Effects of certain carbamate and organophosphorous pesticides on isolated organs of Pila globosa (Gastropoda)

TOXICOLOGY

AND APPLIED PHARMACOLOGY

So, 485-492(1979)

Effects of Certain Carbamate and Organdphosphorous Pesticides on Isolated Organs of Pila globosa (Gastropoda) ONKAR SINGH Department

of Zoology,

AND

R. A. AGARWAL

University of Gorakhpur,

Gorakhpur,

U.P., India

Received September 29, 1978; accepted May 7, 1979 Effects of Certain Carbamate and Organophosphorous Pesticides on Isolated Organs of Pila globosa (Gastropoda). SINGH, O., AND AGARWAL, R. A. (1979). Toxicol. Appl. Pharmacol. SO, 485492. The pharmacological actions of three carbamates, viz. Zectran, Carbaryl, Aldicarb, and two organophosphorous pesticides, viz. Formothion and Trichlorfon, have been studied on isolated heart, median dorsal radula protractor muscle, and rectum of the snail Pilaglobosa, a pest of paddy crops in India, All five pesticides altered the response to exogenously applied acetylcholine (ACh). The order of effectiveness in the case of carbamates was Zectran > Carbaryl > Aldicarb. Among the organophosphates, Formothion was more potent than Trichlorfon. Both groups of pesticides selectively showed cholinomimetic properties on the excitor or inhibitor receptors of ACh in this animal. The effect of organophosphorous compounds was found to be time dependent. The structure-activity relationship of these pesticides has been discussed.

Since all classes of mollusks studied except scaphopoda have been shown to possess cholinergic mechanisms in their bodies (Greenberg et al., 1971; Agarwal et al., 1972), a natural choice for controlling these animals may lie in the use of anticholinesterase agents. Carbamate and organophosphorous compounds are of special interest because of their acetylcholinesterase (AChE)-blocking action (Koelle, 1975). Among the carbamates which have been reported to be effective in controlling molluscan populations are Zectran, 4-(dimethylamino)-3,5-xylenyl N-methylcarbamate (Arnold, 1962; Barry, 1969) ; Carbaryl, I-naphthyl N-methylcarbamate (Barry, 1969; Brar and Simwat, 1973); and Aldicarb, 2-methyl-2-(methylthio)propionaldehyde 0-(methylcarbamoyl)oxime (Judge, 1969; Brar and Simwat, 1973). Of the organophosphorous compounds, Formothion, 0, O485

dimethyl-S-(N-methyl-N-formoylcarbamoylmethyl) dithiophosphate, and Trichlorfon, 2,2,2-trichloro-l-hydroxyethyl phosphate (Thomas et al., 1977; Thompson and Sanderson, 1977), though toxic to insect pests, have not been used on mollusks so far. Most of the toxicological work on mollusks is limited to the determination of the lethal doses of various pesticides, but knowledge regarding the pharmacology of pesticides on this major phylum of pests is almost nonexistent. In the present work studies have been made on the pharmacological action of certain carbamate and organophosphorous compounds on three muscle types of Pilu globosa, a snail pest of the paddy in India, in order to further elucidate the structureactivity

relationship

of molluscicides

may be useful in combatting pest.

which

this gastropod

0041-008X/79/120485-08S02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

486

SINGH

AND

METHODS Collection, maintenance, and procedures for isolating the heart, median dorsal radula protractor muscle (MDRPM), and rectum of the experimental animals have been described in detail in a previous paper (Singh and Agarwal, 1978). The above organs were taken from the body, stretched between a stainless-steel hook and isotonic lever and suspended in an isolated organ bath containing “P&r-saline” formulated by La1 and Agarwal (1968). The isolated organs were allowed to equilibrate for 2-4 hr in a bath, following which responses were recorded on a smoked kymograph drum. Preparation of test solutions. Saturated stock solutions of active ingredients of Zectran (0.1 mg/ml; Dow Chemical, New York), Carbaryl and Aldicarb (0.04 and 6 mg/ml, respectively; Union Carbide India Limited), Formothion (0.25 mg/ml; Sandoz India Limited), and Trichlorfon (10 mg/ml; All India Medical Corporation) were prepared in saline. These solutions were serially diluted in saline in order to obtain the desired concentrations of the drugs. Administration of drugs. Isolated heart, MDRPM, and rectum were challenged with ACh at 3 x 10-6, 3 x 10-4, and 5 x 1O-4 M, respectively, before and after the tissue had been exposed to different concentrations of the pesticides for 5, 10, 30, or 60 min. These test doses of ACh were kept a little higher than the threshold (Singh and Agarwal, 1978) for these organs in order to evoke easily measurable responses. All drugs were added directly to the bath by means of a hypodermic syringe; doses have been expressed as final concentration of drug in the bath. Each experiment was repeated on at least six different preparations. StatisticaI analysis. Responses (amplitude of contraction of the three preparations) produced by test

TABLE ALTERATION

IN

(ACh)

ACETYLCHOLINE

giobosa

Zectran Carbaryl Aldicarb

doses of ACh before application of pesticide were measured and taken to be lOOa%. The alterations produced in the ACh responses following treatment with the experimental drug were also measured similarly and the two were compared. The results have been expressed as mean percentage change+ SE. An analysis of variance was applied to determine whether increase in exposure time (5 to 60 min) to the different pesticides resulted in significant changes in ACh response. The Student-Newman-Keuls test outlined in Woolf (1968) was also used to determine whether changes in mean response from 5 to 10, 10 to 30, and 30 to 60 min were significant.

RESULTS Alteration in the Acetylcholine Responseby Carbamate Compounds Heart. Even though 3 x 1O-6 M ACh applied to isolated hearts of Pila globosa brought about only a small decrease in amplitude, the same concentration of ACh, when applied 5 min after treating with minimally effective doses (threshold dose) of Zectran (5 x 1O-5 mg/ml), Carbaryl (2 x 1O-4 mg/ml), and Aldicarb (3 x lo--* mg/ml), caused either a substantial decrease in response amplitude or, in some preparations, even diastolic arrest (Table 1). Although Zectran was ineffective on the heart in the absence of exogenously applied ACh, 3 to 5 min exposure to higher doses of both Carbaryl (4 x 1O-4 mg/ml) and Aldicarb (6 x

1

RESPONSE AFTER EXPOSURE TO CARBAMATE PESTICIDES

Heart

Pesticides

AGARWAL

OF DIFFERENT FOR 5 min”

ISOLATED

Radula

ORGANS

OF

Pila

Rectum

Threshold dose (mg/ml)

Percentage reduction in amplitude

Threshold dose (mdml)

Percentage increase in ACh response

Threshold dose (m/m0

Percentage increase in ACh response

5x10-5 2 x 10-a 3 x 10-z

87.8Ok4.30 94.46+ 3.52 87.44 + 6.47

10-7 4x lo-” 6 x 10-l

114.44+ 10.50 177.38+11.09 289.02 + 17.30

5 x lo-’ 4x 10-z 6 x 10-l

216.48+ 13.32 177.18+ 30.63 107.00+ 10.16

’ Table showing alteration in response to test dose of ACh after giving the threshold dose of three carbamate pesticides on isolated heart, MDRPM, and rectum of Pila globosa. Values are means + SE (N = 6). Response to ACh without pesticide was taken as 100%.

CARBAMATE

AND

ORGANOPHOSPHOROLJS

TABLE ALTERATION

487

PESTICIDES

2

(ACh) RESPONSE AFI-ER EXPOSURE OF IKBLATED TO ZECTRAN FOR DIFFERENT TIME PERIODS”

IN ACETYLCHOLINE globosu

ORGANS

OF Pila

Percentage change in ACh response Rectum

Radula

Exposure time (min)

Zectran (lo-’ mg/ml)

Zectran (5 x lo-* mg/ml)

Zectran (5 x lo-’ mg/ml)

Zectran (5 x 1O-6 mg/ml)

5 10 30

114.44* 10.50 139.91+ 24.83 164.9Ok32.26

63.44k 7.65 64.65+ 11.55 50.99+ 16.71

216.48+ 13.32 408.10t43.14 580.66k 138.5’

83.02* 13.17 212.42+ 10.02*

E Table showing alteration in response of test dose of ACh after administration of low and high doses of Zectran to isolated MDRPM and rectum of Pih globosu. Values are means + SE (N = 6). Response to ACh without pesticide was taken as 100%. *p
1O-2 mg/ml) produced cardiac arrest. It is also of interest that higher doses of ACh (5 x 1O-4 M) produced a positive tonotropic effect on hearts which had been made quiescent with Aldicarb (6 x 1O-2 mg/ml). MDRPM and rectum. Threshold doses of Zectran, Carbaryl, and Aldicarb substanltially enhanced the contraction produced by ACh in isoIated preparations of MDRPM and rectum (Table 1). Potentiation in the ACh response of the MDRPM showed a tendency to increase if the exposure time of Zectran was increased from 5 to 30 min, but because of large variation in the responses of individual preparations this increase was not ound to be significant. In the case of the ectum, however, administration of threshold oses of Zectran (5 x lo-’ mg/ml) signifiantly increased the ACh response from 16.48 to 580.66% as the Zectran exposure ‘me was increased from 5 to 30 min. Exure for 30 min to a higher dose of Zectran 5 x 166 mg/ml) produced a rise in tone in u rectum, even in the absence of exogenously apphed ACh. It is important to note that high doses of Zectran evoked a lower ACh response in the rectum, and caused partial inhibition of the ACh response in the MDRPM (Table 2).

Alteration in the Acetylcholine Responseby OrganophosphorousCompounds Heart. Hearts

pretreated with threshold (2.5 x lo-’ mg/ml) or higher (2.5 x 1O-6 mg/ ml) doses of Formothion for 5, 10, 30, or 60 min showed a time-dependent reduction in amplitude after administration of ACh (3 x 1O-6 M) (Fig. 1). It was noted, that although 5 min exposure to the threshoId dose (2.5 x lo-’ mg/ml) of Formothion caused a 46% reduction and a higher dose (2.5 x 1O-6 mg/ml) caused a 58 % reduction in amplitude of heart beat, diastolic arrest by both doses could be produced only after 60 min (Fig. 1). The threshold dose of Trichlorfon (5 x 1O-5 mg/ml) caused a 41% reduction in amplitude in 5 min which increased to 100% in 60 min. A lo-fold higher dose of Trichlorfon (5 x 1O-4 mgjml) caused cardiac arrest after 30 min exposure (Fig. 1). An analysis of variance on the above data on Formothion and Trichlorfon showed that an increase in exposure time significantly increased (p ~0.01) the response to ACh in isolated heart preparations of Pila globosa (Fig. 1). Median dorsal radula protractor muscle (MDRPM). In the case of .the MDRPM,

488

SINGH AND AGARWAL

.4 * *

I

_ct

FORM

2.5X10-7

FORM

2.3x10-6

* ----x-x-

TRIG

SXIO-5

~--o--o-

TRIG

SXIO-4

I

I

I

I

5

IO

30

60

TIME

IN MIN

AFTER

PESTICIDE

FIG. 1. Dose-response curves showing percentage reduction in amplitude of heart beat caused by ACh (3 x 10e6 M) after exposure of isolated heart of Pifa globosa to two different concentrations of Formothion and Trichlorfon for varying lengths of time. Values represent mean of at least six experiments in each group. Abbreviations: FORM, Formothion; TRIC, Trichlorfon. *p< 0.01. (Asterisks shows that duration of exposure significantly altered the response at indicated dose when data were analyzed by analysis of variance. Doses expressed as mg/ml.)

threshold doses of Formothion (2.5 x 1O-8 mg/ ml) increased the response of this tissue to ACh from 134 to 189 % as the time was increased from 5 to 60 min but this increase was not significant (Fig. 2). Higher doses of Formothion (2.5 x lo-’ mg/ml), on the other hand, caused a rapid decrease in ACh response as the exposure time of Formothion was increased from 5 to 60 min (p < 0.01). In the case of Trichlorfon (5 x 1O-4 mg/ ml) the ACh response progressively increased up to 30 min, but decreased when the exposure time was increased to 60 min (Fig. 2). This change, however, was found not to be statistically significant. Similar to the effects with higher doses of Formothion, a 5-min treatment with higher doses of Trichlorfon (5 x 1O-3 mg/ml) caused a significant decrease in the ACh response of the

MDRPM (p-=0.05) which was further minished with an increase in exposure ti (Fig. 2). Rectum. In the case of the rectum a mini mum of 7.5 x 1OV mg/ml of Formo needed to enhance the contraction by ACh (5 x 1O-4 M). Even tho of the pesticide showed a increase the ACh response after time was increased from 5 to 60 min, this found not to be statistically (Fig. 3). A higher dose of F (1.3 x lo-’ mg/ml) caused a 230 % mcrease the ACh response after exposure for 30 mq but increase in exposure time to 60 mi$ caused a reduction in ACh response (p < 0.01) (Fig. 3). The threshold dose of Trichlorfon (5 x 1O-3 mg/ml) potentiated the ACh rqonse

CARBAMATE

FORM

AND

ORGANOPHOSPHOROUS

489

PESTICIDES

24XIO”

TRIG

5x10-4

TRIG

5x10-3

5

IO

XJ TIME

60 IN MN

5 AFTER

IO

60

PESTICIDE

FIG. 2. Figure showing percentage change in ACh response of isolated MDRPM of Pi/a globosa caused by pretreatment with different concentrations of Formothion and Trichlorfon for varying lengths of time. Response to ACh without pesticide has been taken as 100%. Note that threshold doses of the two pesticides show a tendency to gradually enhance the ACh effect while higher doses significantly inhibit this action. Inset: Kymograph record showing a typical response of MDRPM to test dose of ACh before and after treatment with pesticide. Abbreviations: ACh, acetylcholine; FORM, formothion; TRIC, trichlorfon; P, treatment with pesticide. Concentrations: ACh, mol/ liter; FORM and TRIC, mg/ml. * p < 0.01, **p < 0.05 (details as in Fig. 1).

for 10 min, after which time the response decreased (Fig. 3). Higher doses of Trichlorfon (1O-2 mg/ml), on the other hand, significantly increased the ACh response for 5 min and then depressed it (p ~0.01) (Fig. 3). DISCUSSION The results of the present investigation clearly show that both carbamate and organophosphorous compounds alter the action of ACh on various isolated tissues of Pila globosa. Also, higher doses of Carbaryl (4 x 1O-4 mg/ml) and Aldicarb (6 x 10e2 mg/

ml) cause diastolic arrest of the heart even without exogenously applied ACh. It appears that high concentrations of these compounds either cause accumulation of large quantities of endogenous ACh at the receptor sites or, as stipulated by Koelle (1975), may act directly at these receptors. That the action of Carbaryl and Aldicarb is perhaps cholinomimetic is borne out by the fact that carbaminoylcholine (carbachol), a carbamic ester of choline, shows ACh-like effects on the hearts of Mytilus edulis, Spisula Solidissima, and Tresuscapax (Greenberg, 1970). Although Aldicarb and Carbaryl mostly showed anticholinesterase properties, in cer-

SINGH AND AGARWAL

I

FORM

7+X IO+

TRI C 5X10-3

219% 195%

TT

70%

TIME

IN

MIN

AFTER

PESTICIDE

FIG. 3. Figures showing Percentage change in ACh response to isolated rectum of Pilaglobosa caused by pretreatment with different concentrations of Formothion and Trichlorfon for varying lengths of time. Inset: Kymograph record showing a typical response of rectum to test dose of ACh before and after treatment with pesticide. Symbols and abbreviations as in Fig. 2. * p < 0.01 (details as in Fig. 1).

tain preparations of the rectum these chemicals partially blocked the effect of even relatively high doses’ of ACh. It has been reported by Paton and Perry (1953) that high doses of physostigmine also have a direct blocking action on cholinergic transmission in autonomic ganglia, by binding it at the cholinoceptive sites. It appears that carbamic acid esters, presumably because of their structural similarity with ACh, sometimes act as competitive blockers of ACh in these tissues. Our data on the cholinomimetic action of carbamate compounds bring out the fact that while Zectran has an ACh-like action on the

rectum but not on the heart, Aldicarb and Carbaryl show a cholinomimetic action only on the heart and not on the MDRPM and rectum. Greenberg (1970) has suggested that the excitor and depressor actions of ACh in the case of mollusks are due to the presence of separate receptor mechanisms. Wilkens and Greenberg (1973) have further shown that increased Na+ conductance is responsible for the excitor action of ACh, while its inhibitor action is due to increased Clconductance. In view of the presence of two distinct receptor mechanisms for inhibition and excitation in mollusks it is not surprising that the muscles which are inhibited (heart)

CARBAMATE AND ORGANOPHOSPHOROUS PESTICIDES

or excited (MDRPM and rectum) by ACh respond differently to different carbamate compounds. A comparison of the doses of Zectran, Carbaryl, and Aldicarb essential to produce a pharmacological response in the three muscles of Pila globosarevealed that the order of efficacy was Zectran > Carbaryl > Aldicarb. It has been stated that, because of their interaction at both the esteratic and anionic sites of the AChE molecule, carbamate compounds having N-methyl and N,Ndimethyl groups possess a high degree of anti-AChE activity (Metcalf, 1971). Indeed, Zectran possesses both N-methyl and 4dimethyl groups; Carbaryl has the N-methyl group only, whereas Aldicarb does not contain either group. Also, Aldicarb, though apparently similar to ACh in molecular dimensions, unlike Zectran and Carbaryl does not possess a quarternary ammonium nitrogen. On the other hand, it even has an S atom which in general reduces the antiAChE activity of carbamates (Metcalf, 197 1). These structural characteristics may therefc e, be responsible for the relative toxicity of the three compounds. Singh and Agarwal(l978) showed that the effects of Phorate, an organophosphorous compound, are time dependent and postulated that the inactive forms of organophosphates undergo metabolic conversion with the help of microsomal mixed-function oxidases. Data on the two organophosphates, Formothion and Trichlorfon, show that like Phorate the effect of these two is also time dependent. However, the time required by Trichlorfon for maximum effect was always less than that for Formothion (Figs. l-3). Julius and Gerald (1977) have pointed out that organophosphates having a P-S group are first converted into a P=O analog before becoming active. Trichlorfon (P = 0), being a phosphate compound, is an active anti-AChE agent in its native form while Formothion (P = S), being a phosphorothionate compound, is inactive and thus requires time for its activation. It can be seen from the above

491

data that Formothion was effective at lower doses as compared to Trichlorfon, in spite of the former being inactive in its native form. It is possible that the carbamoyl group present in Formothion may be responsible for its greater activity. Our data demonstrate that while both high and low doses of Formothion and Trichlorfon potentiated the effect of ACh on the snail heart, only -threshold doses of these drugs potentiated the ACh effect in the MDRPM and rectum. Higher doses of these two drugs, with increase in exposure time, progressively blocked the ACh effect in the MDRPM and rectum. It appears that high concentrations of the two organophosphates have an AChblocking action only on the excitor receptors, without having any such action on the inhibitor receptors. These observations again point toward selective action of pesticides on the two types of ACh receptors in this animal as mentioned earlier for the carbamates.

ACKNOWLEDGMENTS The authors wish to express their gratitude to the following companies for a supply of samples; M/S Dow Chemical, New York, for Zectran; M/S Union Carbide India Limited for Carbaryl and Aldicarb; M/S Sandoz India Limited for Formothion; and M/S All India Medical Corporation for Trichlorfon.

REFERENCES R. A., LIGON, P. J. B., AND GREENBERG, M. J. (1972). The distribution of cardioactive agents among molluscan species and tissues. Camp. Gen. Pharmacol. 3, 249-260. ARNOLD, W. J. (1962). Snail and lawn pest control with zectran pesticide. Down to Earth, Winter, 20-24. BARRY, B. D. (1969). Evaluation of chemicals for control of slugs on field corn in Ohio. J. Econ. Entomol. 62, 1277-1279. BRAR, H. S., AND SIMWAT, G. S. (1973). Control of the common slug, Laevicaulis alre-Ferussae (Gastropoda), with certain chemicals. J. Res. Punjab Agr. Univ. 10. 99-101. AGARWAL,

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AND

M. J. (1970). A comparison of acetylcholine structure activity relations on the hearts of bivalve molluscs. Comp. Biochem. Physiol. 33, 259-294. GREENBERG, M. J., AGARWAL, R. A., WILKENS, L. A., AND LIGON, P. J. B. (1971). Chemical regulation of rhythmical activity in molluscan muscle. In NeurobioIogy of Invertebrutes (J. Salanki, ed.), pp. 1233142. Hung. Acad. Sci., Tihany. JUDGE, F. D. (1969). Preliminary screening of candidate molluscicides. J. Econ. Entomoi.62,1393-1397. JULIUS, J. M., AND GERALD, G. S. (1977). Metabolism of insecticides and herbicides in higher plants. CRC Crit. Rev. Toxicol. 5, l-21. KOELLE, G. B. (1975). Anticholinesterase agents. In The Pharmacological Basis of Therapeutics (L. S. Goodman and A. Gilman, eds.), pp. 445-466. MacMillan Co., New York. LAL, M. B., AND AGARWAL, R. A. (1968). A saline medium for maintaining isolated hearts of Pifa globosa, Swainson. Proc. Indian Sci. 67, l-7. METCALF, R. L. (1971). The chemistry and biology of pesticides. In Pesticides in the Environment (R. White-Stevens, ed.), Vol. 1, pp. 2-140. Dekker, New York. GREENBERG,

AGARWAL W. D. M., AND PERRY, W. L. M. (1953). The relationship between depolarization and block in the cat’s superior cervical ganglion. J. PhysioI.

PATON,

119,43-57. O., AND AGARWAL, R. A. (1978). Pharmacological studies on the moluscicidal activity of phorate on PiIa globosa (Gastropoda). Acta Pharmacol. Toxicol. 42, 365-370. THOMAS, F. L., JACKSON, C. E., WYNHOLDS, P. F., AND COTA, J. A. (1977). Toxicity of selected insecticides applied topically to Lygus hesperus-Miridae. J. Econ. Entomol. 70, 42-44. SINGH,

THOMPSON,

L. S., AND SANDERSON,

J. B. (1977).

Pea

moth control in field peas with insecticides and the effect on crop yield. J. Econ. Entomol. 70, 518520. WILKENS, .L. A., AND GREENBERG, M. J. (1973). Effect of acetylcholine and 5-hydroxytryptamine and their ionic mechanisms of action on the electrical and mechanical activity of molluscan heart smooth muscle. Comp. Biochem. Physiol. Ser. A. 45, 637-651. WOOLF, C. M. (1968). Principles of Biometry, pp. 101-108. Van Nostrand, Princeton, N. J.