Enantioselective interaction with acetylcholinesterase of an organophosphate insecticide fenamiphos

Article abstract of DOI:10.1002/chir.20800

Enantioselectivity in the environmental behavior and ecotoxicity of chiral pesticide is widely observed. However, the investigation of the enantioselective mechanisms remains limited. In this study, we used fenamiphos (FAP), an organophosphorus insecticide, to study enantioselectivity in toxicity to arthropods and the inhibition potential towards acetylcholinesterase (AChE) in the rat pheochromocytoma 12 (PC 12) cell line. Furthermore, we carried out molecular docking to help explain the mechanisms of enantioselective toxicity of FAP. The two enantiomers of FAP were successfully separated and identified as R-(1)-FAP and S-(2)-FAP. Toxicological assays revealed that R-(1)-FAP was 2.4-fold more toxic than S-(2)-FAP to Daphnia magna and approximately threefold more to PC12 cells. Based on molecular docking results, dynamic simulation shows that strong hydrophobic interactions and a key hydrogen bond can only exist between R-(1)-FAP and AChE, which helps explain the preference of R-(1) binding to AChE over that of the S-(2)-enantiomer, and supports our biological results. Our present study considers the impact of stereochemistry on ecotoxicological effects and, ultimately, on development of environmentally safe, insecticidally efficient pesticides.

Full text of DOI:10.1002/chir.20800

CHIRALITY 22:612–617 (2010)
Enantioselective Interaction with Acetylcholinesterase of an
Organophosphate Insecticide Fenamiphos
1
2
1
1
1
1
*
CUI WANG, NA ZHANG, LING LI, QUAN ZHANG, MEIRONG ZHAO, * AND WEIPING LIU
1Research Center of Environmental Science, College of Biological and Environmental Engineering,
Zhejiang University of Technology, Hangzhou 310032, China
2Department of Chemistry, Zhejiang University, Hangzhou 310027, China
ABSTRACT
Enantioselectivity in the environmental behavior and ecotoxicity of chi-
ral pesticide is widely observed. However, the investigation of the enantioselective
mechanisms remains limited. In this study, we used fenamiphos (FAP), an organophos-
phorus insecticide, to study enantioselectivity in toxicity to arthropods and the inhibition
potential towards acetylcholinesterase (AChE) in the rat pheochromocytoma 12 (PC 12)
cell line. Furthermore, we carried out molecular docking to help explain the mecha-
nisms of enantioselective toxicity of FAP. The two enantiomers of FAP were success-
fully separated and identified as R-(1)-FAP and S-(2)-FAP. Toxicological assays
revealed that R-(1)-FAP was 2.4-fold more toxic than S-(2)-FAP to Daphnia magna and
approximately threefold more to PC12 cells. Based on molecular docking results,
dynamic simulation shows that strong hydrophobic interactions and a key hydrogen
bond can only exist between R-(1)-FAP and AChE, which helps explain the preference
of R-(1) binding to AChE over that of the S-(2)-enantiomer, and supports our biological
results. Our present study considers the impact of stereochemistry on ecotoxicological
effects and, ultimately, on development of environmentally safe, insecticidally efficient
C
VV
pesticides. Chirality 22:612–617, 2010.
2009 Wiley-Liss, Inc.
KEY WORDS: enantioselectivity; fenamiphos; molecular docking; toxicity
INTRODUCTION
and critical binding sites for drug-protein interactions. For
example, cytochrome (CYP) P450 mediates the N-dechlor-
oethylation of R-ifosfamide at the N-2 position and the
N-dechloroethylation of S-ifosfamide at the N-3 position.14
However, whether there are similar molecular mecha-
nisms with agrochemicals is a feasible area for research.
Acetylcholinesterase, an important target molecule of
OPs, terminates cholinergic neurotransmission by catalyz-
ing hydrolysis of the neurotransmitter acetylcholine. As
indicated from previous studies, the catalytic triad of this
enzyme consists of serine (Ser) 203, glutamic acid (Glu)
334 and histidine (His) 447 residues located close to the
Chirality is important in many fields of chemistry and
biology and chiral pesticides are a subgroup of agrochemi-
cals that contain one or more chiral centers. In China
more than 40% of marketed pesticides are chiral.1 Over the
past two decades, enantioselectivity of chiral pesticides
has become well-recognized in assessing the environmen-
tal safety of chiral pesticides.2 It is well established that
the opposite enantiomers of chiral pesticides often differ
significantly in environmental impact, including biodegra-
dation,3 bioaccumulation,4,5 aquatic toxicity6 and endo-
crine disruption.7 However, our understanding of the
molecular mechanism of enantioselectivity is still limited.
To understand enantioselectivity in toxicity and its
molecular mechanisms, suitable chemicals and experimen-
tal models are necessary. Organophosphorus pesticides
(OPs), developed in the 1950s as insecticides for fruit,
vegetables and other crops, are one major class of typical
chiral pesticides.8 The OPs, which pose potential human
health risks, were detected in food in China and other
countries.9,10 Studies showed that there was obvious enan-
tioselectivity in their acute toxicity11 and, also, inhibition of
AChE activity.12,13 The basis for the difference between
stereoisomers lies in the three-dimensional nature of
organic drug molecules and their target receptors, which
are most often proteins. This provides a basis for the spa-
tial recognition of stereoisomeric forms of chiral molecules
C
˚
base of a deep active-site gorge that penetrates about 20 A
into the AChE.15 The peripheral anionic site (PAS) is
Contract grant sponsor: National Basic Research Program of China;
Contract grant number: 2009CB421603
Contract grant sponsor: National Natural Science Foundations of China;
Contract grant numbers: 20837002, 20877071
Contract grant sponsor: Program for Changjiang Scholars and Innova-
tive Research Team in Chinese Universities; Contract grant number:
IRT 0653
*Correspondence to: Meirong Zhao, Research Center of Environmental
Science, College of Biological and Environmental Engineering, Zhejiang
University of Technology, Hangzhou 310032, China. E-mail: zhaomr@
zjut.edu.cn or Weiping Liu, Research Center of Environmental Science,
College of Biological and Environmental Engineering, Zhejiang University
of Technology, Hangzhou 310032, China. E-mail: wliu@zjut.edu.cn
Received for publication 17 April 2009; Accepted 13 August 2009
DOI: 10.1002/chir.20800
Published online 6 November 2009 in Wiley InterScience
(www.interscience.wiley.com).
VV
2009 Wiley-Liss, Inc.

ENANTIOSELECTIVE TOXICOLOGY OF FENAMIPHOS
613
located at the rim of the active site gorge and comprises
the aromatic residues tyrosine (Tyr) 72, Tyr 124, trypto-
phan (Trp) 286 and Tyr 341 and the acidic residue aspartic
acid (Asp) 74,16,17 and provides a binding site for allosteric
modulators and PAS inhibitors. Organophosphorus pesti-
cides inactivate AChE by inhibiting its hydrolysis of the
neurotransmitter acetylcholine and, also, the interaction of
OPs with certain oximes used as antidotes for clinical
treatment of OP-intoxications and AChE has been investi-
gated.18,19 The developments of structural biology and
computer-aided drug design methods, molecular model-
ing, ligand docking and molecular dynamics simulation,
have emerged as a powerful tools to investigate stereose-
lective protein-ligand interactions.17,20
In this study, FAP, a broad-spectrum OP insecticide was
used as the model chiral chemical. Using enantiomers sep-
arated by HPLC enabled the establishment of their abso-
lute configurations, their enantioselectivity and concomi-
tant inhibition potency toward AChE in the pheochromo-
cytoma 12 (PC12) pheochromocytoma cell line, as well as
aquatic toxicity to daphnids (Daphnia magna), investi-
gated. To understand the mechanisms of enantioselective
inhibition on AChE by OPs, molecular docking was per-
formed to elucidate and confirm the experimentally
observed enantioselective results. We expect that the pro-
cedures developed herein may serve to better understand
the molecular mechanisms of enantioselective toxicity of
other chiral chemicals. Additionally, our study provides a
platform for developing safe, efficacious and reliable agro-
chemicals grounded on the evaluation of chiral character-
istics relative to their toxicological considerations.
Fig. 1. Chemical structures of fenamiphos enantiomers.
tive phase [amylase tris-(S)-1-methylphenyl-carbamate)]
coated on to 5 lm silica gel. The injection volume was
20 alL, with a flow rate of 0.8 mL min21 for the mobile
phase.
The resolved enantiomers for subsequent bioassays
were manually collected at the column outlet, then evapo-
rated to dryness under nitrogen and redissolved in etha-
nol. The concentrations of the enantiomers were
determined by assuming the same response factor for
enantiomers as for the racemate,12 and also by analyzing
an aliquot on a gas chromatograph coupled with a nitro-
gen-phosphorus detector (GC-NPD). Then, the purity of
each pair of enantiomers was determined to be more than
99% under best HPLC separation conditions.
Chiroptical Detection and Establishment of Absolute
Configurations
The HPLC-CD technique was used to distinguishing
the enantiomers of FAP based upon the (2) or (1)-CD.
CD signals are intrinsically stable to change of tempera-
ture and solvent and are thus gradient compatible. The
(1) or (2) herein indicates the CD signal, and is used to
compare the order of elution between columns since the
chromatographic conditions were equivalent. The CD
detection wavelength was 254 nm. The light source for the
chiral detector was a 150 W Hg-Xe lamp, and the tapered
cell path was 25 mm with a volume of 44 lL. The rotation
sign (‘‘1’’ or ‘‘2’’) was indicated by a positive or negative
peak on the chromatogram and data handling was done
using BORWIN 1.50 for Windows. The lowest energy of
molecular conformation of FAP was calculated on Chem
3D Ultra 8.0 software (CyberChem, Gainesville, FL), when
the octant rule was applied to establish the absolute con-
figuration of FAP.
MATERIALS AND METHODS
Chemicals and Reagents
Fenamiphos, [FAP, 1-(Methylethyl)-ethyl-3-methyl-4-
(methylthio)-phenyl phosphoramidate), purity: 99%,
(Fig. 1)], cetylthiocholine iodide (ATCh-I), 5,50-dithio-bis-2-
nitrobenzoic acid (DNTB) and bovine serum albumin
(BSA) were purchased from Sigma (St. Louis, MO), Other
solvents or chemicals were of HPLC or analytical grade.
Test solutions of the enzymes were made in 0.01 mol L21
potassium phosphate buffer (pH 7.4). FAP was prepared in
HPLC mobile phase at 4000 mg L21 and was stored at 48C
in the dark.
Aquatic Toxicity Assays
Chromatographic Conditions
Daphnids (Daphnia magna) were obtained from the
Chiral separation was performed on a Jasco LC-2000 se- Chinese Academy of Preventive Medicine (Beijing, China)
ries HPLC system (Jasco, Tokyo, Japan) equipped with a which had been reared at the laboratory for several years.
variable-wavelength UV-2075 detector. Enantiomeric iden- The overall testing procedure followed our previous stud-
tification was done by using an on-line variable-wavelength ies.12 Briefly, 5 juveniles aged between 6 and 24 h were
circular dichroism (CD)-2095 detector connected with transferred into glass beakers filled with 20 mL of blank,
HPLC. The chromatographic system consisted of a PU- or 1.4, 2.8, 5.6, 11.2, 22.4 lg L21 for each enantiomer of
2089 quaternary gradient pump, mobile phase vacuum FAP and 1.6, 3.2, 6.4, 12.8, 25.6 lg L21 for rac-FAP. Four
degasser, an AS-2055 intelligent sampler with a 100 lL replicates for each treatment were prepared. The test
loop, a CO-2060 column thermo-stat and a LC-Net II/ADC organisms were fed the alga Scenedesma obliquus 6 h
data collector. Chromatographic data were acquired and before the test began. The mortality of daphnids of all vials
processed with ChromPass software (version 1.7.403.1, was monitored at 24 h intervals for the 48 h exposure
Jasco). Separation was achieved on a Chiralpak1 AD-H period. The concentration that caused 50% mortality of the
column (250 3 4.6 mm i.d.) at 208C with the enantioselec- test population, or LC50 (lg L21) was determined.
Chirality DOI 10.1002/chir

614
WANG ET AL.
Cell Culture and AchE Inhibition in PC12 Cells
rather than the binding affinities. The distance for hydro-
˚
gen bonding was set to 4 A, and the cutoff value for van
In traditional evaluation, the anti-AChE potencies of
samples were determined against Electrophorus electricus
(EE)-AChE or bovine erythrocytes (BE)-AChE by calculat-
ing their respective concentration leading to 50% inhibition
of AChE activity (IC50). In the present study, PC12 cell
line, developed from rat phaeochromocytoma cells21 which
can express some neuronal features including acetyltrans-
ferase22 was chosen. Employing this cell line can provide a
useful model for elaborating the effects of OPs on the
mammalian nervous system in vitro. Thus, we assessed
the potential neurotoxicities to mammals of FAP by meas-
uring AChE IC50 in PC12 cells.
˚
der Waals forces was 2.5 A. The early termination criterion
˚
was that the top ten-ranked solutions are all within 1.5 A
rmsd of each other.
Statistic Analysis
Unless otherwise stated, all data were expressed as
mean 6 SD. The Student’s t-test (P ꢀ 0.05) was used to
compare the differences between groups. The IC50 in
PC12 cells was in accord with the probability statistics cal-
culation.
The PC12 cells was obtained from the cell bank of the
Chinese Academy of Science [Shanghai, China; the origi-
nal source is ATCC (Manassas, VA)]. The cells were cul-
tured in DMEM medium with 10% of FBS and 5% horse se-
rum supplemented with 2 mmol L21 lutamine, 100 U
mL21 penicillin, and 100 lg mL21 streptomycin in a
humidified atmosphere of 5% CO2 and 95% air at 378C. The
culture media was refreshed every 2–3 d, and subculture
at a ratio of 1:4 was performed with routine trypsinization
every 4–5 d. Cells were harvested and the concentration
regulated to 1 3 107 mL21 cells in 0.01 mol L21 phos-
phate-buffered saline (PBS, pH 7.4). Cells (3 3 108) were
incubated with 1.5 lL test compounds (7.25, 12.5, 25, 50,
100 lg L21 for racemic FAP; 73.1, 36.6, 18.8, 9.4, 4.7 lg
L21 for the enantiomers of FAP) and control (1.5 lL etha-
nol) in a humidified atmosphere of 5% CO2 and 95% air at
378C. Then, 50 lL cells suspension was taken to measure
the activity of AChE in 96 well microtiter plate. Concur-
rently, DTNB and ATCh-I were added at 150 lL and 50
lL, to give final concentrations of DTNB and ATCh-I of
0.28 and 0.5 mmol L21, respectively. The enzymatic activ-
ities of the mixtures in microtiter plates were determined
at 405 nm, five times, at an interval of 5 min from the addi-
tion of the enzyme or enzyme-plus inhibitor. All the tests
and measurements were performed in four replicates.
Each enantiomer of FAP was incubated with cells on two
separate occasions, using four wells for each esterase
determination.
RESULTS AND DISCUSSION
Enantiomer Separation and Identification of Absolute
Configuration
To evaluate enantioselectivity of inhibition of AChE by
FAP, we developed a method to separate the two enan-
tiomers of FAP using HPLC, with an AD-H chiral station-
ary phase (CSP) column and different mobile solvent sys-
tems. The mobile system for this separation are shown in
Figure 2A. The FAP enantiomers separated well by
increasing the ratio of hexane to ethanol. Baseline resolu-
tion of the two enantiomers was obtained when the mobile
phase was between hexane: ethanol 98:2 and 95:5. The
corresponding capacity factors (k), separability factor (a)
and resolution (Rs) are listed in Table 1. The stereoiso-
meric separability can be mainly attributed to the steric fit,
hydrogen bonding and dipole–dipole interaction between
CSP and the tested chemicals.24,25 The followed configura-
tion identification was accomplished using the mobile
phase of hexane/ethanol 98:2.
The CD detection was usually used for identifying the
absolute configuration , or optical rotation, of the resolved
enantiomers in our earlier research.26,27 The CD spectra at
254 nm were obtained for identifying enantiomers
(Fig. 2B). The responses obtained at 254 nm conclude
that the (2)-FAP enantiomer was eluted first and (1)-sec-
ond. To facilate the molecular docking modeling, the abso-
lute configuration of these two enantiomers was further
determined using the octant rule for the correlation
between the sign and magnitude of the observed signal.28
The phosphorus group of FAP could be place into the ori-
Molecular Docking
The genetic algorithm (GA) program for ligand docking gin of the octants (Fig. 3). The Cotton effect (CE) bands
3.0 (GOLD),23 which takes protein flexibility into consider- are ascribed to substituted phenyl p?p* of and n?p*
ation, was employed to dock R-(1) and S-(2)-FAP into phosphorus chromophores, respectively, and establish the
AChE PAS site. The atomic coordinates of the receptor absolute configuration of chiral molecules containing
were directly obtained from the Protein Data Bank (PDB these chromospheres. The substituted phenyl p?p* CE
entry is 2GYV).19 The Receptor was prepared for docking band is shifted to a lower wavelength probably due to a
in such a way that all the heteroatoms (i.e., cofactors, stronger electronic effect of two oxygen atoms. Thus,
waters, ions, etc) were removed. The absolute stereo- R-FAP has a negative CE. Figure 3 shows that stable con-
chemistry of FAP was assigned and optimized in Sybyl6.8 figurations of R-(1)-FAP were geometry optimization
(Sybyl version 6.8. Tripos Associates Inc., 2001 (St. Louis, using the MMFF94 force-field calculations.28
MO). The definition of the active site of AChE is from a
The FAP enantiomers were previously resolved on a
file containing a list of residues within a 6.5 A radius of the Chiralpak1 AS column using another mobile phase [hep-
ligand. The standard default settings of GA parameters tane/ethanol 5 90/10 (v/v)]29. A better resolution can be
recommended by the authors were employed to optimize performed on Whelk1-O1 CSP column with n-hexane/
the GoldScore fitness function, which has been more isopropanol 5 95/5 (v/v).11 The order of FAP enantiomers
suited to the prediction of the ligand binding position, eluted order was ‘‘1’’ first and ‘‘2’’ second when the
Chirality DOI 10.1002/chir
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ENANTIOSELECTIVE TOXICOLOGY OF FENAMIPHOS
615
Fig. 2. Chiral chromatograms of fenamiphos on a Chiralpak1 AD-H column at different ratios of mobile phase and CD and UV spectra of separated
enantiomers of fenamiphos. (A) Using mobile phase at ratio of hexane: ethanol 5 90:10, 95:5, 96:4, and 98:2, flow rate 0.8 ml min21; T, 208C. (B) Separa-
tion was performed on Chiralpak1 AD-H (hexane: ethanol 5 98: 2; T, 208C; flow rate 0.8 ml min21). The detection wavelength was 254 nm.
(R, R)-Whelk1-O1 and (S, S)-Whelk1-O1 columns was
Enantioselective Inhibition of AChE in PC12 Cells
used. These results indicated that a different CSP using
It is well known that the OPs are highly toxic to mam-
different mobile systems may separate enantiomers with a
mals and humans through the inhibition of neural AChE
different elution order.
activity, causing respiratory failure. PC 12 cell line is
usually selected as a model system that can be used to
distinguish OPs likely to cause neurotoxicities (because
Enantioselectivity in Aquatic Toxicity
It is well established that D. magna, is very sensitive to of AChE inhibition).30 Thus, we assessed enantio-
OPs and was used to evaluate the enantioselective acute selectivity in potential neurotoxicity to mammals of FAP
toxicity of FAP. A t-test showed significant differences in
LC50 values for R-(1)-FAP and S-(2)-FAP in a 48 h test
(Table 2). The increasing order of acute toxicity was
R-(1)-FAP > rac-FAP > S-(2)-FAP, and R-(1)-FAP was
2.4 times more toxic than the S-(2)-FAP. The activity of
the racemate was attributed primarily to R-(1)-FAP for
TABLE 1. Capacity factors (k), separation factors (a),
and resolutions (Rs) using the Chiralpak1 AD-H column
at different mobile phase ratioa
D.magna. Our results are in good agreement with earlier
research in which the LC50 of R-(1)-FAP, S-(2)-FAP and
theracemate to Daphnia pulex was 1.6, 6.1, and 1.9 lg L21,
respectively, which also shows that the R-(1)-FAP was
mainly responsible for the toxicity of the racemate . There-
fore, although the racemate is commercially used for FAP,
one enantiomer contributes more significantly to acute
toxicity.
Mobile phase
Ratio
k1
k2
a
Rs
n-hexane:ethanol
90:10
95:5
96:4
98:2
1.29
2.57
4.13
7.24
2.46
3.97
4.28
1.40
1.50
1.75
1.91
1.19
1.92
2.39
3.42
10.11
Flow rate 0.8 mL min21
.
aMobile phase ratios: n-hexane: ethanol 5 90:10, 95:5, 96:4, and 98:2.
Chirality DOI 10.1002/chir

616
WANG ET AL.
TABLE 3. Inhibitory potencies of racemate and
enantiomers of fenamiphos toward the
acetylcholinesterase in PC12 cells
Incubating
time (30 min)
R-(1)-FAP
16.4 6 1.06
S-(2)-FAP
46.1 6 6.22
rac-FAP
a
IC50
25.3 6 5.23
aIC50 5 half inhibition of AChE activity (P < 0.05) at 30 min
Different Interaction Modes of R-(1) and S-(2)-FAP
Fig. 3. Application of the octant rule for R-(1)-fenamiphos. [Color fig-
ure can be viewed in the online issue, which is available at www.
interscience.wiley.com.]
Based on the structural information about the binding
site and the inhibitory mechanism, molecular modeling
may be used to gain insight into the ability of AChE to dis-
criminate between enantiomers. As shown in Figure 4,
both R-(1) and S-(2)-FAP fit at the PAS binding pocket
but with distinctly different orientations. Obviously, R-(1)-
FAP embeds in a relative blocked space comprised of Trp
86, Tyr 337, phenylalanine (Phe) 338 and His 477, and
hydrophobic interactions are heavily involved with the aro-
matic side chains of Tyr 337 and Phe 338 and the phenyl
ring of the ligand, as well as the side chain of Trp 86 and
the methyl function. Furthermore, the amide oxygen
forms a hydrogen bond with the side chain of Tyr 124,
which is crucial, and common, in the binding modes of
AChE interacting with small molecules. However, S-(2)-
FAP, although proximate to the binding pocket, could not
form orderly interactions due to the steric hindrance
between Trp 86 and the large N-isopropylamido-phosphate
substituent. Thus, hydrogen bonding and hydrophobic
interactions allow AChE to discriminate the between of
R-(1)- and S-(2)-FAP when binding to AChE, thereby
explaining the greater toxicity of R-(1)-FAP in contrast to
S-(2)-FAP. It is surprising that there have been no reports
on enantiomer-specific affinity to target sites of chiral
chemicals.
by measuring the inhibition of AChE activity (IC50) in the
PC12 cell line. Judging from the IC50 values, the increas-
ing order of toxicity to AChE was R-(1)-FAP > rac-FAP >
S-(2)-FAP. To the best of our knowledge, this is the first
report of inhibition of AChE activity in PC 12 cells by FAP
(Table 3).
Previously, Wang et al.11 confirmed the marked enantio-
selective inhibition of butyrylcholinesterase (BuChE) ac-
tivity by FAP. Acetylcholinesterase is important in the
nervous system for hydrolysing acetylcholine. This is a
suitable endpoint for assessing toxicity, and both enzymes
are type ‘‘B’’ esterase. Taking this into considering, both
our and Wang’s results show that FAP has great potential
in inhibiting the activity of type ‘‘B’’ esterases and R-(1)-
FAP was more toxic than S-(2)-FAP.11
We noted that the FAP enantiomers possessed a similar
toxic trend to both mammalian cells and aquatic inverte-
brates; R-(1)-FAP was more toxic to nontarget organisms
than S-(2)-FAP. These encouraging results motivate our
interest in explore the subject of enantioselectivity. As
mentioned above, the most important reason for OP poi-
soning is inhibition of type ‘‘B’’ esterases by binding to the
active site and phosphorylating the enzyme. The ability of
an enzyme to discriminate between the enantiomers of a
chiral substrate can be the result of kinetic and/or thermo-
dynamic factors and the difference may occur at the bind-
ing site or at the catalytic step.31 Based on this theory, we
hypothesized that the enantioselectivity in toxicity to
D. magna and PC12 cells is due to the different interaction
between enantiomers of FAP and the active centre of cho-
linesterase. Thus, studies on the molecular mechanism of
FAP enantiomers and their interactions with AChE were
necessary.
TABLE 2. The median lethal concentrations (LC50) for
racemate and enantiomers of fenamiphos to Daphnia
magna lg L21
)
Test time (h)
R-(1)-FAP
S-(2)-FAP
rac-FAP
Fig. 4. Different binding modes of R-(1)-(carbon atoms shown in cyan
color) and S-(2)-fenamiphos (carbon atoms displayed in green color) with
residues in the peripheral anionic site (PAS) of acetylcholinesterase
(AChE). [Color figure can be viewed in the online issue, which is available
at www.interscience.wiley.com.]
24
48
5.1 6 0.35
2.7 6 0.42
9.2 6 1.90
6.6 6 0.35
6.4 6 0.00
3.9 6 0.70
Chirality DOI 10.1002/chir

ENANTIOSELECTIVE TOXICOLOGY OF FENAMIPHOS
617
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Chirality DOI 10.1002/chir