Enhancement of Veratridine-Dependent Sodium Channel Activation

Chapter 18

Enhancement of Veratridine-Dependent Sodium Channel Activation by Pyrethroids and DDT Analogs 1

Downloaded by UNIV OF ROCHESTER on June 11, 2018 | https://pubs.acs.org Publication Date: November 9, 1987 | doi: 10.1021/bk-1987-0356.ch018

David M. Soderlund, Jeffrey R. Bloomquist, Syed M. Ghiasuddin , and Adam M. Stuart Department of Entomology, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456

Pyrethroid insecticides (deltamethrin, NRDC 157, cismethrin), DDT analogs (p,p,'-DDT, o,p'-DDT, methoxychlor, EDO), and a DDT-pyrethroid hybrid compound (GH401) enhanced veratridine-dependent sodium uptake by mouse brain synaptosomes. The effectiveness of these compounds in the sodium uptake assay was in good agreement with their acute mammalian toxicities. p,p'-DDT also enhanced veratridine-dependent sodium uptake by fish brain synaptosomes. These findings demonstrate the utility of ion flux assays to study interactions of insecticides with sodium channels in the central nervous system and to explore species differences in insecticide target site sensitivity. Abundant neurophysiological evidence implicates the voltage-dependent sodium channel as an important target s i t e for DDT and p y r e t h r o i d s . These compounds cause r e p e t i t i v e f i r i n g and depolarization i n insect motor nerve terminals (^,^2), v e r t e b r a t e sensory neurons (_3), and arthropod giant axons (4). Voltage clamp studies have shown that a l l these e f f e c t s of pyrethroids and DDT analogs can be a s c r i b e d to a prolongation of sodium channel currents (4^). Electrophysiological studies are well s u i t e d f o r measuring the e f f e c t s of pyrethroids on sodium channel k i n e t i c s , but they are less useful for studying the pharmacology of pyrethroid-receptor i n t e r a c tions. Moreover, these techniques are d i f f i c u l t to employ i n studies of i n s e c t i c i d e effects on neurons i n the c e n t r a l nervous systems of mammals and insects. Radiotracer ion flux measurements have emerged as a useful alternative method for studying the action of pyrethroids on the sodium channel. Investigations using neuroblastoma c e l l s (5) and mouse b r a i n synaptosomes ( 6 0 showed that p y r e t h r o i d s had no s i g n i f i c a n t e f f e c t on Na uptake when applied alone but enhanced the s p e c i f i c s t i m u l a t i o n of sodium uptake caused by e s t a b l i s h e d sodium channel activators such as v e r a t r i d i n e . 1

Current address: IMC Corporation, P.O. Box 207, Terre Haute, IN 47808 0097-6156/87/0356-0251 $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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In t h i s paper, we report that DDT and i t s analogs, as w e l l as p y r e t h r o i d s , enhance v e r a t r i d i n e - d e p e n d e n t sodium uptake i n mouse brain synaptosomes· We a l s o demonstrate the e x t e n s i o n of these methods to study i n s e c t i c i d e e f f e c t s on sodium channel function i n rainbow trout.


Methods Chemicals. j>,£'-DDT was purchased from Chem S e r v i c e , West Chester, PA, and m e t h o x y c h l o r was purchased from A l d r i c h Chemical Co., Milwaukee, WI. DDE and £,j>'-DDT were obtained from Ciba-Geigy Corp., Greensboro, NC. Deltamethrin was a g i f t from J . Martel, RousselU c l a f , R o m a i n v i l l e , France, and c i s m e t h r i n was a g i f t from M. E l l i o t t , Rothamsted Experimental Station, Harpenden, England. GH401 (3-phenoxybenzyl [Sj-2,2-dichloro-l-(4-ethoxyphenyl)c y c l o p r o p a n e c a r b o x y l a t e ) and EDO (2,2-bis_-(4-ethoxyphenyl)-3,3dimethyloxetane; GH149) were g i f t s from G. Holan, CSIRO, Melbourne, A u s t r a l i a . The sources of other chemicals used i n the sodium uptake assay are published elsewhere (7). P r e p a r a t i o n of synaptosomes. Synaptosomes were prepared from the brains of male mice (20-30 g; Blue Spruce Farms, Altamont, NY) either by a modification of the method of Hajos (8) or by the method of Dodd et a l . (9). Both preparations gave q u a l i t a t i v e l y s i m i l a r r e s u l t s , but the magnitude of a l l sodium fluxes per mg of synaptosomal protein was much greater with the l a t t e r preparation. A preparation enriched i n synaptosomes was prepared from the b r a i n s of j u v e n i l e rainbow t r o u t (Salmo g a i r d n e r i ; obtained from the New York S t a t e F i s h Hatchery, Bath, NY) by homogenization i n 14 volumes of 0.7 M sucrose, and centrifugation f i r s t at 2000 £ for 10 min and then at 31000 £ f o r 30 min. The p e l l e t from the second centrifugation was resuspended i n sodium-free buffer i d e n t i c a l to that used i n previous s t u d i e s (7) except that i t also contained 370 mM sucrose. Sodium uptake assay. Assays u s i n g mouse b r a i n synaptosomes were performed as d e s c r i b e d p r e v i o u s l y (6^.7), except that i n s e c t i c i d e s were introduced to resuspended synaptosomes i n 0.2-0.4 u l of ethanol r a t h e r than as a r e s i d u e i n the i n c u b a t i o n tube. This amount of ethanol improved the d e l i v e r y of i n s e c t i c i d e s , thereby increasing the r e p r o d u c i b i l i t y of the assay, and had no measurable e f f e c t on veratridine-dependent sodium channel a c t i v a t i o n . These methods were a l s o used f o r assays with f i s h b r a i n membranes, except that a l l buffers were augmented with sucrose to give o s m o l a r i t i e s e q u i v a l e n t to the 0.7 M sucrose used for membrane i s o l a t i o n . Intracerebral t o x i c i t y determinations. EDO and GH401 were a d m i n i s t e r e d to mice by i n t r a c e r e b r a l i n j e c t i o n i n 3 μΐ of triethylene g l y c o l dimethyl ether under l i g h t ether a n a e s t h e s i a as d e s c r i b e d p r e v i o u s l y (6^). C o n t r o l animals r e c e i v e d v e h i c l e only and were apparently normal following recovery from anaesthesia. Effects of Pyrethroids on Mouse Brain Sodium Channels Our i n i t i a l studies i n t h i s system employed d e l t a m e t h r i n at a high c o n c e n t r a t i o n (10 uM) to characterize the e f f e c t s of pyrethroids on

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


18. SODERLUND ET AL.

Sodium Channel Activation

253

synaptosomal sodium uptake. D e l t a m e t h r i n alone had no e f f e c t on b a s a l l e v e l s of synaptosomal sodium uptake, but i t markedly enhanced the stimulation of sodium uptake caused by the a l k a l o i d v e r a t r i d i n e , a sodium channel a c t i v a t o r (6j Table I ) . Both the v e r a t r i d i n e stimulation and the enhancement of this effect by d e l t a m e t h r i n were completely blocked by t e t r o d o t o x i n (Table I ) , thus confirming that the observed sodium fluxes r e f l e c t a s p e c i f i c e f f e c t on the v o l t a g e dependent sodium channel. The effect of deltamethrin on the potency curve f o r veratridine-dependent sodium channel a c t i v a t i o n i s shown i n F i g u r e 1. Deltamethrin increased the amount of uptake observed at any given veratridine concentration, but i t had no s i g n i f i c a n t e f f e c t on the potency of v e r a t r i d i n e . This finding i s consistent with the e f f e c t s of d e l t a m e t h r i n on v e r a t r i d i n e - d e p e n d e n t sodium channel a c t i v a t i o n i n cultured neuroblastoma c e l l s (5). Maximum enhancement of sodium channel a c t i v a t i o n was observed a t 50 yM v e r a t r i d i n e ( F i g u r e 1); t h i s c o n c e n t r a t i o n was used as the standard activator concentration i n a l l subsequent experiments.

Table I. Effect of Deltamethrin on Basal and Veratridine-Stimulated Sodium Uptake by Mouse Brain Synaptosomes

Preincubation conditions

Uptake, nmol/mg p r o t e i n

Control

1.40+0.18

Deltamethrin (10 PM)

1.35+0.12

Veratridine (50 μΜ)

1.90+0.17°

Deltamethrin + veratridine

2.36+0.15

Deltamethrin + veratridine + tetrodotoxin (5 uM)

1.26+0.09

a

b

d

Data from Ref. 6. Means + standard errors of 4-7 determinations; 15 sec incubations. D i f f e r s s i g n i f i c a n t l y from control uptake (p Ε

ô ε c ο

CO

1.2

0.8

0.4

ο VTD+ DTM

18

1.70

0.0 10

100

1000

[VTD], μΜ F i g u r e 1. E f f e c t s o f v e r a t r i d i n e (VTD) c o n c e n t r a t i o n on veratridine-stimulated sodium uptake by mouse brain synaptosomes i n the presence and absence of deltamethrin (DTM; 10 yM), Data points are means of two r e p l i c a t e experiments using d i f f e r e n t membrane p r e p a r a t i o n s c o r r e c t e d f o r v e r a t r i d i n e independent sodium u p t a k e . Tabulated values a r e : K , c o n c e n t r a t i o n g i v i n g half-maximal uptake i n uM; Ε , maximum uptake at saturating concentrations i n nmol/mg protein. Data are from r e f . 10. n


18.

SODERLUNDETAL.

255


r e l a t i v e acute t o x i c i t i e s measured by intracerebral i n j e c t i o n II).

(Table

Table I I . Effects on Sodium Channel Activation and Acute Intracerebral Toxicity of Deltamethrin, NRDC 157, and Their Enantiomers i n Mice

Compound

Κ


NRDC 157 Enantiomer of NRDC 157

, nM

a

L

D

c > n

k

yg/ S

220

190

>10, ,000

>10, ,500

25

19

>10, ,000

>10 ,500

Deltamethrin Enantiomer of deltamethrin

b

3

Data from Ref. 6. C o n c e n t r a t i o n g i v i n g half-maximal stimulated sodium uptake.

enhancement of v e r a t r i d i n e -

Current e f f o r t s i n these s t u d i e s i n v o l v e a d e t a i l e d c h a r a c t e r i z a t i o n of p y r e t h r o i d - a c t i v a t o r interactions and the examination of a broader range of pyrethroids and other compounds as enhancers of activation. Recently, we have found that cismethrin i s one of the most e f f e c t i v e pyrethroids i n this system, producing enhancement at high c o n c e n t r a t i o n s equal to or g r e a t e r than that observed with deltamethrin (Figure 2). The action of cismethrin i n the mouse brain system i s q u i t e d i f f e r e n t from i t s a c t i o n on neuroblastoma c e l l sodium channels, where i t has no e f f e c t on v e r a t r i d i n e - d e p e n d e n t a c t i v a t i o n but i s able to antagonize the enhancement produced by other pyrethroids (5). Effects of DDT Analogs on Mouse Brain Sodium Channels The similar actions of pyrethroids and DDT analogs on sodium channels i n e l e c t r o p h y s i o l o g i c a l p r e p a r a t i o n s (4) prompted us to evaluate these compounds i n the synaptosomal sodium f l u x assay. Initial experiments using £,£'-DDT at a high concentration ( 100 μΜ) showed an enhancement of veratridine-dependent sodium uptake t h a t was s i m i l a r to t h a t observed with high concentrations of pyrethroids (Figure 2). Enhancement of sodium uptake by £,£'-DDT was concentration-dependent ( F i g u r e 3), but the low s o l u b i l i t y of £,£'-DDT prevented completion of the curve using concentrations higher that 100 μΜ. Nevertheless, the a v a i l a b l e data suggest that the K _ f o r t h i s e f f e c t i s ap proximately 30 μΜ. From these d a t a , i t i s c l e a r that £,£'-DDT i s much less potent i n this system than deltamethrin and NRDC 157 (Table II). However, £,£'-DDT produces low but s i g n i f i c a n t l e v e l s of en hanced a c t i v a t i o n at concentrations as low as 0.1 μΜ. Since altered nerve function can result from modification of only a small p e r c e n t age of t o t a l sodium channels ( LI) , £,£'-DDT may be more potent i n intact nerves than i t s Κ _ value would indicate. n

Λ


256

SITES OF ACTION FOR NEUROTOXIC PESTICIDES Pyrethroids

DDT

analogs

c ο ο lm

α ε

Φ


ο. D +

(0 Ζ

DTM

CM

DDT ο,ρ-DDT

DDE

MC

EDO

GH401

Figure 2. Enhancement of veratridine-stimulated sodium uptake i n mouse brain synaptosomes by p y r e t h r o i d s and DDT analogs. Each v a l u e i s the mean o f t h r e e d e t e r m i n a t i o n s u s i n g d i f f e r e n t membrane preparations corrected f o r veratridine-stimulated uptake i n t h e a b s e n c e of i n s e c t i c i d e ; bars show standard e r r o r s . Compound abbreviations: DTM (deltamethrin); CM (cismethrin) ; MC (methoxychlor). C o n c e n t r a t i o n s were 100 uM f o r a l l compounds except DTM (10 μΜ). 10

Ϊ « σ>

J

6

ο Ε c „ 4

0.1

1

10

100

[DDT], μ M

F i g u r e 3. E f f e c t of DDT c o n c e n t r a t i o n on the enhancement of veratridine-stimulated sodium uptake by mouse brain synaptosomes. Data p o i n t s are means of three determinations using d i f f e r e n t membrane preparations corrected f o r veratridine-stimulated uptake i n the absence of DDT; bars show standard e r r o r s .



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We have a l s o surveyed the e f f e c t s of a l i m i t e d group of DDT analogs on veratridine-dependent sodium uptake (Figure 2)· The o_,j?' isomer of DDT was almost as active as j>,j>'-DDT i n this assay, but the dehydrochlorinated m e t a b o l i t e DDE was much l e s s e f f e c t i v e . The r e l a t i v e effectiveness of j>,jp'-DDT, £,j>'-DDT, and DDE as enhancers of veratridine-dependent sodium uptake i s g e n e r a l l y c o n s i s t e n t with t h e i r acute o r a l t o x i c i t i e s to mammals (J^). Methoxychlor, a DDT analog with much lower mammalian t o x i c i t y (J_2) , a l s o enhanced veratridine-dependent sodium uptake but was somewhat less e f f e c t i v e than DDT. This finding provides further evidence that the low mammalian t o x i c i t y of methoxychlor results from i t s rapid biodégradation r a t h e r than from i t s l a c k of i n t r i n s i c n e u r o t o x i c i t y . EDO, an oxetane analog of DDT with very low mammalian t o x i c i t y (JL3), approached the a c t i v i t y of jp,j>'-DDT i n this assay ( F i g u r e 2). GH401, which was designed as a h y b r i d molecule i n c o r p o r a t i n g s t r u c t u r a l elements of both DDT and p y r e t h r o i d s (]A) and which i s r e l a t i v e l y nontoxic to mammals (JJ^J^A), was weakly active i n this system. A recent study showed that EDO and GH401, u n l i k e p y r e t h r o i d s , were unable to enhance sea anemone t o x i n - s t i m u l a t e d sodium uptake i n cultured neuroblastoma c e l l s (JL3). These authors proposed that the mammalian (neuroblastoma) sodium channel was i n t r i n s i c a l l y i n s e n s i t i v e to EDO and GH401, thus explaining their low acute t o x i c i t i e s to mammals. Since our sodium uptake experiments suggested that EDO might e x h i b i t DDT-like e f f e c t s i n vivo, we undertook a preliminary survey to estimate the i n t r i n s i c neurotoxicity of EDO and GH401 f o l l o w i n g i n t r a c e r e b r a l a d m i n i s t r a t i o n to mice. Of the four animals treated with EDO at 1 mg/kg, two e x h i b i t e d a r a p i d onset of whole body tremor, followed by convulsions and death. The remaining two animals exhibited less severe tremor f o r approximately 30 min. a f t e r t r e a t ment and then recovered. These preliminary findings suggest that EDO produces the Τ or Type I syndrome of i n t o x i c a t i o n i n mammals w i t h a potency somewhat lower than neurotoxic pyrethroids that produce the Τ syndrome (15). In contrast, mice treated with GH401 at 3 mg/kg (n=4) were indistinguishable from vehicle-treated controls, suggesting that this compound has l i t t l e or no neurotoxic a c t i v i t y i n v i v o . These f i n d i n g s , though preliminary i n nature, indicate a good c o r r e l a t i o n for EDO and GH401 between a c t i v i t y i n sodium uptake assays and acute neurotoxicity. These results suggest that the ineffectiveness of EDO on sodium channels i n neuroblastoma c e l l s (13) r e f l e c t s the unique and a t y p i c a l s e l e c t i v i t y of neuroblastoma c e l l sodium channels. The low mammalian t o x i c i t y observed for EDO when administered by other routes probably r e f l e c t s the r a p i d metabolic detoxication of t h i s compound. In contrast, the low t o x i c i t y of GH401 appears to i n v o l v e reduced s e n s i t i v i t y of the target s i t e i n mammals, a phenomenon that has been noted previously f o r several i n s e c t i c i d a l pyrethroids (15)* Effects of DDT

on Fish Brain Sodium Channels

The high acute t o x i c i t y of DDT analogs and most p y r e t h r o i d s to f i s h i s one of the most s i g n i f i c a n t hazards associated with the widespread use of these compounds. The high t o x i c i t y of p y r e t h r o i d s to f i s h appears to result i n part from high i n t r i n s i c s e n s i t i v i t y of the CNS to these compounds (_16>JL7)« Fish are also h i g h l y s e n s i t i v e to some



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i n s e c t i c i d a l p y r e t h r o i d s , such as trans-permethrin, that are com p l e t e l y n o n t o x i c to mammals (_16^). These d a t a s u g g e s t t h a t s i g n i f i c a n t s p e c i e s d i f f e r e n c e s exist between sodium channels from f i s h and mammals. To explore these differences, we have adapted the methods f o r s t u d y i n g synaptosomal sodium uptake to examine the e f fects of i n s e c t i c i d e s on sodium channels i n f i s h brain synaptosomes. Our i n i t i a l s t u d i e s used v e r a t r i d i n e and £,£'-DDT to explore sodium channel pharmacology i n the f i s h b r a i n system. Veratridine (50 μ Μ ) produced a small but r e p r o d u c i b l e and s t a t i s t i c a l l y s i g n i f i c a n t (p

Enhancement of Veratridine-Dependent Sodium Channel Activation

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