Wat. Res. Vol. 25, No. 7, pp. 835-840, 1991 0043-1354/91 $3.00 + 0.

00
Printed in Great Britain. All rights reserved 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 brain acetylcholinesterase preparation. This com-

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 Foun-
dation), for the development of simple and inexpen-
sive 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 depolariz-
ation 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
*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.
munication 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 re-
moved 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 acetylcholin-
esterase fraction. The preparation was kept under refriger-
ation 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 com-
mercial 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 corre-
sponding 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

835
836 V . L . F . CUNHABASTOSet al.

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 incu-
bated 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 acetylcholin-
esterase 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 practi-
cal 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 concen-
trations up to 0.1 ml/1 did not inhibit the enzyme, controls
used in standard inhibition curves also contained corre-
sponding 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

o
2.0 x

~ E
~4 O
"~ 1.5 ~
o ._~

~ o
I

i
~.o
i!
<

#
B
6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2
Initial pH of mixture
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 837

z.J'
1.2
analysis of this parameter was necessary. The activity
expressed by the inverse of time (apparent activity) LO
was compared to the true activity, which was i
expressed in micromoles of acetic acid formed per
minute. In both cases the reaction was continued over - oi~ i
0.6
an interval of 0.20 pH units. As is shown in Fig. 1 the
true activity increased smoothly from pH 6.20 to ~ 0,4

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, de-
creasing thereafter. This peculiar behaviour of the
apparent activity is caused by the increasing neu-
tralizing 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 increas-
ing 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, how-
ever, 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 acetyl-
choline 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).
~ 0.2
c
W
5 10 15 20
Protein (rag)
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 of the enzyme preparation with
pesticides
Organophosphorus insecticides bearing a thiono-
phosphate group (e.g. parathion, malathion, diazi-
non) do not inhibit the cholinesterase directly. Such
compounds are converted in vivo to their oxo-
analogues 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 maxi-
mal 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 immedi-
ately 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 indi-
cate 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-depen-
dent 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
Concentration different preincubationtimes*t
Pesticides (rag/l) 30 rain 60 min 120min 180min
Parathion 0.40 34.0 50.2 58.5 58.0
0.80 64.0 78.5 88.1 90.0
Malathion 0.40 14.0 16.0 28.2 28.0
2.50 47.0 52.6 62.0 62.0
Diazinon 0.04 13.0 30.7 31.6 31.5
Dioxicarb 0.40 45.8 46.0 46.0 45.6
*% Inhibitionrelativeto controls(distilledwater). The enzymewas assayedby the routine
procedure.
tThe maximum SEM value obtained from 5 experimentsof each concentration was 3%.
838 V . L . F . CUNHA BASTOSet al.

60 loo
5o I
-+-

4O

.o 30
"F
C

uJ 2o
u
<
~o

-~
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 incu-
bated at 37°C for 120min. Then, the heat denaturation 0.5 1,o 1.5 2.o 2.5
described above was performed and after addition of Inhibitors (mg/t)
purified AChE (0.1 IU), enzyme activity was assayed by the Fig. 4. Inhibition of acetylcholinesterase by parathion
routine procedure. Each quantity plotted is the mean + SEM (A A); malathion (O O); dioxicarb ( © O)
of 4 experiments. and diazinon ( A &). Toxicants were added to the
routine assay medium. Each point is the mean of 5 exper-
iments. The highest variation obtained was + 4 % (SEM).
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
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
from p a r a t h i o n has been described for b r a i n micro-
communication.
somes a n d is a p p a r e n t l y catalysed by a mixed func-
tion oxidase system also d e p e n d e n t o n N A D P H a n d Enzyme stability
inhibited by C O ( N o r m a n a n d Neal, 1976; F o r s y t h
O u r enzyme p r e p a r a t i o n was quite stable, in
a n d C h a m b e r s , 1989). O u r crude T r i t o n X-100-
c o n t r a s t with the p o o r stability observed in micro-
extracted 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 somal mixed function oxidation in liver microsomes
this activation. A more detailed study o f this acti- ( L e a d b e a t e r a n d Davies, 1964). As s h o w n in Table 2,
vation m e c h a n i s m seen in T r i t o n X-100-extracted 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 prep-
Table 2. Stability of the enzyme preparation
arations kept frozen for 6 m o n t h s as well as
Time Age of % Inhibition 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
of previous the enzyme of acetyl
incubation (min)* preparation (months) cholinesteraset almost intact.
0 59.5
0 2 57.4 Kinetics of acetylcholinesterase inhibition
4 58.1
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
0 58.0 k n o w n to act on acetylcholinesterase by c o m m o n
30 2 60.2 m e c h a n i s m s of irreversible inhibition (Main, 1976).
4 58.9
This fact greatly simplifies the a t t e m p t to detect
0 61.3 minimal c o n c e n t r a t i o n s o f these substances, even
60 2 59.0 using saturating substrate concentrations. Figure 4
4 58.7
0 62.5 Table 3. Pesticide concentration that
120 2 60.9 inhibits 50% of the enzyme activity
4 58.9 (IC~0)
*Enzyme preparation was previously incubated at 37°C for the time Pesticide IC~o (mg/l)*
shown in the presence of Tris--MgCl2, before the addition of a Dioxicarb 0.73
0.4 mg/I solution of parathion (or distilled water in controls). A Malathion 2.06
further 120 rain incubation was then performed, as described in Parathion 0.25
the routine procedure. Diazinon 0.11
tControls were run simultaneously for each experiment. The
maximum SEM value obtained from 7 experiments at each *Calculated by the Dixon plot (Segel,
incubation was 1.8 %. 1975) of the data shown in Fig. 3.
 Cholinesterase detector for insecticides 839

and Table 3 show the inhibition curves as well as the Table 5. Test of the method in inland waters and industrial effluents
ICs0 values (concentration that inhibits 50% of the Sample
Sample site buffering % Inhibition of Parathion
enzyme activity) for the four insecticides tested. It is number* capacityt cholinesterase:I equivalents~
worth noting that all of the pesticides tested inhibited
0 13.9 0.06
the enzyme at least 20-24% at a concentration of 1 II 0 8.0 0.03
0.1 mg/l (the limit recommended by Environmental 0 5.7 <0.03
0 0 --
Protection Agency for public water supplies).
2 0 0 --
0 0 --
Compensation for the interference introduced by the
0 9.0 0.03
presence of extraneous buffers in the sample 3 0 6.0 < 0.03
The presence of strong buffers in an unknown 0 1.0 --
sample would interfere with the results. However, this 1.2 73.0 0.60
4 0.9 75.2 0.63
was overcome with the simple test described in the 0.8 58.3 0.38
Materials and Methods. Table 4 indicates the results
0 0 --
obtained when the enzyme assay was performed 5 0 0 --
in the presence of parathion solutions containing 0 0 --
different concentrations of phosphate buffer. 0 17.4 0.07
6 0.7 62.4 0.44
0.2 12.0 0.05
Practical evaluation of the method
*Three samples from each site were collected at intervals of 3-4
This method has been tested in samples collected at months during the year of 1987.
several sites of the Paraiba do Sul river, in the state 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
of Rio de Janeiro, Brazil. This river is the main water 7.00 in the sample mixture (without substrate) and the volume
source for the city of Rio de Janeiro and for a number inducing the same pH decrease in the control mixture (distilled
of other smaller cities nearby. The area under test water).
~Whenever the presence of buffers was detected in the sample, the
extended for about 100 km, from Furnas dam to the assay was corrected as indicated in the text.
city of Barra do Pirai. It is considered to be critically §Expressed as mg/I of the pesticide parathion.
II1, Water from the confluence of Paraiba do Sul and Piabanha rivers
polluted, not only by city sewage but also by effluent near cultivated area; 2, water supply for the city of Resonde on
from 12 important chemical or metallurgy industries, the Paraiba do Sul river; 3, water supply for the city of Rio de
including some pesticide plants. There, the levels of Janeiro (Guandu reservoir); 4, effluent of a chemical industry
producing organophosphorus pesticides on the Pirapetinga river;
mercury, lead, cadmium, zinc, phenols and cyanide 5, effluent of a metallurgy industry on the Paraiba do Sul river,
are frequently above recommended values, and, near the city of Volta Redonda; 6, effluent of a chemical industry
sometimes, massive fish death occurs. Results from producing insecticides of several classes (Paraiba do Sul river
near Resende).
the acetylcholinesterase test in selected sites within
the area mentioned above are shown in Table 5.
These data indicate that significant buffering interfer- by gas chromatography, showed the presence of
ence was only detected in effluent from some chemical one or several of the organophosphates parathion,
industries. Any weak buffering capacity found in malathion, diazinon, fenitrothion and DDVP in
river water was evidently masked by the internal concentrations around 0.04 mg/1 or less.
buffer of the assay. The proposed test is sensitive, reproducible and not
So far, this enzymatic test has been performed in expensive (as the enzyme preparation can also be
161 water samples (rivers and lakes) from several obtained from bovine brain), only demanding a
sites in the state of Rio de Janeiro, which were digital pH meter and a water bath as equipment. It
also analysed by FEEMA (Environmental Engin- can also be easily adapted to kits and used in small
eering Foundation of the State of Rio de Janeiro) field laboratories or mobile units, which could rou-
for the presence of organophosphorous pesticides tinely cover, all areas of potential hazard. Although
by gas chromatography. Most samples were nega- the method described here is not capable of dis-
tive in both methods; 14 samples did inhibit the tinguishing between individual pesticides in a mixture
enzyme in the range 8-25% and, when analysed (which may be frequently found in environmental

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

Additions Routine assay'[" Corrected assayt % Inhibition
Distilled water 1.065 + 0.027 (8) -- 0
1 mM phosphate -- 1.068 + 0.022 (6) 0
2 mM phosphate -- 1.060 + 0.013 (8) 0
4 mM phosphate -- 1.068 + 0.004 (5) 0
Parathion* 0.894 + 0.019 (5) -- 16.0
I mM phosphate + parathion* 1 0.900 + 0.011 (4) 15.5
2 mM phosphate + parathion* -- 0.881 + 0.024 (7) 17.2
4 mM phosphate + parathion* -- 0.908 __.0.007 (4) 14.7
*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 dis-
carded. 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 .
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