Se4-RH2485R4 and the Se4-20E-R4 subclone, respectively. Archives of Insect Biochemistry and Physiology January 2008 doi: 10.1002/arch. Resistance to Methoxyfenozide and 20E in S. exigua Belgium), S,S,S-tributyl phosphorotrithioate (DEF, 70.5%, Bayer CropScience, Monheim, Germany), and diethyl maleate (DEM, 80%, Janssen Chemica, Beerse, Belgium). A cell viability test with a concentration range of synergists (0.1 nM to 100 µM) was first done to choose a suitable concentration that has no effect on cell proliferation. A concentration up to 10 µM PBO and DEM or 1 µM DEF has no effect (P > 0.05) on cell proliferation (data not shown). The susceptible and the resistant cells (Se4-RH-2485-R4 and Se4-20E-R4) were seeded at the density of 100,000 cells/ml in 96-well plates containing 10 µM PBO or DEM or 1 µM DEF and without synergists. For each treatment, there were at least 4 replications and each experiment was done 2 times. Next, the susceptibility of cells towards methoxyfenozide and 20E after 5 days incubation was evaluated using the MTT assay as described above. 39 also determined. The experiment was performed two times. In parallel, the amount of total protein present in the cell pellets was determined based on Bradford (1976) with the Coomassie staining protein assay kit (Pierce Biotechnology Inc., Erembodegem, Belgium), using bovine serum albumin as the standard. For the protein measurement, cell samples (three samples of each cell type, 106 cells/ml) were centrifuged (5 min, 100g) and the supernatants were removed and the cell pellets resuspended with lysis buffer (0.125 M Tris, 5% β-mercapthoethanol, 2% SDS, and 4 M urea). Next, the samples were put for 15 min in boiled water and then the amount of the total protein was quantified according to the manufacturer’s manual (Pierce Biotechnology Inc.). The test was repeated twice. Effect of Ouabain, an Inhibitor of Active Membrane Transport Retention of Radiolabeled Methoxyfenozide 14 Retention of C-RH-2485 in the Se4-susceptible cells and the Se4-RH-2485-R4 subclone was measured, and compared to Drosophila S2 cells as tested by Sundaram et al. (1998). The cells (three samples of each cell type) were seeded at a density of 106 cells/ml in their respective culture medium. Radiolabeled methoxyfenozide was added to the cells at a level of 900,000 CPM/ml together with 100 µM methoxyfenozide. The cells were incubated for 3 h at 27°C. After the incubation period, samples of 1 ml of cell suspension were washed three times with phosphate buffer saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4 and 1.5 mM KH2PO4, pH 7.3) by resuspension and centrifugation (5 min at 90g). After the final wash, cell pellets were solubilized in 200 µl of 1.5 M NaOH by incubating at 100°C for 1 h. After centrifugation (5 min at 20,000g), 150 µl of supernatant was mixed with 10 ml of scintillation cocktail (Ultima Gold, PerkinElmer Life Sciences, Zaventem, Belgium). Intracellular amounts of 14C-RH-2485 were quantified in a Wallac™ 1400 liquid scintillation counter (PerkinElmer Life Sciences, Zaventem, Belgium). Quenching and background radioactivity were Archives of Insect Biochemistry and Physiology January 2008 doi: 10.1002/arch. In order to determine whether the phenomenon of active transport exists in the resistant subclones, we used ouabain (Sigma Co., Bornem, Belgium), an ATP-dependent transport inhibitor (Sundaram et al., 1998). Firstly, the effect of ouabain on cell proliferation was tested. There was no significant (P > 0.05) effect of ouabain on cell proliferation with concentrations from 10 nM up to 100 µM (data not shown). The resistant cells, Se4-RH-2485R4 and Se4-20E-R4, were seeded at a density of 100,000 cells/ml in 96-well microtiter plates containing 100 µM ouabain and without ouabain. For each treatment, there were at least 4 replications and each experiment was done 2 times. Then, the susceptibility of methoxyfenozide- and 20E-resistant subclones towards methoxyfenozide and 20E was evaluated after 5 days incubation using the MTT assay as described above. Cross-Resistance Test The methoxyfenozide- and 20E-resistant subclones were tested for their susceptibility towards 20E and methoxyfenozide, respectively, to evaluate cross-resistance using the MTT assay. The evalua- 40 Mosallanejad et al. tion of the cross-resistance was conducted between passages 39 and 43 for the methoxyfenozide-resistant cells, and between passages 22 and 25 for the 20E-resistant cells. Stability of Resistance In order to determine the stability of hormone and hormone mimic resistance, the resistant subclones (for Se4-RH2485-R4 after 70 passages and for Se4-20E-R4 after 54 passages) were propagated for over seven passages in the absence of methoxyfenozide and 20E, respectively. Then, the Se4-RH2485-R4 and Se4-20E-R4 cells were bioassayed for their sensitivity towards methoxyfenozide and 20E and compared to the resistant subclones under the pressure of compounds, using the MTT assay as described above. For each treatment, there were at least 4 replications and each experiment was done two times. Statistical Analysis Where appropriate, data were analyzed by Student’s t-test or ANOVA followed by a post-hoc Duncan test, using SPSS version 12 (SPSS Inc., Chicago, IL). RESULTS TABLE 1. Potency of 20E and Non-Steroidal Ecdysteroid Agonists on Cell Proliferation Inhibition of the Se4 Cells Compounds Methoxyfenozide Tebufenozide 20E Halofenozide RH-5849 EC50 (µM) SE (µM) 0.0015 0.0078 0.072 0.357 0.682 0.0003 0.0007 0.018 0.041 0.094 Resistance Towards Methoxyfenozide and 20E The cell susceptibility of obtained resistant subclones to methoxyfenozide and 20E was determined by an in vitro bioassay with the MTT technique. The cells exposed to methoxyfenozide selection developed between 1,700- to 7,900-fold levels of resistance compared to the original sensitive clone within 34–38 passages, which corresponds to about 10 months of selection (Table 2). The cells were selected by continuous exposure to 20E for almost 4 months and developed, during 18–21 passages, a level of resistance yielding 29fold to > 220-fold level of resistance as compared to the original sensitive clone (Table 2). Cell tests with all except one of the 20E-resistant subclones never resulted in more than 40% effect on cell proliferation with the highest concentration tested of 100 µM, and as a consequence we could not calculate EC50 values in these subclones. The methoxyfenozide- and 20E-resistant subclones were kept Comparative Potency of Ecdysteroid Agonists The Se4 cell line is sensitive to ecdysteroid activity stimulated by 20E and the dibenzoylhydrazine compounds, showing cell proliferation inhibition and developing filamentous extensions and aggregating into clumps. The median effective concentrations (EC50) of the test compounds were found in the range between 0.001 and 1 µM (Table 1). Our data showed that these compounds affected the cell proliferation in a dose-dependent fashion. The relative order of potency of the compounds on cell proliferation inhibition is methoxyfenozide (EC50 = 0.0015 µM) > tebufenozide (0.0078 µM) > 20E (0.072 µM) > halofenozide (0.357 µM) > RH- 5849 (0.682 µM). TABLE 2. Susceptibility of the Se4-RH2485-R1-5 Subclone to Methoxyfenozide (After 34–38 Passages) and of the Se4-20E-R1-4 Subclone to 20E (After 18–21 Passages) in Comparison With the Se4 Sensitive Cells Compounds Cell population EC50 (95%CL) (µM) RR* Methoxyfenozide Se4 sensitive clone Se4-RH2485-R1 Se4-RH2485-R2 Se4-RH2485-R3 Se4-RH2485-R4 Se4-RH2485-R5 0.0079 (0.0009–0.07) 14 (0.9–200) 28 (9–83) 63 (14–270) 45 (26–77) 57 (39–82) — 1770 3540 7970 5700 7215 20E Se4 sensitive clone Se4-20E-R1 Se4-20E-R2 Se4-20E-R3 Se4-20E-R4 0.452 (0.17–1.14) >100 (44%) 13 (2.4–74) >100 (40%) >100 (28%) — >220 29 >220 >220 *RR: resistance ratio calculated by dividing the EC50 value of the resistant subclones by the EC50 of the susceptible one. 95%CL, 95% confidence limits. Archives of Insect Biochemistry and Physiology January 2008 doi: 10.1002/arch. Resistance to Methoxyfenozide and 20E in S. exigua under pressure and after 60 and 70 passages, respectively, the concentration of methoxyfenozide and 20E reached 100 µM. The resistant cells were maintained at this high concentration of 100 µM until the present (February 2007), representing passages 100 and 90 for the methoxyfenozide- and 20-resistant subclones, respectively. It was clear that the latter cells have kept their loss of susceptibility to the compounds and they are still resistant for the high concentration of 100 µM. This represents a resistance ratio of at least 2,000-fold for the methoxyfenozide-resistant cells, and of at least 220fold for the 20E-resistant cells. During the time of this project, we measured an altered susceptibility of the sensitive cells towards methoxyfenozide and 20E in 2006 (resistance towards methoxyfenozide and 20E) compared with the results of 2005 (comparative potency of ecdysteroid agonists). In this period, we changed the culture medium because of the unavailability of the old version of EX-CELL™ 401 after the experiments with the different ecdysteroid agonists. To date, we cannot explain the exact reason for the shift in susceptibility. Effect of Metabolism Synergists None of the test synergists (DEF, PBO, and DEM) had influenced the potency of methoxyfenozide and 20E in the susceptible and the resistant cells (Figs. 2 and 3). PBO is an inhibitor of cytochrome-P450 monooxygenases, DEF is a strong esterase inhibitor, and DEM is an inhibitor of glutathione S-transferases (Jang et al., 1992; Jones, 1998). Our data indicate that these enzyme inhibitors cannot counteract the observed resistance towards 20E and methoxyfenozide in the Se4 cells. Retention of Methoxyfenozide We measured the accumulation of 14C-RH-2485 in Se4-RH2485-R4 and susceptible Se4 cells. We also tested Drosophila S2 cells as a control because Sundaram et al. (1998) reported that the retention of 14C-RH-5992 in lepidopteran (CF-203 and MDArchives of Insect Biochemistry and Physiology January 2008 doi: 10.1002/arch. 41 66) was higher than in dipteran Drosophila cells (DM-2 and Kc). The respective absorption in the Se4-RH2485-R4 and Se4 sensitive cells yielded significantly equal amounts of 14C-RH-2485 (P > 0.05) of 2.2 ± 0.4 and 1.5 ± 0.1 fg per cell, respectively. In contrast, in Drosophila S2 cells 0.2 ± 0.05 fg per cells was recorded, 8–9 times lower than in the lepidopteran Se4 cells (P < 0.05). Taken together, the current results indicated that there is no significant difference in the uptake pattern of 14 C-RH-2485 in the sensitive Se4 cells and methoxyfenozide-resistant Se4 subclone. There is no significant difference (P > 0.05) of cell protein concentrations (µg/ml) between the Se4 sensitive (2,480 ± 150) and Se4-RH2485-R4 (2,780 ± 100), while the amount of protein in the S2 cells is about 3 times lower (950 ± 140). Effect of Ouabain, an Inhibitor of Active Membrane Transport In a previous study, Sundaram et al. (1998) reported a low absorption in Drosophila cells that was due to an efflux of tebufenozide from Drosophila DM-2 cells and this active transport could be blocked by 10 µM ouabain, an inhibitor of Na+– K+–ATPase. The activities of methoxyfenozide and 20E in the susceptible and the resistant subclones were not influenced by ouabain (Fig. 4). Cross-Resistance for 20E and Methoxyfenozide The methoxyfenozide- and 20E-resistant subclones were tested for their respective susceptibility towards 20E and methoxyfenozide, to evaluate cross-resistance. Methoxyfenozide-resistant subclones showed resistance towards 20E, and the 20Eresistant subclones showed resistance towards methoxyfenozide (Table 3). The cross-resistance ratio yielded between 2,025- to 6,580-fold for the 20E-resistant subclones and >220-fold for the methoxyfenozide-resistant subclones. Cell tests with methoxyfenozide-resistant subclones to 20E did not result in more than 35% effect on cell proliferation, so we could not calculate EC50 values. 42 Mosallanejad et al. Fig. 2. Susceptibility to methoxyfenozide in the presence and absence of the synergists (A: PBO; B: DEF; C: DEM) in the sensitive cells and the Se4-RH2485-R4 subclone. The error bars represent the standard errors based on two independent experiments. Stability of Resistance To test the stability of resistance, the resistant subclones Se4-RH-2485-R4 and Se4-20E-R4 were propagated in the absence of methoxyfenozide and 20E for seven passages, respectively. For Se4RH2485-R4, this was after 70 passages and for Se420E-R4 after 54 passages. The results of the stability test of resistance showed that in the absence of the hormone and hormone mimic in the culture medium, the resistance is stable and no changes in sensitivity to compounds were seen (Fig. 5). DISCUSSION Insect cell lines can be used to predict the possible mechanisms for resistance against a given compound, which may arise under field conditions after prolonged treatment (Grebe et al., 2000). Archives of Insect Biochemistry and Physiology January 2008 doi: 10.1002/arch. Resistance to Methoxyfenozide and 20E in S. exigua 43 Fig. 3. Susceptibility to 20E in the presence and absence of the synergists (A: PBO; B: DEF; C: DEM) in the sensitive cells and the Se4-20E-R4 subclone. The error bars represent the standard errors based on two independent experiments. The Se4 cell line is sensitive for ecdysteroid activity stimulated by 20E and tebufenozide (Decombel et al., 2005) and can be used as a model to study the mode of action of ecdysteroids and their agonists. First, we investigated the relative potency of the four ecdysteroid agonists on cell proliferation in comparison with 20E. Methoxyfenozide and tebufenozide are the most potent followed by 20E, halofenozide, and RH-5849. These results are Archives of Insect Biochemistry and Physiology January 2008 doi: 10.1002/arch. consistent with various in vivo and in vitro studies (Trisyono and Chippendale, 1998; Trisyono et al., 2000; Smagghe et al., 2001). We selected the cells for resistance against methoxyfenozide and 20E in order to investigate the possible resistance mechanisms towards these compounds. Cell proliferation cessation by 20E and methoxyfenozide was used to select resistant subclones. We obtained five methoxyfenozide-resistant and four 20E-resis- 44 Mosallanejad et al. Fig. 4. Susceptibility to methoxyfenozide (A) and to 20E (B) in the presence and absence of ouabain in the sensitive Se4 cells, the Se4RH2485-R4 and Se4-20E-R4 subclone, respectively. The error bars represent the standard errors based on two independent experiments. tant subclones. During the selection period we increased the concentration 1,000,000-fold for methoxyfenozide (from 0.1 nM to 100 µM) and 10,000-fold for 20E (from 10 nM to 100 µM). The speed of selection was much faster than reported TABLE 3. Cross-resistance in 20E- (Se4-20E-R1-4, after 22–25 Passages) and Methoxyfenozide-Resistant (Se4-RH2485-R1-5, After 39–43 Passages) Subclones Towards Methoxyfenozide (RH-2485) and 20E, Respectively Cell population Se4-20E-R1 Se4-20E-R2 Se4-20E-R3 Se4-20E-R4 Se4-RH2485-R1 Se4-RH2485-R2 Se4-RH2485-R3 Se4-RH2485-R4 Se4-RH2485-R5 EC50 (95%CL) for RH-2485 (µM) EC50 for 20E (µM)a Cross-RR* 52 (24–110) 38 (13–100) 36 (8.7–150) 16 (1.5–150) — — — — — — — — — >100 (35%) >100 (17%) >100 (26%) >100 (21%) >100 (13%) 6580 4810 4557 2025 >220 >220 >220 >220 >220 *Cross-RR: resistance ratio calculated by dividing the EC50 value of the resistant subclones by the EC50 of the susceptible one. a The EC50 is higher than the highest concentration tested, i.e., 100 µM, as the response did not reach 50%. The response with 100 µM is given in parentheses. 95%CL, 95% confidence limits. by Grebe et al. (2000). The latter authors selected subclones of Chironomus dilutus, formerly known as C. tentans (Shobanov et al., 1999), from 0.1 nM tebufenozide to a final concentration of 0.1 µM and from 1 nM 20E to a final concentration of 5 µM during a period of about two years. The resistance towards the moulting hormone and methoxyfenozide in this insect cell line could be due to various changes: (1) it may be due to metabolic inactivation of the compounds, thus decreasing their effective concentration at the target site; (2) to reduced uptake or active exclusion of these compounds of the cell; (3) or to defective hormone receptors or downstream in their signaling pathway(s) (Dhadialla et al., 2005). Metabolism is a powerful factor, which frequently leads to a decreased toxicity of the applied insecticide. Enhanced metabolism of 20E is associated with hormone resistance in clones of the epithelial cell line from the dipteran C. dilutus, selected under the continuous presence of 20E (Kayser et al., 1997; Archives of Insect Biochemistry and Physiology January 2008 doi: 10.1002/arch. Resistance to Methoxyfenozide and 20E in S. exigua 45 Fig. 5. Stability test of resistance in the Se4-RH-2485-R4 subclone (A), after 70 passages, and in the Se4-20E-R4 subclone (B), after 54 passages. The cells were kept free of compound for seven passages. Next, susceptibility of the cells towards methoxyfenozide (A) and 20E (B) was evaluated using the MTT assay and compared to the resistant population under pressure of compound. The error bars represent the standard errors based on two independent experiments. Spindler-Barth and Spindler, 1998). However, the latter authors suggested that such enzyme-based resistance mechanism may not be the primary cause of resistance but a consequence of other changes that happened somewhere in a regulatory pathway. Therefore, to investigate the metabolic resistance in the current study, we used three synergists, PBO, DEF, and DEM, inhibiting monooxygenases, esterases, and glutathione-S-transferases, respectively. Our results showed no significant effect of these synergists in the susceptible and resistant cells, suggesting no significant role of the latter metabolism enzymes in the resistance process. This agrees with Grebe et al. (2000) who reported that metabolism did not play a significant role in the tebufenozideresistant C. dilutus cells. Nakagawa et al. (1995) also reported that most dibenzoylhydrazines are only marginally synergized in vivo with metabolic inhibitors (PBO and DEF), while no synergistic effect was observed in the cultured integument in vitro system. On the other hand, in the in vivo system of a whole insect body, previous studies (Smagghe et al., Archives of Insect Biochemistry and Physiology January 2008 doi: 10.1002/arch. 1998, 2003; Waldstein and Reissig, 2000; Smagghe, 2004) indicated oxidative and glutathione-S-transferase metabolism as the primary reason for development of resistance to tebufenozide and methoxyfenozide. In addition, amidase-mediated metabolism may occur for the dibenzoylhydrazines because of the presence of an amide bond in the structure of these compounds (Waldstein and Reissig, 2000; Smagghe, 2004). Sundaram et al. (1998) demonstrated that active exclusion of tebufenozide accounted for resistance in dipteran Drosophila cells (DM-2 and Kc) compared to lepidopteran cells (CF-203 and MD66). They showed that while the accumulation and active exclusion of ponasterone A was similar between lepidopteran and dipteran cell lines, the lepidopteran cells retained higher levels of 14 C-RH-5992 than the dipteran cells. This was an active efflux mechanism as it could be blocked with 10 µM ouabain, an inhibitor of Na+–K+–ATPase. Retnakaran et al. (2001) hypothesized that a membrane pump such as an ATP-binding cassette trans- 46 Mosallanejad et al. porter (ABC transporter) protein might be involved in the exclusion mechanism of the dipteran cells. These transporters have been referred to as multidrug resistance (MDR) genes in mammals and pleiotropic drug resistance (PDR) genes in yeast. Hu et al. (2004) using 20E- and tebufenozideresistant cells (CF-203) also reported that the resistant cells might take up less toxic compounds or actively pump them out as is the case for the Drosophila cell line (DM-2) (Sundaram et al., 1998). In this respect and in order to find whether an active transport is involved in the resistance process of the resistant Se4 cells, we evaluated this phenomenon using ouabain. Our results indicated that ouabain has no ability to change the susceptibility of the resistant subclones to methoxyfenozide and 20E. Also, the results of the kinetics experiments revealed no significant differences between the uptake pattern of the 14C-RH-2485 in the sensitive and methoxyfenozide-resistant cells. In this study, we also found cross-resistance in either methoxyfenozide- and 20E-resistant subclones. Wing (1988) also reported cross-resistance towards 20E and RH-5849 in the Kc D. melanogaster cells selected for resistance against both compounds. Spindler-Barth and Spindler (1998) reported that cells of C. dilutus selected in the presence of 20E were resistant only to the moulting hormone, but still responded to tebufenozide, whereas subclones selected in the presence of tebufenozide showed cross-resistance to 20E. Related to the in vivo system, some cross-resistance has been reported between dibenzoylhydrazinetype insecticides and other insecticides in many insects (Sauphanor and Bouvier, 1995; Wearing, 1998; Waldstein et al., 1999; Moulton et al., 2002; Smirle et al., 2002; Cao and Han, 2006). The selective toxicity of dibenzoylhydrazine-type ecdysteroid agonist insecticides is primarily determined by the different binding affinity of ligands to the ecdysteroid receptors (Dhadialla et al., 1998; Minakuchi et al., 2005). However, a modified insect ecdysteroid hormone receptor complex in vivo has not been documented as a cause of the resistance for ecdysteroid agonists. At the cellular level, Grebe et al. (2000) reported that selection of tebufenozide-resistant cells in the continuous presence of this compound in the C. dilitus cell line caused clones with defects in ecdysteroid receptor function. In these resistant clones, the receptor function, but not the expression level, was changed. While the profile of the ecdysteroid receptor in these cells is approximately normal, some lines show a modest decline in the abundance of phosphorylated USP forms, which is not reversible by addition of 20E. A variety of differences in ecdysteroid receptor ligand-binding characteristics were noted among the cell lines that presumably reflect the role of unknown factors underlying the resistance. Moreover, Stevens and O’Connor (1982) reported that in 20E-resistant Kc cells, the resistance is associated with a quantitative, rather than a qualitative, change in the receptor content. Koelle et al. (1991) also reported that the resistant Drosophila Kc and S2 cells are insensitive to ecdysteroid action by reducing their titer of ecdysteroid receptor. Indeed, they are deficient in both ecdysteroid-binding activity and ecdysteroid responsiveness. Cherbas and Cherbas (1996) also reported that ecdysteroidresistant cells arise spontaneously in Drosophila Kc cell populations at a significant frequency (around 2%), but this spontaneous resistance is in general derived from mechanisms other than a defect in ecdysteroid receptor; thus, the ecdysteroid response cannot be fully restored by expression of ecdysteroid receptor from a plasmid source in most spontaneous ecdysteroid receptor-deficient lines. Based on our results of the current selection for resistance, the potential for Se4 cells to develop resistance to methoxyfenozide and to 20E exists because the sensitive cells achieved high resistance during the selection period. The acquisition of resistance was stable and could not be reversed even after the compounds were removed. It means that the resistance appears to be inherited in the cells. Previous in vivo studies also imply the development of resistance to methoxyfenozide in a relatively short period of time in this insect (Smagghe et al., 1998; Moulton et al., 2002; Gore and Adamczyk, 2004). If the 20E- and methoxyfenozide-resistant cells have the same mechanisms of Archives of Insect Biochemistry and Physiology January 2008 doi: 10.1002/arch. Resistance to Methoxyfenozide and 20E in S. exigua resistance, it is unlikely that this resistance mechanism can occur in vivo because insects need ecdysteroids for their successful growth and development like moulting, metamorphosis, and also reproduction. So the mechanism of resistance that we selected in these lepidopteran cells will probably only be possible in vitro. 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