Brain acetylcholinesterase as an in vitro detector of organophosphorus and carbamate insecticides in water

Wat. Res. Vol. 25, No. 7, pp. 835-840, 1991 Printed in Great Britain. All rights reserved

0043-1354/91 $3.00 + 0.00 Copyright © 1991 Pergamon Press plc

BRAIN ACETYLCHOLINESTERASE AS AN IN VITRO DETECTOR OF ORGANOPHOSPHORUS A N D CARBAMATE INSECTICIDES IN WATER V. L. F. CUNHA BASTOS, J. CUNHA BASTOS*, J. S. LIMA and M. V. CASTRO FARIA Department of Biochemistry, Biology Institute, Rio de Janeiro State University, 20551 Rio de Janeiro, Brazil

(First received February 1990; accepted in revised form January 1991) Abstract--An inexpensive but accurate enzymatic method is proposed for the detection of carbamate and organophosphorus pesticides contaminating water supplies. The method uses an acetylcholinesterase preparation obtained after extraction of rat brain microsomal fraction with Triton X-100. The method is based on inhibition of acetylcholinesterase in the presence of the pesticides. Some phosphorothionate insecticides (e.g. parathion, malathion), which are not direct acetylcholinesterase inhibitors, can also be activated by preincubation with the enzyme preparation. Enzyme assay is performed by a potentiometric method based on the formation of acetic acid in the incubation mixture. Interference of any eventual buffering capacity of the sample can be easily corrected. Malathion, parathion, diazinon and deoxicarbamate inhibited the enzyme at least 20% when they were added to the medium in the limit concentration recommended for public water supplies (0.1 mg/1). The method was evaluated in samples collected from selected locations of Paraiba do Sul river, Rio de Janeiro, Brazil, and it proved to be sufficiently practical and accurate as an alarm routine test for such pesticide classes.

Key words--pollutants, acetylcholinesterase, detection, water, organophosphorus, carbamate

INTRODUCTION The use of pesticides in agriculture and in insect control has been creating a potential danger to aquatic life and to human health. Determination of these substances normally involves methods like gas chromatography and mass spectroscopy, which are available only in sophisticated laboratories and are expensive and time consuming. In the course o f a project in cooperation with F E E M A (Environmental Engineering State Foundation), for the development of simple and inexpensive techniques that could be used routinely in some critical areas, we proposed an in vitro enzymatic method which could be useful as a warning of the presence of organophosphorus and carbamates in the water. Organophosphorus and carbamate insecticides have acetylcholinesterase as the main target (Main, 1976). Inhibition of this enzyme causes depolarization o f axon plasma membrane as a consequence of acetylcholine accumulation in cholinergic synapses (Benke et al., 1974). Studies concerning biological effects and metabolism of such compounds are well documented (Murphy, 1967; Nakatsugawa and Dahn, 1967; Norton, 1975; Sultatus et al., 1985). Our enzymatic method uses a stable rat or ox

brain acetylcholinesterase preparation. This communication describes the method, its response to some insecticides and its use in natural waters. MATERIALS AND METHODS

Preparation of the acetylcholinesterase enriched fraction Our preparation was a modification of a procedure used for acetylchohnesterase purification (Raconczay et al., 1981). Albino rats were decapitated and brains were removed and homogenized in 5.5 vol of distilled water (15-20 strokes in a Potter type apparatus). The homogenate was centrifuged at 53,000 g for 60 min. The supernatant was discarded and the pellet was suspended in 3.3 vol of 0.5 g% Triton X-100. This suspension was stirred for 30min at room temperature and centrifuged again as described above. The pellet was discarded and, after addition of 0.05 g% sodium azide, the supernatant was used as the acetylcholinesterase fraction. The preparation was kept under refrigeration and enzyme activity remained stable for at least 6 months. Considering the economy, we have also tested and used ox brains to prepare the enzyme. The use of commercial pure enzyme (e.g. from Sigma Chemical Co.) is not recommended, since pure enzyme preparations do not activate phosphorothionate pesticides.

Acetylcholinesterase assay The method is based on the potentiometric measurement of the acetic acid formed from the enzymatic hydrolysis of acetylcholine. The assay routinely performed contained: 0.5 ml of 1.6 M MgC12 (Merck) in 0.16 M Tris-HC1 (Merck) buffer solution, pH 7.6; 3 ml of enzyme preparation corresponding to 20rag of protein and 16ml of the sample or distilled water (in the control mixture). The pH was then checked and, if necessary, corrected to 7.5-7.6 with dilute acetic acid or NaOH solution. Even though the enzyme activity is to be measured in the pH range 7.30-7.00, as

*All correspondence should be addressed to: Jayme Cunha Bastos, Instituto de Biologia--UERJ, Av. 28 de Setembro, 87 fundos, Vila Isabel, Rio de Janeiro, CEP 20551--Brazil. 835

V . L . F . CUNHABASTOSet al.

836

described below, the somewhat higher starting pH of the mixture allows the reaction to reach complete stability for the time the pH falls to 7.30. To achieve complete activation of phosphorothionate pesticides, these mixtures were incubated at 37°C for 120 min under continuous stirring. After incubation, the pH electrode was immersed in the mixture, the pH was checked again (7.5-7.6) and the acetylcholinesterase reaction was then started at room temperature by the addition of 0.5 ml of 0.28 M acetylcholine chloride (Merck). When the pH reached 7.30, the time (in seconds) for a further 0.30 unit decrease in pH (7.30-7.00) was recorded (we used a pH meter equipped with a digital display). Enzyme activity was considered proportional to the inverse of this time (apparent activity). When the influence of pH on the true reaction velocity was determined (as in Fig. 1), the amount of acetic acid formed was calculated by measuring the volume of 0.01 M acetic acid solution necessary to promote a decrease of 0.2 pH units in the incubation mixture, in the absence of substrate.

Elimination of the interference caused by unknown buffers in the sample In the presence of buffering capacity in the sample to be analysed, a previous test must be done. Firstly, the sample pH was adjusted to 7.30. Then, the volume of a 0.01 M acetic acid solution which induces a drop in pH from 7.30 to 7.00 in the control mixture (distilled water without substrate), was registered. Usually this volume was around I ml (10 #mol of acetic acid). Secondly, this volume of the acetic acid solution was added to the sample mixture (without substrate), which was also preadjusted to pH 7.30, and the final pH was registered. Then, the enzyme apparent activity was assayed in another aliquot of the same sample by measuring the time necessary for the pH to drop, after substrate addition, from 7.30 to this final registered pH. In some experiments enzyme inhibition was expressed in percentual units (relative to controls). However, for practical purposes, the enzyme inhibition capacity of unknown

samples can be expressed as concentration of a reference pesticide (e.g. parathion) by interpolating the values of percent inhibition into the values achieved in an appropriate standard inhibition curve of the enzyme using different concentrations of the reference pesticide (as in Table 5).

Protein assay Protein was determined by mixing a 0.1 ml protein sample with 4.4 ml of water, 0.2 ml of 20 g% NaOH and 0.3 ml of Follin--Ciocalteau reagent. The absorbance was read at 660 nm against a proper blank. Bovine serum albumin standards were run simultaneously.

Pesticide solutions Starting solutions of pesticides were freshly prepared by dissolving 10 mg of the compound in 0.I ml of benzene. Water was then added under vigorous stirring to a final volume of 1000 ml. This solution was diluted with water to the desired concentrations. Although benzene concentrations up to 0.1 ml/1 did not inhibit the enzyme, controls used in standard inhibition curves also contained corresponding amounts of this solvent. ChemicaLs' Insecticides (technical grade) were kindly donated by the Environmental Engineering Foundation of the State of Rio de Janeiro, Brazil. A high purity parathion, kindly donated by the Environmental Protection Agency, U.S.A., was used in one experiment. Pure acetylcholinesterase from electric eel was purchased from Sigma. All other reagents were of analytical grade. RESULTS AND DISCUSSION

Influence o['pH on enz.vme activity As the p r o p o s e d assay was based on a fixed p H decrease in the incubation mixture, a careful

A

o 2.0

~

x

E

"~ 1.5

~4 ~

o

._~

~

O

o

I i!

~.o <

1

# 6.4

6.6

6.8

7.0

7.2

Initial pH of mixture

7.4

i 7.6

B 7.8

8.0

8.2

Fig. 1. Effect of the pH on acetylcholinesterase activity. Apparent activity is the inverse of time necessary for a 0.2 pH unit decrease in a complete incubation mixture. True activity (#moles acetic acid/min) is the ratio between the amount of acetic acid that induces a 0.2 pH unit decrease of an incubation mixture (containing distilled water) where substrate was omitted and the time for the same pH drop in a complete mixture. Buffering capacity is the difference represented by the amount (in #moles) of acetic acid that induces a 0.2 pH unit fall of an incubation mixture (without substrate) in presence of the Tris-HCl buffer minus that amount which induces the 0.2 pH unit fall in absence of the same buffer. Each quantity plotted is the mean__+SEM of 5 experiments.

Cholinesterase detector for insecticides analysis of this parameter was necessary. The activity expressed by the inverse of time (apparent activity) was compared to the true activity, which was expressed in micromoles of acetic acid formed per minute. In both cases the reaction was continued over an interval of 0.20 pH units. As is shown in Fig. 1 the true activity increased smoothly from pH 6.20 to 8.20, as would be expected, since intrinsic activity is pH-dependent for all enzymes. The apparent activity also increased from pH6.40 up to 6.80, but it remained constant from pH 6.80 up to 7.40, decreasing thereafter. This peculiar behaviour of the apparent activity is caused by the increasing neutralizing capacity of the buffering system (Tris-HC1), as the pH increases. As a result, the time required for a pH change of 0.2 units increases with increasing pH. Increments in the true enzyme activity and in the buffering capacity are of the same magnitude from pH 6.60 to 7.40, as is also depicted in Fig. 1. Consequently, in the pH range 7.30-7.00, which was chosen for the routine assay, the apparent activity remained approximately constant. Difficulty with changing activity and buffering capacity could have been avoided by assaying at higher pH (where enzyme activity is constant) and using a buffer with p K near assay pH. This, however, is an experimental refinement. The presence of the Tris-HCl buffer in the assay not only facilitates the work, avoiding sudden pH alterations, but is also able to minimize the interference of weak buffers eventually found in unknown samples of natural waters. Linearity between apparent activity and enzyme concentration In the proposed assay conditions, the apparent activity, as expressed by the inverse of the time necessary to drop the pH from 7.30 to 7.00, is directly proportional to the enzyme concentration as is shown in Fig. 2. Furthermore, reported Km values for acetylcholine vary between 0.92 x 10 - 4 and 6.3 x 10 -4 M for acetylcholinesterase (Main, 1976), and in the control assay no more than 10 pmol of acetic acid is produced from the initial 140/~mol of substrate. As a result, it is assumed that the reaction follows a zero-order kinetics (i.e. saturation conditions hold over the entire assay period).

837

z.J'

1.2 LO i

0.6 ~

0,4

~

0.2

-

oi~

i

c W

5

10

Malathion Diazinon Dioxicarb

0.40 0.80 0.40 2.50 0.04 0.40

34.0 64.0 14.0 47.0 13.0 45.8

20

Fig. 2. Linearity between apparent enzyme activity and enzyme protein concentration. The assay was performed as described in the routine procedure using different protein concentrations of enzyme preparation. Each point is the mean + SEM of 4 experiments. Preincubation pesticides

of

the

enzyme

preparation

with

Organophosphorus insecticides bearing a thionophosphate group (e.g. parathion, malathion, diazinon) do not inhibit the cholinesterase directly. Such compounds are converted in vivo to their oxoanalogues which are the active inhibitors (Main, 1976). However, as shown in Table 1, the substances cited above did inhibit the activity of the enzyme preparation upon a preincubation period (2 h for maximal inhibition). A carbamate pesticide needed less than 30 min of preincubation to reach the maximal inhibition. Since the technical grade of pesticides that we were using could be contaminated by active derivatives, or some spontaneous activation could be taking place during preincubation, we designed the experiment shown in Fig. 3, in which the enzyme preparation was heat denatured before or immediately after preincubation with a parathion standard (obtained from EPA). Following preincubation, 0.1 IU of purified acetylcholinesterase (Sigma) was added and its activity assayed. Results clearly indicate that an intact preparation is necessary for parathion activation. Conversion of thionophosphate insecticides to their oxo-analogues is mainly performed by the liver, through the microsomal cytochrome P-450-dependent mixed function oxidase system (Neal, 1967; Ludke et aL, 1972; Sultatus et al., 1985; Tsuda et al., 1987; Sultatus, 1987). Other tissues, however,

Table 1. Inhibition of acetylcholinesteraseby some pesticidesfor different preincubation times % Inhibitionof acetylcholinesterasefor different preincubationtimes*t Concentration Pesticides (rag/l) 30 rain 60 min 120min 180min Parathion

15

Protein (rag)

50.2 78.5 16.0 52.6 30.7 46.0

58.5 88.1 28.2 62.0 31.6 46.0

58.0 90.0 28.0 62.0 31.5 45.6

*% Inhibitionrelativeto controls(distilledwater). The enzymewas assayedby the routine procedure. tThe maximum SEM value obtained from 5 experimentsof each concentration was 3%.

V . L . F . CUNHA BASTOSet al.

838

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o

A B C Fig. 3. Effect of heat denaturation on parathion activation. The experimental conditions were as follows: (A) parathion (0.4 rag/l) and enzyme preparation (20 nag) were incubated at 37°C for 120min. Subsequently, enzyme activity was assayed by the routine procedure. (B) Parathion (0.4 rag/l), enzyme preparation previously denatured at 100°C for 5 rain and purified AChE (0.1 IU) were incubated at the same conditions described above. Enzyme activity was subsequently assayed by the routine procedure. (C) Parathion (0.4mg/l) and enzyme preparation were incubated at 37°C for 120min. Then, the heat denaturation described above was performed and after addition of purified AChE (0.1 IU), enzyme activity was assayed by the routine procedure. Each quantity plotted is the mean + SEM of 4 experiments. including lung a n d brain, are also able to do so (Poore a n d Neal, 1972). The f o r m a t i o n o f p a r o x o n from p a r a t h i o n has been described for b r a i n microsomes a n d is a p p a r e n t l y catalysed by a mixed function oxidase system also d e p e n d e n t o n N A D P H a n d inhibited by C O ( N o r m a n a n d Neal, 1976; F o r s y t h a n d C h a m b e r s , 1989). O u r crude T r i t o n X-100extracted brain cholinesterase p r e p a r a t i o n u n d o u b t edly contains some adequate enzyme system for this activation. A more detailed study o f this activation m e c h a n i s m seen in T r i t o n X-100-extracted Table 2. Stability of the enzyme preparation Time Age of % Inhibition of previous the enzyme of acetyl incubation (min)* preparation (months) cholinesteraset 0 59.5 0 2 57.4 4 58.1 30

0 2 4

58.0 60.2 58.9

60

0 2 4

61.3 59.0 58.7

0 62.5 2 60.9 4 58.9 *Enzyme preparation was previously incubated at 37°C for the time shown in the presence of Tris--MgCl2, before the addition of a 0.4 mg/I solution of parathion (or distilled water in controls). A further 120 rain incubation was then performed, as described in the routine procedure. tControls were run simultaneously for each experiment. The maximum SEM value obtained from 7 experiments at each incubation was 1.8 %. 120

0.5

1,o Inhibitors

1.5

2.o

2.5

(mg/t)

Fig. 4. Inhibition of acetylcholinesterase by parathion (A A); malathion (O O); dioxicarb ( © O) and diazinon ( A &). Toxicants were added to the routine assay medium. Each point is the mean of 5 experiments. The highest variation obtained was + 4 % (SEM). brain p r e p a r a t i o n s will be the subject o f a n o t h e r communication.

Enzyme stability O u r enzyme p r e p a r a t i o n was quite stable, in c o n t r a s t with the p o o r stability observed in microsomal mixed function oxidation in liver microsomes ( L e a d b e a t e r a n d Davies, 1964). As s h o w n in Table 2, the p a r a t h i o n t r a n s f o r m i n g capacity was u n c h a n g e d when the enzyme itself was p r e i n c u b a t e d at 37°C before the addition o f the pesticide. Enzyme preparations kept frozen for 6 m o n t h s as well as lyophilized p r e p a r a t i o n s m a i n t a i n e d this property almost intact.

Kinetics of acetylcholinesterase inhibition B o t h c a r b a m a t e s and o r g a n o p h o s p h a t e s are k n o w n to act on acetylcholinesterase by c o m m o n m e c h a n i s m s of irreversible inhibition (Main, 1976). This fact greatly simplifies the a t t e m p t to detect minimal c o n c e n t r a t i o n s o f these substances, even using saturating substrate concentrations. Figure 4 Table 3. Pesticide concentration that inhibits 50% of the enzyme activity (IC~0) Pesticide IC~o (mg/l)* Dioxicarb 0.73 Malathion 2.06 Parathion 0.25 Diazinon 0.11 *Calculated by the Dixon plot (Segel, 1975) of the data shown in Fig. 3.

Cholinesterase detector for insecticides and Table 3 show the inhibition curves as well as the ICs0 values (concentration that inhibits 50% of the enzyme activity) for the four insecticides tested. It is worth noting that all of the pesticides tested inhibited the enzyme at least 20-24% at a concentration of 0.1 mg/l (the limit recommended by Environmental Protection Agency for public water supplies).

Table 5. Test of the method in inland waters and industrial effluents Sample site number* 1 II

2

Compensation for the interference introduced by the presence of extraneous buffers in the sample

3

Sample buffering capacityt

% Inhibition of cholinesterase:I

Parathion equivalents~

0 0 0 0

13.9 8.0 5.7 0

0.06 0.03 <0.03 --

0 0

0 0

0 0

9.0 6.0

0

The presence of strong buffers in an unknown sample would interfere with the results. However, this was overcome with the simple test described in the Materials and Methods. Table 4 indicates the results obtained when the enzyme assay was performed in the presence of parathion solutions containing different concentrations of phosphate buffer.

4

5

6

Practical evaluation of the method This method has been tested in samples collected at several sites of the Paraiba do Sul river, in the state of Rio de Janeiro, Brazil. This river is the main water source for the city of Rio de Janeiro and for a number of other smaller cities nearby. The area under test extended for about 100 km, from Furnas dam to the city of Barra do Pirai. It is considered to be critically polluted, not only by city sewage but also by effluent from 12 important chemical or metallurgy industries, including some pesticide plants. There, the levels of mercury, lead, cadmium, zinc, phenols and cyanide are frequently above recommended values, and, sometimes, massive fish death occurs. Results from the acetylcholinesterase test in selected sites within the area mentioned above are shown in Table 5. These data indicate that significant buffering interference was only detected in effluent from some chemical industries. Any weak buffering capacity found in river water was evidently masked by the internal buffer of the assay. So far, this enzymatic test has been performed in 161 water samples (rivers and lakes) from several sites in the state of Rio de Janeiro, which were also analysed by FEEMA (Environmental Engineering Foundation of the State of Rio de Janeiro) for the presence of organophosphorous pesticides by gas chromatography. Most samples were negative in both methods; 14 samples did inhibit the enzyme in the range 8-25% and, when analysed

839

1.2 0.9 0.8

1.0

0

0

--

0 0

0 0

---

0

17.4

0.07

0.7 0.2

62.4 12.0

0.44 0.05

*Three samples from each site were collected at intervals of 3-4 months during the year of 1987. tExpressed as the difference between the volume (ml) of a 0.01 M acetic acid solution which induces a pH decrease from 7.30 to 7.00 in the sample mixture (without substrate) and the volume inducing the same pH decrease in the control mixture (distilled water). ~Whenever the presence of buffers was detected in the sample, the assay was corrected as indicated in the text. §Expressed as mg/I of the pesticide parathion. II1, Water from the confluence of Paraiba do Sul and Piabanha rivers near cultivated area; 2, water supply for the city of Resonde on the Paraiba do Sul river; 3, water supply for the city of Rio de Janeiro (Guandu reservoir); 4, effluent of a chemical industry producing organophosphorus pesticides on the Pirapetinga river; 5, effluent of a metallurgy industry on the Paraiba do Sul river, near the city of Volta Redonda; 6, effluent of a chemical industry producing insecticides of several classes (Paraiba do Sul river near Resende).

by gas chromatography, showed the presence of one or several of the organophosphates parathion, malathion, diazinon, fenitrothion and DDVP in concentrations around 0.04 mg/1 or less. The proposed test is sensitive, reproducible and not expensive (as the enzyme preparation can also be obtained from bovine brain), only demanding a digital pH meter and a water bath as equipment. It can also be easily adapted to kits and used in small field laboratories or mobile units, which could routinely cover, all areas of potential hazard. Although the method described here is not capable of distinguishing between individual pesticides in a mixture (which may be frequently found in environmental

Routine assay'["

Corrected assayt

% Inhibition

-1.068 + 0.022 (6) 1.060 + 0.013 (8) 1.068 + 0.004 (5) -0.900 + 0.011 (4)

0 0 0 0 16.0 15.5

phosphate + parathion*

--

0.881 + 0.024 (7)

17.2

4 mM phosphate + parathion*

--

0.908 __.0.007 (4)

14.7

2 mM

-0.60 0.63 0.38

1.065 + 0.027 (8) ---0.894 + 0.019 (5) 1

Distilled water 1 mM phosphate 2 mM phosphate 4 mM phosphate Parathion* I mM phosphate + parathion*

0.03 < 0.03

73.0 75.2 58.3

Table 4. Enzyme assay in the presence of buffers in the water Additions

---

*Parathion concentration = 0.08 mg/I. tResults are the m e a n _ SEM and are expressed as apparent activity (s- x 102). The number of experiments is shown in parentheses.

840

V . L . F . CUNI-tAB^STOSet al.

samples) it can be used specially as a practical alarm system a n d also to select water samples to be analysed t h r o u g h gas c h r o m a t o g r a p h y for determining the exact c o n c e n t r a t i o n a n d the chemical structure of the pesticides which have inhibited the enzyme in the test. The m e t h o d c a n n o t be used as a confirmatory test for the presence of o r g a n o p h o s p h o r u s or c a r b a m a t e c o m p o u n d s in the water, since the possibility of an eventual enzyme inhibition by a high c o n c e n t r a t i o n o f some other water c o n t a m i n a n t c a n n o t be discarded. Then, the a p p r o p r i a t e a p p r o a c h for the identification of such c o n t a m i n a n t s must be taken. Even in this case, this enzymatic test still holds as an indicator o f water c o n t a m i n a t i o n . REFERENCES

Benke G. M., Cheever K. L., Mirer F. E. and Murphy S. D. (1974) Comparative toxicity, anticholinesterase action and metabolism of methyl parathion and parathion in sunfish and mice. Toxic. appl. Pharmac. 28, 97-109. Forsyth C. S. and Chambers J. E. (1989) Activation and degradation of the phosphorothionate insecticides parathion and EPN by rat brain. Biochem. Pharrnac. 38, 1597-1603. Leadbeater L. and Davies D. R. (1964) The stability of the drug metabolizing enzymes of liver microsomal preparations. Biochem. Pharmac. 13, 1607-1617. Ludke L. J., Gibson J. R. and Luske C. I. (1972) Mixed function oxidase activity in freshwater fishes: aldrin epoxidation and parathion activation. Toxic appl. Pharmac. 21, 89-97. Main A. R. (1976) Structure and inhibitors of cholinesterase. In Biology of Cholinergic Function (Edited by

Goldberg M. and Hanin I.), p. 269. Raven Press, New York. Murphy S. D. (1967) Malathion inhibition of esterases as a determinant of malathion toxicity. J. Pharmac. exp. Ther. 156, 352-365. Nakatsugawa T. and Dahm P. A. (1967) Microsomal metabolism of parathion. Biochem. Pharmac. 16, 25-38. Neal R. A. (1967) Studies on the metabolism of diethyl-4nitrophenyl phosphorothionate (parathion) in vitro. Biochem. J. 108, 183-191. Norman B. J. and Neal R. A. (1976) Examination of the metabolism in vitro of parathion (diethyl p-nitrophenyl phosphorothionate) by rat lung and brain. Biochem. Pharmac. 25, 37-45. Norton T. R. (1975) Metabolism of toxic substances. In Toxicology-The Basic Science of Poisons (Edited by Casarett L. J. and Doull J.). Macmillan, New York. Poore R. E. and Neal R. A. (1972) Evidence for extrahepatic metabolism of parathion. Toxic. appl. Pharmac. 22, 759--768. Rakonczay Z., Mallot J., Schenk H., Vincendon G. and Zametta J. P. (1981) Purification and properties of the membrane-bound acetylcholinesterase from adult rat brain. Biochim. biophys. Acta 657, 243-256. Segel I. H. (1975) Enzyme Kinetics, pp. 134-~135. Wiley. New York. Sultatus L. G. (1987) The role of liver in mediating the acute toxicity of the pesticide methyl parathion in the mouse. Drug Metabol. Dispos. 15, 613~517. Sultatus L. G., Minor L. D. and Murphy S. D. (1985) Metabolic activation of phosphorothioate pesticides. Role of liver. J. Pharmac. exp. Ther. 232, 624-4i28. Tsuda S., Scherman W., Rosenberg A., Timoszyk J., Becker J. M., Keadtisuke S. and Nakatsugawa T. (1987) Rapid periportal uptake and translobular migration of parathion with concurrent metabolism in rat liver in vivo. Pest. Biochem. Physiol. 28, 201-215.