kinetic analysis of acetylcholinesterase inhibition by combinations of acephate and methamidophos

Toxicology, 42 (1986) 143--156 Elsevier Scientific Publishers Ireland Ltd.

KINETIC ANALYSIS OF ACETYLCHOLINESTERASE INHIBITION BY COMBINATIONS OF ACEPHATE AND METHAMIDOPHOS

ASHOK K. SINGH Department of Veterinary Diagnostic Investigation, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108 (U~S.A.)

(Received March 19th, 1986) (Accepted July 21st, 1986)

SUMMARY

Acephate pre-exposure provided protection against the inhibition of RBC and brain acetylcholinesterase (ACHE), and plasma cholinesterase (ChE) activities in rats exposed to both acephate and methamidophos. In vitro addition of acephate to AChE prior to or with methamidophos also provided complete protection against AChE inhibition by methamidophos. When acephate was added to the enzyme after methamidophos, its protective effect decreased with increasing time between the additions. Since acephate has greater affinity than does methamidophos for the AChE active site (Singh, A.K., Toxicol. Appl. Pharmacol., 81 (1985) 302), it is proposed that acephate provided protection by binding with the AChE active site and, therefore, preventing methamidophos from binding with the enzyme. It is also proposed that acephate prevented the initial competitive binding of methamidophos to the AChE active site and delayed the initial sequence of events essential for phosphorylation of ACHE.

Key words: Acephate; Methamidophos; Organophosphates; Acetylcholin- esterase

INTRODUCTION

Acephate and methamidophos are organophosphorus (OP) insecticides widely used to control agricultural and forestry pests. Both insecticides are known to increase the concentration of acetylcholine (ACh) in brain, blood diaphragm, and other tissues primarily by inhibiting the enzyme acetylcholinesterase (ACHE) [1]. The increase in ACh is believed to be

0300-483X/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

143

resEonsible for many of the observed symptoms of toxicity from OP compounds in mammals [2]. Acephate is produced by the N-acetylation of methamidophos, and the anti-cholinesteratic (anti-AChE) potency and mammalian toxicity of acephate are 75--100 times less than that of methamidophos [3]. Recent studies have proposed that: (1) acephate can be partially metabolized in some plants and animals to yield the relatively toxic methamidophos [4]; and (2) mammalian blood may contain certain enzymes capable of metabolizing acephate to methamidophos [3]. The toxicological consequences of the presence of methamidophos in acephate- exposed animals are not known. It is also not known whether the presence of methamidophos in acephate exposed rats alters the anti-AChE potency of acephate. Several workers have observed that the biological response following simultaneous exposure to a combination of anti-AChE insecticides may be different than would be expected from the separate additive action of each [5--7]. Therefore, the aims of this investigation were to study AChE inhibition in rats exposed to acephate, methamidophos and acephate plus methamidophos; and the kinetics of in vitro inhibition of AChE activity by a combination of acephate and methamidophos.

MATERIALS AND METHODS

Materials Bovine erythrocyte ACHE, acetylt_hiocholine iodide (ATCh), butyrylthio-

choline iodide (BTCh), 5,5'<tithiobis-2-nitrobenzoic acid (DTNB) were purchased from Sigma Chemicals. Acephate and methamidophos were supplied as a gift by Cheveron Chemicals.

In vivo s t u d y Fifty rats (100 g) were divided into 4 groups. Rats from each group were

injected (intraperitoneal (i.p.)) with acephate, methamidophos, or acephate plus methamidophos as described below:

Group 1 (n = 5, control): These rats were injected with 0.5 ml isotonic saline {solvent for acephate and methamidophos).

Group 2 in = 5): These rats received 0.5 mmol acephate/kg body weight. Group 3 (n = 20): These rats were divided into 4 subgroups with 5 rats

in each. Subgroup 1 received 0.05 mmol, subgroup 2 received 0.1 mmol, subgroup 3 received 0.2 mmol, and subgroup 4 received 0.3 mmol methamidophos/kg body weight.

Group 4 (n = 20): These rats received 0.5 mmol acephate/kg body weight. At 30 min after acephate injection the acephate exposed rats were further exposed to various doses of methamidophos as described in group 3.

At 60 min after treatment, each rat was anesthetized with ether. Blood was collected in a heparinized tube from the abdominal aorta. Immediately

144

after blood collection, the skull was removed by decapitation, the brain was dissected out, homogenized in 0.32 M sucrose (1 :4 w/v), and centrifuged {1000 g) at 4°C for 30 min. The supernatant was transferred into another centrifuge tube and centrifuged again at 17 000 g for 30 min [8]. The clear supernatant {Cytoplasmic fraction) was collected and used in this study. Blood samples were centrifuged at 4°C, and plasma and RBC were separated. RBC samples were washed twice with isotonic saline, and the hematocrit was adjusted to the original blood hematocrit with isotonic saline. Plasma and RBC samples were stored at 4°C and used within 8--10 h.

Determination o f brain and RBC-A ChE activity Before analysis, RBC ssamples were diluted 10 times with isotonic saline.

One hundred microliters of brain or RBC samples were mixed with 2.9 ml of phosphate buffer (pH 7.6, 0.1 M) containing 1.0 mM DTNB. Reaction was started by adding 20 pl of substrate (ATCh iodide) solution. AChE activity was determined by a method described by Ellman et al. [9].

Determination o f plasma-ChE activity One hundred microliters of plasma was diluted with 2.9 ml of phosphate

buffer (pH 7.6, 0.1 M) containing 1.0 mM DTNB. The enzyme activity was determined as described by Ellman et al. [9].

In vitro studies Supernatant samples obtained from the control brains were used in

the following in vitro experiments:

Inhibition o f AChE by acephate, methamidophos, and acephate plus methamidophos

One hundred microliters of enzyme samples were incubated with 100/~l of various concentrations of acephate, prepared in buffered isotonic saline (final concentration of acephate in the mixture ranged 1.0--10.0 nmol/ml. The mixture was incubated for 1 min at 25°C. Aliquots (20 ~l) of acephate- exposed samples were pipet ted into another test tube. Twenty microliters of methamidophos in isotonic saline {final concentration 0.8 nmol/ml), or 20 pl of isotonic saline (for control activity) were added to the test tube. The enzyme samples containing acephate or acephate plus methamidophos were diluted to 0.3 ml by phosphate buffer (pH 7.6, 0.1 M) containing 1.0 mM DTNB and 1.0 mM ATCh. The enzyme activity was determined as described by Ellman et al. [9]. Residual enzyme activity {calculated as % of control enzyme activity) was plot ted against the concentrat ion of acephate.

Establishment o f the timecourse o f AChE inhibition by acephate, me thamidophos, and acephate plus methamidophos

The concentrat ion of acephate which provided maximum protect ion was used in this experiment. Twenty microliters of enzyme samples were incubated with acephate (5.0 nmol/ml) and/or methamidophos (0.8 nmol/ml).

145

The following incubation protocols were used when both acephate and methamidophos were added to the enzyme: (1) acephate and methamidophos were simultaneously added; (2) acephate was added to the enzyme 30 s after the addition of methamidophos; and (3) acephate was added to the enzyme 60 s after the addition of methamidophos. At various t ime intervals (15 s to 4 min) after the addition of methamidophos to the enzyme, each sample was diluted with 0.3 ml of phosphate buffer (pH 7.5, 0.1 M) containing 1.0 mM DTNB. The enzyme activity was determined by the method described by EUman et al. [9].

Determination o f k i values Twenty microliters of enzyme samples were incubated with acephate

(5.0 nmol/ml) and/or various concentrations of methamidophos (0.1--1.4 nmol/ml). The following incubation protocols were used when both acephate and methamidophos were added to the enzyme: (1) acephate and methamidophos were simultaneously added; {2) acephate was added 1 min prior to the addition of methamidophos; (3) acephate was added 30 s prior to the addition of methamidophos; (4) methamidophos was added 1 min prior to the addition of acephate; and (5) methamidophos was added 30 s prior to the addition of acephate. Enzyme activity was determined at 10, 30, 60, 120, and 300 s after the addition of methamidophos by a method described previously [9]. A value of k i was calculated for each time interval by the equation of Aldridge [10] and Main [11]:

k i -- 2 . 3 0 3 A l o g v 1

At i

where 'v' is the rate at t ime 't ' , and 'i' is the inhibitor concentration. Statis- tics was done by the "one-way" program adapted from the Statistical Package for Social Sciences (SPSS).

Determination o f affinity constant (Ka) and phosphorylat ion rate (kp) values

Twenty microliters of brain samples were incubated with acephate (5 nmol/ml) + various concentrat ion of methamidophos (0.1--1.4 nmol/ml), or with methamidophos alone {0.1--1.4 nmol/ml). The concentrat ion of acephate (5 nmol/ml) used in this study did not inhibit the brain AChE activity. The protocols used, when bo th acephate and methamidophos were added to the enzyme, has been described earlier {determination of ki section). At the end of incubation period, each sample was diluted to 0.3 ml with phosphate buffer (pH 7.6, 0.1 M) containing 1.0 mM DTNB and 1.0 mM ATCh. The absorbance change at 410 nm was recorded, and the initial reaction rate was measured as residual enzyme activity. The reaction rate was measured for at least 10 s after each incubation period. The Ka and kp values were calculated by a method described by Singh [12], Main [11] , Main and Iverson [13] , and Wang and Murphy [14].

146

Lineweaver-Burk plot for AChE in the presence of acephate, methamidophos, and acephate + methamidophos

1. Twenty microliters of enzyme samples were incubated with 0.3 ml of phosphate buffer (pH 7.5, 0.1 M) containing 1.0 mM DTNB at room temperature. The reaction was started by adding 20 pl of various concentration of substrate (final concentration of ATCh ranged from 0.8 to 10.0 mM) to the mixture. Enzyme activity was determined by recording the absorbance change at 410 nm as described by Ellman et al. [9]. The Line- weaver-Burk plot was drawn by plotting 1Iv against 1/[s] , where [s] is the final substrate concentration.

2. Various concentrations of ATCh (final concentrat ion ranging from 0.8 to 10.0 mM) were mixed with acephate* (10.0 nmol/ml) and/or methamidophos (0.3 nmol/ml), and 20 gl of enzyme solution were added to the mixture. Samples were incubated for 5 s at room temperature. After incubation, each sample was diluted to 0.3 ml with phosphate buffer (pH 7.5, 0.1 mM) containing 1.0 mM DTNB. Enzyme activity and the initial rates (v) were determined as described by Ellman et al. [9]. The Lineweaver- Burk plot was drawn by plotting 1/v against 1/Is] , where [s] is the substrate concentration.

3. Acephate* (10.0 nmol/ml) and/or methamidophos (0.3 nmol/ml) were added to 20 gl of enzyme and incubated for 60 s at room temperature. At the end of the incubation period, 20 gl of various concentrations of ATCh (ranging from 0.8 to 10.0 mM) were added to the sample. Each sample was diluted with 2.9 ml of phosphate buffer (pH 7.5, 0.1 M) con- mining 1.0 mM DTNB. Enzyme activity was determined by the method of Ellman et al. [9]. The Lineweaver-Burk plot was drawn by plotting 1Iv against 1/[s] .

RESULTS

In vivo study Control values for brain, RBC, and plasma enzyme activities were 110.0 +

10.3 nmol/min/mg protein, 72.6 + 8.1 nmol/min/ml, and 107.0 + 7.6 nmol/min/ml plasma, respectively. The AChE and ChE activities in acephate exposed rats (group 2) were similar to the control rats. The enzyme activity decreased gradually by increasing the dose of methamidophos from 0.05 mmol/kg to 0.3 mmol/kg (Fig. 1). In rats exposed to 0.3 mmol methamidophos/kg, the enzyme activity was 10--30% of the control enzyme activity (indicating 70--90% inhibition) (Fig. 1). However, when acephate-treated rats were exposed to methamidophos, the brain and RBC-AChE activities were 90--98% of the control enzyme activities (indicating only 2--10% inhibition). The plasma ChE activity was 80--90% of the control enzyme

*The concentration of acephate used in this experiment was twice the concentration used in other in vitro experiments. This was done to achieve inhibition in AChE by acephate exposure.

147

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M-PLASMA

M-BRAIN

0.05 0.10 0.15 0.20 0.25 0.30

METHAMIDOPHOS (m mol/kg)

Fig. 1. Brain and RBC-AChE and Plasma ChE act ivi t ies in ra ts exposed to m e t h a m i d o - phos , and acepha t e plus m e t h a m i d o p h o s . M-Brain, M-RBC and M-Plasma are samples o b t a i n e d f rom m e t h a m i d o p h o s exposed rats. A + M-Brain, A + M-RBC and A + M- Plasma are samples o b t a i n e d f r o m ra ts exposed to b o t h acepha t e plus m e t h a m i d o p h o s . (Values are m e a n + S.D.).

activity. These observations indicate that a dose of acephate which did not inhibit AChE activity, provided significant protection against the inhibition of brain and RBC-AChE, and plasma ChE by methamidophos.

In vitro studies Inhibition of AChE by acephate + methamidophos or by methamido-

phos: The residual AChE activity in samples incubated with various doses of acephate in the presence or absence of methamidophos is shown in

148

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Fig. 2. The residual AChE activity in samples incubated with various doses of acephate in the presence or absence of methamidophos. ( , ) samples exposed to acephate; and (o) samples exposed to both acephate plus methamidophos in vitro.

Fig. 2. AChE activity was not inhibited for concentrations up to 8.0 nmol/ ml. At 10.0 nmol/ml acephate concentration, 10% inhibition in AChE activity occurred. Addition of methamidophos (0.8 nmol/ml) to the enzyme samples resulted in >80% inhibition in AChE activity (Fig. 2). When samples were exposed to both acephate and methamidophos, increasing the concentration of acephate decreased the degree of inhibition of AChE by methamidophos (Fig. 2). Maximum protection of AChE from inhibition by methamidophos was noted at 5.0 nmol/ml acephate concentration.

Inhibition of AChE by various doses of methamidophos in presence or absence of acephate is shown in Fig. 3. AChE activity in samples exposed to 0.1 nmol methamidophos was 65% of the control enzyme activity. Increasing the concentration of methamidophos increased the degree of AChE inhibition (Fig. 3). However, when the enzyme samples were exposed to 0.1 nmol methamidophos after being first treated with acephate (5.0 nmol/ml), only 3--5% inhibition in the AChE activity occurred (Fig. 3). In the presence of acephate, the potency of methamidophos to inhibit

149

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METHAMIDOPHOS (n mol/ml) Fig. 3. Residual AChE activity in samples incubated with various doses of methamidophos in presence or absence of acephate. I, --~--, samples exposed to methamidophos; II, ---~--, samples exposed to acephate plus methamidophos together; III, --a--, samples exposed to acephate 30 s after methamidophos; and IV, --A--, samples exposed to acephate 60 s after methamidophos.

AChE was significantly reduced at all the methamidophos concentrat ions used in this s tudy (Fig. 3). The protective effect of acephate was lost when it was added to the enzyme after the addition of methamidophos (Fig. 3).

The time course of inhibition by methamidophos (0.8 nmol/ml) of the AChE is shown in Fig. 4I. A gradual decrease in AChE activity occurred for up to 2 min when approximately 85% of the enzyme activity was inhibited (Fig. 4I). When enzyme samples were simultaneously exposed to acephate (5.0 nmol/ml) and methamidophos (0.8 nmol/ml), only 20% inhibition in the AChE activity occurred (Fig. 4II). The time courses of

150

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Fig. 4. The time course of inhibi t ion of acetytcholinesterase (ACHE) by acephate and/or methamidophos in vitro. I. Enzyme incubated wi th methamidophos. II. Enzyme incubated with both acephate and methamidophos. IIIo Methamidophos added to the enzyme 30 s before acephate. IV. Methamidophos added to the enzyme 1 rain before acephate. V. Enzyme incubated wi th acephate.

inhibition of AChE simultaneously exposed to acephate (5.0 nmol/ml) and methamidophos (0.8 nmol/ml), or exposed to acephate (5.0 nmol/ml) alone were similar (Fig. 4II, 4V). When acephate was added to the enzyme 30 s after methamidophos, 60% inhibition in AChE activity occurred (Fig. 4III). The protective effect of acephate was lost when it was added to the enzyme 60 s after methamidophos (Fig. 4I, 4IV).

Lineweaver-Burk plot The results of this study indicated that the Lineweaver-Burk plot was

linear at all the substrate concentrations used in this study (Fig. 5I). Simul- taneous addition of both substrate and methamidophos (0.8 nmol/ml) to the enzyme changed the slope of the plot but did not change the Vmax values (Fig. 5II), indicating a competitive type of inhibition. When substrate was added to the enzyme 60 s after the enzyme was exposed to methamidophos, the Km values did not change, but the Vmax values decreased significantly (Fig. 5III), indicating a non-competitive type of inhibition. Non-competitive type inhibition was also observed in the following cases: (1) when the enzyme was exposed to acephate (10.0 nmol/ml) (Fig. 5IV); (2) when the enzyme was simultaneously exposed to acephate (10.0 nmol/ml), methamidophos (0.8 nmol/ml) and substrate (Fig. 5V); and (3) when the enzyme was exposed to acephate 60 s after methamidophos

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Fig. 5. Lineweaver-Burk plot for AChE in the presence or absence of inhibitors. I. No inhibitor. II. Methamidophos and substrate simultaneously added to the enzyme. III. Substrate added to the enzyme 1 rain after methamidophos. IV. Acephate. V. Acephate, methamidophos, and substrate added simultaneously to the enzyme. VI. Acephate added 1 rain after methamidophos.

(Fig. 5V). The Vma x values in samples exposed to acephate or simultaneously exposed to acephate and methamidophos were similar (Fig. 5IV, 5V).

Bimolecular rate constant (ki) values. The k i values of AChE in presence of methamidophos, when this insecticide was added to AChE simultaneously with the substrate, was 15.0 -+ 1.6 min {Table I). Values increased gradually with longer enzyme-methamidophos incubation t ime (Table I, Fig. 6). However, the tei values were significantly lower when enzyme samples were first exposed to acephate before they were exposed to methamidophos {Table I). Pre~exposure with acephate also abolished the time depen- dent increase in the k i values of AChE for methamidophos (Fig. 6). The effect of acephate exposure was lost when acephate was added to the enzyme after the addition of methamidophos (Table I).

Af f in i t y constant (Ka) and phosphorylation constant (kp) values: The Ka values of AChE for methamidophos, when this insecticide was added to the enzyme simultaneously with the substrate, was 3.6 + 0.3 pM. Values increased significantly when enzyme samples were first exposed to acephate (a dose which did not inhibit AChE activity) prior to the exposure to methamidophos (Table I). These observations indicate that prior exposure to acephate decreased the affinity of methamidophos to AChE active site. The Ka values did not change significantly when acephate exposure occurred after the exposure of AChE to methamidophos (Table I).

kp values of AChE in presence of methamidophos was 75--100 times

152

T A B L E I

KINETICS OF I N H I B I T I O N OF AChE BY M E T H A M I D O P H O S IN P R E S E N C E OR A B S E N C E OF A C E P H A T E ( V A L U E S A R E MEAN + S.D.).

I I n c u b a t i o n P ro toco l kp K a k i Type of (rain -1) (~M) (~M -1 rain -1) i nh ib i t i on

M M + S a d d e d t o ' E ' 15.0 _+1.6 3 . 6 + 0 . 3 0 s imu l t aneous l y

M 'M' added to ' E ' 1 min 31.6 + 3.2 3.3 _+ 0 .20 pr ior to t he a d d i t i o n of 'S '

M + A M + A + S a d d e d t o 2.9 _+0.3 3 .0_+0.07 'E ' s imu l t aneous l y

M + A 'A ' was added to ' E ' 0 . 1 5 _ + 0 . 0 2 8.3 ± 0 . 3 7 1 rain pr ior to the add i t i on of 'M'

M + A 'A ' was added to ' E ' 0 .17 ± 0 . 0 1 8.2 ± 0.7 30 s p r io r to t h e add i t i on o f 'M'

M + A 'M' was added to ' E ' 30.3 _+2.0 3 .0+_0 .1 1 min pr ior to t he add i t i on of ' A '

M + A 'M' was added to ' E ' 20 .0 _+1.3 3 .3_+0.27 30 s pr ior to t he a d d i t i o n of ' A '

4.1 _+ 0.9 C

10.3 _+ 1.0 NC

1.3 _+ 0.2 NC

0.02 + 0 .005 NC

0.03 ± 0 .003 NC

10.3 i 2.3 NC

7.9 _+ 2.2 NC

M: m e t h a m i d o p h o s ; A: acepha t e ; E: bra in sample ; S: subs t ra te , ATCh; C: compe t i t i ve i n h i b i t i o n ; NC: n o n - c o m p e t i t i v e i nh ib i t i on ; kp: p h o s p h o r y l a t i o n c o n s t a n t ; Ka: af f in i ty c o n s t a n t and ki: b imolecu la r ra te cons t an t .

greater than the kp values in presence of acephate {Table I). The kp values of AChE in samples which were exposed to acephate, and in samples which were simultaneously exposed to both acephate and methamidophos, did not differ significantly (Table I). These observations indicated that acephate reduced the AChE phosphorylating power of methamidophos. The protective effect of acephate was lost when methamidophos was added to AChE 60 s prior to the addition of acephate (Table I).

DISCUSSION

Although the mammalian toxicity of acephate is 75--100 times less than that of methamidophos [12], the overall toxicity might increase with time because of the conversion of acephate into relatively toxic methamidophos [3]. Contrary to this belief, results of the present in vivo and in vitro studies have conversely indicated that a controlled concentration of acephate which did not inhibit AChE activity, provided protection against the inhibition of AChE by methamidophos. Main [11], Main and Iverson [13] and Aldridge [10] proposed that the inhibitory power of OP-insecticides is governed by the affinity of insecticides for the enzyme active site and/or by the rate of phosphorylation. Previous studies have shown that acephate

153

30

25

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15

10

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Fig. 6. Bimolecular rate (ki) and phosphorylation rate (kp) of AChE in presence of rnethamidophos (o) and acephate plus methamidophos (o).

has greater affinity than does methamidophos for the AChE active site [12]. Based on the results of Singh [12] and the results of this study, it is proposed that acephate may provide protection by preventing methamidophos from binding with the AChE active site. The observation that a dose of acephate which did not inhibit AChE activity but did provide protection against the inactivation of AChE by methamidophos suggest the possibility that the acephate~enzyme binding alters the structure of the active site so that substrate-enzyme binding is not affected, but that methamidophos- enzyme binding is reduced. Singh et al. [8] have proposed a similar mech- anism for the protection provided by quinidine against the inhibition of AChE by Satin, a potent organophosphorus compound.

This study indicates that treatment of enzyme samples with acephate prior to their exposure to methamidophos reduced the AChE phosphorylation capacity of methamidophos. Along with preventing methamidophos from binding to the AChE active site, the protective effect of acephate could also be explained by observing differences in the kinetics of AChE inhibition between these two insecticides. Similar to the observations of Ando and Wakamatsu [15] for plasma cholinesterase, acephate was found

154

to be a n o n - c o m p e t i t i v e inh ib i to r o f ACHE. Unl ike acepha t e , t he initial inh ib i t ion o f AChE by m e t h a m i d o p h o s was c o m p e t i t i v e and a f t e r several seconds , b e c a m e n o n - c o m p e t i t i v e . Previous s tudies have shown t h a t a cepha t e has a lower kp value fo r AChE t h a n does m e t h a m i d o p h o s [12] . These obse rva t ions suggest t h a t the initial c o m p e t i t i v e b inding of m e t h a m i d o p h o s to AChE migh t be an essential s tep for the p h o s p h o r y l a t i o n of this e n z y m e . The re fo re , in samples which were first t r ea t ed wi th acepha t e be fo re be ing exposed to m e t h a m i d o p h o s , a cepha t e migh t m o d i f y the s t ruc tu re o f AChE so t h a t the initial c o m p e t i t i v e b inding o f m e t h a m i d o p h o s to the AChE act ive site, and the p h o s p h o r y l a t i o n of AChE by m e t h a m i d o p h o s are prevented .

In conc lus ion , a c e p h a t e p re -exposu re p rov ided c o m p l e t e p r o t e c t i o n against the inh ib i t ion o f AChE b y m e t h a m i d o p h o s . The p ro t ec t ive e f fec t s o f a c e p h a t e we re r educed if a cepha t e was added a f t e r the exposu re of AChE to m e t h a m i d o p h o s . This decrease in the p ro t ec t i ve e f fec t o f a c e p h a t e migh t be because p r io r add i t ion o f m e t h a m i d o p h o s p h o s p h o r y l a t e d and inac t iva ted the ACHE.

REFERENCES

1 Koelle, G.B. (1981) Organophosphate poisoning -- An overview. Fund. Appl. Toxicol., 1, 129--134.

2 O'Brien, R.D. (1969). In "Annals to the New York Academy of Science", Ed. H.F. Kraybill. 160,204--210.

3 Singh, A.K. (1984). Improved analysis of acephate and methamidophos in biological samples by selective ion monitoring gas-chromatography-mass spectrometry. J. Chro- matogr. 301,465--469.

4 Szeto, S.Y., Yee, J., Brown, M.J., Oloffs, P.C. (1982). Simplified method for deter- mining acephate and methamidophos residues in several substrates. J. Chromat. 240, 525--531.

5 Frawley, J.P., Fuyat, H.N., Hagan, E.C., Blake, J.R., Fitzhugh, O.G. (1957). Marked potentiation in mammalian toxicity from simultaneous administration of two anti- cholinesterase compounds. J. Pharm. Exp. Ther. 121, 96--100.

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