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acaricide diafenthiuron has a novel mode of action: Inhibition of mitochondrial respiration by its carbodiimide product
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
41,207-219
(1991)
The Thiourea Insecticide/Acaricide Diafenthiuron Has a Novel Mode of Action: Inhibition of Mitochondrial Respiration by Its Carbodiimide Product FRANZ J. RUDER,'WALTERGUYER,JACK R + D Plant Protection, Agricultural
A. BENSON,AND HARTMUT KAYSER
Division, Ciba-Geigy Ltd., CH-4002 Basel, Switzerland
Received April 26, 1991; accepted June 17, 1991 The thiourea diafenthiuron (CGA 106630) is a novel insecticide/acaricide. The present paper is concerned with the molecular mechanism underlying its action. So far, we have not found any direct target for diafenthiuron itself to explain its toxic effect. Since it is known that diafenthiuron is rapidly desulfurated abiotically to the pesticidal carbodiide CGA 140408 in the presence of sunlight and singlet oxygen, CGA 140408 is probably the toxic agent mediating the in vivo action of diafenthiuron. Indeed, CGA 140408 is one of the major in vivo products of diafenthiuron in Calliphora. Furthermore, there is evidence that cytochrome P450 mediates this desulfuration, biotically, since diafenthiuron binds to cytochrome P450 as a substrate in rat liver microsomes. None of the known molecular targets of current commercial insecticides are affected by either diafenthiuron or CGA 140408 (e.g., acetylcholinesterase, sodium channel, acetylcholine receptor, chitin synthesis). Further, we cannot confirm a proposed octopaminergic action of CGA 140408. However, we have been able to demonstrate that CGA 140408, but not diafenthiuron, is a potent in vitro inhibitor of mitochondrial respiration in rat liver and Calliphora flight muscles. It selectively blocks the coupling site in a time-dependent manner which is different from the action of other pesticides. The resemblance in the chemical structure to the well-known mitochondrial inhibitor dicyclohexylcarbodiimide suggests that CGA 140408 inhibits mitochondrial ATPase. Further experimental details are reported in a forthcoming paper. o IM Academic press, IIIC.
INTRODUCTION
Thioureas are a relatively new class of insecticides of a hitherto unknown mode of action. As one of these, diafenthiuron (CGA 106630; 3-(2,6-diisopropyl-4phenoxyphenyl)-1-t-butyl-thiourea; Fig. l), a product especially effective against sucking pests (l), has recently been introduced into the market. There are several lines of evidence suggesting that thioureas are proinsecticides, being transformed into the corresponding carbodiimides, which are considerably more active. A previous report on the chemodynamic behavior of diafenthiuron suggested that the insecticidal proper-
’ To whom requests for reprints dressed.
should be ad-
ties of diafenthiuron are correlated with its transformation into CGA 140408 (3-(2,6diisopropyl-4-phenoxyphenyl)-l-t-butylcarbodiimide; Fig. 1) by sunlight and singlet oxygen (2). Further support for a propesticide role for diafenthiuron comes from the observation that the carbodiimide CGA 140408 is more toxic to insects than the thiourea. Moreover, both diafenthiuron and CGA 140408 are broadly acting insecticides/acaricides. These parallel properties are consistent with a common mode of action. In an attempt to identify the mode of action of diafenthiuron, we carried out a series of in viva and in vitro assays, including assays of the known targets of the common insecticides, for effects by diafenthiuron and its carbodiimide product, CGA 140408. of all the systems tested, only mitochon207 0048-3575/91 $3.00 Copyright 8 1991 by Academic FVess, Inc. All rights of reproduction in any form rese~cd.
RUDER ET AL.
208
respectively, were labeled at one of the isopropyl groups and had a sp act of 0.65 GBq/ mmol (18 mCi/mmol). [23,24-3H]Ecdysone with a specific radioactivity of 3.3 TBq/ mmol (90 Ci/mmol) was purchased from New England Nuclear and [14C]glucose (10 GBq/mmol; 270 mCi/mmol) was from Amersham.
CGA1404UE
CH3 \
CH-CH3
@O@
CH.,
N=C=N CH-
+CH3
C”3
CH3
/ CH3
Animals, Topical Application, Toxicity Determination
CGA 106630, diafenthiuon
Locusta migratoria,
C”3 \ CH-CH3
CH-
@o-Q.-NH-CS-
NH-!ICH~ CH-
CH3
CH3
/ CH3 FIG. 1. Structural diafenthiuron.
formulae
drial ATP2 synthesis activity. MATERIALS
of CGA 140408 and
revealed
significant
AND METHODS
Chemicals
Unless otherwise indicated, chemicals were obtained from Fluka, Switzerland. NADPH was from Boehringer Mannheim. Acetylcholinesterase type XII-S from bovine erythrocytes was obtained from Sigma. Diafenthiuron (CGA 106630, N-(2,6-diisopropyl-4-phenoxyphenyl)-N’tert-butylthiourea) and CGA 140408 (N(2,6-diisopropyl-4-phenoxyphenyl)-N’-tertbutylcarbodiimid) are Ciba-Geigy products. [‘4C]Diafenthiuron and [i4C]CGA 140408 with radiochemical purities of 99 and 95%, 2 Abbreviations used: AChE, acetylcholinesterase; ADP, adenosinediphosphate; ATP, adenosinetriphosphate; DCCD, dicyclohexylcarbodiimide; DDT, 2,2bist.p-chlorophenyl)-l,l, I-trichloroethane; DMSO, dimethyl sulfoxide; DTNB, 5,5’-dithiobis(2nitrobenzoic acid); EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol-bis(2aminoethylether)tetraacetic acid; FCCP, carbonylcyanide-4trifluormethoxyphenylhydrazone; GABA, y-aminobutyric acid; HPLC, high-pressure liquid chromatography; NADPH, reduced nicotinamide adenine dinucleotide phosphate; PBO, piperonylbutoxide.
and
Musca domestica,
and Calliphora erythrocephala were from in-house strains. Limulus polyphemus was obtained from the Marine Biological Laboratory (Woods Hole, MA). For injection assays, CGA 140408, suspended in 5 ~1 Locusta physiological saline (3), was injected into adult Locusta. For topical assays, 0.5 to 20 p.g of the test substance dissolved in acetone was applied to the abdomen (Locusta adult; 5 ~1) or dorsal thorax (Calliphora adult; 2 ~1). The animals were observed for 2 days or until they died, and both behavioral abnormalities and mortalities were determined. In Vivo Distribution and Metabolites Calliphora adults were topically treated with 4 p,g of either [14C]diafenthiuron or [i4C]CGA 140408 (2 ~1 acetone solution). For the study of distribution of radioactivity, the insects were frozen on dry ice after 12 hr. The thorax cuticle was carefully removed from the flight muscles, and the head and abdomen were isolated (10 animals/experiment). Total radioactivity was determined in head, abdomen, thorax, and thorax cuticle using Insta-Gel and a Packard Tri-Carb scintillation counter. For analysis of metabolism of diafenthiuron, treated flies (10 animals/experiment) were incubated for 16 hr in the dark until they showed clear signs of intoxication. Following this, metabolites were extracted with methanol (100 @body part). The extracts were diluted with 1 vol of water and concentrated on a C18-BondElut column (Analytichem International, Harbor City, CA). Eluates
DIAFENTHIURON
HAS
A NOVEL
obtained with 100% acetonitrile were subjected to HPLC separation using a Shimadzu system and a 5-p,m Cl8 column (Ciba-Geigy) and eluted with a gradient of 75-100% acetonitrile in water. Radioactivity was quantitatively monitored on line with a Ramona apparatus (Ray test, Straubenhardt, Germany). Tests on Cuticle Formation
The “tearing test” measures the mechanical stability of the cuticle. One-day-old Calliphora adults were treated topically with 1 pg of test substance (2 ~1 acetone). After 6 days, the living insect was fixed between a pulling motor and a weight on a balance. The recorded decrease in the weight measured by the balance is identical to the pulling force of the motor. The motor was started and the force increased, stretching the animal, until the tearing point was reached and the force dropped to zero. The tearing point is a measure of the mechanical strength of the cuticle. The data of eight animals were collected for one experiment. To quantify chitin synthesis, 3 hr after molting, eight Spodoptera larvae (fifth instar) per experiment were injected with 25 ng of test substance in 5 ~1 water (CGA 140408 or diafenthiuron; the benzoylurea chlorlluazurone was used as a positive control). After 2 and 24 hr, respectively, 100 kBq (2.7 pCi) [14C]glucose was injected. One hour later the insects were frozen on dry ice. For preparation of chitin, the cuticles of eight larvae were treated in 10% KOH for 1 hr at 95°C. After centrifugation at 15OOg for 10 min and resuspending in KOH twice, the chitin pellet was homogenized in water. 14C label incorporation in chitin was quantified on a Packard scintillation counter using Packard Insta-Gel to prevent sedimentation of the cuticle particles. Acetylcholinesterase
Assay
AChE activity was determined colorimetrically by a modified Ellman assay us-
MODE
OF
209
ACTION
ing acetylthiocholine as the substrate (4). Briefly, the test compounds at various concentrations were preincubated with bovine erythrocyte AChE (12 cl.g of protein/ml) in potassium phosphate buffer, pH 8.0, at 25°C. The preincubation time was usually 10 min. Enzyme activity was monitored by following the extinction change at 412 nm at 37°C after the addition of acetylthiocholine and DTNB in final concentrations of 1.3 and 0.14 mM, respectively. Carbofuran was run in parallel as a standard inhibitor. Ecdysone
20-hydroxylase
Assay
Locust malpighian tubule microsomes were used as a source for this P450 enzyme (5). The assay was based on the procedures described by Weirich (6). The details are given elsewhere (H. Kayser, in preparation). Test compounds were dissolved in methanol. The assay was carried out in phosphate buffer (pH 7.4) at 25°C for 60 min with 0.03 PM [3H]ecdysone, 1.2 mM NADPH, and about 100 kg of microsomal protein. After prepurification on a Bond Elut Cl8 cartridge, the samples were subjected to HPLC on a Cl8 column with online quantification of radioactivity. Preparation of Mitochondria and Microsomes
Rat liver mitochondria were prepared as described by Gazzotti et al. (7). Rat liver microsomes were obtained from the postmitochondrial supematant by sedimentation at 100,000g for 1 hr. Thoracic flight muscle mitochondria from Calliphora were isolated according to the method of Slack and Bursell (8). Protein was determined as described by Bradford (9). Spectroscopy
of Liver Cytochrome
P450
Difference spectra of rat liver microsomes were run on a Perkin-Elmer Lambda 15 spectrometer at a cytochrome P450 concentration of 1.5 PM. Concentrations of P450 were determined according to Omura and Sato (10). For recording binding spectra of diafenthiuron and CGA 140408,
210
RUDER
the chemicals were added in methanolic solution to the sample cuvette up to a final concentration of 30 pJ4 (about IO-fold the KS of diafenthiuron). The reference cuvette received the same amount of solvent. Methanol never exceeded a 1% final concentration. Mitochondrial Respiration Calliphora mitochondria were preincubated on ice in 320 mM sucrose, 1 mM EDTA, 5 miV potassium phosphate, pH 7.4, for the time indicated and with the amount of carbodiimide indicated. Rat liver mitochondria were preincubated in 250 mM sucrose 5 mM Tris/HCl, pH 7.4, 1 mM EGTA. For preincubation, carbodiimides were added as methanolic solutions, methanol not exceeding 1% in the final medium. Oxygen consumption of mitochondria, with glutamate (rat liver mitochondria, 2 mg protein/25 ml incubation chamber) or pyruvate (Calliphora flight muscle mitochondria, 0.5 mg protein) as the substrate, was measured by a Clark-type oxygen electrode (7). When compounds were added to the incubation chamber (2.5 ml), 0.5 ymol ADP (5 111H,O), 1 nmol FCCP (5 ~1 ethanol), 2.5 pg oligomycin (5 t.~l ethanol), or 0.6 nmol CGA 140408 (5 ~1 ethanol) was used. Isolated Limulus Heart The method for isolating the heart and recording its contractions in vitro was as reported previously (11). The test compounds were dissolved first in DMSO and then diluted with physiological saline. DMSO by itself was without effect on the heart beat at concentrations IO-fold those used here (0.1%). The experimental protocols were as follows. First, CGA 140408 was tested at 10 PM for transmittermimetic effects on the heart beat frequency. The control frequency was taken from the 3 min preceding the application of the test compound, and the response was measured over the last 3 min of application. CGA 140408 was then tested for any effect on the magnitude of the chronotropic re-
ET
AL.
sponse to a standard test transmitter dose (at approximately the ECso values: octopamine, 0.1 PM; dopamine, 3 FM; serotonin, 0.1 PM; GABA, 20 $V) in order to detect antagonistic or modulatory influences. The standard transmitter dose was applied once for 10 min to establish the magnitude of the control response. After a 30-min perfusion with control saline, the test compound was applied at 10 PM for 30 min, followed, without washout, by the same concentration of test compound together with the standard transmitter dose for 10 min. The mixture was then washed out and the preparation was super-fused with control saline for 30 min after which the standard transmitter dose was tested again to determine the reversibility of any effects by the test compound. Isolated Locusta Thoracic Neuronal Somata The method used for isolating and recording transmitter-evoked responses in somata from the thoracic ganglia was as reported previously (3) and based on the method of Usherwood et al. (12). The three thoracic ganglia were dissected free from adult specimens of the locust, L. migratoria, desheathed, and aspirated three or four times through a Pasteur pipette tip. The resulting suspension of dissociated neuronal somata was left in a few drops of physiological saline in a Petri dish for 2 to 4 hr. The cells were impaled and voltageclamped using conventional single-electrode means. The test compounds were dissolved in DMSO and then in Locusta physiological saline (3), and bath-applied via the continuous perfusion of the recording chamber. Physiological saline solutions of the test transmitters were pressure applied to the voltage-clamped somata via a micropipette. Cockroach Giant Axon The method used to record the resting and action potentials from the giant axons in the isolated nerve cord of the cockroach,
DIAFENTHIURON
HAS
A NOVEL
Periplaneta americana, was based on that of Boistel and Coraboeuf (13). The nerve cord was stretched by about 30% so that a pair of desheathed, interganglionic connectives spanned the recording chamber which was continuously perfused with cockroach physiological saline (14). A giant axon in one of these connectives was impaled with a thin-walled glass microelectrode (9-12 Mfl, 1 M KCl) and the membrane and action potentials were measured. Musca Larval Body Wall Neuromuscular Junction The insect neuromuscular junction glutamate receptor assay was based on the preparation of Irving and Miller (15). The viscera and central nervous system were removed from third instar larvae of the housefly, Musca domestica, and the preparation was super-fused with physiological saline in a 1.25ml recording chamber. Intracellular recordings were made from muscle 7A. Only muscle fibers with stable resting potentials of between - 60 and - 70 mV were used. The stimulating system consisted of 2 cm of PE-10 polyethylene tubing (Intramedic) mounted on a syringe. A segmental nerve innervating muscle 7A was drawn up into the PE tubing by means of gentle negative pressure which was then discontinued. The nerve was stimulated via silver wires with a 1-msec pulse of l-2 V every 5 sec. RESULTS
In Vivo Observations
and Toxicity
Adults of Locusta and Calliphora were treated with diafenthiuron or CGA 140408. The treated animals were observed until they died. The time of first appearance of effects on the behavior of Calliphora and Locusta due to topical treatment with either diafenthiuron or CGA 140408 depends on the dose of the pesticide. The first effects are clear signs of disorientation and paralysis. No differences can be observed between the effects of diafenthiuron and
MODE
211
OF ACTION
CGA 140408 on the behavior of the insects, except that CGA 140408 acts faster than diafenthiuron. In Calliphora, it takes at least 2.5-3 hr with CGA 140408 (20 pg/fly; average weight 50-70 mg, depending on sex) or 4 hr with diafenthiuron (25 pg/fly) until the first effects on behavior can be seen. In the flies, the flight activity is reduced and the legs tense up. After several more hours, movements cease and the animals die. The shortest time until death after treatment with either 25 Fg diafenthiuron or 20 pg CGA 140408 per Calliphora is 4 to 5 hr. Higher amounts of pesticide do not accelerate the lethal effect. The LD,, value (for death within 48 hr) for Calliphora adults is about 2-3 pg (6-9 nmol) for diafenthiuron and 1.1 p.g (3.3 nmol) for CGA 140408 by topical application to the thorax. To kill adult Locusta (average weight 1.6-2.2 g, depending on sex) by injection into the abdomen, 0.5 pg of CGA 140408 (1.5 nmol) is necessary. All of the data mentioned above, in addition to results obtained during biological screening and field tests (data not shown), indicate that both diafenthiuron and CGA 140408 are slowly acting insecticides with a broad spectrum of target species. In Vivo Distribution
in Calliphora
In order to estimate how much carbodiimide reaches its potential target sites, we measured the amount of substance (diafenthiuron or CGA 140408) that remains on the thorax cuticle, how much enters the thorax, and how much penetrates as far as the head and abdomen, following topical application (Table 1). Approximately 3050% of the xenobiotics are lost, probably due to evaporation or cleaning movements by the animal. As an estimate, of the remaining amount, in each experiment more than two-thirds remain on the thorax cuticle or in tissue associated with the cuticle after separation of the thorax cuticle from the interior. The other one-third of the remaining xenobiotic penetrates the thorax cuticle and is distributed into the thorax
212
RUDER
TABLE 1 Distribution of Recovered Radioactivity after Topical Application of Radiolabeled Diafenthiuron and CGA 140408 on Calliphora Adults (in Percentage of Recovered Radioactivity; Combined Results of Two Experiments, Each Including 10 Animals; Results from the Two Experiments Did Not Deviate by More Than J.5-20% of Counts from Each Other)
Diafenthiuron Head + cuticle Thorax content Abdomen + cuticle Thorax cuticle Recovery of applied radioactivity
CGA 140408
2 3 16 79
5 4 19 73
48
53
cavity or is transferred to the head and the abdomen. Surprisingly, most of the latter one-third of the detected xenobiotics can be found in the abdomen and head, perhaps because the animal transfers the compound from the thorax during cleaning movements. From these results it is concluded that only a small amount of xenobiotics reach potential targets within the insect. Metabolism of Diafenthiuron to Cytochrome P450
and Binding
To obtain some preliminary information about the metabolism of diafenthiuron, we analyzed radiolabeled compounds after treatment of Calliphora with diafenthiuron (Table 2). Diafenthiuron is metabolized to three major products: the corresponding urea, carbodiimide, and at least one uniden-
ET
AL.
tified polar substance (Table 2). A high proportion of the carbodiimide CGA 140408 is generated in the thorax (34% of the thorax metabolites). This might be an underestimate since a considerable part of the carbodiimide might bind covalently to mitochondrial proteins (16). A large amount of the polar substance is detectable in the abdomen. In view of a possible enzymatic in vivo desulfuration of diafenthiuron, difference spectroscopy was performed with rat liver microsomes in order to detect any interactions of the two chemicals with P450. The addition of diafenthiuron reproducibly results in a type I difference spectrum with a maximum at 382 nm and a minimum at 420 nm. Type I spectra are typically observed with compounds representing substrates for P450 enzymes (17). Hence, we expect that diafenthiuron can be metabolized by rat liver P450. On the other hand, there are no hints of inhibitory interactions by diafenthiuron with P450. In contrast, CGA 140408 does not produce any type of difference spectrum with liver P45Os, implying that it does not bind to P450 in the heme region that is part of the active site. Tests on Inhibition of Cuticle Formation, AChE, and Ecdysone 20-hydroxylase To test whether either diafenthiuron or CGA 140408 affects cuticle formation, we applied two different tests. In the first experiment, we measured incorporation of
TABLE 2 Metabolites of Diafenthiuron after Topical Application on Calliphora Thorax (in Percentage of Recovered Radioactivity from the Respective Body Part); CGA 197430, the Urea Analogue of CGA 106630, Is a Hydrolysis Product of Both Diafenthiuron and CGA 140408. Data Derived from a Typical Experiment (10 Animals; Two Further Experiments Confirmed the Generation of the Same Metabolic Products in the Respective Body Parts, although in Varying Concentrations)
Head + cuticle Thorax cuticle Thorax content Abdomen + cuticle
Unknown polar products
Urea (CGA 197430)
Thiourea (CGA 106630)
Carbodiimide (CGA 140408)
25 19 40 79
15 30 26 5
39 20 0 8
22 30 34 8
DIAFENTHIURON
HAS
A NOVEL
i4C-labeled glucose into chitin of Spodoptera larvae (Table 3). Neither diafenthiuron nor CGA 140408had a significant effect on incorporation of radioactivity into chitin. In another experiment, we used Calliphora adults to measure the mechanical properties of the cuticle after insecticide treatment. Neither diafenthiuron nor CGA 140408had any significant effect on the mechanical stability of the cuticle. In both tests, the benzoylurea chlorfluazurone, a known chitin synthesis inhibitor (18), was highly effective. The effects of diafenthiuron and CGA 140408on bovine AChE were studied up to their limits of solubility under assay conditions. With a preincubation time of 10 min, 1C50values of about 0.55 and 1.2 mM are obtained, by extrapolation, for diafenthiuron and CGA 140408,respectively. Under identical conditions, the corresponding value for carbofuran as a standard inhibitor is 1.5 Q4. Increasing the preincubation time to 2 hr produces no more effect. We conclude that neither compound can be regarded as an inhibitor of AChE. Ecdysone 20-hydroxylase was examined as an example of a specific insect P450 enzyme. Diafenthiuron and CGA 140408were tested for inhibition of the hydroxylase at 0.03 to 300 pike final concentrations. With both compounds, no inhibition was ob-
MODE
OF
213
ACTION
served even at the highest concentrations which led to precipitation of the test compounds (especially of the carbodiimide, which is less soluble than the thiourea). On the contrary, there is a trend to a slight stimulation of P450 activity, which is more pronounced with CGA 140408. The reason for this effect was not studied further. Inhibition of Mitochondrial in Vitro
Respiration
CGA 140408, but not diafenthiuron, was found to be a potent inhibitor of respiration both in isolated rat liver and Calliphora flight muscle mitochondria. Because of its structural similarity with dicyclohexylcarbodiimide (DCCD), both being hydrophobic carbodiimides, we hypothesized that CGA 140408 might inhibit mitochondrial respiration in the same manner as DCCD [for a review of proteins affected by DCCD, see Ref. (19)]. Both carbodiimides block ADP-dependent respiration of Culliphoru mitochondria (Fig. 2; Table 4). After 10 min of preincubation, Calliphoru mitochondria required 1 nmol DCCD/mg protein and 1.8 nmol CGA 140408/mgprotein during preincubation for complete inhibition. After 2 hr preincubation, only 0.3 nmol DCCD/mg protein and 0.18 nmol CGA 140408/mgprotein are necessary, although no exact titration of the required amount of carbodiimide
TABLE 3 Effects of Test Substances on Chitin Synthesis and Cuticle Formation Incorporation of [‘4C]glucose into chitin in ffth instar Spodoptera larvae (radioactivity in % of controBa Hours after injection of test substance Test substance
2
Diafenthiuron CGA 140408 Chlorfhtazurone Solvent control
106 91 17 100
24
Cuticle formation in % of control as derived from the tearing test”
93 13 4
9-I 98 22
100
100
Note. For details see under Materials and Methods. LICombined results from two experiments; the two sets of results did not show more than 15% deviation from each other which is within the accuracy range expected for this method. b Results from one experimental series of eight tlies.
*
,.
^
,‘
.
,
,,
214
RUDER
A
ET
AL.
TABLE 4 Respiration in Calliphora Mitochondria
d PlDP --T
*AoP \
\ 2. Oxygen consumption of Calliphora thorax mitochondria. Calliphora thorox mitochondria were isolated and incubated with 5 mMpyruvate in incubation buffer. ADP, FCCP, oligomycin (OLJ), and CGA 140408 were added at the time indicated by the triangle. (A) Experiment with CGA 140408. (B) Experiment with oligomycin. FIG.
was undertaken in these experiments (tested concentrations for CGA 140408: 6, 1.8, 0.6, 0.18, and 0.06 nmohmg; for DCCD: 10, 3, 1, 0.3, and 0.1 nmol/mg). We tried to locate the site of inhibition of mitochondrial respiration and found that in Culliphoru mitochondria, under the incubation conditions described above, both CGA 140408 and DCCD interfere exclusively with the coupling site in a manner similar to ohgomycin (Fig. 2; Table 4). Carbodiimidetreated mitochondria do not increase respiration after the addition of ADP, but addition of the uncoupler (FCCP) increases oxygen consumption identically to untreated mitochondria. Oxygen consumption of uncoupled mitochondria is inhibited completely only when preincubation is at a high carbodiimide concentration (2 10 nmol carbodiimide/mg protein; data not shown). Therefore, at low carbodiimide concentrations (approx 1 nmoVmg protein) only the coupling site and not the respiratory chain is inhibited in Calliphora mitochondria. These data indicate that ATP synthesis is
Substance
ADPlstate 4
FCCPIstate 4
Control Oligomycin CGA 140408 (nmol/mg protein) 0.06 0.18 0.6 DCCD (nmohmg protein) 0.1 0.3 1.0
8.4 1.0
7.5 7.6
7.1 2.3 1.0
7.6 6.6 6.0
8.2 2.0 1.2
7.0 7.6 8.1
Note. Mitochondria were preincubated with DCCD or CGA 1404@3for 30 min as indicated under Materials and Methods. The given indices were calculated from the rates of oxygen consumption after and before addition of ADP or FCCP (ADP/state 4 or FCCPktate 4; state 4, respiration before addition of ADP or FCCP) (data derived from one typical experiment using the same mitochondrial preparation; five more experiments from two mitochondrial preparations gave similar results, although preincubation of the carbodiimide with the mitochondria was for varying times and carbodiimide concentrations).
blocked in carbodiimide-treated mitochondria. The results obtained in Cufliphoru mitochondria also apply to rat liver mitochondria. ADP-dependent respiration is severely inhibited by CGA 140408 (Table 5). Moreover, CGA 140408 blocks rat liver mitochondria at the coupling site, as in Calliphoru mitochondria (data not shown). Diafenthiuron, at concentrations of up to 5 nmoVmg protein, has no effect on mitochondrial respiration in either rat liver or Calliphora (data not shown). Isolated
Limuius
Heart
When isolated, the heart of the horseshoe crab, Limulus polyphemus, continues to beat rhythmically under the control of its endogenously active cardiac ganglion. The heartbeat responds to octopamine, dopamine, serotonin, and GABA with characteristic changes in both its rate and amplitude of contraction that can be reversed by
DIAFENTHIURON TABLE
HAS A NOVEL
5
Respirationin Rat Liver Mitochondria CGA 140408 (nmol/mg protein) Control 0.06 0.18 0.6 1.8
ADP/state 4 16 12.5 9.5 I
MODE OF ACTION
215
GABA,-like (3), an uncharacterized dopaminergic (Kaufmann and Benson, unpublished data), and three serotonergic receptors (26). None of these receptors is sensitive to 10 or 1 fl CGA 140408applied for between 20 and 120 min. Cockroach
Giant Axon
1.0
Overshooting action potentials of constant amplitude (105 to 110 mV) and rising Note. Mitochondria were preincubated with DCCD from a stable resting membrane potential or CGA 140408 for 6 hr as indicated under Materials and Methods. The ratio of the rate of oxygen con( - 65 to - 70 mV) were recorded for at sumption (ADPktate 4) was determined (for details least 4 hr from cockroach giant axons stimsee Table 4) (data derived from one typical experiment ulated continually by electric shock at 0.1 with the same mitochondrial preparation; six more exHz. Superfusion of the nerve cord by diperiments from two mitochondrial preparations gave afenthiuron and CGA 140408for up to 4 hr similar results, although preincubation of the carbodiimide with the mitochondria was for varying times and has no effect on either the resting or the carbodiimide concentrations). action potential. The Na channel blocker, tetrodotoxin, reversibly reduces the action washing in control saline (20). The octo- potential by 50% within 10 min under these pamine effects are mediated by cyclic AMP experimental conditions, and the pyre(21, 22). throids cypermethrin and permethrin proCGA 140408 is without an agonistic ef- duce total block in 35 and 90 min, respecfect on the heartbeat frequency or ampli- tively. CGA 140408is also without effect on tude when applied to the isolated heart at the Nat and K+ potential-dependent cur10 TV&for up to 40 min. When tested for rents in voltage-clamped, cockroach giant antagonism of the responses to octopa- axons (M. Pelhate, unpublished data). mine, dopamine, serotonin, and GABA, Musca Larval Body Wall CGA 140408 has no effect. Neuromuscular
Isolated Locusta
Neuronal
Thoracic Somata
Octopamine activates an inward current in neuronal somata clamped at a holding potential of -60 mV. This response is blocked by bath application of 10 pJ4 phentolamine for 20 min (23, 24). The resting membrane current of the clamped somata and the octopamine response are both unaffected by bath application of 1and 10 PM CGA 140408for up to 2 hr. CGA 140408is thus neither an agonist nor an antagonist at the Locusta thoracic neuronal soma octopamine receptor. Acetylcholine, muscarine, GABA, dopamine, and serotonin also evoke characteristic responses in the voltage-clamped Locusta thoracic neuronal somata, mediated by a nicotinic (29, muscarinic (25), a
Junction
Composite excitatory postsynaptic potentials (EPSPs) were recorded from the muscle fibers of larval Musca in response to stimulation of the motor nerve. These EPSPsare mediated by glutamate receptors located in the muscle postsynaptic membrane. Diafenthiuron and CGA 140408were without direct agonist effects on the membrane potential of the muscle fibers and did not alter the amplitude or time course of the EPSP during applications of 10 ELMfor an hour or more. Under the experimental conditions used in these experiments, the ion channel blocker, chlorisondamine, for example, reduces the EPSP to a new steady state amplitude in less than 5 min. DISCUSSION
Diafenthiuron probably exerts its lethal
216
RUDER
effect via its desulfuration product, the carbodiimide CGA 140408. In addition to the abiotic desulfuration of diafenthiuron reported previously (2), our metabolism studies show that a significant proportion of diafenthiuron is also converted to CGA 140408 in vivo. We have observed that piperonylbutoxide (PBO), an inhibitor of P450, decreases the toxicity of diafenthiuron in Spodoptera (H. Kayser and F. J. Ruder, in preparation). Kadir and Knowles (27) demonstrated the formation of CGA 140408 from diafenthiuron in bulb mites and found that PBO decreases the toxicity of diafenthiuron in the diamond-backed moth, Plutella xylostella. Further evidence that diafenthiuron is desulfurated by a P450dependent reaction is provided by the observation that diafenthiuron, but not CGA 140408, binds to rat liver P450 as a substrate and can be converted to CGA 140408 in a
ET
AL.
P450-dependent reaction in vitro (H. Kayser and F. J. Ruder, in preparation). A summary of the results obtained with diafenthiuron and CGA 140408 in all test systems applied is given in Table 6. The carbodiimide CGA 140408 has no effect at concentrations up to 10 p.M on any of the established target sites of the major current commercial insecticides (the Na channel, acetylcholinesterase, the nicotinic acetylcholine receptor, cuticle formation) nor on the Locusta muscarinic acetylcholine receptor and the Musca larva neuromuscular glutamate receptor. It was also without effect on the receptors for GABA, dopamine, and serotonin in Locusta and in the heart of the primitive arthropod, L. polyphemus. Neither diafenthiuron nor CGA 140408 acts on the action potential or potentialdependent currents of the cockroach giant axon over a period of hours, under exper-
TABLE 6 Potential Target Sites Assayed for Effects of Diafenthiuron Target Mitochondria
P450 spectroscopy Ecdysone 20-hydroxylase Cuticle formation Chitin synthesis Acetylcholinesterase Axonal Na+ channel Nicotinic cholinergic receptor Muscarinic cholinergic receptor GABA receptor Dopamine receptor Serotonin receptor Octopamine receptor Glutamate receptor
Assay system
and CGA 140408
Diafenthiuron
Calliphora Rat liver
NE
Rat liver Locusta Calliphora Spodoptera Bovine erythrocytes Periplaneta Locusta Locusta Locusta Limulus Locusta Limulus Locusta Limulus Locusta Limulus Musca
Type 1 spectrum NE NE NE NE NE NE -b
NE -
NE
Note. The neurobiological transmitter receptor preparations were tested electrophysiologically the ligand recognition site and the associated ionophore were assayed. LINo effect. b Not tested.
CGA 1404OfI Block of the coupling site Block of the coupling site NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE so that both
DIAFENTHIURON
HAS
A NOVEL
imental conditions in which tetrodotoxin and the pyrethroids cypermethrin and permethrin rapidly block action potential conduction. Kadir and Knowles (28) have proposed that the toxic effect of CGA 140408 is mediated by octopamine receptors. Our electrophysiological data show that CGA 140408 is without agonistic or antagonistic effects on the octopamine receptors of Locusfu neurones and in the heart of the arthropod Limulus. The evidence presented for an octopaminergic mode of action for CGA 140408 was derived from experiments on bulb mites and fireflies (28). The differences between the two sets of findings might be accounted for by a remarkably high specificity for a particular subtype of octopamine receptor. Evans (29) has shown that octopamine receptors in insects belong to at least three pharmacologically distinct subtypes. However, extended investigations of possible effects by CGA 140408 on adenylate cyclase failed to reveal any evidence that CGA 140408 acts as an octopamine agonist or antagonist in any of the mite and insect systems studied (H. Kayser et al., in preparation). CGA 140408, but not diafenthiuron, is a potent inhibitor of mitochondrial respiration in rat liver and Culfiphoru thorax muscle mitochondria in vitro. Its target is the coupling site since it blocks ADP-dependent respiration and this block can be abolished by the uncoupler FCCP. CGA 140408 interferes specifically with the coupling site at low concentrations (<2 nmol/mg protein). The threshold quantity required to block the coupling site decreases with the increased preincubation time of the carbodiimide with the mitochondria. At 10 min preincubation, a CGA 140408 concentration of 1.8 nmol/mg protein, and at 2 hr preincubation of 0.18 nmol/mg protein, is sufficient for complete block of the coupling site. This observation indicates that the inhibition is irreversible and is due to a chemical reaction of the carbodiimide with the coupling site. Only at a concentration of 310 nmol CGA 1404081mg mitochondrial
MODE
OF
ACTION
217
protein do the respiratory chain enzymes become inhibited. Theoretically, there are at least two explanations for the experimental data. First, CGA 140408 could block the ATP/ADP antiporter of the inner mitochondrial membrane in the same way as atractyloside. Second, the FO/Fl mitochondrial ATPase could be affected in the same way as by oligomycin or DCCD. Since the carbodiimide DCCD is chemically related to CGA 140408 and the time dependence of the inhibition indicates a covalent, irreversible reaction, we believe that these compounds inhibit mitochondrial ATPase in the same manner [for a review on DCCD, see Refs. (30, 31)]. In a forthcoming paper we show that CGA 140408 covalently reacts with the FO proteolipid of the proton channel moiety of the ATPase (16). Recently, Kadir and Knowles (32) have also shown that CGA 140408 is an inhibitor of mitochondrial ATPase in a mite. Many insecticides act on mitochondrial respiration. Rotenone is a specific inhibitor of NADH (site I)-dependent respiration (33). Substances, like dinitrophenols or arsenate, uncouple respiration from oxidative phosphorylation (34, 35). The fumigants phosphine and hydrogen cyanide are inhibitors of cytochrome oxidase, thereby blocking the respiratory chain (35, 36). Reversible inhibition of mitochondrial ATPase has been observed with chlorinated hydrocarbons (e.g., DDT) (37) and organotin compounds (35). Evidence has been presented that DDT binds to the same proteolipid as DCCD (38), although it appears unclear how far ATPase inhibition contributes to the pesticidal effect of DDT in vivo (37). The major site of action of DDT is probably the sodium channel. The acaricide tetradifon inhibits mitochondrial ATPase similarly to oligomycin (39). In conclusion, the modes of action of all of these insecticides, at the level of the molecular mechanism, appear different from what we know about CGA 140408 since this carbodiimide is an irreversible inhibitor of mitochondrial ATPase, covalently binding
218
RUDER
to the FO proteolipid (16). Although some pesticides inhibit mitochondrial ATPase, there is no evidence for a covalent reaction with the proteolipid. The data presented in this paper are consistent with the possibility that the insecticidal and acaricidal effects of diafenthiuron result from the irreversible and time-dependent inhibition of mitochondrial ATP synthesis by CGA 140408, the active metabolite of diafenthiuron. ACKNOWLEDGMENTS
The authors thank C. Dang, F. Glanzmann, Dr. L. Kaufmann, C. Kramer, K. Leuenberger, and F. Schilrmann for technical assistance. REFERENCES
1. H. P. Streibert, J. Drabek, and A. Rindlisbacher, Diafenthiuron: A new type of acaricidel insecticide for the control of the sucking pest complex in cotton and other crops, Brighton Crop Protection Conference-Pest and Diseases, 25 (1988). 2. A. Steinemann, E. Stamm, and B. Frei, The effect of chemodynamic parameters on pesticide performance, Aspects Appl. Biol. 21, 203 (1989). 3. G. Lees, D. J. Beadle, R. Neumann, and J. A. Benson, Responses to GABA by isolated insect neuronal somata: Pharmacology and modulation by a benzodiazepine and a barbiturate, Brain Res. 401, 267 (1987). 4. G. L. Ellman, K. D. Courtney, V. Andres, Jr., and R. M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7, 88 (l%l). 5. R. Feyereisen and F. Durst, Ecdysterone biosynthesis: A microsomal cytochrome-P-4SO-linked ecdysone 20-monooxygenase from tissues of the african migratory locust, Eur. J. Biochem. 88, 37 (1978). 6. G. F. Weirich, Ecdysone 20-monooxygenase, in “Methods in Enzymology” (J. Law and H. Rilling, Eds.), Vol. 111, pp. 454-l58, Academic Press, New York, 1985. 7. P. Gazzotti, K. Malmstrom, and M. Crompton, Preparation and assay of animal mitochondria and submitochondrial vesicles, in “Membrane Biochemistry: A Laboratory Manual on Transport and Bioenergetics” (E. Carafoli and G. Semenza, Eds.), pp. 62-76, Springer-Verlag, New York, 1979. 8. E. N. Slack and E. Bursell, The isolation of mitochondria from dipteran Bight muscle, Biochim. Biophys. Acta 449, 491 (1976).
ET AL.
9. M. M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72, 248 (1976). 10. T. Omura and R. Sato, The carbon monoxidebinding pigment of liver microsomes. I. Evidence for its hemoprotein nature, J. Biol. Chem. 239, 2379 (1964). 11. J. A. Benson, R. E. Sullivan, W. H. Watson, and G. J. Augustine, The neuropeptide proctolin acts directly on Limulus cardiac muscle to increase the amplitude of contraction, Brain Res. 213, 449 (1981). 12. P. N. R. Usherwood, D. Giles, and C. Suter, Studies of the pharmacology of insect neurones in vitro, in “Insect Neurobiology and Pesticide Action (Neurotox ‘79),” pp. 115-128, Society of Chemical Industry, London, 1980. 13. J. Boistel and E. Coraboeuf, Potentiels de membrane et potentiels d’action de nerf d’insecte recueillis a l’aide de microelectrodes intracellulaires, C. R. Acad. Sci. Paris 238, 2116 (1954). 14. R. M. Pitman, The ionic dependence of action potentials induced by colchicine in an insect motoneurone cell body, J. Physiol. (London) 247, 511 (1975). 15. S. N. Irving and T. Miller, Ionic differences in “fast” and “slow” neuromuscular transmission in body wall muscles of Musca domestica larvae, J. Comp. Physiol. 135, 291 (1980). 16. F. J. Ruder and H. Kayser, The carbodiimide product of diafenthiuron reacts covalently with two mitochondrial proteins, porin and the FO-proteolipid, and inhibits mitochondrial ATPases in vitro, submitted for publication. 17. J. B. Schenkman, H. Remmer, and R. W. Estabrook, Spectral studies of drug interaction with hepatic microsomal cytochrome, Mol. Pharmacol. 3, 113 (1967). 18. R. Neumann and W. Guyer, Biochemical and toxicological differences in the modes of action of the benzoylureas, Pestic. Sci. 20, 147 (1987). 19. A. Azzi, R. P. Casey, and M. J. Nalecz, The effects of N,N’-dicyclohexylcarbodiimide on enzymes of bioenergetic relevance, Biochim. Biophys. Acta 768, 209 (1984). 20. G. J. Augustine, R. Fetterer, and W. H. Watson, Amine modulation of the neurogenic Limulus heart, J. Neurobiol. 13, 61 (1982). 21. J. R. Groome and W. H. Watson, Mechanism for amine modulation of the neurogenic Limulus heart: Evidence for involvement of CAMP, .I. Neurobiol. 18, 417 (1987). 22. J. R. Groome and W. H. Watson, Involvement of cyclic AMP in multiple, excitatory actions of biogenic amines on the cardiac ganglion of the horseshoe crab Limulus polyphemus, .I. Exp. Biol. 152, 313 (1990).
DIAFENTHIURON
HAS A NOVEL
23. C. Suter, The action of octopamine and other biogenie amines on locust central neurones, Comp. Biochem. Physiol. C 84, 181 (1986). 24. L. Kaufmarm and J. A. Benson, Characterisation of a locust neuronal octopamine response, Sot. Neurosci. Abs., in press. 25. J. A. Benson, Transmitter receptors on insect neuronal somata: GABAergic and cholinergic pharmacology, in “The Molecular Basis of Drug and Pesticide Action: Neurotox ‘88” (G. G. Lunt, Ed.), pp. 193-206, Elsevier, Amsterdam, 1988. 26. I. Bermudez, D. J. Beadle, and J. A. Benson, Multiple serotonin-activated currents in isolated, neuronal somata from locust thoracic ganglia, Sue. Neurosci. Abs. 16, 857 (1990). 27. H. A. Kadii and C. 0. Knowles, Toxicological studies of the thiourea diafenthiuron in diamond back moths (Lepidoptera: Yponomeutidae), twospotted spider mites (Atari: Tetranychidea), and bulb mites (Atari: Acaridiae), in press. 28. H. A. Kadir and C. 0. Knowles, Octopaminergic action of the carbodiimide metabolite of diafenthiuron, Pestic. Biochem. Physiol. 39, 261 (1991). 29. P. D. Evans, Multiple receptor types for octopamine in the locust, J. Physiol. (London) 318,99 (1981). 30. W. Sebald and J. Hoppe, On the structure and genetics of the proteolipid subunit of the ATP synthase complex, Curr. Top. Bioenerg. 12, 1 (1981). 31. J. Hoppe and W. Sebald, The proton conducting FO-part of bacterial ATP-synthases, Biochim. Biophys. Actu 768, 1 (1984).
MODE OF ACTION
219
32. H. A. Kadir and C. 0. Knowles, Inhibition of ATP dephosphorylation by acaricides with emphasis on the anti-ATPase activity of the carbodiimide metabolite of diafenthiuron, in press. 33. J. I. Fukami, Rotenone and rotenoids, in “Comprehensive Insect Physiology, Biochemistry, and Pharmacology” (G. A. Kerkut and L. I. Gilbert, Eds.), Vol. 12, pp. 291-312, Pergamon, New York, 1985. 34. R. D. O’Brien, “Insecticides: Action and Metabolism,” p. 286, Academic Press, New York, 1%7. 35. J. Fukami, Insecticides as inhibitors of respiration, in “Insecticide Biochemistry and Physiology” (C. F. Wilkinson, Ed.), pp. 3533%, Plenum, New York, 1976. 36. K. P. Kashi and W. Chefurka, The effect of phosphine on the absorption and circular dichroic spectra of cytochrome c and cytochrome oxidase, Pestic. Biochem. Physiol. 6, 350 (1976). 37. M. P. Osborne, DDT, y-HCH and the cyclodienes, in “Comprehensive Insect Physiology, Biochemistry, and Pharmacology” (G. A. Kerkut and L. I. Gilbert, Eds.), Vol. 12, pp. 131-182, Pergamon, New York, 1985. 38. C. C. Dary, S. C. Buessow, H. Wenzler, and L. K. Cutkomp, Binding of (14C)-DDT and (‘4C)-dicyclohexylcarbodiimide to a lipoprotein of the membrane sector of mitochondrial ATPase, Pestic. Sci. 16, 605 (1985). 39. E. Bustamente and P. L. Pedersen, Tetradion: An oligomycin-like inhibitor of energy-linked activities of rat liver mitochondria, Biochem. Biophys. Res. Commun. 51, 292 (1973).
BIOCHEMISTRY
AND
PHYSIOLOGY
41,207-219
(1991)
The Thiourea Insecticide/Acaricide Diafenthiuron Has a Novel Mode of Action: Inhibition of Mitochondrial Respiration by Its Carbodiimide Product FRANZ J. RUDER,'WALTERGUYER,JACK R + D Plant Protection, Agricultural
A. BENSON,AND HARTMUT KAYSER
Division, Ciba-Geigy Ltd., CH-4002 Basel, Switzerland
Received April 26, 1991; accepted June 17, 1991 The thiourea diafenthiuron (CGA 106630) is a novel insecticide/acaricide. The present paper is concerned with the molecular mechanism underlying its action. So far, we have not found any direct target for diafenthiuron itself to explain its toxic effect. Since it is known that diafenthiuron is rapidly desulfurated abiotically to the pesticidal carbodiide CGA 140408 in the presence of sunlight and singlet oxygen, CGA 140408 is probably the toxic agent mediating the in vivo action of diafenthiuron. Indeed, CGA 140408 is one of the major in vivo products of diafenthiuron in Calliphora. Furthermore, there is evidence that cytochrome P450 mediates this desulfuration, biotically, since diafenthiuron binds to cytochrome P450 as a substrate in rat liver microsomes. None of the known molecular targets of current commercial insecticides are affected by either diafenthiuron or CGA 140408 (e.g., acetylcholinesterase, sodium channel, acetylcholine receptor, chitin synthesis). Further, we cannot confirm a proposed octopaminergic action of CGA 140408. However, we have been able to demonstrate that CGA 140408, but not diafenthiuron, is a potent in vitro inhibitor of mitochondrial respiration in rat liver and Calliphora flight muscles. It selectively blocks the coupling site in a time-dependent manner which is different from the action of other pesticides. The resemblance in the chemical structure to the well-known mitochondrial inhibitor dicyclohexylcarbodiimide suggests that CGA 140408 inhibits mitochondrial ATPase. Further experimental details are reported in a forthcoming paper. o IM Academic press, IIIC.
INTRODUCTION
Thioureas are a relatively new class of insecticides of a hitherto unknown mode of action. As one of these, diafenthiuron (CGA 106630; 3-(2,6-diisopropyl-4phenoxyphenyl)-1-t-butyl-thiourea; Fig. l), a product especially effective against sucking pests (l), has recently been introduced into the market. There are several lines of evidence suggesting that thioureas are proinsecticides, being transformed into the corresponding carbodiimides, which are considerably more active. A previous report on the chemodynamic behavior of diafenthiuron suggested that the insecticidal proper-
’ To whom requests for reprints dressed.
should be ad-
ties of diafenthiuron are correlated with its transformation into CGA 140408 (3-(2,6diisopropyl-4-phenoxyphenyl)-l-t-butylcarbodiimide; Fig. 1) by sunlight and singlet oxygen (2). Further support for a propesticide role for diafenthiuron comes from the observation that the carbodiimide CGA 140408 is more toxic to insects than the thiourea. Moreover, both diafenthiuron and CGA 140408 are broadly acting insecticides/acaricides. These parallel properties are consistent with a common mode of action. In an attempt to identify the mode of action of diafenthiuron, we carried out a series of in viva and in vitro assays, including assays of the known targets of the common insecticides, for effects by diafenthiuron and its carbodiimide product, CGA 140408. of all the systems tested, only mitochon207 0048-3575/91 $3.00 Copyright 8 1991 by Academic FVess, Inc. All rights of reproduction in any form rese~cd.
RUDER ET AL.
208
respectively, were labeled at one of the isopropyl groups and had a sp act of 0.65 GBq/ mmol (18 mCi/mmol). [23,24-3H]Ecdysone with a specific radioactivity of 3.3 TBq/ mmol (90 Ci/mmol) was purchased from New England Nuclear and [14C]glucose (10 GBq/mmol; 270 mCi/mmol) was from Amersham.
CGA1404UE
CH3 \
CH-CH3
@O@
CH.,
N=C=N CH-
+CH3
C”3
CH3
/ CH3
Animals, Topical Application, Toxicity Determination
CGA 106630, diafenthiuon
Locusta migratoria,
C”3 \ CH-CH3
CH-
@o-Q.-NH-CS-
NH-!ICH~ CH-
CH3
CH3
/ CH3 FIG. 1. Structural diafenthiuron.
formulae
drial ATP2 synthesis activity. MATERIALS
of CGA 140408 and
revealed
significant
AND METHODS
Chemicals
Unless otherwise indicated, chemicals were obtained from Fluka, Switzerland. NADPH was from Boehringer Mannheim. Acetylcholinesterase type XII-S from bovine erythrocytes was obtained from Sigma. Diafenthiuron (CGA 106630, N-(2,6-diisopropyl-4-phenoxyphenyl)-N’tert-butylthiourea) and CGA 140408 (N(2,6-diisopropyl-4-phenoxyphenyl)-N’-tertbutylcarbodiimid) are Ciba-Geigy products. [‘4C]Diafenthiuron and [i4C]CGA 140408 with radiochemical purities of 99 and 95%, 2 Abbreviations used: AChE, acetylcholinesterase; ADP, adenosinediphosphate; ATP, adenosinetriphosphate; DCCD, dicyclohexylcarbodiimide; DDT, 2,2bist.p-chlorophenyl)-l,l, I-trichloroethane; DMSO, dimethyl sulfoxide; DTNB, 5,5’-dithiobis(2nitrobenzoic acid); EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol-bis(2aminoethylether)tetraacetic acid; FCCP, carbonylcyanide-4trifluormethoxyphenylhydrazone; GABA, y-aminobutyric acid; HPLC, high-pressure liquid chromatography; NADPH, reduced nicotinamide adenine dinucleotide phosphate; PBO, piperonylbutoxide.
and
Musca domestica,
and Calliphora erythrocephala were from in-house strains. Limulus polyphemus was obtained from the Marine Biological Laboratory (Woods Hole, MA). For injection assays, CGA 140408, suspended in 5 ~1 Locusta physiological saline (3), was injected into adult Locusta. For topical assays, 0.5 to 20 p.g of the test substance dissolved in acetone was applied to the abdomen (Locusta adult; 5 ~1) or dorsal thorax (Calliphora adult; 2 ~1). The animals were observed for 2 days or until they died, and both behavioral abnormalities and mortalities were determined. In Vivo Distribution and Metabolites Calliphora adults were topically treated with 4 p,g of either [14C]diafenthiuron or [i4C]CGA 140408 (2 ~1 acetone solution). For the study of distribution of radioactivity, the insects were frozen on dry ice after 12 hr. The thorax cuticle was carefully removed from the flight muscles, and the head and abdomen were isolated (10 animals/experiment). Total radioactivity was determined in head, abdomen, thorax, and thorax cuticle using Insta-Gel and a Packard Tri-Carb scintillation counter. For analysis of metabolism of diafenthiuron, treated flies (10 animals/experiment) were incubated for 16 hr in the dark until they showed clear signs of intoxication. Following this, metabolites were extracted with methanol (100 @body part). The extracts were diluted with 1 vol of water and concentrated on a C18-BondElut column (Analytichem International, Harbor City, CA). Eluates
DIAFENTHIURON
HAS
A NOVEL
obtained with 100% acetonitrile were subjected to HPLC separation using a Shimadzu system and a 5-p,m Cl8 column (Ciba-Geigy) and eluted with a gradient of 75-100% acetonitrile in water. Radioactivity was quantitatively monitored on line with a Ramona apparatus (Ray test, Straubenhardt, Germany). Tests on Cuticle Formation
The “tearing test” measures the mechanical stability of the cuticle. One-day-old Calliphora adults were treated topically with 1 pg of test substance (2 ~1 acetone). After 6 days, the living insect was fixed between a pulling motor and a weight on a balance. The recorded decrease in the weight measured by the balance is identical to the pulling force of the motor. The motor was started and the force increased, stretching the animal, until the tearing point was reached and the force dropped to zero. The tearing point is a measure of the mechanical strength of the cuticle. The data of eight animals were collected for one experiment. To quantify chitin synthesis, 3 hr after molting, eight Spodoptera larvae (fifth instar) per experiment were injected with 25 ng of test substance in 5 ~1 water (CGA 140408 or diafenthiuron; the benzoylurea chlorlluazurone was used as a positive control). After 2 and 24 hr, respectively, 100 kBq (2.7 pCi) [14C]glucose was injected. One hour later the insects were frozen on dry ice. For preparation of chitin, the cuticles of eight larvae were treated in 10% KOH for 1 hr at 95°C. After centrifugation at 15OOg for 10 min and resuspending in KOH twice, the chitin pellet was homogenized in water. 14C label incorporation in chitin was quantified on a Packard scintillation counter using Packard Insta-Gel to prevent sedimentation of the cuticle particles. Acetylcholinesterase
Assay
AChE activity was determined colorimetrically by a modified Ellman assay us-
MODE
OF
209
ACTION
ing acetylthiocholine as the substrate (4). Briefly, the test compounds at various concentrations were preincubated with bovine erythrocyte AChE (12 cl.g of protein/ml) in potassium phosphate buffer, pH 8.0, at 25°C. The preincubation time was usually 10 min. Enzyme activity was monitored by following the extinction change at 412 nm at 37°C after the addition of acetylthiocholine and DTNB in final concentrations of 1.3 and 0.14 mM, respectively. Carbofuran was run in parallel as a standard inhibitor. Ecdysone
20-hydroxylase
Assay
Locust malpighian tubule microsomes were used as a source for this P450 enzyme (5). The assay was based on the procedures described by Weirich (6). The details are given elsewhere (H. Kayser, in preparation). Test compounds were dissolved in methanol. The assay was carried out in phosphate buffer (pH 7.4) at 25°C for 60 min with 0.03 PM [3H]ecdysone, 1.2 mM NADPH, and about 100 kg of microsomal protein. After prepurification on a Bond Elut Cl8 cartridge, the samples were subjected to HPLC on a Cl8 column with online quantification of radioactivity. Preparation of Mitochondria and Microsomes
Rat liver mitochondria were prepared as described by Gazzotti et al. (7). Rat liver microsomes were obtained from the postmitochondrial supematant by sedimentation at 100,000g for 1 hr. Thoracic flight muscle mitochondria from Calliphora were isolated according to the method of Slack and Bursell (8). Protein was determined as described by Bradford (9). Spectroscopy
of Liver Cytochrome
P450
Difference spectra of rat liver microsomes were run on a Perkin-Elmer Lambda 15 spectrometer at a cytochrome P450 concentration of 1.5 PM. Concentrations of P450 were determined according to Omura and Sato (10). For recording binding spectra of diafenthiuron and CGA 140408,
210
RUDER
the chemicals were added in methanolic solution to the sample cuvette up to a final concentration of 30 pJ4 (about IO-fold the KS of diafenthiuron). The reference cuvette received the same amount of solvent. Methanol never exceeded a 1% final concentration. Mitochondrial Respiration Calliphora mitochondria were preincubated on ice in 320 mM sucrose, 1 mM EDTA, 5 miV potassium phosphate, pH 7.4, for the time indicated and with the amount of carbodiimide indicated. Rat liver mitochondria were preincubated in 250 mM sucrose 5 mM Tris/HCl, pH 7.4, 1 mM EGTA. For preincubation, carbodiimides were added as methanolic solutions, methanol not exceeding 1% in the final medium. Oxygen consumption of mitochondria, with glutamate (rat liver mitochondria, 2 mg protein/25 ml incubation chamber) or pyruvate (Calliphora flight muscle mitochondria, 0.5 mg protein) as the substrate, was measured by a Clark-type oxygen electrode (7). When compounds were added to the incubation chamber (2.5 ml), 0.5 ymol ADP (5 111H,O), 1 nmol FCCP (5 ~1 ethanol), 2.5 pg oligomycin (5 t.~l ethanol), or 0.6 nmol CGA 140408 (5 ~1 ethanol) was used. Isolated Limulus Heart The method for isolating the heart and recording its contractions in vitro was as reported previously (11). The test compounds were dissolved first in DMSO and then diluted with physiological saline. DMSO by itself was without effect on the heart beat at concentrations IO-fold those used here (0.1%). The experimental protocols were as follows. First, CGA 140408 was tested at 10 PM for transmittermimetic effects on the heart beat frequency. The control frequency was taken from the 3 min preceding the application of the test compound, and the response was measured over the last 3 min of application. CGA 140408 was then tested for any effect on the magnitude of the chronotropic re-
ET
AL.
sponse to a standard test transmitter dose (at approximately the ECso values: octopamine, 0.1 PM; dopamine, 3 FM; serotonin, 0.1 PM; GABA, 20 $V) in order to detect antagonistic or modulatory influences. The standard transmitter dose was applied once for 10 min to establish the magnitude of the control response. After a 30-min perfusion with control saline, the test compound was applied at 10 PM for 30 min, followed, without washout, by the same concentration of test compound together with the standard transmitter dose for 10 min. The mixture was then washed out and the preparation was super-fused with control saline for 30 min after which the standard transmitter dose was tested again to determine the reversibility of any effects by the test compound. Isolated Locusta Thoracic Neuronal Somata The method used for isolating and recording transmitter-evoked responses in somata from the thoracic ganglia was as reported previously (3) and based on the method of Usherwood et al. (12). The three thoracic ganglia were dissected free from adult specimens of the locust, L. migratoria, desheathed, and aspirated three or four times through a Pasteur pipette tip. The resulting suspension of dissociated neuronal somata was left in a few drops of physiological saline in a Petri dish for 2 to 4 hr. The cells were impaled and voltageclamped using conventional single-electrode means. The test compounds were dissolved in DMSO and then in Locusta physiological saline (3), and bath-applied via the continuous perfusion of the recording chamber. Physiological saline solutions of the test transmitters were pressure applied to the voltage-clamped somata via a micropipette. Cockroach Giant Axon The method used to record the resting and action potentials from the giant axons in the isolated nerve cord of the cockroach,
DIAFENTHIURON
HAS
A NOVEL
Periplaneta americana, was based on that of Boistel and Coraboeuf (13). The nerve cord was stretched by about 30% so that a pair of desheathed, interganglionic connectives spanned the recording chamber which was continuously perfused with cockroach physiological saline (14). A giant axon in one of these connectives was impaled with a thin-walled glass microelectrode (9-12 Mfl, 1 M KCl) and the membrane and action potentials were measured. Musca Larval Body Wall Neuromuscular Junction The insect neuromuscular junction glutamate receptor assay was based on the preparation of Irving and Miller (15). The viscera and central nervous system were removed from third instar larvae of the housefly, Musca domestica, and the preparation was super-fused with physiological saline in a 1.25ml recording chamber. Intracellular recordings were made from muscle 7A. Only muscle fibers with stable resting potentials of between - 60 and - 70 mV were used. The stimulating system consisted of 2 cm of PE-10 polyethylene tubing (Intramedic) mounted on a syringe. A segmental nerve innervating muscle 7A was drawn up into the PE tubing by means of gentle negative pressure which was then discontinued. The nerve was stimulated via silver wires with a 1-msec pulse of l-2 V every 5 sec. RESULTS
In Vivo Observations
and Toxicity
Adults of Locusta and Calliphora were treated with diafenthiuron or CGA 140408. The treated animals were observed until they died. The time of first appearance of effects on the behavior of Calliphora and Locusta due to topical treatment with either diafenthiuron or CGA 140408 depends on the dose of the pesticide. The first effects are clear signs of disorientation and paralysis. No differences can be observed between the effects of diafenthiuron and
MODE
211
OF ACTION
CGA 140408 on the behavior of the insects, except that CGA 140408 acts faster than diafenthiuron. In Calliphora, it takes at least 2.5-3 hr with CGA 140408 (20 pg/fly; average weight 50-70 mg, depending on sex) or 4 hr with diafenthiuron (25 pg/fly) until the first effects on behavior can be seen. In the flies, the flight activity is reduced and the legs tense up. After several more hours, movements cease and the animals die. The shortest time until death after treatment with either 25 Fg diafenthiuron or 20 pg CGA 140408 per Calliphora is 4 to 5 hr. Higher amounts of pesticide do not accelerate the lethal effect. The LD,, value (for death within 48 hr) for Calliphora adults is about 2-3 pg (6-9 nmol) for diafenthiuron and 1.1 p.g (3.3 nmol) for CGA 140408 by topical application to the thorax. To kill adult Locusta (average weight 1.6-2.2 g, depending on sex) by injection into the abdomen, 0.5 pg of CGA 140408 (1.5 nmol) is necessary. All of the data mentioned above, in addition to results obtained during biological screening and field tests (data not shown), indicate that both diafenthiuron and CGA 140408 are slowly acting insecticides with a broad spectrum of target species. In Vivo Distribution
in Calliphora
In order to estimate how much carbodiimide reaches its potential target sites, we measured the amount of substance (diafenthiuron or CGA 140408) that remains on the thorax cuticle, how much enters the thorax, and how much penetrates as far as the head and abdomen, following topical application (Table 1). Approximately 3050% of the xenobiotics are lost, probably due to evaporation or cleaning movements by the animal. As an estimate, of the remaining amount, in each experiment more than two-thirds remain on the thorax cuticle or in tissue associated with the cuticle after separation of the thorax cuticle from the interior. The other one-third of the remaining xenobiotic penetrates the thorax cuticle and is distributed into the thorax
212
RUDER
TABLE 1 Distribution of Recovered Radioactivity after Topical Application of Radiolabeled Diafenthiuron and CGA 140408 on Calliphora Adults (in Percentage of Recovered Radioactivity; Combined Results of Two Experiments, Each Including 10 Animals; Results from the Two Experiments Did Not Deviate by More Than J.5-20% of Counts from Each Other)
Diafenthiuron Head + cuticle Thorax content Abdomen + cuticle Thorax cuticle Recovery of applied radioactivity
CGA 140408
2 3 16 79
5 4 19 73
48
53
cavity or is transferred to the head and the abdomen. Surprisingly, most of the latter one-third of the detected xenobiotics can be found in the abdomen and head, perhaps because the animal transfers the compound from the thorax during cleaning movements. From these results it is concluded that only a small amount of xenobiotics reach potential targets within the insect. Metabolism of Diafenthiuron to Cytochrome P450
and Binding
To obtain some preliminary information about the metabolism of diafenthiuron, we analyzed radiolabeled compounds after treatment of Calliphora with diafenthiuron (Table 2). Diafenthiuron is metabolized to three major products: the corresponding urea, carbodiimide, and at least one uniden-
ET
AL.
tified polar substance (Table 2). A high proportion of the carbodiimide CGA 140408 is generated in the thorax (34% of the thorax metabolites). This might be an underestimate since a considerable part of the carbodiimide might bind covalently to mitochondrial proteins (16). A large amount of the polar substance is detectable in the abdomen. In view of a possible enzymatic in vivo desulfuration of diafenthiuron, difference spectroscopy was performed with rat liver microsomes in order to detect any interactions of the two chemicals with P450. The addition of diafenthiuron reproducibly results in a type I difference spectrum with a maximum at 382 nm and a minimum at 420 nm. Type I spectra are typically observed with compounds representing substrates for P450 enzymes (17). Hence, we expect that diafenthiuron can be metabolized by rat liver P450. On the other hand, there are no hints of inhibitory interactions by diafenthiuron with P450. In contrast, CGA 140408 does not produce any type of difference spectrum with liver P45Os, implying that it does not bind to P450 in the heme region that is part of the active site. Tests on Inhibition of Cuticle Formation, AChE, and Ecdysone 20-hydroxylase To test whether either diafenthiuron or CGA 140408 affects cuticle formation, we applied two different tests. In the first experiment, we measured incorporation of
TABLE 2 Metabolites of Diafenthiuron after Topical Application on Calliphora Thorax (in Percentage of Recovered Radioactivity from the Respective Body Part); CGA 197430, the Urea Analogue of CGA 106630, Is a Hydrolysis Product of Both Diafenthiuron and CGA 140408. Data Derived from a Typical Experiment (10 Animals; Two Further Experiments Confirmed the Generation of the Same Metabolic Products in the Respective Body Parts, although in Varying Concentrations)
Head + cuticle Thorax cuticle Thorax content Abdomen + cuticle
Unknown polar products
Urea (CGA 197430)
Thiourea (CGA 106630)
Carbodiimide (CGA 140408)
25 19 40 79
15 30 26 5
39 20 0 8
22 30 34 8
DIAFENTHIURON
HAS
A NOVEL
i4C-labeled glucose into chitin of Spodoptera larvae (Table 3). Neither diafenthiuron nor CGA 140408had a significant effect on incorporation of radioactivity into chitin. In another experiment, we used Calliphora adults to measure the mechanical properties of the cuticle after insecticide treatment. Neither diafenthiuron nor CGA 140408had any significant effect on the mechanical stability of the cuticle. In both tests, the benzoylurea chlorfluazurone, a known chitin synthesis inhibitor (18), was highly effective. The effects of diafenthiuron and CGA 140408on bovine AChE were studied up to their limits of solubility under assay conditions. With a preincubation time of 10 min, 1C50values of about 0.55 and 1.2 mM are obtained, by extrapolation, for diafenthiuron and CGA 140408,respectively. Under identical conditions, the corresponding value for carbofuran as a standard inhibitor is 1.5 Q4. Increasing the preincubation time to 2 hr produces no more effect. We conclude that neither compound can be regarded as an inhibitor of AChE. Ecdysone 20-hydroxylase was examined as an example of a specific insect P450 enzyme. Diafenthiuron and CGA 140408were tested for inhibition of the hydroxylase at 0.03 to 300 pike final concentrations. With both compounds, no inhibition was ob-
MODE
OF
213
ACTION
served even at the highest concentrations which led to precipitation of the test compounds (especially of the carbodiimide, which is less soluble than the thiourea). On the contrary, there is a trend to a slight stimulation of P450 activity, which is more pronounced with CGA 140408. The reason for this effect was not studied further. Inhibition of Mitochondrial in Vitro
Respiration
CGA 140408, but not diafenthiuron, was found to be a potent inhibitor of respiration both in isolated rat liver and Calliphora flight muscle mitochondria. Because of its structural similarity with dicyclohexylcarbodiimide (DCCD), both being hydrophobic carbodiimides, we hypothesized that CGA 140408 might inhibit mitochondrial respiration in the same manner as DCCD [for a review of proteins affected by DCCD, see Ref. (19)]. Both carbodiimides block ADP-dependent respiration of Culliphoru mitochondria (Fig. 2; Table 4). After 10 min of preincubation, Calliphoru mitochondria required 1 nmol DCCD/mg protein and 1.8 nmol CGA 140408/mgprotein during preincubation for complete inhibition. After 2 hr preincubation, only 0.3 nmol DCCD/mg protein and 0.18 nmol CGA 140408/mgprotein are necessary, although no exact titration of the required amount of carbodiimide
TABLE 3 Effects of Test Substances on Chitin Synthesis and Cuticle Formation Incorporation of [‘4C]glucose into chitin in ffth instar Spodoptera larvae (radioactivity in % of controBa Hours after injection of test substance Test substance
2
Diafenthiuron CGA 140408 Chlorfhtazurone Solvent control
106 91 17 100
24
Cuticle formation in % of control as derived from the tearing test”
93 13 4
9-I 98 22
100
100
Note. For details see under Materials and Methods. LICombined results from two experiments; the two sets of results did not show more than 15% deviation from each other which is within the accuracy range expected for this method. b Results from one experimental series of eight tlies.
*
,.
^
,‘
.
,
,,
214
RUDER
A
ET
AL.
TABLE 4 Respiration in Calliphora Mitochondria
d PlDP --T
*AoP \
\ 2. Oxygen consumption of Calliphora thorax mitochondria. Calliphora thorox mitochondria were isolated and incubated with 5 mMpyruvate in incubation buffer. ADP, FCCP, oligomycin (OLJ), and CGA 140408 were added at the time indicated by the triangle. (A) Experiment with CGA 140408. (B) Experiment with oligomycin. FIG.
was undertaken in these experiments (tested concentrations for CGA 140408: 6, 1.8, 0.6, 0.18, and 0.06 nmohmg; for DCCD: 10, 3, 1, 0.3, and 0.1 nmol/mg). We tried to locate the site of inhibition of mitochondrial respiration and found that in Culliphoru mitochondria, under the incubation conditions described above, both CGA 140408 and DCCD interfere exclusively with the coupling site in a manner similar to ohgomycin (Fig. 2; Table 4). Carbodiimidetreated mitochondria do not increase respiration after the addition of ADP, but addition of the uncoupler (FCCP) increases oxygen consumption identically to untreated mitochondria. Oxygen consumption of uncoupled mitochondria is inhibited completely only when preincubation is at a high carbodiimide concentration (2 10 nmol carbodiimide/mg protein; data not shown). Therefore, at low carbodiimide concentrations (approx 1 nmoVmg protein) only the coupling site and not the respiratory chain is inhibited in Calliphora mitochondria. These data indicate that ATP synthesis is
Substance
ADPlstate 4
FCCPIstate 4
Control Oligomycin CGA 140408 (nmol/mg protein) 0.06 0.18 0.6 DCCD (nmohmg protein) 0.1 0.3 1.0
8.4 1.0
7.5 7.6
7.1 2.3 1.0
7.6 6.6 6.0
8.2 2.0 1.2
7.0 7.6 8.1
Note. Mitochondria were preincubated with DCCD or CGA 1404@3for 30 min as indicated under Materials and Methods. The given indices were calculated from the rates of oxygen consumption after and before addition of ADP or FCCP (ADP/state 4 or FCCPktate 4; state 4, respiration before addition of ADP or FCCP) (data derived from one typical experiment using the same mitochondrial preparation; five more experiments from two mitochondrial preparations gave similar results, although preincubation of the carbodiimide with the mitochondria was for varying times and carbodiimide concentrations).
blocked in carbodiimide-treated mitochondria. The results obtained in Cufliphoru mitochondria also apply to rat liver mitochondria. ADP-dependent respiration is severely inhibited by CGA 140408 (Table 5). Moreover, CGA 140408 blocks rat liver mitochondria at the coupling site, as in Calliphoru mitochondria (data not shown). Diafenthiuron, at concentrations of up to 5 nmoVmg protein, has no effect on mitochondrial respiration in either rat liver or Calliphora (data not shown). Isolated
Limuius
Heart
When isolated, the heart of the horseshoe crab, Limulus polyphemus, continues to beat rhythmically under the control of its endogenously active cardiac ganglion. The heartbeat responds to octopamine, dopamine, serotonin, and GABA with characteristic changes in both its rate and amplitude of contraction that can be reversed by
DIAFENTHIURON TABLE
HAS A NOVEL
5
Respirationin Rat Liver Mitochondria CGA 140408 (nmol/mg protein) Control 0.06 0.18 0.6 1.8
ADP/state 4 16 12.5 9.5 I
MODE OF ACTION
215
GABA,-like (3), an uncharacterized dopaminergic (Kaufmann and Benson, unpublished data), and three serotonergic receptors (26). None of these receptors is sensitive to 10 or 1 fl CGA 140408applied for between 20 and 120 min. Cockroach
Giant Axon
1.0
Overshooting action potentials of constant amplitude (105 to 110 mV) and rising Note. Mitochondria were preincubated with DCCD from a stable resting membrane potential or CGA 140408 for 6 hr as indicated under Materials and Methods. The ratio of the rate of oxygen con( - 65 to - 70 mV) were recorded for at sumption (ADPktate 4) was determined (for details least 4 hr from cockroach giant axons stimsee Table 4) (data derived from one typical experiment ulated continually by electric shock at 0.1 with the same mitochondrial preparation; six more exHz. Superfusion of the nerve cord by diperiments from two mitochondrial preparations gave afenthiuron and CGA 140408for up to 4 hr similar results, although preincubation of the carbodiimide with the mitochondria was for varying times and has no effect on either the resting or the carbodiimide concentrations). action potential. The Na channel blocker, tetrodotoxin, reversibly reduces the action washing in control saline (20). The octo- potential by 50% within 10 min under these pamine effects are mediated by cyclic AMP experimental conditions, and the pyre(21, 22). throids cypermethrin and permethrin proCGA 140408 is without an agonistic ef- duce total block in 35 and 90 min, respecfect on the heartbeat frequency or ampli- tively. CGA 140408is also without effect on tude when applied to the isolated heart at the Nat and K+ potential-dependent cur10 TV&for up to 40 min. When tested for rents in voltage-clamped, cockroach giant antagonism of the responses to octopa- axons (M. Pelhate, unpublished data). mine, dopamine, serotonin, and GABA, Musca Larval Body Wall CGA 140408 has no effect. Neuromuscular
Isolated Locusta
Neuronal
Thoracic Somata
Octopamine activates an inward current in neuronal somata clamped at a holding potential of -60 mV. This response is blocked by bath application of 10 pJ4 phentolamine for 20 min (23, 24). The resting membrane current of the clamped somata and the octopamine response are both unaffected by bath application of 1and 10 PM CGA 140408for up to 2 hr. CGA 140408is thus neither an agonist nor an antagonist at the Locusta thoracic neuronal soma octopamine receptor. Acetylcholine, muscarine, GABA, dopamine, and serotonin also evoke characteristic responses in the voltage-clamped Locusta thoracic neuronal somata, mediated by a nicotinic (29, muscarinic (25), a
Junction
Composite excitatory postsynaptic potentials (EPSPs) were recorded from the muscle fibers of larval Musca in response to stimulation of the motor nerve. These EPSPsare mediated by glutamate receptors located in the muscle postsynaptic membrane. Diafenthiuron and CGA 140408were without direct agonist effects on the membrane potential of the muscle fibers and did not alter the amplitude or time course of the EPSP during applications of 10 ELMfor an hour or more. Under the experimental conditions used in these experiments, the ion channel blocker, chlorisondamine, for example, reduces the EPSP to a new steady state amplitude in less than 5 min. DISCUSSION
Diafenthiuron probably exerts its lethal
216
RUDER
effect via its desulfuration product, the carbodiimide CGA 140408. In addition to the abiotic desulfuration of diafenthiuron reported previously (2), our metabolism studies show that a significant proportion of diafenthiuron is also converted to CGA 140408 in vivo. We have observed that piperonylbutoxide (PBO), an inhibitor of P450, decreases the toxicity of diafenthiuron in Spodoptera (H. Kayser and F. J. Ruder, in preparation). Kadir and Knowles (27) demonstrated the formation of CGA 140408 from diafenthiuron in bulb mites and found that PBO decreases the toxicity of diafenthiuron in the diamond-backed moth, Plutella xylostella. Further evidence that diafenthiuron is desulfurated by a P450dependent reaction is provided by the observation that diafenthiuron, but not CGA 140408, binds to rat liver P450 as a substrate and can be converted to CGA 140408 in a
ET
AL.
P450-dependent reaction in vitro (H. Kayser and F. J. Ruder, in preparation). A summary of the results obtained with diafenthiuron and CGA 140408 in all test systems applied is given in Table 6. The carbodiimide CGA 140408 has no effect at concentrations up to 10 p.M on any of the established target sites of the major current commercial insecticides (the Na channel, acetylcholinesterase, the nicotinic acetylcholine receptor, cuticle formation) nor on the Locusta muscarinic acetylcholine receptor and the Musca larva neuromuscular glutamate receptor. It was also without effect on the receptors for GABA, dopamine, and serotonin in Locusta and in the heart of the primitive arthropod, L. polyphemus. Neither diafenthiuron nor CGA 140408 acts on the action potential or potentialdependent currents of the cockroach giant axon over a period of hours, under exper-
TABLE 6 Potential Target Sites Assayed for Effects of Diafenthiuron Target Mitochondria
P450 spectroscopy Ecdysone 20-hydroxylase Cuticle formation Chitin synthesis Acetylcholinesterase Axonal Na+ channel Nicotinic cholinergic receptor Muscarinic cholinergic receptor GABA receptor Dopamine receptor Serotonin receptor Octopamine receptor Glutamate receptor
Assay system
and CGA 140408
Diafenthiuron
Calliphora Rat liver
NE
Rat liver Locusta Calliphora Spodoptera Bovine erythrocytes Periplaneta Locusta Locusta Locusta Limulus Locusta Limulus Locusta Limulus Locusta Limulus Musca
Type 1 spectrum NE NE NE NE NE NE -b
NE -
NE
Note. The neurobiological transmitter receptor preparations were tested electrophysiologically the ligand recognition site and the associated ionophore were assayed. LINo effect. b Not tested.
CGA 1404OfI Block of the coupling site Block of the coupling site NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE so that both
DIAFENTHIURON
HAS
A NOVEL
imental conditions in which tetrodotoxin and the pyrethroids cypermethrin and permethrin rapidly block action potential conduction. Kadir and Knowles (28) have proposed that the toxic effect of CGA 140408 is mediated by octopamine receptors. Our electrophysiological data show that CGA 140408 is without agonistic or antagonistic effects on the octopamine receptors of Locusfu neurones and in the heart of the arthropod Limulus. The evidence presented for an octopaminergic mode of action for CGA 140408 was derived from experiments on bulb mites and fireflies (28). The differences between the two sets of findings might be accounted for by a remarkably high specificity for a particular subtype of octopamine receptor. Evans (29) has shown that octopamine receptors in insects belong to at least three pharmacologically distinct subtypes. However, extended investigations of possible effects by CGA 140408 on adenylate cyclase failed to reveal any evidence that CGA 140408 acts as an octopamine agonist or antagonist in any of the mite and insect systems studied (H. Kayser et al., in preparation). CGA 140408, but not diafenthiuron, is a potent inhibitor of mitochondrial respiration in rat liver and Culfiphoru thorax muscle mitochondria in vitro. Its target is the coupling site since it blocks ADP-dependent respiration and this block can be abolished by the uncoupler FCCP. CGA 140408 interferes specifically with the coupling site at low concentrations (<2 nmol/mg protein). The threshold quantity required to block the coupling site decreases with the increased preincubation time of the carbodiimide with the mitochondria. At 10 min preincubation, a CGA 140408 concentration of 1.8 nmol/mg protein, and at 2 hr preincubation of 0.18 nmol/mg protein, is sufficient for complete block of the coupling site. This observation indicates that the inhibition is irreversible and is due to a chemical reaction of the carbodiimide with the coupling site. Only at a concentration of 310 nmol CGA 1404081mg mitochondrial
MODE
OF
ACTION
217
protein do the respiratory chain enzymes become inhibited. Theoretically, there are at least two explanations for the experimental data. First, CGA 140408 could block the ATP/ADP antiporter of the inner mitochondrial membrane in the same way as atractyloside. Second, the FO/Fl mitochondrial ATPase could be affected in the same way as by oligomycin or DCCD. Since the carbodiimide DCCD is chemically related to CGA 140408 and the time dependence of the inhibition indicates a covalent, irreversible reaction, we believe that these compounds inhibit mitochondrial ATPase in the same manner [for a review on DCCD, see Refs. (30, 31)]. In a forthcoming paper we show that CGA 140408 covalently reacts with the FO proteolipid of the proton channel moiety of the ATPase (16). Recently, Kadir and Knowles (32) have also shown that CGA 140408 is an inhibitor of mitochondrial ATPase in a mite. Many insecticides act on mitochondrial respiration. Rotenone is a specific inhibitor of NADH (site I)-dependent respiration (33). Substances, like dinitrophenols or arsenate, uncouple respiration from oxidative phosphorylation (34, 35). The fumigants phosphine and hydrogen cyanide are inhibitors of cytochrome oxidase, thereby blocking the respiratory chain (35, 36). Reversible inhibition of mitochondrial ATPase has been observed with chlorinated hydrocarbons (e.g., DDT) (37) and organotin compounds (35). Evidence has been presented that DDT binds to the same proteolipid as DCCD (38), although it appears unclear how far ATPase inhibition contributes to the pesticidal effect of DDT in vivo (37). The major site of action of DDT is probably the sodium channel. The acaricide tetradifon inhibits mitochondrial ATPase similarly to oligomycin (39). In conclusion, the modes of action of all of these insecticides, at the level of the molecular mechanism, appear different from what we know about CGA 140408 since this carbodiimide is an irreversible inhibitor of mitochondrial ATPase, covalently binding
218
RUDER
to the FO proteolipid (16). Although some pesticides inhibit mitochondrial ATPase, there is no evidence for a covalent reaction with the proteolipid. The data presented in this paper are consistent with the possibility that the insecticidal and acaricidal effects of diafenthiuron result from the irreversible and time-dependent inhibition of mitochondrial ATP synthesis by CGA 140408, the active metabolite of diafenthiuron. ACKNOWLEDGMENTS
The authors thank C. Dang, F. Glanzmann, Dr. L. Kaufmann, C. Kramer, K. Leuenberger, and F. Schilrmann for technical assistance. REFERENCES
1. H. P. Streibert, J. Drabek, and A. Rindlisbacher, Diafenthiuron: A new type of acaricidel insecticide for the control of the sucking pest complex in cotton and other crops, Brighton Crop Protection Conference-Pest and Diseases, 25 (1988). 2. A. Steinemann, E. Stamm, and B. Frei, The effect of chemodynamic parameters on pesticide performance, Aspects Appl. Biol. 21, 203 (1989). 3. G. Lees, D. J. Beadle, R. Neumann, and J. A. Benson, Responses to GABA by isolated insect neuronal somata: Pharmacology and modulation by a benzodiazepine and a barbiturate, Brain Res. 401, 267 (1987). 4. G. L. Ellman, K. D. Courtney, V. Andres, Jr., and R. M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7, 88 (l%l). 5. R. Feyereisen and F. Durst, Ecdysterone biosynthesis: A microsomal cytochrome-P-4SO-linked ecdysone 20-monooxygenase from tissues of the african migratory locust, Eur. J. Biochem. 88, 37 (1978). 6. G. F. Weirich, Ecdysone 20-monooxygenase, in “Methods in Enzymology” (J. Law and H. Rilling, Eds.), Vol. 111, pp. 454-l58, Academic Press, New York, 1985. 7. P. Gazzotti, K. Malmstrom, and M. Crompton, Preparation and assay of animal mitochondria and submitochondrial vesicles, in “Membrane Biochemistry: A Laboratory Manual on Transport and Bioenergetics” (E. Carafoli and G. Semenza, Eds.), pp. 62-76, Springer-Verlag, New York, 1979. 8. E. N. Slack and E. Bursell, The isolation of mitochondria from dipteran Bight muscle, Biochim. Biophys. Acta 449, 491 (1976).
ET AL.
9. M. M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72, 248 (1976). 10. T. Omura and R. Sato, The carbon monoxidebinding pigment of liver microsomes. I. Evidence for its hemoprotein nature, J. Biol. Chem. 239, 2379 (1964). 11. J. A. Benson, R. E. Sullivan, W. H. Watson, and G. J. Augustine, The neuropeptide proctolin acts directly on Limulus cardiac muscle to increase the amplitude of contraction, Brain Res. 213, 449 (1981). 12. P. N. R. Usherwood, D. Giles, and C. Suter, Studies of the pharmacology of insect neurones in vitro, in “Insect Neurobiology and Pesticide Action (Neurotox ‘79),” pp. 115-128, Society of Chemical Industry, London, 1980. 13. J. Boistel and E. Coraboeuf, Potentiels de membrane et potentiels d’action de nerf d’insecte recueillis a l’aide de microelectrodes intracellulaires, C. R. Acad. Sci. Paris 238, 2116 (1954). 14. R. M. Pitman, The ionic dependence of action potentials induced by colchicine in an insect motoneurone cell body, J. Physiol. (London) 247, 511 (1975). 15. S. N. Irving and T. Miller, Ionic differences in “fast” and “slow” neuromuscular transmission in body wall muscles of Musca domestica larvae, J. Comp. Physiol. 135, 291 (1980). 16. F. J. Ruder and H. Kayser, The carbodiimide product of diafenthiuron reacts covalently with two mitochondrial proteins, porin and the FO-proteolipid, and inhibits mitochondrial ATPases in vitro, submitted for publication. 17. J. B. Schenkman, H. Remmer, and R. W. Estabrook, Spectral studies of drug interaction with hepatic microsomal cytochrome, Mol. Pharmacol. 3, 113 (1967). 18. R. Neumann and W. Guyer, Biochemical and toxicological differences in the modes of action of the benzoylureas, Pestic. Sci. 20, 147 (1987). 19. A. Azzi, R. P. Casey, and M. J. Nalecz, The effects of N,N’-dicyclohexylcarbodiimide on enzymes of bioenergetic relevance, Biochim. Biophys. Acta 768, 209 (1984). 20. G. J. Augustine, R. Fetterer, and W. H. Watson, Amine modulation of the neurogenic Limulus heart, J. Neurobiol. 13, 61 (1982). 21. J. R. Groome and W. H. Watson, Mechanism for amine modulation of the neurogenic Limulus heart: Evidence for involvement of CAMP, .I. Neurobiol. 18, 417 (1987). 22. J. R. Groome and W. H. Watson, Involvement of cyclic AMP in multiple, excitatory actions of biogenic amines on the cardiac ganglion of the horseshoe crab Limulus polyphemus, .I. Exp. Biol. 152, 313 (1990).
DIAFENTHIURON
HAS A NOVEL
23. C. Suter, The action of octopamine and other biogenie amines on locust central neurones, Comp. Biochem. Physiol. C 84, 181 (1986). 24. L. Kaufmarm and J. A. Benson, Characterisation of a locust neuronal octopamine response, Sot. Neurosci. Abs., in press. 25. J. A. Benson, Transmitter receptors on insect neuronal somata: GABAergic and cholinergic pharmacology, in “The Molecular Basis of Drug and Pesticide Action: Neurotox ‘88” (G. G. Lunt, Ed.), pp. 193-206, Elsevier, Amsterdam, 1988. 26. I. Bermudez, D. J. Beadle, and J. A. Benson, Multiple serotonin-activated currents in isolated, neuronal somata from locust thoracic ganglia, Sue. Neurosci. Abs. 16, 857 (1990). 27. H. A. Kadii and C. 0. Knowles, Toxicological studies of the thiourea diafenthiuron in diamond back moths (Lepidoptera: Yponomeutidae), twospotted spider mites (Atari: Tetranychidea), and bulb mites (Atari: Acaridiae), in press. 28. H. A. Kadir and C. 0. Knowles, Octopaminergic action of the carbodiimide metabolite of diafenthiuron, Pestic. Biochem. Physiol. 39, 261 (1991). 29. P. D. Evans, Multiple receptor types for octopamine in the locust, J. Physiol. (London) 318,99 (1981). 30. W. Sebald and J. Hoppe, On the structure and genetics of the proteolipid subunit of the ATP synthase complex, Curr. Top. Bioenerg. 12, 1 (1981). 31. J. Hoppe and W. Sebald, The proton conducting FO-part of bacterial ATP-synthases, Biochim. Biophys. Actu 768, 1 (1984).
MODE OF ACTION
219
32. H. A. Kadir and C. 0. Knowles, Inhibition of ATP dephosphorylation by acaricides with emphasis on the anti-ATPase activity of the carbodiimide metabolite of diafenthiuron, in press. 33. J. I. Fukami, Rotenone and rotenoids, in “Comprehensive Insect Physiology, Biochemistry, and Pharmacology” (G. A. Kerkut and L. I. Gilbert, Eds.), Vol. 12, pp. 291-312, Pergamon, New York, 1985. 34. R. D. O’Brien, “Insecticides: Action and Metabolism,” p. 286, Academic Press, New York, 1%7. 35. J. Fukami, Insecticides as inhibitors of respiration, in “Insecticide Biochemistry and Physiology” (C. F. Wilkinson, Ed.), pp. 3533%, Plenum, New York, 1976. 36. K. P. Kashi and W. Chefurka, The effect of phosphine on the absorption and circular dichroic spectra of cytochrome c and cytochrome oxidase, Pestic. Biochem. Physiol. 6, 350 (1976). 37. M. P. Osborne, DDT, y-HCH and the cyclodienes, in “Comprehensive Insect Physiology, Biochemistry, and Pharmacology” (G. A. Kerkut and L. I. Gilbert, Eds.), Vol. 12, pp. 131-182, Pergamon, New York, 1985. 38. C. C. Dary, S. C. Buessow, H. Wenzler, and L. K. Cutkomp, Binding of (14C)-DDT and (‘4C)-dicyclohexylcarbodiimide to a lipoprotein of the membrane sector of mitochondrial ATPase, Pestic. Sci. 16, 605 (1985). 39. E. Bustamente and P. L. Pedersen, Tetradion: An oligomycin-like inhibitor of energy-linked activities of rat liver mitochondria, Biochem. Biophys. Res. Commun. 51, 292 (1973).