Sites of Action for Neurotoxic Pesticides - American Chemical Society

Chapter 20

Methyllycaconitine

Downloaded by EAST CAROLINA UNIV on January 15, 2018 | http://pubs.acs.org Publication Date: November 9, 1987 | doi: 10.1021/bk-1987-0356.ch020

A Potent Natural Insecticide Active on the Cholinergic Receptor K. R. Jennings, D. G. Brown, D. P. Wright, Jr., and A. E. Chalmers Agricultural Research Division, American Cyanamid, P.O. Box 400, Princeton, NJ 08540

A chloroform extract from the seeds of Delphinium plants was shown to be toxic to a number of insect species, and to have a very high affinity for the nicotinic acetylcholine receptor of the housefly. The major active fraction of the extract was an alkaloid, methyllycaconitine (MLA). A pure sample of MLA.citrate showed similar insecticidal activity to the crude extract, and had a similarly high affinity for the acetylcholine receptor. Previous experiments on a vertebrate preparation had indicated an action of MLA on the nicotinic acetylcholine receptor, but the concentrations used were many times greater than those affecting the housefly receptor. There may therefore be significant differences between the acetylcholine receptors of insects and vertebrates, and MLA could be a very useful tool for studying these differences. A large number of commercially important insecticides act on the nervous system of the insect. By investigating c e r t a i n areas of this system i n d e t a i l , i t may be possible to find new classes of chemicals which can disrupt the normal nervous a c t i v i t y , and this may lead to the development of new i n s e c t i c i d e s . I t i s important to look not only at chemicals which k i l l the insect, but also at those groups of chemicals which can disrupt the insect's behavior s u f f i c i e n t l y to prevent i t s survival i n the f i e l d . One target of interest i n the search for new insecticides i s the n i c o t i n i c acetylcholine receptor. Chemicals which act at this s i t e are known to k i l l insects, and two are used as i n s e c t i c i d e s . These are nicotine, which has been used for over 200 years, mainly against aphids and thrips, and Padan, an i n s e c t i c i d e used against r i c e pests, which was derived from a toxin produced by marine worms of the genus Nereis (1_) · The fact that one of these insecticides was derived from a plant and one from an animal underlines the 0097-6156/87/0356-0274$06.00/0 © 1987 American Chemical Society Hollingworth and Green; Sites of Action for Neurotoxic Pesticides ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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importance of investigating natural products as p o t e n t i a l sources of new i n s e c t i c i d e s .


Studies on the Nicotinic Acetylcholine Receptor One way of investigating the effect of chemicals at the n i c o t i n i c acetylcholine receptor i s to isolate the receptor and measure the a b i l i t y of chemicals to bind to i t . In the experiments described here, housefly heads were used as a source of nervous tissue, and a crude membrane preparation was obtained using a method based on that described by Jones et a l . (2). The snake toxin, orbungarotoxin (α-BGTx), i s known to bind to this receptor with a very high a f f i n i t y (KD=1.1 +/- 0.1nM.(3)) Chemicals of interest are incubated with the housefly head receptor preparation, then the radioligand [3H]- propionyl- -bungarotoxin (3Hlycoctonine>aconitine, while that i n the rat phrenic diaphragm preparation was aconitine>MLA>lycoctonine (10). Insecticidal Activity The a c t i v i t y of MLA.citrate against insects and mites was tested and compared with the a c t i v i t y of the Delphinium seed extract (Table 1). The properties were found to be very similar, the main difference being that the MLA.citrate was not active against Tetranychus urticae or Anopheles quadrimaculatus. It i s thought that

Hollingworth and Green; Sites of Action for Neurotoxic Pesticides ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

20.

JENNINGS ET AL.

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Table I. I n s e c t i c i d a l and a c a r i c i d a l a c t i v i t y of Delphinium extracts compared with MLA

Spodoptera eridania Aphis fabae Tetranychus u r t i c a e Diabrotica undecimpunctata howardi Anopheles quâdrimaculatus

Empoasca abrupta H e l i o t h i s virescens Musca domestica

Larvae

Larvae Adult Larvae Eggs Larvae Eggs

Chloroform extract

Alkaloid fraction

MLA. citrate

+

+

+

+

N.D.

-

N.D. N.D.

-

+ N.D.

+ + + + +

-

+ + + N.D.

+ + + +

(+) active, (-) inactive, (N.D. ) not determined. A c t i v i t y on eggs indicates a contact o v i c i d a l a c t i v i t y , reducing v i a b l e egghatch. Active denotes s i g n i f i c a n t (50% +) m o r t a l i t y at a screening rate of 1000 ppm (Spodoptera, H e l i o t h i s larvae, Musca), 300 ppm (Heliothis eggs) or 100 ppm (Empoasca). Copyright 1986 American Cyanamid Co. Reprinted with permission. HO

R = H Lycoctonine

Aconitine

ο Methyllycaconitine

Figure 1. Structures of aconite alkaloids investigated. Copyright 1986 American Cyanamid Co. Reprinted with permission.



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these organisms are k i l l e d by the saponins i n the seed extract, and this would explain why they were unaffected by the MLA. The similar spectrum of a c t i v i t y of the seed extract and the MLA.citrate implies that MLA i s the main i n s e c t i c i d a l agent i n the Delphinium plant. I t seems l i k e l y that i t i s the very potent action of MLA at the n i c o t i n i c cholinergic receptor that causes the t o x i c i t y . The symptoms caused by MLA i n Spodoptera larvae are consistent with the hypothesis that i t i s acting on the nervous system. When the larvae were fed on treated bean leaves, observed mortality was rapid, with s i g n i f i c a n t numbers of larvae being k i l l e d within 24 hours at concentrations as low as 100 ppm. MLA gave s i g n i f i c a n t protection against feeding damage at levels of 300 ppm and above. Figure 3 i l l u s t r a t e s the protection afforded at twenty-four hours. After 72 hours, feeding damage i n the treated leaves was approxi mately 5%, whereas controls showed over 95% feeding damage. An approximate LC50 for MLA.citrate was calculated by dipping lima beans i n d i f f e r e n t concentrations of MLA i n 2:1 acetone:water, and exposing groups of ten larvae to the treated leaves. This experi ment gave an LC50 value of 308 +/- 48 ppm. The LC50 for nicotine in the same assay was much greater than 1000 ppm. Physiological Experiments The binding studies on the housefly head preparation indicate that MLA binds to the n i c o t i n i c acetylcholine receptor with a very high a f f i n i t y , but they do not show whether i t acts as an agonist or an antagonist of the natural neurotransmitter. A useful preparation for examining the physiological effects of drugs at cholinergic synapses i s the cereal giant synapse of the cockroach. Sensory nerves from the c e r c i form synapses i n the sixth abdominal ganglion with the giant fibres i n the ventral nerve cord, and these synapses are thought to be cholinergic (11). The effects of bungarotoxin and nicotine on this preparation have previously been investigated, using mannitol gap or o i l gap techniques (12). I n i t i a l experiments on MLA were carried out using the mannitol gap technique described by Callec et a l . (12). However, comparable results could be obtained using a much simpler preparation, i n which the sixth abdominal ganglion and cereal nerves were placed in a saline f i l l e d chamber, and the ventral nerve cord was draped over a wax p a r t i t i o n into a separate chamber (Fig.4). The saline contained 210 mM NaCl, 3.1 mM KC1, 5.4 mM CaCl , 0.1 mM NaH2P04, 2.0 mM NaHC03 and 5 mM MgCl2. The nerve was embedded i n vaseline as i t passed over the p a r t i t i o n . One cereal nerve was picked up in a suction electrode, and stimulated at 10 sec i n t e r v a l s . The r e s u l t i n g excitatory post-synaptic potential (epsp) was recorded using a d i f f e r e n t i a l amplifier. Stable recordings could be obtain ed for over two hours. The effects of nicotine, MLA.citrate and a-BGTx on the e l e c t r i c a l a c t i v i t y of this preparation were investigated. As α-BGTx i s not water soluble, i t was f i r s t dissolved i n DMSO, then added to saline to give a f i n a l concentration of 1% DMSO. 1% DMSO was there fore included i n a l l salines, to abolish any differences i n response which might be due to solvent e f f e c t s . 2



JENNINGS ET AL.

Methyllycaconitine

LOG MOLAR C O N C E N T R A T I O N s

Figure 2. Inhibitory effects of test compounds on binding of HaBGTx to Musca domestica head homogenates. Test compounds were preincubated for 20 min with the tissue homogenate at 22-24 *C in pH 7.4 sodium phosphate buffer containing 1 mg/mL bovine serum albumin and 9% DMSO. The binding reaction was carried out for 30 min with 5 nM Ha-BGTx and terminated by filtration on Whatman GF/C filters. Data shown are percentage inhibition of binding ± SD. Δ, MLA.citrate; V, MLA purified from Delphinium seed; a-BGTx; Ο» lycoctonine; • , aconitine. (Reproduced with permission from American Cyanamid Co. Copyright 1986.) 8

Figure 3. The insecticidal effect of methyllycaconitine against Spodoptera eridania. Lima bean leaves were dipped in 2:1 acetonerwater solutions containing 1000 ppm MLA.citrate (A), 300 ppm MLA.citrate (B), acetonerwater control (C). Leaves were exposed to 5 third instar Spodoptera eridania larvae for 24 h at an ambient temperature of 27 *C. (Reproduced with permission from American Cyanamid Co. Copyright 1986.)



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The e f f e c t of nicotine (1 μΜ) on this preparation was to cause a massive increase i n spontaneous a c t i v i t y , which tended to obscure the synaptic response. I f the concentration was increased to 10 μΜ, a l l synaptic a c t i v i t y was rapidly blocked. Both MLA and o-BGTx caused a gradual block of synaptic response. Concentrations of MLA above 10 μΜ also caused a s l i g h t increase i n spontaneous a c t i v i t y in approximately 50% of preparations tested, but neither MLA nor BGTx ever caused the violent bursts of a c t i v i t y observed i n response to n i c o t i n e . α-BGTx had no effect i n preparations with an intact sheath around the ganglion. Even i n desheathed preparations, i t took over t h i r t y minutes fora-BGTx to have an e f f e c t . 100 ηΜα-BGTx caused synaptic block i n 52 +/- 4.9 min, but increasing the concentration one-hundred fold did not s i g n i f i c a n t l y reduce the time to block. MLA was also inactive i n preparations with an intact ganglionic sheath, but i t s effects i n desheathed preparations were more dose dependent (Fig.5). The lowest concentration ever to cause block within one hour was 10 nM fororiBGTx and 100 nM for MLA. The simi l a r i t y of action of MLA and BGTx indicates that MLA may be acting as an antagonist. The concentration of MLA to cause a physiological e f f e c t i n the cockroach was s i g n i f i c a n t l y higher than that i n h i b i t i n g

Sites of Action for Neurotoxic Pesticides - American Chemical Society

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