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Selection for resistance to methoxyfenozide and 20-hydroxyecdysone in cells of the beet armyworm Spodoptera exigua.

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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: guy.smagghe@ugent.be
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. Further in vivo assays using the insect as a target for determining
mode of resistance are required to determine
whether a good correlation with the cell line examined in our assays does exist.
ACKNOWLEDGMENTS
H. Mosallanejad is recipient of a doctoral grant
from the Ministry of Science, Research and Technology (SRT), and Agricultural Research Organization of Iran (Plant Pest and Disease Research
Institute).
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