Enantioselective acetylcholinesterase inhibition of the organophosphorous insecticides profenofos, fonofos, and crotoxyphos

Article abstract of DOI:10.1897/07-001R.1

A large number of organophosphorous insecticides (OPs) are chiral compounds, and yet enantioselectivity in their environmental fate and effects is rarely addressed. In the present study, we isolated individual enantiomers of three OPs, profenofos, fonofos, and crotoxyphos, and evaluated enantioselectivity in their inhibition of acetylcholinesterase (AChE). Acetylcholinesterase inhibition by the enantiomers and racemates was determined in vivo in the aquatic invertebrate Daphnia magna and in Japanese medaka (Oryzias latipes) as well as in vitro with electric eel (Electrophorus electricus) and human recombinant AChEs. The overall results showed variable sensitivity between AChE enzymes from different species as well as variable magnitude of enantioselectivity in enzyme inhibition. The (-)-enantiomer of profenofos was 4.3- to 8.5-fold more inhibitory to AChE in vivo, whereas (-)-fonofos was 2.3- to 29-fold more potent than the corresponding (+)-enantiomer. The (+)-enantiomer of crotoxyphos was 1.1- to 11-fold more inhibitory to AChE than the (-)-enantiomer. In contrast, the in vitro results showed (+)-profenofos to be 2.6- to 71.8-fold more inhibitory than the (-)-enantiomer and (-)-crotoxyphos to be 1.6- to 1.9-fold more active than the (+)-enantiomer. The reversed direction of enantioselectivity observed between the in vivo and in vitro assays suggests enantioselectivity within toxicodynamic processes such as uptake, biotransformation, or elimination. Findings from the present study provide evidence of enantioselectivity in the AChE inhibition of chiral OPs in nontarget organisms and indicate the need to consider enantiomers individually when assessing environmental risk of these chiral pesticides.

Full text of DOI:10.1897/07-001R.1

Environmental Toxicology and Chemistry, Vol. 26, No. 9, pp. 1949–1954, 2007
2007 SETAC
Printed in the USA
0730-7268/07 $12.00 .00
᭧
ϩ
ENANTIOSELECTIVE ACETYLCHOLINESTERASE INHIBITION OF THE
ORGANOPHOSPHOROUS INSECTICIDES PROFENOFOS, FONOFOS,
AND CROTOXYPHOS
M
AE
G
RACE
N
ILLOS,*† GABRIELA
R
ODRIGUEZ-FUENTES,‡ JAY
G
AN,† and DANIEL
S
CHLENK
†
†Environmental Toxicology Program, ‡Department of Environmental Sciences, University of California, Riverside, California 92521, USA
(
Received 1 January 2007; Accepted 12 April 2007)
Abstract—A large number of organophosphorous insecticides (OPs) are chiral compounds, and yet enantioselectivity in their
environmental fate and effects is rarely addressed. In the present study, we isolated individual enantiomers of three OPs, profenofos,
fonofos, and crotoxyphos, and evaluated enantioselectivity in their inhibition of acetylcholinesterase (AChE). Acetylcholinesterase
inhibition by the enantiomers and racemates was determined in vivo in the aquatic invertebrate Daphnia magna and in Japanese
medaka (Oryzias latipes) as well as in vitro with electric eel (Electrophorus electricus) and human recombinant AChEs. The overall
results showed variable sensitivity between AChE enzymes from different species as well as variable magnitude of enantioselectivity
in enzyme inhibition. The (
was 2.3- to 29-fold more potent than the corresponding (
more inhibitory to AChE than the ( )-enantiomer. In contrast, the in vitro results showed (
more inhibitory than the ( )-enantiomer and ( )-crotoxyphos to be 1.6- to 1.9-fold more active than the (ϩ
Ϫ
)-enantiomer of profenofos was 4.3- to 8.5-fold more inhibitory to AChE in vivo, whereas (
)-enantiomer. The ( )-enantiomer of crotoxyphos was 1.1- to 11-fold
)-profenofos to be 2.6- to 71.8-fold
)-enantiomer. The
Ϫ)-fonofos
ϩ
ϩ
Ϫ
ϩ
Ϫ
Ϫ
reversed direction of enantioselectivity observed between the in vivo and in vitro assays suggests enantioselectivity within toxi-
codynamic processes such as uptake, biotransformation, or elimination. Findings from the present study provide evidence of
enantioselectivity in the AChE inhibition of chiral OPs in nontarget organisms and indicate the need to consider enantiomers
individually when assessing environmental risk of these chiral pesticides.
Keywords—Acetylcholinesterase
Enantioselectivity
Chiral pesticides
Organophosphorus
Chirality
INTRODUCTION
steadily increasing in recent years [5,9–12]. Chiral OP com-
pounds also have been shown to stereoselectively inhibit cho-
linesterase enzymes in a variety of species [9,13]. However,
the magnitude of stereoselectivity in enzyme inhibition may
vary and the preferred configuration could reverse between
animal species or between in vitro and in vivo determinations
[1,14–16].
The importance of stereoselectivity in the bioactivity of
natural and synthetic pharmaceutical products has long been
recognized [1,2]. More recently, pesticides consisting of active
ingredients with chiral centers have increasingly attracted at-
tention [3]. Because biochemical receptors and enzymes gen-
erally are stereoselective, enantiomers of the same pesticide
are expected to behave as completely different compounds in
biologically mediated environmental processes, such as bio-
transformation and toxicity [4]. Because chiral pesticides are
physically and chemically identical in an achiral environment,
they often are considered as single compounds, and risk as-
sessment generally is not carried out for individual enantio-
mers [5]. Their widespread use, however, calls for a better
understanding about the importance of enantioselectivity in
the environmental fate and effects of these pesticides.
Organophosphorous (OP) insecticides continue to be an
important chemical protection against agricultural and house-
hold pests in many countries [6,7]. They are potent acetyl-
cholinesterase (AChE) inhibitors and are less persistent in the
environment than their chlorinated predecessors. More than
30% of the modern-use OP insecticides are chiral, with a ster-
eogenic center on a carbon, sulfur, pentavalent phosphorus, or
other substituent atom of phosphorus [4,8]. Chiral OPs are
mostly marketed and used as racemic mixtures (equimolar
mixture of its enantiomers).
Both enantioselective and nonselective degradation of
several chiral OPs have been reported recently [3,4,12,17];
however, research concerning enantioselectivity in the tox-
icity of chiral OPs to nontarget and nonmammalian species,
particularly aquatic organisms, is still inadequate. The limited
research may be attributed to the challenges in chiral sepa-
ration and analysis and to unavailability of enantiomer stan-
dards. To date, studies of the differential AChE activities of
OP enantiomers have been conducted mostly in mammalian
systems and target insects, and have been contingent with
the synthesis and availability of enantiomer standards. Recent
advances in chiral separation using high-performance liquid
chromatography (HPLC) offer an opportunity to prepare en-
antiopure samples that could be used in bioassays to improve
our understanding of the ecotoxicological risk associated with
chiral pesticides [18]. The purpose of the present study was
to evaluate the enantioselective inhibition of AChE by in-
dividual enantiomers of three chiral OPs (profenofos, fono-
fos, and crotoxyphos) in an aquatic invertebrate (Daphnia
magna) and a juvenile fish, Japanese medaka (Oryzias la-
tipes). In addition, in vitro studies using Electrophorus elec-
tricus AChE (EE-AChE) and human recombinant AChE (HR-
AChE) were carried out to compare in vitro and in vivo
biological activities.
The number of investigations regarding the occurrence of
enantioselectivity in the aquatic toxicity of chiral OPs has been
* To whom correspondence may be addressed
(mae.nillos@email.ucr.edu).
1949

1950
Environ. Toxicol. Chem. 26, 2007
M.G. Nillos et al.
MATERIALS AND METHODS
AChE inhibition assay
Chemicals and enzymes
In vivo AChE inhibition by individual OP enantiomers and
racemates was determined in juvenile medaka following a
96-h exposure and in D. magna following a 24-h exposure.
The organisms were exposed to various aqueous concentra-
tions derived from the literature or determined from range-
finding experiments with the OP racemates. All exposures were
performed in triplicate. The exposure procedure was based on
established protocols [19]. Following aqueous exposure, ho-
mogenates were prepared from pooled whole animals in so-
dium phosphate buffer with 3% Triton-X 100 (Sigma; 1 ml
homogenization buffer/g tissue) using a tissue tearer, followed
by centrifugation of the homogenate at 12,500 rpm for 15 min
Analytical standards of racemic profenofos (purity, 94.9%;
O
-4-bromo-2-chlorophenyl-
rothioate), fonofos (purity, 98.5%;
ethylphosphonodithioate), and crotoxyphos (
O
-ethyl
S
O
-[R,S]-propyl phospho-
-ethyl -phenyl [R,S]-
-methylbenzyl-
S
␣
3-hydroxycrotonate-dimethylphosphate) were purchased from
Chem Service (West Chester, PA, USA). All other solvents or
chemicals used in the present study were of HPLC or analytical
grade and were purchased from Fisher (Fair Lawn, NJ, USA).
Acetylthiocholine iodide (ATC), 5,5Ј-dithiobis-2-nitrobenzoic
acid (DTNB), EE-AChE (type VI-S), HR-AChE (expressed in
Human Embryonic Kidney 293 cells), and all other chemicals
were purchased from Sigma-Aldrich (St. Louis, MO, USA).
at 4ЊC. The supernatant was then used to assay for AChE
activity using a modified Ellman method adapted to the mi-
croplate technique [20,21]. The total reaction volume was 225
Animals
␮
l, consisting of 5
mM final concentrations of ATC and DTNB, respectively. In
all cases the homogenate was preincubated at pH 7.5 and 20
␮l of homogenate and 1.5 mM and 0.22
Juvenile Japanese medaka (O. latipes; age, four to five
weeks posthatch; length, 10–12 mm; wt, 11.2–12.8 mg) were
obtained from the culture at the University of California–Riv-
erside (CA, USA). The fish were kept in glass aquaria in a
Њ
C
for 10 min with DTNB to allow completion of the background
binding of DTNB to the sulfhydryl groups in the sample. All
assays were performed in 96-well microtiter plates and read
in a SOFTmax Pro microplate reader (Molecular Devices, Sun-
nyvale, CA, USA) at 405 nm for 2 min. Residual AChE activity
was normalized to protein content by the Bradford method,
using Coomasie Plus-200 Protein Assay reagent and bovine
serum albumin standard (Pierce, Rockford, IL, USA) [21].
The in vitro inhibition of AChE was evaluated using EE-
AChE and also with HR-AChE for comparison. Briefly, test
chemical-free room with the temperature maintained at 25
Ϯ
2
Њ
C and a 16:8-h light:dark photoperiod. Dechlorinated water
was used for all stock cultures and experiments. Water-quality
parameters were constantly monitored, and fish were fed live
brine shrimp twice daily. Fish were not fed during exposure.
Daphnia magna were purchased from Aquatic Biosystems
(Fort Collins, CO, USA), and cultures were maintained in a
chemical-free room at 22
Ϯ 2ЊC with 16:8-h light:dark pho-
toperiod. Reconstituted moderately hard water was used for
all stock cultures and experiments. Daphnia magna was fed
with yeast-cerophylla-trout chow and Selenastrum capricor-
nutum. Test organisms were not fed during exposure.
solutions (5
were added to microplate wells, followed by the addition of
AChE-DTNB solution (205 l). The mixture was incubated
at room temperature (25 C) for 30 min, and then the residual
AChE activity was determined with the microplate reader at
405 nm for 2 min following the addition of ATC (20 l). The
␮l) at various concentrations of each enantiomer
␮
Њ
Preparation of enantiomers
␮
Enantiomers were resolved on an 1100 Series HPLC (Agi-
lent, Wilmington, DE, USA) equipped with an in-line laser
polarimeter detector (PDR-Chiral, Lake Park, FL, USA). Col-
umns with different chiral stationary phases (CSPs), including
a Sumichiral OA-2500-I column (Sumika Chemical Service,
Tokyo, Japan) as well as Chiralcel OD-R and Chiralcel OJ
columns (Daicel Chemical Industries, Tokyo, Japan), were pre-
viously tested in our laboratory [5,10]. Optimal resolution and
isolation of enantiomers were achieved for the test OPs on a
final concentrations of AChE, ATC, and DTNB were 0.005 U/
ml, 1.5 mM, and 0.22 mM, respectively. All assays were con-
ducted with at least three replicates. The median inhibitory
concentration (IC50) based on nominal aqueous concentrations
was determined by linear interpolation analysis using Tox-
Calc
௢ Version 5.0 (Tidepool Scientific Software, Mc-
Kinleyville, CA, USA).
RESULTS AND DISCUSSION
Chiralcel OJ column. Each OP sample (20
␮l) was injected
Enantiomer resolution
into the HPLC and eluted with hexane (100%), hexane/ethanol
(90:10, v/v), and hexane/2-propanol (95:5, v/v) as the mobile
phase for the separation of profenofos, crotoxyphos, and fon-
ofos enantiomers, respectively. All analyses were carried out
The structures of the chiral OP compounds investigated in
the present study are shown in Figure 1. Fonofos, a phos-
phonodithioate insecticide also known as dyfonate, is primarily
applied to control insects in a number of crops and in turfs
[4,17]. Profenofos is a phosphorothiolate OP compound that
has been widely used in cotton-growing areas in most countries
worldwide [22]. Crotoxyphos is a phosphate used as contact
and stomach poison, primarily for pests on lactating dairy and
beef cattle [4]. Both fonofos and profenofos have a pentavalent
phosphorus stereogenic center (denoted by an asterisk in Fig.
1), whereas crotoxyphos has a chiral carbon substituent of
phosphorus.
Resolution of enantiomers was highly column-specific [5].
Successful separation of enantiomers of profenofos, fonofos,
and crotoxyphos was achieved only on the Chiralcel OJ column
(Fig. 2). The corresponding retention (
resolution (RS) factors are given in Table 1. The separated
at room temperature (25ЊC). The flow rate of the mobile phase
was fixed at 0.8 ml/min. The ultraviolet detection wavelength
was set at 230 nm for all analyses. The specific rotation of the
resolved enantiomers was determined at 675 nm, and the cell
path was 50 mm. The rotation sign (
ϩ or Ϫ) was directly given
by a positive or negative peak on the polarimeter. Enantiopure
samples for AChE assays were manually collected at the HPLC
outlet, evaporated to dryness under a stream of pure nitrogen,
and then redissolved in acetone (carrier solvent). Stock solu-
tion concentrations were determined on HPLC assuming the
same response factor for enantiomers originating from the
same compound. The purity of the derived enantiomers was
checked with reanalysis on HPLC and found to be greater than
99% in all cases.
k), separation (␣), and

Acetylcholinesterase inhibition by chiral OP enantiomers
Environ. Toxicol. Chem. 26, 2007
1951
complexity of investigating the chiral recognition mechanisms
of CSPs, the present study did not attempt to elucidate any
structural features of the selected OPs or the CSP that corre-
lated with the resolution. Several researchers, however, have
suggested hydrogen bonding, ␲–␲ interaction, and dipole–di-
pole stacking, determined by the degree of steric fit of the
enantiomers in the chiral cavity, to be responsible for the chiral
discrimination [18]. The adequate resolution of the selected
OPs in the present study allowed individual enantiomers to be
recovered for use in bioassays.
In vivo inhibition of AChE
Acetylcholinesterase inhibition of profenofos, fonofos, and
crotoxyphos enantiomers and racemates were investigated in
vivo in the aquatic invertebrate D. magna and in juvenile
Japanese medaka. The AChE IC50 of the individual enantio-
mers and racemic mixtures of the selected chiral OPs are given
in Table 2. The selectivity ratios of inhibition between enan-
tiomers are given in Table 3. The more active enantiomer is
assigned the value of unity. The (
and fonofos were observed to be more potent AChE inhibitors
than the ( )-enantiomers in vivo. The ( )-enantiomer of pro-
fenofos was an approximately 8.5-fold more potent inhibitor
than the corresponding ( )-enantiomer to D. magna AChE
(Table 3). Similarly, the ( )-enantiomer of profenofos was
more inhibitory to the medaka AChE than ( )-profenofos, but
Ϫ)-enantiomers of profenofos
ϩ
Ϫ
ϩ
Ϫ
Fig. 1. Structures of test compounds (the asterisk indicates the chiral
position). (A) Profenofos. (B) Fonofos. (C) Crotoxyphos.
ϩ
the magnitude of enantioselectivity was only 4.3-fold. In both
species, the racemic profenofos had near intermediate toxicity
enantiomers were identified by determining their optical ro-
between the (ϩ)- and (Ϫ)-enantiomers. This observation clear-
tation. For fonofos and profenofos, the (
eluted before the corresponding ( )-enantiomer, whereas for
crotoxyphos, the ( )-enantiomer was eluted before the ( )-
ϩ
)-enantiomer were
ly indicates that most of the AChE activity in both aquatic
invertebrate and fish in the racemic mixture can be attributed
to the more potent enantiomer of profenofos in vivo.
Ϫ
Ϫ
ϩ
enantiomer under the described conditions (Table 1). Fonofos,
profenofos, and crotoxyphos have been previously separated
on the same or similar CSP column under slightly different
conditions or using different systems [4,5,23]. Because of the
R
-(
tivity than S-
to mice in vivo [16]. In the present study, the (
of fonofos was approximately twofold as inhibitory to a non-
Ϫ
)-fonofos was reported to have higher biological ac-
)-fonofos to housefly and mosquito fly and also
)-enantiomer
(
ϩ
Ϫ
Fig. 2. High-performance liquid chromatogram and optical rotation spectra for the enantiomeric separation of (A) crotoxyphos, (B) profenofos,
and (C) fonofos on Chiralcel OJ column (Daicel Chemical Industries, Tokyo, Japan). i optical rotation; ii chromatogram of the separated
enantiomers; iii chromatogram of the first-eluting enantiomer; iv chromatogram of the second-eluting enantiomer.
ϭ
ϭ
ϭ
ϭ

1952
Environ. Toxicol. Chem. 26, 2007
Table 1. The separation (
M.G. Nillos et al.
␣
) and resolution (RS) factors of profenofos, fonofos, and crotoxyphos on the Chiralcel OJ columna
Retention time (min)
Peak width (min)
Retention factor
kϩ kϪ
Rotation
( pk1/p2)
b
T0
Tϩ
TϪ
wϩ
wϪ
Ј
Ј
␣
RS
Profenofos
Fonofos
Crotoxyphos
4.91
4.94
4.68
35.06
12.85
24.19
41.86
16.85
19.37
1.32
0.33
0.80
1.60
0.47
0.55
6.14
1.60
4.17
7.53
2.41
3.14
1.23
1.50
1.33
1.12
2.39
1.81
ϩ
ϩ
Ϫ
/
/
/
Ϫ
Ϫ
ϩ
a Daicel Chemical Industries, Tokyo, Japan.
kϩЈ ϭ Tϩ T0)/T0 kϪЈ ϭ TϪ T0)/T0 ␣ ϭ
b pk1/p2
peak 1/peak 2.
T
0 ϭ solvent retention time; the subscripts
pk2)/ pk1); RS 1.18[ pk2
ϩ
and
Ϫ
indicate the (
ϩ
)- and (
Ϫ
)-enantiomers, respectively.
(
Ϫ
;
(
Ϫ
;
k
Ј
(
k
Ј
(
ϭ
k
Ј
(
)
Ϫ
k
Ј
(
pk1)]/0.5(wϪ
ϩ
wϩ).
ϭ
target aquatic invertebrate (D. magna) AChE and more than
29-fold more active to medaka AChE than the ( )-enantiomer
(Tables 2 and 3). Interestingly, the racemic fonofos was almost
equipotent to the ( )-enantiomer in medaka and approximately
in vivo (Tables 2 and 3). Additionally, the (ϩ)-enantiomer of
crotoxyphos was more than 11-fold more inhibitory to D. mag-
na AChE. Such selectivity, however, was not as clearly defined
ϩ
Ϫ
in medaka, although the IC50 was not reached for the (Ϫ)-
fivefold more potent to D. magna AChE than the active en-
antiomer. The results of the in vivo AChE inhibition by pro-
fenofos and fonofos in D. magna are qualitatively consistent
with the previously observed acute toxicity of profenofos and
enantiomer at the highest concentration tested (Tables 2 and
3). A wide range of inhibition selectivity ratios have been
reported for the inhibition of different AChE enzymes, sug-
gesting that enantioselectivity in AChE inhibition may be de-
pendent on the species of test organisms [14].
fonofos, in which the (
Ϫ)-enantiomers were found to be more
toxic than the ( )-enantiomers to the aquatic invertebrate [5].
ϩ
In vitro inhibition of AChE
Similar relationships were previously reported for other chiral
OPs. In the acute toxicity test with Daphnia pulex, no sig-
nificant difference in the median lethal concentrations was
In contrast to the in vivo results, the (
ϩ)-enantiomer of
profenofos and the ( )-enantiomer of crotoxyphos were found
Ϫ
observed between (
ϩ
)-fenamiphos and its racemate, but the
to have higher activity against both EE-AChE and HR-AChE
in the in vitro assays (Table 4). As observed in vivo, however,
the magnitude of difference in activity between the two en-
antiomers of crotoxyphos was relatively small in both EE-
(
Ϫ
)-enantiomer showed significantly lower toxicity [12]. In
another study, no significant difference was observed in both
AChE IC50 in housefly heads and acute toxicity to D. pulex
between the more active (
ϩ
)-enantiomer and racemic lepto-
AChE and HR-AChE in the in vitro assay (Table 3). The (
enantiomer of crotoxyphos was only 1.6- and 1.9-times more
inhibitory than the ( )-enantiomer to EE-AChE and HR-
AChE, respectively. Larger differences in toxicity were ob-
served between the enantiomers of profenofos (Table 3). The
differences in the magnitude of selectivity may be related to
the differences in the stereogenic centers between the two
chiral OPs [16,25].
Ϫ)-
phos [11]. These findings suggest that for certain chiral OPs,
chirality may affect the toxicodynamic processes, so a straight-
forward relationship between enantiomers and racemates may
not always be found [24].
Enantiomers of both malathion and malaoxon exist because
of an asymmetric carbon center in the thiosuccinyl substituent
[16]. Rodriguez et al. [14] showed significant enantioselectiv-
ity in activity of malaoxon against cholinesterase enzymes,
ϩ
Comparison of in vivo and in vitro inhibition
where the
R-enantiomer was up to 22-fold more inhibitory
than the -enantiomer to bovine erythrocyte AChE. Similar to
malaoxon, crotoxyphos chirality is a result of an asymmetric
carbon center in a substituent group (Fig. 1).
In the present study, stereoselectivity also was observed,
with (ϩ)-crotoxyphos being the more potent AChE inhibitor
S
Regardless of the magnitude of enantioselectivity, the ap-
parent reversal in potency of enantiomers between in vivo and
in vitro assays may be explained by the possible occurrence
of stereoselectivity in metabolic processes in vivo [16]. It is
likely that one enantiomer is preferentially bioactivated to a
Table 2. In vivo acetylcholinesterase median inhibitory concentration (IC50;
␮mol/L) of the enantiomers and racemic profenofos, fonofos, and
crotoxyphosa
(
ϩ
)
(
Ϫ
)
Racemate
Profenofos
Daphnia magnabc
Japanese medakade
0.102
0.226
Ϯ
Ϯ
0.007
0.019
0.012
0.053
Ϯ
Ϯ
0.002
0.010
0.020
0.094
Ϯ
Ϯ
0.0004
0.003
Fonofos
Daphnia magnabe
Japanese medakade
Ͼ
Ͼ
0.406
0.812
0.176
0.028
Ϯ
Ϯ
0.025
0.0004
0.037
0.029
Ϯ
Ϯ
0.006
0.0004
Crotoxyphos
Daphnia magnabc
Japanese medakade
0.029
0.894
Ϯ
Ϯ
0.007
0.010
Ͼ
Ͼ
0.318
0.954
0.0727
0.781
Ϯ
Ϯ
0.009
0.017
a All values are presented as the mean
b Assayed following 24-h exposure.
Ϯ standard error.
c n
ϭ
6.
d Assayed following 96-h exposure of juvenile medaka (age, four to five weeks posthatch).
e n
3.
ϭ

Acetylcholinesterase inhibition by chiral OP enantiomers
Environ. Toxicol. Chem. 26, 2007
1953
Table 3. Comparison of in vivo and in vitro inhibition of
acetylcholinesterase between enantiomers of the chiral
organophosphorous (OP) compounds profenofos, fonofos, and
crotoxyphosa
of the inability of the system to form fonofos-oxon. The mode
of action of fonofos that contains a P S moiety requires met-
abolic activation by oxidative desulfuration to the P O (oxon)
form for enzyme inhibition to take place [14]. Preliminary
assays with the fonofos racemate showed no significant in-
hibition of EE-AChE at the highest concentration assayed
ϭ
ϭ
In vivob
In vitroc
EE-AChE HR-AChE
Daphnia
magna
Japanese
medaka
(1,000
of fonofos and fonofos-oxon enantiomers in housefly, mos-
quito fly, and mice [24], results showed the -enantiomer of
fonofos to be 1.8- to 4.0-fold more toxic than the -enantiomer.
-oxon as 2.6- to 12.2-fold
-oxon to the same animals.
␮M). In an earlier study regarding the acute toxicity
OP
Profenofos
Racemic
R
1.7
8.5
1.0
1.8
4.3
1.0
0.88
1.0
2.6
2.03
1.0
71.8
S
(
(
ϩ
Ϫ
)
)
The same study also reported the
more toxic than the corresponding
S
R
Ͼ
Fonofos
Racemic
In addition, in an in vitro metabolic activation study of fonofos
in rat liver microsomes [28], it was found that -fonofos was
predominantly converted to the -oxon, which explains the
greater toxicity of -fonofos over -fonofos.
0.21
2.3
1.0
1.03
29
1.0
R
(
ϩ
)
Ͼ
Ͼ
NAd
NA
S
(Ϫ)
R
S
Crotoxyphos
Racemic
2.5
1.0
11.0
0.87
1.0
1.1
0.83
1.6
1.0
2.06
1.9
1.0
CONCLUSION
(
(
ϩ
Ϫ
)
)
The successful chiral chromatographic separation and iso-
lation of enantiomers of the selected chiral OPs made possible
the evaluation of enantioselectivity in the inhibition of AChE
in the present study. The in vivo and in vitro results with
aquatic organisms suggest that for the selected OPs, enantio-
selectivity for AChE inhibition does occur. The magnitude of
enantioselectivity, however, varied greatly between species,
from aquatic invertebrates to fish to humans. In addition, the
present results also suggest that in vivo AChE inhibition by
chiral OPs is influenced by biological processes other than the
compound’s mode of action. Findings from the present study
provide evidence for enantioselectivity in the AChE inhibition
of chiral OPs in nontarget organisms in the aquatic environ-
ment. The observed enantioselectivity indicates the need to
consider enantiomers individually when assessing environ-
mental risk of chiral pesticides. In addition, the reversed di-
rection of enantioselectivity observed between the in vivo and
in vitro AChE inhibition assays suggests significant enantio-
selective contributions of toxicodynamic processes, such as
biotransformation, uptake, or elimination.
Ͼ
Ͼ
a Selectivity
ϭ IC50 of less active enantiomer (or racemate)/IC50 of
the more active enantiomer, where IC50 is the median inhibitory
concentration.
b Calculated from the in vivo IC50.
c Calculated from the in vitro IC50. EE-AChE
ϭ
Electrophorus elec-
tricus acetylcholinesterase; HR-AChE
ϭ human recombinant ace-
tylcholinesterase.
d NA
ϭ Not assayed in vitro.
metabolite more inhibitory to AChE, whereas the other is pref-
erentially detoxified and its capacity to inhibit AChE reduced.
An earlier study with rat liver microsomes coupled with AChE
inhibition quantification [26] indicated that the individual en-
antiomers reacted in an opposite manner. In that study, ste-
reospecific metabolic activation resulted in the (
of profenofos becoming 34-fold more inhibitory to AChE,
whereas ( )-profenofos was preferentially detoxified and its
Ϫ)-enantiomer
ϩ
capacity to inhibit the enzyme reduced by twofold. Such a
shift in enantioselectivity in AChE inhibition following me-
tabolism was attributed to either differences in rates of oxi-
dation or the intrinsic potency of metabolite enantiomers. Re-
sults similar to that observed in the present study have been
reported in housefly and mice (in vivo) and in bovine eryth-
rocyte, housefly head, and EE-AChE (in vitro) for profenofos
[15,16,26]. In addition, differential metabolism also was dem-
onstrated for the chiral OP, isofenfos, in rat liver microsomes,
Acknowledgement—We acknowledge the Fulbright-Philippine Agri-
culture Scholarship Program and the University of California Toxic
Substances Research and Teaching Program for the support of M.G.
Nillos. This study was supported by a U.S. Department of Agriculture–
National Research Initiatives grant 2005-35107-16189.
REFERENCES
in which (
than the (
function oxidases-dependent metabolism of the oxon was high-
er for the ( )-enantiomer [27].
The enantioselectivity in AChE inhibition of fonofos en-
antiomers was not assayed in vitro in the present study because
Ϫ
)-isofenfos produced nearly fourfold more oxon
1. Kuriahara N, Miyamoto J, Paulson GD, Zeeh B, Skidmore MW,
Hollingworth RM, Kuiper HA. 1997. Chirality in synthetic ag-
rochemicals: Bioactivity and safety considerations. Pure Appl
Chem 69:2007–2025.
2. Ladik J, Szekeres Z. 2002. A study on chirality in biomolecules:
The effect of the exchange of l-amino acids to d ones in Sso7d
ribonuclease. Journal of Molecular Modeling 12:462–467.
ϩ
)-enantiomer, although AChE inhibition and mixed-
ϩ
Table 4. In vitro acetylcholinesterase median inhibitory concentration (
Profenofosb
␮
mol/L) of the enantiomers and racemic profenofos and crotoxyphosa
Crotoxyphosb
(
ϩ
)
(
Ϫ
)
Racemate
(
ϩ
)
(
Ϫ
)
Racemate
EE-AChEc
HR-AChEd
193.2
2.26
Ϯ
Ϯ
23.4
0.16
Ͼ
162.3
500
6.58
169.84
4.60
Ϯ
Ϯ
2.94
0.27
14.06
5.58 Ϯ
Ϯ
0.19
0.18
8.95
2.92
Ϯ
Ϯ
0.57
0.15
7.46
6.02
Ϯ
Ϯ
0.54
0.40
Ϯ
a All values are presented as the mean
Ϯ standard error.
b n
ϭ 3.
c EE-AChE
d HR-AChE
ϭ
Electrophorus electricus acetylcholinesterase.
ϭ
human recombinant acetylcholinesterase.

1954
Environ. Toxicol. Chem. 26, 2007
M.G. Nillos et al.
3. Garrison AW. 2004. On the issue of enantioselectivity of chiral
pesticides: A green chemistry opportunity. Green Chemistry 6:
G77–G78.
4. Ellington JJ, Evans JJ, Prickett KB, Champion WL. 2001. High-
performance liquid chromatographic separation of the enantio-
mers of organophosphorous pesticides on polysaccharide chiral
stationary phases. J Chromatogr A 928:145–154.
5. Liu W, Gan J, Schlenk D, Jury W. 2005. Enantioselectivity in
environmental safety of current chiral insecticides. Proc Natl
Acad Sci USA 102:701–706.
6. Scholz N, Hopkins W. 2006. Ecotoxicology of acetylcholines-
terase pesticides: Data gaps and research challenges. Environ Tox-
icol Chem 25:1185–1186.
agrochemicals. In Kurihara N, Miyamoto J, eds, Chirality in Ag-
rochemicals. Wiley, Chichester, UK, pp 85–139.
17. Jarman JL, Jones WJ, Howell LA, Garrison AW. 2005. Appli-
cation of capillary electrophoresis to study the enantioselective
transformation of five chiral pesticides in aerobic soil slurries.
J
Agric Food Chem 53:6175–6182.
18. Lao W, Gan J. 2006. High-performance liquid chromatographic
separation of imidazolinone herbicide enantiomers and their
methyl derivatives on polysaccharide coated chiral stationary
phases. J Chromatogr A 1117:184–193.
19. U.S. Environmental Protection Agency. 2002. Methods for mea-
suring the acute toxicity of effluents and receiving waters to fresh-
water and marine organisms. EPA-821-R-02-012. Washington,
DC.
7. Singh B, Walker A. 2006. Microbial degradation of organophos-
phorous compounds. FEMS Microbiol Rev 30:428–471.
8. Garrison AW. 2006. Probing the enantioselectivity of chiral pes-
ticides. Environ Sci Technol 40:16–23.
9. Lin K, Zhou S, Xu C, Liu W. 2006. Enantiomeric resolution and
biotoxicity of methamidophos. J Agric Food Chem 54:8134–
8138.
10. Liu W, Lin K, Gan J. 2006. Separation and aquatic toxicity of
enantiomers of the organophosphorous insecticide trichloronate.
Chirality 18:1–4.
11. Yen J, Tsai C, Wang Y. 2003. Separation and toxicity of enan-
tiomers of organophosphorous insecticide leptophos. Ecotoxicol
Environ Saf 55:236–242.
12. Wang P, Tai K, Yen J. 2004. Separation, bioactivity and dissipation
of enantiomers of the organophosphorous insecticide fenamiphos.
Ecotoxicol Environ Saf 57:346–353.
13. Thompson C, Ryu S, Berkman C. 1992. Consequence of phos-
phorus stereochemistry upon the postinhibitory reaction kinetics
of acetylcholinesterase poisoned by phosphorothiolates. J Am
Chem Soc 114:10710–10715.
14. Rodriguez OP, Muth GW, Berkman CE, Kim K, Thompson C.
1997. Inhibition of various cholinesterases with the enantiomers
of malaoxon. Bull Environ Contam Toxicol 58:171–176.
15. Leader H, Casida J. 1982. Resolution and biological activity of
20. Ellman G, Courtney D, Andres V, Featherstone R. 1961. A new
and rapid colorimetric determination of acetylcholinesterase ac-
tivity. Biochem Pharmacol 7:88–95.
21. Sandahl J, Jenkins J. 2002. Pacific steelhead (Oncorhynchus my-
kiss) exposed to chlorpyrifos: Benchmark concentration estimates
for acetylcholinesterase inhibition. Environ Toxicol Chem 21:
2452–2458.
22. Kumar A, Chapman JC. 1998. Profenofos toxicity to the eastern
rainbow fish (Melanotaenia duboulayi). Environ Toxicol Chem
17:1799–1806.
23. Wang P, Jiang S, Liu D, Zhang H, Zhou Z. 2006. Enantiomeric
resolution of chiral pesticides by high performance liquid chro-
matography. J Agric Food Chem 54:1577–1583.
24. Lee P, Allahyari R, Fukuto TR. 1978. Studies on the chiral isomers
of fonofos and fonofos oxon: I. Toxicity and antiesterase activ-
ities. Pestic Biochem Physiol 8:146–157.
25. Doorn JTT, Thompson C, Richardson R. 2001. Probing the active
site of butyrylcholinesterase and cholesterol esterase with iso-
malathion: Conserved stereoselective inactivation of serine hy-
drolases structurally related to acetylcholinesterase. Chem Res
Toxicol 14:807–813.
26. Wing K, Glickman A, Casida J. 1983. Oxidative bioactivation of
S
-alkyl phosphorothiolate pesticides: Stereospecificity of profen-
ofos insecticide activation. Science 219:63–65.
27. Ueji M, Tomizawa C. 1987. Metabolism of chiral isomers of
isofenphos in the rat liver microsomal system. J Pestic Sci 12:
269–272.
28. Lee P, Allahyari R, Fukuto TR. 1978. Studies on the chiral isomers
of fonofos and fonofos oxon: II. In vitro metabolism. Pestic
Biochem Physiol 8:158–169.
the chiral isomers of
O-(4-bromo-2-chlorophenyl) O-ethyl S-pro-
pyl phosphorothioate (profenofos insecticide). J Agric Food
Chem 30:546–551.
16. Sasaki M. 1998. Importance of chirality in organophosphorous