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Membrane binding and uptake of diflubenzuron in a cell line from Manduca sexta (L.)

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Archives of Insect Biochemistry and Physiology 4:169-182 (1987)
Membrane Binding and Uptake of
Diflubenzuron in a Cell Line From
Manduca sexfa (L.)
Gabriele E. Klitschka, Wolfgang Witt, and Richard T. Mayer
U.S. Horticultural Research Lab, Agricultural Research Service, U.S. Department of Agriculture,
Orlando, Florida
The binding and accumulation of the chitin synthesis inhibitor diflubenzuron
(DFB) by a cell line derived from embryonic tissue of the tobacco hornworm,
Manduca sexta (L.), was analyzed. A rapid and reversible binding to viable
and nonviable cells suspended in the culture medium was observed at
soluble concentrations of DFB for short exposure periods. Scatchard analysis
gave no indication of a saturable uptake mechanism. The DFB-binding
capacity of intact cells was found to be similar to that of a crude membrane
preparation (70,OOOg pellet); however, plasma membrane-enriched fractions
bound almost three times as much DFB as the homogenate. Repetitive shorttime incubations (up to 3 h) of suspended cells with DFB resulted in a
stepwise intracellular accumulation of DFB. Treatment of growing cells with
DFB at high concentrations (50 pM) of DFB for longer periods (up to 7 days)
resulted in elevated intracellular accumulation of DFB, which exceeded the
binding capacity of the cell membranes and the aqueous solubility of DFB.
These results indicate that the intracellular crystals detected by transmission
electron microscopy are precipitated DFB. N o metabolites or other chemically
modified products of intracellular DFB were detected by high pressure liquid
chromatography (HPLC) after a 7-day incubation.
Key words: insect growth regulators, binding, plasma membrane, chitin
INTRODUCTION
The chitin synthesis inhibitor diflubenzuron (N-[[(4-chlorophenyl)amino]carbonyl]2,6-difluorobenzamide)interferes with the deposition of chiAcknowledgments: We thank Mr. Robert E. Droleskey for the electron microscopic material
and Mr. Randy Smith for excellent technical assistance. G.E. Klitschka was supported by the
Alexander von Humbolt-Foundation as a Feodor Lynen-Postdoctoral Fellow.
This paper reports the results of research only. Mention of a pesticide in this paper does not
constitute a recommendation by the U.S. Department of Agriculture nor does it imply
registration under FIFRA.
Received May 16,1986; accepted July 7,1986.
Address reprint requests to Dr. R.T. Mayer, USDA, ARS, 2120 Camden Road, Orlando FL 32803.
0 1987 Alan R. Liss, Inc.
170
Klitschka, Witt, and Mayer
tin in the insect cuticle [I]; however, there is disagreement about the exact
biochemical mode of action [2-9]. Initially investigators suggested that DFB*
directly interacted with chitin synthase [2]. The direct interaction of DFB with
chitin synthase was disproven by Mayer et al. [5] and Cohen and Casida
[lo], who found that cell-free preparations of this enzyme were not inhibited
by DFB.
Recently, another site of action for DFB was proposed. Based on the
finding that the thymidine incorporation into the DNA of pupae of Stornoxys
cuEcitvarzs (L.) was inhibited, Deloach et al. [8] suggested that DFB modified
membrane properties such that the permeability for certain substances, eg,
nucleosides, was changed. This idea was supported by the finding that DFB
inhibited the uptake of nucleosides into Harding-Passey melanoma cells [ll]
and into cells from Murzducu sexta (L.) [l2].
Bishai and Stoolmiller [13] showed that DFB was taken up by rat glial cells
and probably bound to cell membranes. They also found that DFB was not
further metabolized during the 5-day incubation period. But, thus far, data
on the affinity of DFB for biological membranes are unavailable.
The cell line used here responds to DFB in several ways. In addition to the
effect on the nucleoside transport [ll, 121, chitin synthesis is inhibited [14],
growth rate is reduced, and morphological changes are observed (ie, normal
flat cells withdrew to clumps of cells upon exposure to DFB) [12]. In a recent
communication [12], crystals were observed via transmission electron microscopy in cells treated with high concentrations of DFB. The results presented
here indicate that these crystals are DFB. In addition, the mechanism of the
intracellular accumulation of DFB and data on the membrane affinity are
presented and discussed.
MATERIALS AND METHODS
Cell Line and Culture Condition
The insect cell line CH-MRRL was established from embryonic tissue of
the tobacco hornworm, Manducu sexta (L.) [15] and was obtained from E.P.
Marks (Fargo, ND). The cells were grown in Yunker’s variation [16] of
Grace’s medium (Gibco, Grand Island, NY) containing 100 IUlml penicillinG-potassium, 100 pglml streptomycin sulfate, and 0.1% gentamycin in 150cm2 flasks at 27-29°C. Antibiotics were purchased from Sigma (St. Louis,
MO) or Gibco.
Preparation of Crude Cell Membranes
Cells grown for 7 days were harvested by centrifugation at 200g. The
pelleted cells were washed twice by resuspending in 20 ml Grace’s medium
at room temperature and recentrifuging. Finally, pelleted cells were resus-
*Abbreviations: DFB = diflubenzuron; DMSO = dimethylsulfoxide; HPLC = high pressure
liquid chromatography; SDS = sodium dodecylsulfate; PAGE = polyacrylamide gel electrophoresis; TEM = transmission electron microscopy; TRIS = tris (hydroxymethyl)
aminomethane.
Diflubenzuron in a Manduca sexfa Cell Line
171
pended in 5 ml Grace's medium. Aliquots of the cell suspension were
removed for the determination of cell populations, viability, and the DFB
binding andlor content.
Membranes were pre ared by homogenizing an aliquot of the cell suspension (4.8 ml, 16-22x106 F cellslml) twice for 20 s at 0°C with a Polytron PT
homogenizer equipped with a 10l35 generator (Brinkman Instruments, Westbury, NY). Three milliliters of the homogenate were diluted with 5 ml of cold
Grace's medium and centrifuged for 30 min at 70,OOOg. The pellet was
resuspended in 8 ml Grace's medium by rehomogenizing (as above) and
centrifuged at 70,OOOg for 1h. The membranes were washed once more by
repeating this last step. The membrane pellet was finally resuspended in
Grace's medium and the volume adjusted to 6 ml.
Preparation of Plasma Membranes
Cells, 7 days old, were harvested by gently pipetting them from the flasks,
and were washed three times in Puck's saline A by centrifugation at 2008 for
5 min at 2°C [lq.The pellet was resuspended in 10 mM TRISIHCI (pH 7.4)
at 2 x 10' cellslml, and the cells were allowed to swell for 30 min at 2°C. Cells
were disrupted by sonication (Kontes, micro ultrasonic cell disrupter) for 30
s, and the unbroken cells were separated by centrifugation at 1,5008. The
supernatants (homogenates) were adjusted to 1mM EDTA and layered on
top of a sucrose gradient composed of 50%, 40%, 30%, and 20% (wlw)
sucrose in 10 mM TRISlHCl (pH 7.4) and 1 mM EDTA. Centrifugation
(50,OOOg) was performed in a Beckman L8-70 ultracentrifuge, using an SW 28
rotor, for 16 h at 4°C (50,OOOg). The fractions were collected and dialyzed 20
h against 10 mM TRISIHCI (pH 7.4) containing 1 mM EDTA. Finally, the
fractions were pelleted at 50,OOOg and resuspended in a known volume of 10
mh4 TRIS/HCI (pH 7.4), 1mM EDTA.
The identification and purity of the fractions were determined with marker
enzymes. Acid phosphatase (EC 3.1.3.2.), a presumptive marker of lysosomes, was tested with a kit from Sigma. NADPH cytochrome c reductase
(EC 1.6.2.4.) was measured according to Weirich and Adams [IS], and cytochrome c oxidase (EC 1.9.3.1) activity was determined using 100 pM cytochrome c in 50 mM potassium phosphate at pH 7.8. The ouabain-sensitive
Na+lK+-ATI'ase (EC 3.6.1.3.) assay was adapted from Buff and Brundl [19]
with 3.5 mM ATP, 1mM ouabain, and 20 mM TRISIHCI, pH 7. The cholesterol content was measured with a test kit from BoehringerlMannheim (West
Germany).
Long-Term Exposure of Growing Cells to DFB
Cells (3x lo6) were seeded in 25-cm2 tissue culture flasks containing 3 ml
culture medium. On day 1, 7.5 pl of a solution of [3H]DFB (230 mCilg;
DuPhar-Solvay, B.V., The Netherlands, purified by TLC) in DMSO (50 pM,
0.06-0.18 pCi/ml) were added to cell cultures.Controls containing only DFB
pure; Thompson-Hayward Chemical Co., Kansas City, KS) and culture
media were run in parallel to detect any nonspecific adsorption of DFB to
culture flasks or test tubes. After an incubation period of 6 days at 27-29"C,
172
Klitschka, Witt, and Mayer
the cells were washed twice with 3 ml culture medium, harvested, and
counted using a hemocytometer. In some cases they were allowed to continue growing in the same medium but without DFB.
The harvested cells were washed four times by resuspension in Grace’s
medium (4 ml) and recenhifugation at 5008 for 5 min. Concentrations of
[3H]DFB in the pellets and the supernatants were determined by liquid
scintillation counting.
Short-Term Incubations of Cells, Crude Membranes, and
Cell Fractions With DFB
The insect cells were cultured, harvested, washed, and resuspended in
Grace’s medium at room temperature as described above. Aliquots were
removed for cell counting and protein determinations. The cells were either
stored at -20°C or were used within 4 h for incubation with DFB.
Cells used for this purpose were pelleted by centrifugation at 1,OOOg for 10
min and resuspended in culture medium containing [SHIDFB. Culture medium solutions of DFB were prepared by adding aliquots of [3H]DFB stock
solutions in DMSO to Grace’s medium at 29°C for 10-60 min. Actual DFB
concentrations were determined by liquid scintillation counting. This procedure was necessary since significant amounts of DFB adsorbed to the surface
of the glass tubes at the low concentrations applied here. Concentrations and
specific activities of C3H]DFB in the repetitive incubation experiments were
0.23 and 0.44 pM (56.7 and 28.3 pCi1mmol) at the beginning of the tests. In
all other short-term incubation experiments [3H]DFB with a specific activity
of 56.7 pcilmmol was used.
The cells or the cell fractions ( 2 . 5 ~ 1 0cells
~ or the equivalent amount of
protein (0.165 mg) from the membrane suspensions) were incubated at 29°C
in 5 ml Grace’s medium, containing 0.2 pM f3H]DFB.After 5 min, 2 ml were
applied to glass fiber filters (Gelman type AIE, 25mm, Gelman Science Inc.,
Ann Arbor, MI). The filters themselves and the filtrates were analyzed for
radioactivity. Controls (radioisotopes and medium only) were performed to
determine adventitious binding of [3H]DFJ3. Efficacy of the filtration process
was controlled by determination of protein in the filtrates.
Analysis of DFB by HPLC
DFB-treated and control (without DFB) cells were washed twice with
Puck’s saline A and homogenized in 5 ml acetonitrile with a Polytron PT
homogenizer equipped with a 10135 generator. The homogenates were filtered through a sintered glass funnel, washed twice with acetonitrile (5 ml),
and evaporated to dryness under vacuum. The residues were dissolved in
acetonitrile: H20 (10190) applied to a Sep-Pak C18 cartridge (Waters Associates, Milford, MA). After the cartridge was washed with 5 ml acetonitrile:
H20 (10/90), DFB was eluted with 5 ml acetonitrile. The solvent was evaporated and the residue dissolved in 200 pl acetonitrile for HPLC analysis [20]
using an LDClMilton Roy HPLC (Riviera Beach, FL) equipped with a
3.9 x300-mm p-Bondapak C18 (Phenomenex, Palos Verdes Estades, CA) column. Twenty microliters of the cell extract were injected and fractions (30 s)
Diflubenzuron in a Manduca sexta Cell Line
173
were collected after elution with acetonitrile: H20 (57/43)at a flow rate of 1.5
mllmin. Fractions were collected in scintillation vials, evaporated, and analyzed for radioactivity after adding scintillation cocktail. When analyzed by
HPLC, standard solutions of DFB in acetonitrile gave a major peak (97%)
with a retention time of 7.5 min and a minor peak (3%) at 9.1 min, which
may be either an impurity or an isomer.
Analytical Methods
Radioactivity was measured with an LKB 1219 Rackbeta scintillation
counter and was quench-corrected. [3H]DFB samples (G0.75 ml) were mixed
with 10 ml scintillation fluid (PCS, Amersham Co, Arlington Heights, IL).
Counting efficiency was 48%.
~)
to Ways and
Lipids were extracted from frozen cells ( 1 0 0 ~ 1 0according
Hanahan following extraction procedure 2 [21]. Phospholipids were determined by the procedure of Broekhuyse [22].
SDS-polyacrylamide slab gel electrophoresis followed a modified version
of the discontinuous system described by Laemmli [23] with 8% running gels
~241.
Protein was determined by the procedure of Bradford [25] using bovine yglobulin standards. Transmission electron microscopy of cells grown for 7
days with DFB was conducted as described recently [12].
RESULTS
Purification and Characterization of Plasma Membranes
The centrifugation of sonicated cells on the discontinuous sucrose gradient
led to three distinct bands, located at the 5040% (fraction l), 40-30% (fraction 2), and 30-20% (fraction 3) interfaces. A minor band was present at the
top of the 20% sucrose layer and was numbered fraction 4. Analysis of the
fractions showed ouabain-sensitive Na+/K+ -ATPase to be concentrated in
fraction 3 (Table l), which also contained the highest amount of cholesterol.
Cytochrome c oxidase, a marker for mitochondria, was found to be enriched
in fractions 1 and 2. NADPH cytochrome c reductase, a marker for endoplasmic reticulum, was concentrated in fraction 2. Acid phosphatase was
TABLE 1. Analysis of Total Homogenates and Cell Fractions of Munducu sextu L. Cells
Fractiona
Homogenate
1
2
3
4
NADPH
Ouabain-sensitive
Cytochrome
Cytochrome
Acid
c reductase
c oxidase
phosphatase
AWase
Cholesterol
(nmol/min/mg)b (pg/mg)b (pmol/min/mg)b (pmol/min/mg)b (pmol/min/mg)b
1.4
9.9
2.6
16.4
2.2
4.4 f 2.3
6.1 f 1.5
15.1 It 6.2
26.4 f 9.9
12.6 f 0.5
4.75 f 0.8
9.85 f 0.2
15.45 f 1.6
8.95 f 2.9
8.90 f 2.6
12.5 f 0.2
87.6 f 26.0
53.0 f 7.0
5.6 f 1.5
12.7 f 6.6
0.83 It- 0.14
0.52 f 0.10
1.54 f 0.11
0.79 f 0.12
1.90 f 0.88
'Fractionations were performed four times with two to five replicate enzymes or lipid analyses per
fractionation.
bmg = mg protein.
174
Klitschka, Witt, and Mayer
equally distributed in fractions 2 and 4. This one purification step led to a
sixfold enrichment in cholesterol and 11.7-fold in ouabain-sensitive Na+/K+
-ATPase, indicating the presence of plasma membranes in fraction 3. Fraction
3 was almost free of lysosomal and mitochondria1 contamination and contained some endoplasmic reticulum (Table 1).
Short-Term Incubations With DFB to Determine Accumulation, Loss, and
Uptake Mechanism
Binding of DFB to cells and crude membrane preparations is compared in
Table 2. Almost the same binding capacity was found with fresh, viable cells
and frozen, nonviable cells. Nearly 75% of the DFB in fresh cells also bound
to membranes prepared from these cells when incubated similarly. Table 2
also shows the binding of DFB to the cell fractions. Fractions 1and 4 had a
binding rate similar to that of the homogenate, but in fractions 2 and 3, a 22.5-fold increase of binding was observed. This suggests that plasma membranes (fraction 3) have a high affinity for DFB. Considering that this was
not a pure plasma membrane preparation, the increase in binding is fairly
large. Fraction 2, identified as endoplasmic reticulum enriched, also shows a
high level of cholesterol, which is indicative of the presence of membranes.
The DFB-binding capacity of the isolated crude membranes and the cell
fractions may be underestimated, since ca 18% of the membrane protein
applied to the filters was detected in the filtrate.
The amount of intracellular DFB for fresh cells could be increased by
repeated incubations with low concentrations of DFB (Fig. 1).After the fifth
incubation, the DFB content of the cells was raised about 1.5-fold the amount
of DFB available at each incubation. A concomitant reduction of the DFB
TABLE 2. Binding of DFB to Fresh, Frozen, and
Cell Fractions of Munducu sextu (L.) Cells*
pmol DFB/106cells
Fresh cellsa
Frozen cells
Crude membranes
Homogenate
Fraction 1
Fraction 2
Fraction 3
Fraction 4
85.2 f 5.8
73.8 f 4.3
63.6 f 5.5
19.8 f 6.4
27.5 f 7.7b
39.3 f 7.5c
53.2 f 10.0'
22.2 f 5.9b
*DFB concentration was 200 nM. Significance for
the fractions was calculated by Student's t-test
using the homogenate as the control value. Results
reported as X f SD, n = 4.
aFresh and frozen cells and crude membrane
preparations were obtained using a polytron. All
other preparations were obtained using a sonic
cell disruptor. The two methods caused differences
in the binding data.
b~ < .5, not significant.
'P < .OOl, considered significantly different from
control value.
Diflubenzuron in a Manduca sexfa Cell Line
FRESH CELLS
175
1
-0.6
-!
4
c
z
4
c
-0.42
a
W
a
a
v)
f
m
u.
-0.2
51.
E
Fig. 1. Repetitive incubation of fresh, viable insect cells with DFB and subsequent wastiings
~ ) incubated in 1 ml Grace's
to determine accumulation and loss of DFB. Cells ( 3 ~ 1 0 were
medium as described in Methods with 13H]DFB (230 nM, open symbols; 440 nM, filled
symbols) for 5 min. Twenty microliters of the cell suspension were removed during this time
for the determination of radioactivity. The cells were pelleted by centrifugation at 500g for 5
min. Aliquots (200 pl) of the supernatant were removed to determine the DFB concentration
( A ) by liquid scintillation counting. Subsequently, the supernatant was removed completely
and the cells were resuspended in Grace's medium with the same DFB concentrations.
Twenty microliters of the suspension were removed to determine the amount of [3H]DFB.
After a second incubation for 5 min, the cells were centrifuged as before to measure the DFB
concentration in the supernatant. These incubation/centrifugation cycles were repeated 10
times. Then the incubation continued for another five cycles at otherwise unchanged conditions except that DFB was omitted (arrow). The cell-bound DFB (0)
was calculated for every
incubation cycle by subtraction of the 3H content of the cell suspensions and the corresponding supernatants.
concentrations in the supernatants was observed. The DFB concentration of
the supernatants was constant and equal to the concentration of the added
DFB solution for the next five incubations (ie, incubations 6 to 10). The cells
showed no further change in the DFB content (ie, for incubations 6-10),
indicating that the net accumulation had ceased. The reduced DFB content
of fresh cells (Fig. 1)during the last incubation cycles probably resulted from
fractionation and incomplete centrifugation of broken cells. The maximum
amounts of cell-bound DFB obtained by this procedure were 0.10 nmol/106
cells at an external concentration of 230 nM and 0.21 nmol/106 cells at 440
nM. Five subsequent incubation cycles without DFB almost completely released the radioactivity (Fig. 1).Similar results were obtained for frozen cells
(data not shown). The analysis of the binding of DFB to fresh or frozen cells
by Scatchard plots gave no indication for a saturable uptake mechanism (Fig.
2, frozen cell plots not shown). In accordance with the finding that shortterm incubations at low concentrations resulted in a rapid, reversible bind-
176
Klitschka, Witt, and Mayer
5 -
4.
8
:
0
0
v
.,
A
0
32-
1-
Fig. 2. Scatchard-plot of the DFB binding to insect cells to determine if a saturable uptake
mechanism exists. The binding of 13H]DFB to freshly harvested cells was analyzed by the
procedures described in Materials and Methods. Cells (3x 106/ml) were incubated with
[3H]DFB in concentrations of 43-520 nM.
ing, this result suggests that DFI3 occupies cellular binding sites by simple
diffusion.
Long-Term Incubations With DFB in Cell Cultures to Determine
Accumulation and Loss of DFB
In another series of experiments, cells were grown at standard culture
conditions and were treated with DFB in concentrations that affect nucleoside
uptake [12]. After an incubation of 6 days with 50 pM DFB, 52% of the
applied radioactivity was recovered in the cells (Fig. 3). This means that lo6
cells contained 14.7 nmol of DFB at these conditions. DFB was not as easily
removed from the cells as were the smaller amounts incorporated during the
short-term experiments. Four washings by centrifugation and resuspension
of cells grown for 6 days in the presence of DFB reduced the intracellular
content by 40%, but a large amount (32%of the originally applied DFB) was
still retained (Fig. 3A). Nearly the same reduction of the DFB content was
found when the cells were grown for an additional 7 days in fresh growth
medium without DFB with five intermediary changes of medium (Fig. 3B).
Diflubenzuron in a Manduca sexfa Cell Line
177
-
*
60
m 50LL
n
n
40-
a
a
0
30-
A
20-
B
0
0
-
z
$
10-
2
8
1
I
s
2
3
WASHINGS
8
4
Fig. 3. Accumulation and removal of DFB in cultured insect cells after long-term incubations
with DFB. Cells were grown in the presence of [3H]DFB for 6 days as described in Materials
and Methods and harvested immediately (A). They were then grown for another 7 days in
DFB-free medium with five intermediary changes of the growth medium and then harvested
(B), or were harvested after a growth of 14 days with ten intermediary changes in DFB-free
medium following the initial growth period with [3H]DFB(C). The DFB content of the freshly
harvested cells and after four washings by centrifugation and resuspension in Grace’s medium, expressed as percentage of the originally applied amount of DFB, is presented.
The DFB content of these cells was further reduced by subsequent washings,
but less rapidly than after the 6 days‘ incubation. Even when the experiment
was extended to 14 days with ten changes of the growth medium after the
initial 6 days’ incubation with DFB, a large amount (16% of the originally
applied 150 nmol DFB) was detectable. This amount was only slightly reduced by subsequent washings of the harvested cells (Fig. 3C).
These results show that DFB incorporated during long-term incubations at
high concentrations is not freely exchanged with extracellular DFB. The
results also indicate that DFB is stored differently in the cells for the longterm studies than for the short-term, low-concentration experiments. In
addition, cell growth (ie, increase in cell population) had no effect on the
DFB content.
DFB Metabolism in Cultured Cells
DFB was extracted from cells grown in the presence of 50 pM DFB and
analyzed by HPLC to determine whether a chemical or metabolic modification of DFB occurred under long-term incubation conditions. Figure 4 shows
178
Klitschka, Witt, and Mayer
i
20000
Fig. 4. Chromatogram of HPLC analysis of a DFB extract from Manduca sexta cells. Cells were
grown for 7 days with 50 pM [3H]DFB, harvested, washed, and extracted with acetonitrile.
Details of the extraction and analysis procedures are given in Materials and Methods.
Absorbance at 254 nm. Radioactivity =
-a-.
the elution pattern of the separation with a reversed phase column. The
radioactivity eluted as a single peak with a retention time of 7.5 min, which
corresponded to the major DFB peak in the standard. The other minor UVabsorbing peaks represent cellular substances, which were coextracted with
DFB during the procedure. No major UV-absorbing peaks were detected
between retention times of 3-13 min in extracts from cells grown without
DEB (not shown). The same results were obtained with extracts from cells
grown with DFB for 6 days and cultured subsequently for 14 days in a DFBfree medium with ten intermediary changes of the growth medium.
Further Characterizationof DFB-Treated Cells
Lipids were extracted from the cells and quantified to estimate the hydrophobic binding capacity of the cells. The phospholipid content was determined to be 0.32 nmol/106cells.
Analysis of complete cell protein, membrane protein, and soluble protein
by SDS-PAGE at reducing conditions gave no indication for changes in the
overall protein pattern of cells treated with DFB in long-term incubations
(not shown).
The transmission electron microscopy studies of a recent communication
[12] were continued to further elucidate the intracellular storage of DFB. It
was previously assumed [12] that the crystals in DFB-treated cells repre-
Diflubenzuron in a Manduca sexta Cell Line
179
sented precipitated DFB. This assumption was based on the evaluation of a
large number of specimens. Long-term incubations of the cells with high
concentrations of DFB always produced observable, intracellular crystals. In
addition, crystals associated with lysosomes were also frequently observed
(Fig. 5 ) . The accumulation and initial precipitation of DFB in the lysosomes
may result in the formation of the large crystals reported previously [12].
DISCUSSION
A rapid and completely reversible binding of DFB was observed in shortterm incubations with low concentrations of DFB. No indications for the
existence of a specific and saturable uptake mechanism were found by Scatchard analysis. These results support the view that a free diffusional exchange of the external, soluble, and the cell-bound DFB occurs at these
conditions.
Fig. 5. Transmission EM of cells grown for 7 days in the presence of
inside lysosomes, b: Crystals in cytoplasm. x 10,336.
DFB (50 pM). a: Crystals
180
Klitschka, Witt, and Mayer
Since DFB is a hydrophobic substance which has a low water solubility,
the intracellular binding sites should be mostly membrane lipids and other
hydrophobic membrane areas. The cell-fraction binding experiments showed
that the purified plasma membrane fraction bound almost three times as
much DFB as the homogenate. Also the binding to the endoplasmic reticulum-enriched fraction was significantly higher.
The high level of DFB binding to plasma membranes supports the theory
that DFB affects the cell membrane in such a way that chitin synthesis/
deposition is inhibited [6-81. The observation that DFB appears to have an
affinity for plasma membranes becomes more significant when one realizes
that chitin synthase is probably attached to the plasma membrane of insect
epidermal cells. This conclusion is supported by microscopic studies of insect
epidermal cells which suggest that chitin microfibrils originate at the cell
surface [26] and by reports on yeast chitin synthase which conclusively show
that the enzyme is attached to the plasma membrane [27,28].
Cells containedlbound larger amounts of DFB when incubated long-term
with high concentrations of DFB. The DFB concentration used exceeded the
solubility of DFB in water almost 100-fold and resulted in an almost complete
precipitation of DFB in the culture flasks. Consequently, the cells were
continuously exposed to a saturated DFB solution. This situation was simulated by the repetitive incubation experiments of Figure 1.The precise extent
of incorporation during the long-term incubation cannot be determined from
these experiments, since the cells fragmented after several consecutive incubations. The experiment does demonstrate that the continuous contact to
DFB in soluble concentrations results in a cellular accumulation.
We assume that during long-term incubations with high DFB concentrations the incorporation of DFB proceeds first by diffusion into cellular hydrophobic domains. However, this process cannot explain the accumulation of
the large amounts of DFB after a 6-day incubation, since the cells do not have
adequate hydrophobic binding sites. The amount of intracellular DFB exceeded the phospholipid content of the cells 42-fold.
Klitschka et al. [12] suggested that the intracellular crystals represent
precipitated DFB. The results presented here support this assumption. The
simple fact that the hydrophobic binding capacity of the cells for a highly
insoluble compound such as DFB is limited has already been mentioned
above. In addition, the large amount of intracellular DFB was not as rapidly
exchanged with the extracellular medium as was the small amount bound to
the cells after short-term incubations with low DFB concentrations (Fig. 3).
Growth of the cells for 14 days in DFB-free medium with ten intermediary
changes of the growth medium reduced the DFB content of the cells by 84%.
Probably, small amounts of the intracellular-precipitated DFB were dissolved
and exited the cell by diffusion with each change of the medium until a new
equilibrium was established. The metabolic or chemical modification of DFB
to more water-soluble or reactive compounds can be excluded since DFB
itself was the only radiolabeled compound detected by HPLC in extracts of
cells treated for 6 days with DFB (Fig. 4).
The mechanism of the intracellular crystal formation is not yet clear.
Phagocytosis of extracellular crystals as the main mechanism can be excluded
Diflubenzuron in a Manduca sexfa Cell Line
181
since this event was only rarely observed by TEM. We suggest another
membrane-dependent process. Extracellular DFB may occupy hydrophobic
binding sites in the plasma membrane and is then further transported into
the cell. There the accumulated amount of DFB exceeds the solubility limit
and DFB starts precipitating out of the cytoplasm. It may also be transported
to lysosomes and then form crystals inside the lysosomes, a situation that
was detected frequently by TEM (Fig. 5). The high affinity of DFB supports
the suggestion that DFB interferes with membrane processes and that the
primary site of action will be found here and not in the inhibition of the
chitin synthesis itself [8,11,12].
LITERATURE CITED
1. Verloop A, Ferrell CD: Benzoylphenylureas-a new group of larvicides interfering with
chitin deposition. In: Pesticide Chemistry in the 20th Century. Plimmer, JR, ed. Am.
Chem. SOC.,Washington, DC, Vol. 37, p 237 (1977).
2. Duel DH, DeJong BJ, Kortenbach JAM: Inhibition of chitin synthesis by two 1-(2,6disubstituted benzoyl)-3-phenylurea insecticides. Pest Biochem Physiol4, 98 (1978).
3. Hajjar NP, Casida JE: Structure-activity relationships of benzoylphenyl ureas as toxicants
and chitin synthesis inhibitors in Oncopeltus fasciatus. Pest Biochem Physiol 22, 33 (1979).
4. Maas W,Van Hes R, Grosskurt AC, Deul DH: Benzoylphenylurea insecticides. Chemie
der Pflanzenschutz- und Schaedlingsbekaempfungsmittel6,423-470(1983).
5. Mayer RT, Chen AC, DeLoach JR: Characterization of a chitin synthase from the stable
fly, Stornoxys calcitrans (L.). Insect Biochem 10, 549 (1980).
6. Meola SM, Mayer RT: Inhibition of cellular proliferation of imaginal epidermal cells by
diflubenzuron in pupae of the stable fly. Science 207, 985 (1980).
7. Mayer QT, Chen AC, DeLoach FR: Chitin synthesis inhibiting insect growth regulators do
not inhibit chitin synthase. Experientia 37, 337 (1981).
8. DeLoach JR, Meola SM, Mayer RT, Thompson JM: Inhibition of DNA synthesis by
diflubenzuron in pupae of the stable fly Stomoxys calcitrans (L.). Pest Biochem Physiol 25,
172 (1981).
9. Chen AC, Mayer RT, DeLoach JR: Purification and characterization of chitinase from the
stable fly, Stomoxys calcitrans (L.). Arch Biochem Biophys 226, 314 (1982).
10. Cohen E, Casida JE: Inhibition of Trilobium gut chitin synthase. Pest Biochem Physiol 23,
129 (1980).
11. Mayer RT, Netter KJ, Leising HB, Schachtschabel DO: Inhibition of the uptake of nucleosides in cultured Harding-Parrey melanoma cells by diflubenzuron. Toxicology 30, 1
(1984).
12. Klitschka GE, Mayer RT, Droleskey RE, Norman JO, Chen AC: Effects of chitin synthesis
inhibitors on incorporation of nucleosides into DNA and RNA in a cell line from Manduca
sexta (L.). Toxicology 39, 307 (1986).
13. Bishai WR, Stoolmiller AC: Uptake of diflubenzuron (N-[[(4-~hlorophenyl)amino]
carbonyl]-2,6-difluorobenzamide)
by rat C 6 glial cells in vitro. Pest Biochem Physiol 12,
258 (1979).
14. Marks EP, Balke J, Klosterman H: Evidence for chitin synthesis in an insect cell line. Arch
Insect Biochem Physiol 1, 225 (1984).
15. Eide PE, Caldwell JM, Marks EP: Establishment of two cell lines from embryonic tissue of
the tobacco hornworm, Manduca sexfa (L.). In Vitro 12, 395 (1975).
16. Yunker CE, Vaughn JL, Cory J: Adaption of a cell line (Grace’s Antheraea cells) to a
medium free of insect hemolymph. Science 155, 1565 (1967).
17. Butte TD, Hughes RC: Isolation and characterization of mosquito cell membrane glycoproteins. Biochim Biophys Acta 640, 655 (1981).
18. Weirich GR, Adams JR: Microsomal marker enzymes of Manduca sexfa (L.) Midgut. Arch
Insect Biochem Physiol 1, 311 (1984)
182
Klitschka, Witt, and Mayer
19. Buff U, Briindl A: Selective alterations of membrane properties of cultured human liver
cells caused by the insecticide DDT. Pest Biochem Physiol22, 36 (1984).
20. Oehler DD, Holman GM: Residue determination of Thompson-Hayward 6040 in bovine
manure by high performance liquid scintillation chromatography. J-AgricFood Chem 23,
590 (1975).
21. Ways P, Hanahan DJ: Characterization and quantification of red cell lipids in normal men.
J Lipid Res 5, 318 (1964).
22. Broekhuyse RM: Phospholipids in tissues of the eye. I. Isolation, characterization and
quantitative analysis by two-dimensional thin-layer chromatography of diacyl- and vinylether phospholipids. Biochim Biophys Acta 152, 307 (1968).
23. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 (1970).
24. Witt W, Schweingruber ME, Mertsching A: Phospholipase B from the plasma membrane
of Saccharornyces cerevisiae. Separation of two forms with different carbohydrate content.
Biochim Biophys Acta 795, 108 (1984).
25. Bradford MM: A rapid and sensitive method for the quantification of microgram quantities
of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248 (1976).
26. Locke M: The role of plasma membrane plaques and golgi complex vesicles in cuticle
deposition during the moultlintermoult cycle. In: The Insect Integument. Hepburn HR,
ed. Elsevier, New York, pp. 237-258 (1976).
27. Duran A, Bowers B, Cabib E: Chitin synthetase zymogin is attached to the yeast plasma
membrane. Proc Natl Acad Sci USA 72, 3952 (1975).
28. Duran A, Cabib E, Bowers B: Chitin synthetase distribution on the yeast plasma membrane. Science 203, 363 (1979).
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