Full text of DOI:10.1002/etc.5620211109

Environmental Toxicology and Chemistry, Vol. 21, No. 11, pp. 2319–2325, 2002
2002 SETAC
Printed in the USA
᭧
0730-7268/02 $9.00
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LIZARD CHOLINESTERASES AS BIOMARKERS OF PESTICIDE EXPOSURE:
ENZYMOLOGICAL CHARACTERIZATION
J
UAN C. SANCHEZ-HERNANDEZ* and BEATRIZ
MORENO SANCHEZ
Laboratory of Ecotoxicology, Department of Environmental Science, University of Castilla–La Mancha, Toledo, Spain
(
Received 24 October 2001; Accepted 4 April 2002)
Abstract—Here we report the results of a study conducted to elucidate the enzymological characteristics of lizard cholinesterases
(ChEs) in order to use them as potential biomarkers for pesticide exposure. Serum and brain tissue of the lizard Gallotia galloti
were used as ChE sources and in vitro assays were performed to identify acetylcholinesterase (AChE) and butyrylcholinesterase
(BChE) activities. The pH, substrate concentration, and specificity for ChE assays as well as the response of serum BChE to the
reactivating agent pyridine-2-aldoxime methochloride (2-PAM) were also investigated in order to assess the possibilities of this
methodology in biomonitoring programs. By the use of selective substrates and the inhibitor tetraisopropyl pyrophosphoramide,
AChE and BChE activities were identified in lizard serum, while brain contained solely AChE. Likewise, butyrylthiocholine iodide
was the optimum substrate for determining BChE activity and acetylthiocholine iodide for assaying both serum and brain AChE
activities. The optimal ranges of pH and substrate concentrations were 7.5 to 8.0 and 5 to 10 mM, respectively. Serum was incubated
with different doses of the organophosphorus (OP) compounds dichlorvos and paraoxon and subsequently incubated in the presence
Ϫ
2
Ϫ4
of two concentrations of 2-PAM (2
ϫ 10 or 2 ϫ 10 M). Reactivation rate of phosphorylated BChE was related to the degree
Ϫ
of inhibition of BChE and the dose of 2-PAM. It was found that a 90-min incubation time with 2
ϫ
10 4 M of 2-PAM satisfactorily
increased the OP-inhibited BChE activity. The enzymological properties of serum BChE activity and its in vitro reactivation in the
presence of 2-PAM represent the initial justification for its use in monitoring OP contamination in the field.
Keywords—Lizard
Acetylcholinesterase
Butyrylcholinesterase
Enzymological properties
Pyridine-2-aldoxime
methochloride
INTRODUCTION
choline, such as butyryl- and propionylesters [2,5]. The use
of selective substrates and inhibitors makes it possible to dis-
tinguish between AChE and BChE activities when both en-
zymes occur in the same target tissue.
Cholinesterases constitute a group of hydrolases highly sen-
sitive to toxicity by organophosphorus (OP) and carbamate
pesticides [1,2]. Because of this, inhibition of cholinesterases
(ChEs) is commonly used as an index of pesticide exposure
in biomonitoring programs [3,4]. Acetylcholinesterase (AChE;
EC 3.1.1.7) is the widely used ChE in the assessment of pes-
ticide exposure. It participates in the regulation of cholinergic
synapses of the nervous system and the neuromuscular end-
plate. Its inhibition by anti-ChE pesticides (OPs and carba-
mates) originates a wide range of clinical manifestations (e.g.,
convulsions, tremors, lacrimation, coma). In the last decade,
blood butyrylcholinesterase (BChE; EC 3.1.1.8) emerged as a
suitable biomarker for exposure to ChE-inhibiting pesticides
[2,4–6] due to its high sensitivity to inhibition by this class
of pesticides and its stability in various measurement tech-
niques.
Birds, fish, and aquatic invertebrates are the organisms most
often used to assess exposure to anti-ChE agrochemicals in
field studies [7–14]. Tissue distribution of ChEs and the op-
timal conditions for their analysis are reported for some species
[15–19]. Several sources of variability linked to experimental
conditions, such as homogenization of tissues or factors as-
sociated with instrumentation, can affect ChE measurement
[19]. In addition, the biochemical properties and tissue distri-
bution of ChEs must be taken into account to avoid errors
during processing and interpretation of data. For example, it
is well known that BChE is a nonspecific ChE capable of
hydrolyzing a number of cholinesters in addition to acetyl-
In general, field studies of ChE activity related to pesticide-
polluted environments compare the mean ChE activities among
sampling areas or sampling times. Usually, a diagnostic thresh-
old (2 SD below the mean ChE activity) is calculated in a
nonexposed group of individuals [6,20]. Individuals having
lower ChE activity than the threshold are considered exposed
to ChE-inhibiting pesticides. It is therefore necessary to an-
alyze a good representative control group in order to obtain
an accurate diagnostic threshold. Reactivation of ChE activity
using chemical agents such as pralidoxime (2-PAM) has be-
come a complementary indicator of pesticide exposure, par-
ticularly for OPs [2]. Yet this presents a set of limitations—
reactivation of OP-inhibited ChE depends mainly on OP type
and time elapsed since exposure [2,5,21]. Despite these dis-
advantages and limited data availability, chemical reactivation
of ChE activity has been recognized as a potential indicator
of wildlife exposure to anti-ChE pesticides [20].
From an ecotoxicological perspective, reptiles, like am-
phibians, have not been studied in depth [22]. Several com-
prehensive reviews have stressed the need for increasing the
knowledge about toxic effects of environmental pollutants on
herpetofauna [23–25]. Data on toxic effects of OP pesticides
on the lizard Gallotia galloti provide evidence that this or-
ganism shows an array of desirable features that make it a
suitable sentinel of pollution. Serum BChE activity of G. gal-
loti is a very sensitive biomarker of pesticide exposure, with
a slow recovery rate (over weeks) after acute OP exposure
[26]. In addition, serum BChE activity shows a significant
* To whom correspondence may be addressed
(jcsanche@amb-to.uclm.es).
2319

2320
Environ. Toxicol. Chem. 21, 2002
J.C. Sanchez-Hernandez and B.M. Sanchez
relationship with brain AChE activity after acute OP exposure,
which appears to be dependent on the type of OP [27]. This
may ultimately facilitate predicting inhibition of brain AChE
activity in a nonlethal manner.
Effect of pH and substrate concentration
Both AChE and BChE activities were assayed using a 25
mM Tris-HCl, 1 mM CaCl2 buffer at pH values from 5.0 to
11.0. The AChE activity was assayed using AcSCh substrate
The aim of this study was to characterize the AChE and
BChE activities in the lizard G. galloti in order to use them
as potential biochemical biomarkers. Distribution of ChEs was
determined in the target tissues, serum, and brain, commonly
used for ChE activity assays, and optimal conditions for ChE
measurements were established. We also investigated the re-
sponse of serum ChE activity of G. galloti to the reactivating
ability of 2-PAM as a diagnostic for OP exposure. Factors that
could affect enzyme reactivation such as concentration of 2-
PAM, inhibition of serum ChE, and OP type were examined.
after incubation of the samples for 30 min at 25
Њ
C in the
Ϫ
4
presence of 5
ϫ 10 M iso-OMPA; this concentration was
found to be sufficient for full inhibition of BChE activity (see
Results section).
Initial conditions for assaying ChE activities (25 mM Tris-
HCl, 1 mM CaCl2, pH 7.6, 25ЊC) were used to investigate the
effect of different substrate concentrations. The BChE activity
was determined using BuSCh (a selective substrate for BChE
activity), and AChE activity was assayed using AcSCh after
iso-OMPA incubation. Concentrations of substrates (BuSCh
Ϫ
3
Ϫ3
and AcSCh) ranged between 0.01
ϫ
10 and 50
ϫ
10
M
MATERIALS AND METHODS
FC. Butyrylthiocholine iodide was dissolved in 1:1 (v/v) dH2O:
ethanol, and the concentration of ethanol was always below
5% in the incubation medium. All determinations were made
Reagents and tissue preparation
Acetylthiocholine iodide (AcSCh), butyrylthiocholine io-
dide (BuSCh), propionylthiocholine iodide (PrSCh), tetraiso-
by triplicate at 25ЊC.
propyl pyrophosphoramide (iso-OMPA), 2-PAM, and 5,5Ј-di-
thiobis-2-nitrobenzoic acid were supplied by Sugelabor (Bar-
celona, Spain). Organophosphate pesticides or metabolites
Chemical reactivation of ChEs
Chemical reactivation of serum BChE activity in the pres-
ence of 2-PAM was investigated in order to develop it as a
methodology for assessing OP exposure in the lizard G. galloti
Time to full recovery of OP-inhibited enzyme was examined
under different concentrations of 2-PAM, two types of OPs
(dichlorvos and paraoxon), and different degrees of BChE in-
hibition. Two aliquots of serum were separately incubated with
(Ͼ98% purity) paraoxon (O,O-diethyl-p-nitrophenyl phos-
.
phate) and dichlorvos (2,2-dichlorovinyl dimethyl phosphate)
were purchased from Chem Service (West Chester, PA, USA).
Dimethyl sulfoxide and ethanol were obtained from Panreac
Quimica (Barcelona, Spain). Solutions of each substrate, in-
hibitor, and pesticide were prepared daily as required. Pesticide
solutions were initially dissolved in dimethyl sulfoxide and
the final concentration of the solvent was kept below 5% in
the incubation medium.
Ϫ
6
Ϫ
6
Ϫ5
dichlorvos or paraoxon (2.2
M FC) for 15 min at 25 C. A third aliquot was spiked with
an equal volume of dH2O as control. Subsequently, a solution
ϫ 10 , 5.4 ϫ 10 , or 1.1 ϫ 10
Њ
Ϫ
Ϫ
of 2-PAM (2
ϫ ϫ
10 2 or 2 10 4 M FC) was added and BChE
Samples of serum and brain were obtained from six adult
activity was measured every 10 min over a 90-min time period.
specimens of G. galloti (10.0
caught in the Tenerife Island (Canary Islands, Spain). Serum
was separated by centrifugation at 10,000 for 10 min at 4 C.
Ϯ 1.4 cm [mean length Ϯ SD])
RESULTS
g
Њ
Brain samples were homogenized in 0.1% Triton X-100 (Pan-
reac Quimica) in 25 mM Tris-HCl (pH 8.0). Homogenates were
Tissue distribution of ChEs
The use of selective substrates and inhibitors allowed us
to distinguish between AChE and BChE activities in the an-
alyzed tissues. The treatment of the serum samples with dif-
ferent concentrations of iso-OMPA demonstrated the presence
of both AChE and BChE (Fig. 1A). There was an iso-OMPA-
sensitive ChE fraction and an iso-OMPA-resistant fraction, the
activity of which remained unalterable at iso-OMPA concen-
then centrifuged at 5,000
g
for 5 min at 4
Њ
C. Serum and brain
homogenates were then kept at
Ϫ80ЊC until analysis.
Cholinesterase activity assay
Cholinesterase activities were determined colorimetrically
by the Ellman method [28] using a Spectronic Genesis-5 spec-
trophotometer (Spectronic Instruments, Rochester, NY, USA).
Ϫ
4
trations higher than 10 M. In addition, the iso-OMPA-sen-
sitive fraction preferentially cleaved BuSCh, a substrate be-
lieved to be specific for BChE activity, while the iso-OMPA-
resistant fraction hydrolyzed AcSCh at a higher rate than
PrSCh and did not hydrolyze BuSCh. These results indicate
that the iso-OMPA-resistant fraction was probably AChE ac-
tivity, whereas the iso-OMPA-sensitive fraction could be
BChE activity. Brain ChE activity was not affected over a
Samples were preincubated for 2 min with 5,5
Ј
-dithiobis-2-
10 4 M final concentration [FC]) in 25
mM Tris-HCl, 1 mM CaCl2 (pH 7.6) before the substrate was
Ϫ
nitrobenzoic acid (3
ϫ
Ϫ
added (2
recorded at 410 nm for 2 min and enzyme kinetics were mea-
sured in triplicate at 25 C. Serum ChE activity was expressed
ϫ
10 3 M FC). The variations in optical density were
Њ
as micromoles substrate hydrolyzed per minute per milliliter
and brain ChE activity as micromoles per minute per milligram
of total protein, using the extinction coefficient 13,600/cm/M.
Protein concentrations were determined by the Bradford meth-
od [29] using bovine serum albumin as a standard.
Ϫ
3
Ϫ6
wide range of iso-OMPA concentrations (10 to 10 M), its
activity remaining invariable on both substrates, AcSCh and
PrSCh (Fig. 1B).
In order to determine the contribution of each ChE (AChE
and BChE) to total ChE activity and to ascertain which sub-
strate is preferentially hydrolyzed by ChEs, six aliquots of
Under these initial conditions, AChE and BChE activities
were determined with the aid of selective substrates (BuSCh,
AcSCh, or PrSCh) and the inhibitor iso-OMPA (believed to
be selective for BChE activity). Sample aliquots were incu-
serum and brain homogenates were incubated with 10 4 M iso-
Ϫ
OMPA and ChE activities assayed using AcSCh, BuSCh, and
PrSCh. Table 1 summarizes mean (Ϯ SD) ChE activities in
Ϫ
6
Ϫ3
bated with varying concentrations (from 10 to 10 M) of
iso-OMPA for 30 min at 25
substrates was recorded.
Њ
C and ChE activity on the different
both tissues. Butyrylcholinesterase represented the 83% of to-
tal serum ChE activity, while in the brain, AChE is the prin-

Characterization of lizard cholinesterases
Environ. Toxicol. Chem. 21, 2002
2321
Fig. 2. Effect of pH on serum (A) and brain (B) cholinesterase ac-
tivities of Gallotia galloti. Each point corresponds to the average of
three determinations.
Fig. 1. Dose–response curves for the inhibitory action of tetraisopro-
pyl pyrophosphoramide (iso-OMPA), a selective inhibitor for butyr-
ylcholinesterase (BChE) activity, on serum (A) and brain (B) cholin-
esterase (ChE) activities of Gallotia galloti. Enzyme activity is ex-
pressed as percentage of activity using AcSCh (acetylthiocholine io-
dide), BuSCh (butyrylthiocholine iodide), and PrSCh
Effect of pH and substrate concentrations
(propionylthiocholine iodide) after a 30-min incubation at 30ЊC with
iso-OMPA. Brain ChE activity using BuSCh was not detected. Each
point corresponds to the average of three determinations (coefficient
Figure 2 shows the effect of pH on the ChE activities. Serum
BChE and AChE exhibited a similar pattern of activity, with
maximum activities at pH higher than 9.0. However, brain
AChE activity showed a maximum activity at pH 9.0. Spon-
taneous nonenzymatic hydrolysis of the substrates BuSCh and
of variation
Ͻ 4%).
AcSCh was observed at pH
surements were therefore corrected for spontaneous hydrolysis
of the substrates.
Variations in serum AChE and BChE activities related to
substrate concentrations were established using AcSCh and
BuSCh, respectively, while substrate kinetics of brain AChE
were investigated using AcSCh. All ChE activities showed an
apparent Michaelis–Menten kinetic (Fig. 3). The values of
apparent Km and Vmax were calculated from the Lineweaver–
Ͼ8.0. Cholinesterase activity mea-
cipal ChE, as previously observed in the dose–response re-
lationship experiment with iso-OMPA (Fig. 1). The AChE
activity in serum (0.27
lower than BChE activity (1.33
␮
mol/min/ml of serum, on AcSCh) was
mol/min/ml, computed as the
␮
difference between total ChE activity on AcSCh and the re-
maining ChE activity after iso-OMPA treatment). Serum BChE
activity using BuSCh was completely depressed after iso-
OMPA treatment. This substrate was therefore sufficient to
estimate BChE activity in serum samples. Brain ChE activity
Burk plots. For brain AChE activity, Km was 12.3
␮
M and
1.22
], r2
10 [1/S ϭ 0.89). Likewise, serum ChE
was considered to be exclusively AChE, with a mean (
Ϯ SD)
Vmax was 820
␮
ϫ
mol/min/mg of total protein ([1/V0
]
ϭ
ϫ
activity of 801 115 mol/min/mg of total protein. No ac-
Ϯ
␮
Ϫ3
Ϫ5
10
ϩ
1.50
tivity using BuSCh was found in brain. The substrate AcSCh
was cleaved at a higher rate than PrSCh by brain and serum
AChE activities.
activities showed Km and Vmax values of 1.0 mM and 11.6
mol/min/ml, respectively, for BChE ([1/V0 0.086
␮
]
ϭ
ϩ
Table 1. Distribution and substrate specificity of cholinesterase activitya in brain and serum of Gallotia galloti
AcSCh BuSCh PrSCh
Ϫ4
Ϫ4
Ϫ4
Tissue
Basal activity
10 M iso-OMPA
Basal activity
10 M iso-OMPA
Basal activity
10 M iso-OMPA
Serum
Brain
1.60
801
Ϯ
Ϯ
0.38
115
0.27
797
Ϯ
Ϯ
0.04
90
6.68
Ϯ
—
1.02
—
—
5.53
596
Ϯ
Ϯ
1.10
120
0.18
588
Ϯ
Ϯ
0.03
86
a Cholinesterase (ChE) activity (mean
protein for brain ChE. AcSCh acetylthiocholine iodide; BuSCh
tetraisopropyl pyrophosphoramide.
Ϯ
standard deviation) is expressed as
␮
mol/min/ml of serum for serum ChE or
␮mol/min/mg of total
ϭ
ϭ
butyrylthiocholine iodide; PrSCh ϭ
propionylthicholine iodide; iso-OMPA
ϭ

2322
Environ. Toxicol. Chem. 21, 2002
J.C. Sanchez-Hernandez and B.M. Sanchez
Fig. 3. Serum butyrylcholinesterase (BChE, (A)) and acetylcholin-
esterase (AChE, (B)) activities of Galliotia galloti as a function of
substrate concentration. Each point corresponds to the average of three
determinations. Insets are Lineweaver–Burk plots of both ChE activ-
ities for butyrylthiocholine iodide (BuSCh) and acetylthiocholine io-
dide (AcSCh) concentrations.
Fig. 4. Reactivation of paraoxon-inhibited serum butyrylcholinester-
ase (BChE) activity in the presence of pralidoxime (2-PAM). Aliquots
Ϫ
Ϫ6
of serum were previously incubated with 2.2
ϫ
10 6 (A), 5.4
ϫ
10
Ϫ
5
(B), and 1.1
ϫ
10 M (C) of paraoxon for 15 min at 25
Њ
C and
Ϫ
2
subsequently treated with two concentrations of 2-PAM (2
ϫ
10
0.086[1/
for AChE ([1/V0
S
], r2
ϭ
0.92), and 3.50 mM and 0.42
␮mol/min/ml
Ϫ
and 2
ϫ
10 4 M, final concentration). Enzyme activity was determined
]
ϭ
2.34 8.32[1/ 0.97).
ϩ
S
], r2
ϭ
serially for varying time periods after addition of 2-PAM and is ex-
pressed as percentage of remaining activity. Each point corresponds
to the average of three determinations.
The BuSCh substrate suggests the optimal concentration
for determining serum BChE activity is 10 mM while the
AcSCh optimal concentration for assaying serum and brain
AChE activity is 5 mM. Nonenzymatic hydrolysis was ob-
2-PAM had a pronounced effect on the recovery rate of BChE.
This enzyme activity was almost completely recovered with
the lowest dose of 2-PAM for the three OP doses tested, while
served at high concentrations of substrate (Ͼ25 mM BuSCh
or 8 mM AcSCh). Furthermore, no inhibition of BChE and
Ͼ
Ϫ
2
AChE activities was detected at high substrate concentrations
(up to 50 mM and 17 mM for BuSCh and AcSCh, respec-
tively).
it remained below 80% of normal activity when 2
ϫ
10
M
of 2-PAM was used or even 40% in the samples previously
Ͻ
treated with the highest dose of paraoxon (Fig. 4C).
In a second experiment, serum was incubated with 2.2
ϫ
Chemical reactivation of BChE with 2-PAM
Ϫ6
Ϫ
6
Ϫ5
10 , 5.4
ϫ 10 , or 1.1 ϫ 10 M FC of dichlorvos (Fig. 5).
The possibility of serum BChE reactivation in the presence
of a reactivating agent such as 2-PAM was investigated. In a
first experiment, aliquots of serum were incubated with three
As for paraoxon-phosphorylated BChE, positive reactivation
was observed in the samples previously incubated with di-
chlorvos at the three different concentrations, and the lowest
Ϫ
6
Ϫ6
Ϫ2
concentrations of paraoxon (2.2
ϫ
10 , 5.4
ϫ
10 , or 1.1
ϫ
dose of 2-PAM appeared to be more effective than 2
ϫ 10
Ϫ
10 5 M FC) and the reactivation rate was examined using two
M. A delay in the reactivation of 2-PAM was found when the
Ϫ
Ϫ
Ϫ5
concentrations of 2-PAM (2
ϫ
10 2 or 2
ϫ
10 4 M FC). Figure
highest dose (1.1
ϫ 10 M FC) of dichlorvos was used (Fig.
4 shows that BChE activity increased progressively in the
presence of 2-PAM over 90 min of incubation, irrespective of
the degree of BChE inhibition. However, the concentration of
5C). An increase of BChE activity was not observed after 20
Ϫ
4
Ϫ2
and 40 min of incubation with 2
2-PAM, respectively.
ϫ 10 and 2 ϫ 10 M of

Characterization of lizard cholinesterases
Environ. Toxicol. Chem. 21, 2002
2323
necessary to characterize both ChEs. To our knowledge, this
is the first report on enzymological characterization of lizard
ChEs. Current results point out the existence of AChE and
BChE activities in the analyzed tissues of G. galloti. Both
AChE and BChE activities were found in serum, the latter
being the primary ChE activity. Brain contained exclusively
AChE activity. Regardless of the tissue, both ChE activities
showed features typical of mammalian AChE and BChE, i.e.,
AChE hydrolyzed AcSCh but not BuSCh (a selective substrate
for BChE activity) and was resistant to the inhibitory action
of iso-OMPA (a specific inhibitor for BChE activity) and BChE
activity hydrolyzed BuSCh at a higher rate than AcSCh or
PrSCh and furthermore was drastically inhibited by iso-
OMPA.
Measurement of serum BChE activity did not require the
use of iso-OMPA pretreatment. The use of the substrate BuSCh
was sufficient to estimate this enzyme activity because AChE
activity was not able to hydrolyze it. Serum BChE activity is
suggested to be a better biomarker than serum AChE activity
for assessing exposure to anti-ChE pesticides. The high activity
(on BuSCh) of serum BChE with respect to AChE, together
with the previously reported [26] response to the inhibitory
effect from OPs (high sensitivity and slow recovery rate after
acute OP exposure), justify the use of serum BChE activity as
an acceptable biomarker of pesticide exposure in G. galloti
.
Effects of pH and substrate concentration
Nonenzymatic hydrolysis of BuSCh and AcSCh takes place
at pH above 8.0 as well as at relatively high substrate con-
centrations. Lizard ChEs, however, showed maximum activi-
ties at pH higher than this value. Despite this, it is preferable
to use pH values between 7.5 and 8.0 for the ChE determi-
nations to avoid undesirable interferences by nonenzymatic
hydrolysis of substrates. The reason for a different pH-depen-
dent pattern of response of brain and serum AChE compared
with mammals is not clear.
Like ChEs in other organisms, lizard BChE and AChE ac-
tivities showed a Michaelis–Menten behavior with pronounced
differences in Km and Vmax between them. Although compar-
ison with data reported in the literature is difficult because of
differences in the analytical procedures, brain AChE activity
Fig. 5. Reactivation of dichlorvos-inhibited serum butyrylcholines-
terase (BChE) activity in the presence of pralidoxime (2-PAM). Al-
Ϫ
6
iquots of serum were previously incubated with 2.2
ϫ
10 (A), 5.4
10 M (C) of dichlorvos for 15 min at 25ЊC
Ϫ2
Ϫ
6
Ϫ5
ϫ
10 (B), and 1.1
ϫ
and subsequently treated with two concentrations of 2-PAM (2
ϫ
10
Ϫ
4
and 2
ϫ 10 M, final concentration). Enzyme activity was serially
determined for varying time periods after addition of 2-PAM and
expressed as percentage of remaining activity. Each point corresponds
to the average of three determinations.
showed a relatively high affinity by AcSCh (Km
ϭ 12.3 ␮M)
in comparison with Km values (160–650 M) of brain AChE
␮
activity of teleost fish, for instance [30]. However, it was in
the same order of magnitude of the ChE Km for mollusks (30
DISCUSSION
␮
M, [16]).
The AChE activity, but not BChE activity, is generally
Characterization and tissue distribution of ChEs
inhibited by high substrate concentrations [2,17,30]. This prop-
erty can help distinguish AChE from BChE in addition to the
use of selective substrates and inhibitors. However, other au-
thors found that ChE activity of marine mussels is not inhibited
by high substrate concentration (up to 20 mM AcSCh). Brain
and serum AChE activities of G. galloti did not show inhibition
by high concentrations of AcSCh (up to 50 mM; Fig. 3). Al-
though initial experimental conditions for lizard ChE deter-
minations were carried out using a substrate concentration of
2 mM, current results support that the optimal substrate con-
centration is 10 mM for determining serum BChE activity and
5 mM for serum and brain AChE activities. Substrate con-
centrations higher than these values could lead to nonenzy-
matic hydrolysis of substrates.
Both AChE and BChE are commonly used as biomarkers
for pesticide exposure. They can be distinguished easily by
the use of specific substrates and inhibitors [2,30,31]. In order
to validate these enzymes as reliable biomarkers, it is necessary
to know their tissue distribution, their enzymological behavior,
optimal conditions for their activity, and even other features
(e.g., relationship between field OP exposure and inhibition of
ChEs, in vivo sensitivity of ChEs to OPs, stable inhibition
response of ChEs, etc). For example, muscle tissue in several
species of teleost fish have both AChE and BChE activities,
while other species have AChE solely [30]. Both types of ChEs
are present in the serum of many vertebrate species, with dif-
ferent sensitivity to the inhibitory action of OPs [4]. In order
to use the most suitable enzyme as a biomarker, it is therefore

2324
Environ. Toxicol. Chem. 21, 2002
J.C. Sanchez-Hernandez and B.M. Sanchez
Table 2. Optimal conditions for cholinesterase measurements in Gallotia galloti; data of some species are also reported for comparisons
Incubation
temperature
Substrate
concn.
(mM)
Suitable tissue
or organ
Incubation
pH range
Species
(
Њ
C)
Substratea
Reference
Reptiles
Gallotia galloti
Brain
Serum
Serum
25
25
25
7.5–8.0
7.5–8.0
7.5–8.0
AcSCh
BuSCh
AcSCh
5
10
5
This study
This study
This study
Fish
Cyprinus carpio
Rutilus rutilus
Cyprinodon variegatus
Perca fluviatilis
Cymatogaster aggregata
Acerina cernua
Brain
Brain
Brain
Brain
Brain
Brain
25
37
22
37
37
37
7.5–8.5
7.1–8.5
7.0–7.5
7.1–8.5
7.0–7.5
7.1–8.5
AcSCh
AcSCh
AcSCh
AcSCh
AcSCh
AcSCh
2
1.8
10
1.8
1.8
2
[30]
[30]
[30]
[30]
[30]
[30]
Invertebrates
Mytilus edulis
Gills
Muscle
20–34
20–34
20–34
20–34
37–42
28–40
25
6.5–8.5
6.5–8.5
6.5–8.5
6.5–8.5
6.0–8.5
6.0–8.5
7.2–9.2
8.0–9.2
8.0–8.5
AcSCh
AcSCh
AcSCh
AcSCh
AcSCh
AcSCh
AcSCh
PrSCh
AcSCh
—
—
—
—
—
—
2
[33]
[33]
[33]
[33]
[15]
[15]
[16]
[16]
[18]
Palaemon serratus
Crangon crangon
Nereis sp.
Mytilus galloprovincialis
Perna perna
Mytilus galloprovincialis
Corbicula fluminea
Chironomus riparius
Cephalothorax
Whole animal
Digestive gland, gills
Digestive gland, muscle
Gills
Whole animal
Whole animal
25
30
5
4
a AcSCh
ϭ acetylthiocholine iodide; BuSCh ϭ butyrylthiocholine iodide; PrSCh ϭ propionylthicholine iodide; — ϭ data not reported.
2-PAM reactivation
In summary, in vitro experiments have demonstrated that
chemical reactivation of phosphorylated BChE in the presence
of 2-PAM is a good index of OP exposure. A 90-min incu-
bation time was sufficient to detect a significant increase of
BChE activity after OP exposure, and 10 M 2-PAM con-
centration was optimal for satisfactory enzyme reactivation.
Furthermore, optimal conditions for ChE measurements in G.
galloti are in the pH range of 7.5 to 8.0 and the substrate
concentrations of 5 mM (serum and brain AChE) or 10 mM
(serum BChE) (Table 2). Although not examined in this study,
Oximes, in particular 2-PAM, are known reactivating agents
of phosphorylated ChEs. They are clinically used, in addition
to atropine, as enzyme reactivators for OP intoxications. This
property has been tentatively used for diagnosing field ex-
posure to OP pesticides [14,20]. However, this methodology
presents a set of limitations. The ability of 2-PAM to reactivate
phosphorylated ChE is dependent on the time elapsed since
OP exposure. The progressive aging of the phosphorylated
enzyme, leading to the loss of one OP alkyl group, interferes
with the ability of 2-PAM to reactivate the enzyme. The re-
activation of OP-inhibited ChEs is, at least in birds, dependent
on the OP type [21]. However, our results with two OPs did
not support this assumption because no substantial differences
were observed among OPs in reactivation.
The concentration of 2-PAM was a determinant in achieving
full recovery of enzyme activity. A concentration 2
2-PAM in the incubation medium was considered as optimum.
In fact, large doses of oximes can cause inhibition of AChE
activity [32]. Inhibition of serum BChE was observed by
Thompson et al. [21] when samples of starlings exposed to
Ϫ
4
an incubation temperature of 25ЊC was considered as optimal
for ChE measurements, as previously reported for this lizard
species [26]. Likewise, data on the most favorable conditions
for determining ChE activity in some species of aquatic in-
vertebrates and fish are also reported in Table 2 for comparison.
We have chosen assay conditions for the lizard that yield sat-
isfactory ChE activities and minimize nonenzymatic hydro-
lysis of substrates. These optimal conditions appear to be in
line with those reported for other organisms (Table 2).
Ϫ
ϫ
10 4 M
Acknowledgement—The authors thank J.M. Siverio for his sugges-
tions and for the use of his laboratory during the sampling campaign.
Special thanks to F.J. Milan Cabrera and A. Valido. This study was
supported in part by funds from the University of Castilla–La Mancha
(Spain).
demeton-S-methyl were incubated with 12.5 mM of 2-PAM.
These investigators concluded that this enzyme inhibition in
the presence of 2-PAM might be due to a combination of two
factors, i.e., aging of enzyme and/or high residual levels of
demeton-S-methyl or its metabolites. No decrease in phos-
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