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Brain Acetylcholinesterase As A N Detector of Organophosphorus A N D Carbamate Insecticides in Water
Brain Acetylcholinesterase As A N Detector of Organophosphorus A N D Carbamate Insecticides in Water
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Printed in Great Britain. All rights reserved Copyright © 1991 Pergamon Press plc
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.
835
836 V . L . F . CUNHABASTOSet al.
described below, the somewhat higher starting pH of the samples can be expressed as concentration of a reference
mixture allows the reaction to reach complete stability for pesticide (e.g. parathion) by interpolating the values of
the time the pH falls to 7.30. To achieve complete activation percent inhibition into the values achieved in an appropriate
of phosphorothionate pesticides, these mixtures were incu- standard inhibition curve of the enzyme using different
bated at 37°C for 120 min under continuous stirring. After concentrations of the reference pesticide (as in Table 5).
incubation, the pH electrode was immersed in the mixture,
the pH was checked again (7.5-7.6) and the acetylcholin- Protein assay
esterase reaction was then started at room temperature by Protein was determined by mixing a 0.1 ml protein sample
the addition of 0.5 ml of 0.28 M acetylcholine chloride with 4.4 ml of water, 0.2 ml of 20 g% NaOH and 0.3 ml of
(Merck). When the pH reached 7.30, the time (in seconds) Follin--Ciocalteau reagent. The absorbance was read at
for a further 0.30 unit decrease in pH (7.30-7.00) was 660 nm against a proper blank. Bovine serum albumin
recorded (we used a pH meter equipped with a digital standards were run simultaneously.
display). Enzyme activity was considered proportional to
the inverse of this time (apparent activity). When the Pesticide solutions
influence of pH on the true reaction velocity was determined Starting solutions of pesticides were freshly prepared by
(as in Fig. 1), the amount of acetic acid formed was dissolving 10 mg of the compound in 0.I ml of benzene.
calculated by measuring the volume of 0.01 M acetic acid Water was then added under vigorous stirring to a final
solution necessary to promote a decrease of 0.2 pH units in volume of 1000 ml. This solution was diluted with water to
the incubation mixture, in the absence of substrate. the desired concentrations. Although benzene concen-
trations up to 0.1 ml/1 did not inhibit the enzyme, controls
Elimination of the interference caused by unknown buffers in used in standard inhibition curves also contained corre-
the sample sponding amounts of this solvent.
In the presence of buffering capacity in the sample to be ChemicaLs'
analysed, a previous test must be done. Firstly, the sample
pH was adjusted to 7.30. Then, the volume of a 0.01 M Insecticides (technical grade) were kindly donated by the
acetic acid solution which induces a drop in pH from 7.30 Environmental Engineering Foundation of the State of Rio
to 7.00 in the control mixture (distilled water without de Janeiro, Brazil. A high purity parathion, kindly donated
substrate), was registered. Usually this volume was around by the Environmental Protection Agency, U.S.A., was used
I ml (10 #mol of acetic acid). Secondly, this volume of the in one experiment. Pure acetylcholinesterase from electric
acetic acid solution was added to the sample mixture eel was purchased from Sigma. All other reagents were of
(without substrate), which was also preadjusted to pH 7.30, analytical grade.
and the final pH was registered. Then, the enzyme apparent
activity was assayed in another aliquot of the same sample RESULTS AND DISCUSSION
by measuring the time necessary for the pH to drop, after
substrate addition, from 7.30 to this final registered pH. Influence o['pH on enz.vme activity
In some experiments enzyme inhibition was expressed in
percentual units (relative to controls). However, for practi- As the p r o p o s e d assay was based on a fixed
cal purposes, the enzyme inhibition capacity of unknown 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
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
samples) it can be used specially as a practical alarm Goldberg M. and Hanin I.), p. 269. Raven Press, New
York.
system a n d also to select water samples to be analysed
Murphy S. D. (1967) Malathion inhibition of esterases as a
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 determinant of malathion toxicity. J. Pharmac. exp. Ther.
exact c o n c e n t r a t i o n a n d the chemical structure of the 156, 352-365.
pesticides which have inhibited the enzyme in the test. Nakatsugawa T. and Dahm P. A. (1967) Microsomal
The m e t h o d c a n n o t be used as a confirmatory test metabolism of parathion. Biochem. Pharmac. 16, 25-38.
Neal R. A. (1967) Studies on the metabolism of diethyl-4-
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
nitrophenyl phosphorothionate (parathion) in vitro.
c o m p o u n d s in the water, since the possibility of an Biochem. J. 108, 183-191.
eventual enzyme inhibition by a high c o n c e n t r a t i o n Norman B. J. and Neal R. A. (1976) Examination of the
o f some other water c o n t a m i n a n t c a n n o t be dis- metabolism in vitro of parathion (diethyl p-nitrophenyl
carded. Then, the a p p r o p r i a t e a p p r o a c h for the phosphorothionate) by rat lung and brain. Biochem.
Pharmac. 25, 37-45.
identification of such c o n t a m i n a n t s must be taken. Norton T. R. (1975) Metabolism of toxic substances. In
Even in this case, this enzymatic test still holds as an Toxicology-The Basic Science of Poisons (Edited by
indicator o f water c o n t a m i n a t i o n . Casarett L. J. and Doull J.). Macmillan, New York.
Poore R. E. and Neal R. A. (1972) Evidence for extrahepatic
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