Reaction of cresyl saligenin phosphate, the organophosphorus agent implicated in aerotoxic syndrome, with human cholinesterases: Mechanistic studies employing kinetics, mass spectrometry, and X-ray structure analysis

Article abstract of DOI:10.1021/tx100447k

Aerotoxic syndrome is assumed to be caused by exposure to tricresyl phosphate (TCP), an antiwear additive in jet engine lubricants and hydraulic fluid. CBDP (2-(ortho-cresyl)-4H-1,2,3-benzodioxaphosphoran-2-one) is the toxic metabolite of triortho-cresylphosphate, a component of TCP. Human butyrylcholinesterase (BChE; EC 3.1.1.8) and human acetylcholinesterase (AChE; EC 3.1.1.7) are irreversibly inhibited by CBDP. The bimolecular rate constants of inhibition (k_i), determined under pseudo-first-order conditions, displayed a biphasic time course of inhibition with k_i of 1.6 ×10⁸ M^-1 min^-1 and 2.7 ×10 ⁷ M^-1 min^-1 for E and E′ forms of BChE. The inhibition constants for AChE were 1 to 2 orders of magnitude slower than those for BChE. CBDP-phosphorylated cholinesterases are nonreactivatable due to ultra fast aging. Mass spectrometry analysis showed an initial BChE adduct with an added mass of 170 Da from cresylphosphate, followed by dealkylation to a structure with an added mass of 80 Da. Mass spectrometry in ¹⁸O-water showed that ¹⁸O was incorporated only during the final aging step to form phospho-serine as the final aged BChE adduct. The crystal structure of CBDP-inhibited BChE confirmed that the phosphate adduct is the ultimate aging product. CBDP is the first organophosphorus agent that leads to a fully dealkylated phospho-serine BChE adduct.

Full text of DOI:10.1021/tx100447k

ARTICLE
pubs.acs.org/crt
Reaction of Cresyl Saligenin Phosphate, the Organophosphorus
Agent Implicated in Aerotoxic Syndrome, with Human
Cholinesterases: Mechanistic Studies Employing Kinetics, Mass
Spectrometry, and X-ray Structure Analysis
†
‡
†
§
Eug ꢀe nie Carletti, Lawrence M. Schopfer, Jacques-Philippe Colletier, Marie-Th ꢀe r ꢁe se Froment,
§
†
‡
,†,‡,§
Florian Nachon, Martin Weik, Oksana Lockridge, and Patrick Masson*
†
Laboratoire de Biophysique Mol ꢀe culaire, Institut de Biologie Structurale, 41 Rue Jules Horowitz, 38027 Grenoble, France
‡
Eppley Institute and Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha,
Nebraska 68198-5950, United States
§
D ꢀe partement de Toxicologie, Institut de Recherche Biom ꢀe dicale des Arm ꢀe es (IRBA)-Centre de Recherches du Service de Sant ꢀe des
Arm ꢀe es (CRSSA), 24 av des Marquis du Gr ꢀe sivaudan, 38702 La Tronche, France
ABSTRACT:
Aerotoxic syndrome is assumed to be caused by exposure to tricresyl phosphate (TCP), an antiwear additive in jet engine lubricants
and hydraulic fluid. CBDP (2-(ortho-cresyl)-4H-1,2,3-benzodioxaphosphoran-2-one) is the toxic metabolite of triortho-cresylphos-
phate, a component of TCP. Human butyrylcholinesterase (BChE; EC 3.1.1.8) and human acetylcholinesterase (AChE; EC
3
.1.1.7) are irreversibly inhibited by CBDP. The bimolecular rate constants of inhibition (k ), determined under pseudo-first-order
i
8 7
ꢁ1
ꢁ1
ꢁ1
ꢁ1
0
conditions, displayed a biphasic time course of inhibition with k of 1.6 ꢀ 10 M min and 2.7 ꢀ 10 M min for E and E
i
forms of BChE. The inhibition constants for AChE were 1 to 2 orders of magnitude slower than those for BChE. CBDP-
phosphorylated cholinesterases are nonreactivatable due to ultra fast aging. Mass spectrometry analysis showed an initial BChE
adduct with an added mass of 170 Da from cresylphosphate, followed by dealkylation to a structure with an added mass of 80 Da.
18
18
Mass spectrometry in O-water showed that O was incorporated only during the final aging step to form phospho-serine as the
final aged BChE adduct. The crystal structure of CBDP-inhibited BChE confirmed that the phosphate adduct is the ultimate aging
product. CBDP is the first organophosphorus agent that leads to a fully dealkylated phospho-serine BChE adduct.
5
,6
’
INTRODUCTION
in vivo. CBDP is formed in vivo by two consecutive reactions:
6
(
1) liver microsomal cytochrome P450-catalyzed oxidation and (2)
serum albumin-catalyzed cyclization of the oxidation product
Scheme 1).
TOCP is a component of tricresyl phosphate (TCP) that is a
combination of ten tricresyl phosphate isomers (triortho, tripara,
trimeta, and mixtures of the three). TOCP is more toxic than the
para and meta isomers. TCP is used as an antiwear/extreme
The cyclic organophosphorus agent 2-(o-cresyl)-4H-1,3,2-
7
benzodioxaphosphoran-2-one (CBDP), also called cresyl sali-
(
genin phosphate, has been implicated in the development o1f
organophosphate-induced delayed neuropathy (OPIDN).
OPIDN is a paralytic condition that is distinct from the toxicity
associated with organophosphate inhibition of acetylcholinester-
ase (AChE). Well known incidents of OPIDN have been attributed
to the consumption of Jamaica ginger, a folk medicine, adulterated
with tri-o-cresyl phosphate (TOCP), and to the consumption of
cooking oil adulterated with jet engine oil containing TOCP. The
toxicity of TOCP has been attributed to its conversion into CBDP,
8
2
pressure agent and flame retardant in jet hydraulic fluids and
3
4
Received: December 21, 2010
Published: March 25, 2011
r 2011 American Chemical Society
797
dx.doi.org/10.1021/tx100447k
|Chem. Res. Toxicol. 2011, 24, 797–808

Chemical Research in Toxicology
ARTICLE
Scheme 1. Metabolic Activation of TOCP to CBDP
likely plays a role in protection against the toxicity of TOCP by
scavenging CBDP from the human bloodstream. Both X-ray
crystallography and mass spectrometry confirm that the ultimate
product from the reaction of CBDP with BChE is a novel
phosphorylated adduct of the catalytic serine, Ser198.
Kinetics for the reaction of CBDP with human AChE indicate
that AChE is significantly sensitive to CBDP, although less than
BChE by at least 1 order of magnitude.
8
,9
engine oils. Because of its toxicity, the level of TOCP in
’ MATERIALS AND METHODS
8
commercial TCP mixtures has been reduced over time. How-
Caution: CBDP is a highly toxic organophosphorus compound. Hand-
ling requires suitable personal protection, training, and facilities. These
requirements are the same as those for other poisonous organophosphorus
compounds.
Chemicals. CBDP was a gift from Dr. D. Lenz (USAMRICD,
Aberdeen PG, MD, USA); this compound is 99.5% pure and was custom
synthesized by Starks Associates Buffalo, NY, USA. A 0.1 M stock solution
of CBDP was made in acetonitrile and stored at ꢁ70 ꢀC. Working
solutions of CBDP were made in anhydrous methanol, acetonitrile, or
dimethylsulfoxide (DMSO) and stored at ꢁ70 ꢀC. 2-PAM (pralidoxime
methiodide, N-methyl-pyridin-1-ium 2-aldoxime methiodide) was Gold-
label-grade from Ega-Chemie (Steinheim, Germany) and HI-6 (1-[[[4-
ever, it is unclear whether this precaution has been sufficient to
prevent toxic exposure to TOCP because safe levels of exposure
1
0
are still in dispute.
Over the past 30 years, an increasing number of reports have
appeared documenting the occurrence of neurological signs
10
associated with air travel, both commercial and military.
Short-term symptoms include blurred vision, dizziness, confu-
sion, headache, tremors, nausea, vertigo, shortness of breath,
increased heart rate, and irritation of the eyes and nose. Long-
term symptoms include memory loss, numbness, lack of co-
ordination, sleep disorders, severe headaches, nausea, diarrhea,
susceptibility to upper respiratory infection, chest pain, skin
blisters, signs of immunosuppression, muscle weakness, muscle
(
aminocarbonyl) pyridinio] methoxy] methyl]-2-[(hydroxyimino)-
methyl] pyridinium dichloride monohydrate) was a gift from Dr. D.
1
0,11
pain, and fatigue.
The term aerotoxic syndrome has recently
18
Genkin (Pharmsynthez, Saint Petersburg, Russia). Water- O (99 atom
10
been coined to describe this condition. Fumes escaping from
the engine through leaky oil seals into the bleed air of the aircraft
cabin are suspected to be the source of the toxicants that cause
18
%
O) was from Isotec (a member of the Sigma-Aldrich group St. Louis,
MO; catalog number 487090). Sequencing grade modified trypsin was
from Promega (Madison, WI, catalog number V5111). Alpha-cyano-2-
hydroxy cinnamic acid (CHCA) was purchased from Fluka (a member
of the Sigma-Aldrich group St. Louis, MO, catalog number 70990) and
prepared as a saturating solution in 50% acetonitrile/water plus 0.3%
trifluoroacetic acid. Acetonitrile used for mass spectrometry was of DNA
sequencing grade from Fisher (Pittsburgh, PA, catalog number BP-1170),
and trifluoroacetic acid used for mass spectrometry was of sequencing
grade (>99.9%) from Beckman (Brea, CA, catalog number 290204).
All other chemicals were of biochemical grade.
8
,10
aerotoxic syndrome.
Toxic components of these fumes include hexane, CO, CO2,
and TOCP. TOCP after bioactivation to CBDP is suspected of
being responsible for the symptoms. A key gap in the evidence
trail between fumes and syndrome is a quantitative biomarker for
exposure to TOCP. CBDP has long been known as an inhibitor
15
1
2ꢁ14
of carboxylesterases
Animal studies have shown that CBDP is also an irreversible
and of neuropathy target esterase.
12,16
inhibitor of both AChE and BChE.
However, in vitro studies
Enzyme Sources. Both native and recombinant human BChE
(hBChE; accession number P06276) were used in this work. Native
have indicated that CBDP reacts slowly with mammalian
1
4,17
21
hBChE purified from plasma as described was used for the kinetic
ChEs.
Modifications to the structure of CBDP have led to
studies and mass spectrometry. hBChE used for the kinetic studies
consisted mostly of the tetrameric form. It was 61% pure with an activity
of 41 units/mL, using BTC as the substrate at pH 8.0 and 25 ꢀC (unit = 1
μmol of substrate hydrolyzed per min). The hBChE that was used for
mass spectrometry had an activity of 73,400 units/mL (100 mg/mL)
and was >50% pure. Both batches of enzyme were stored for months at
þ4 ꢀC without activity loss in 30 mM Tris/HCl, pH 7.5, containing
the development of insecticides, such as salioxon (2-methoxy-4H-
1
,3,2-benzodioxaphosphorin 2-oxide), that display strong antic-
1
8ꢁ20
holinesterase activity.
Recently, we established that human
BChE reacts with CBDP. The organophosphorylated adduct
undergoes two consecutive dealkylation reactions, i.e., aging,
forming an ultimate phosphate adduct on the active site serine
1
(
Ser198). Thisphosphorylated derivativeisunique inthe study of
0.1 M NaCl and 0.02% sodium azide. The recombinant hBChE was used
organophosphate (OP) reactions with BChE and therefore would
be an ideal candidate for use as a biomarker of exposure to TOCP.
Interest in BChE as a biomarker for exposure to TOCP has
prompted us to investigate the mechanism and kinetics of the
reaction of CBDP with BChE and AChE in more detail.
for crystallization. It was a truncated monomer, containing residues
ꢁ529 and 5 carbohydrate chains rather than the 9 in native hBChE.
Recombinant human BChE was expressed in Chinese hamster ovary
CHO) cells and purified by affinity and ion exchange chromatogra-
1
(
22
phies, as described previously. Its concentration was 6.47 mg/mL.
Recombinant human AChE (hAChE; accession number P22303) was
expressed in stably transfected Chinese hamster ovary cells (CHO) and
In the present article, we (1) investigated the kinetics of
phosphorylation, inhibition, and aging of highly purified human
AChE and human BChE by CBDP; (2) examined the chemistry
for the formation of the postphosphorylation adducts, using mass
23
purified as described previously. Highly purified AChE (>90%) was
300-fold diluted into 0.1 M phosphate buffer at pH 8.0 supplemented
18
spectrometry and O-water; and (3) determined the X-ray
structure of the ultimate aged conjugate of CBDP-phosphorylated
human BChE.
with 0.1% bovine serum albumin (BSA) and 0.01% sodium azide for all
kinetic studies.
Enzyme Activity and Inhibition. Cholinesterase activity was
2
4
The kinetics for the reaction of CBDP with human BChE
indicate that CBDP is one of the most potent OP inhibitors for
BChE heretofore discovered and that postphosphorylation aging
is extremely rapid. Because of this high reactivity, BChE very
measured by the method of Ellman at pH 8.0, at 25 ꢀC in 50 mM
phosphate buffer, using acetylthiocholine (ATC) iodide (1 mM) as the
substrate for AChE and butyrylthiocholine (BTC) iodide (1 mM) as the
substrate for BChE.
7
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Chemical Research in Toxicology
ARTICLE
Scheme 2. Reaction of OPs with Cholinesterases
The reactions were incubated at room temperature for 30 min, after which
time there was no measurable BChE activity remaining (1 μL from each
sample was used for measuring activity). Parallel incubations of BChE
were made without CBDP. All samples were prepared in siliconized,
1
.7 mL microfuge tubes (Avant from Midwest Scientific, St. Louis, MO,
Progressive inhibition of enzymes by CBDP was performed in 50 mM
sodium phosphate buffer at pH 8.0 at 25 ꢀC, 10% methanol, or 5%
acetonitrile. For inhibition studies of hBChE and hAChE, the final
CBDP concentrations ranged from 0.5 nM to 10 nM and 0.1 to 5.5 μM,
respectively. Ten percent MeOH has no denaturing effect on BChE and
cat# AVSC1510). After the incubations, all four samples were denatured
by boiling for 10 min, then dried under vacuum in a Savant SpeedVac
(Thermo Scientific, Waltham, MA). The purpose for drying was to
1
8
18
remove the O-water. Removal of the O-water before trypsinolysis
simplifies interpretation of the mass spectra because trypsin would
introduce two O-oxygens into the C-terminus of every peptide during
proteolysis if trypsinolysis were conducted in an O-medium. The
pellets were redissolved in 106 μL of 10 mM ammonium bicarbonate
25,26
does not significantly alter its reactivity.
Despite the presence of
1
8
0.1% BSA, the addition of 10% methanol or 5% acetonitrile caused a
1
8
33
decrease in AChE activity that stabilized in a few minutes at 75ꢁ80%
residual activity. This solvent effect does not interfere with the inhibition
mechanism of AChE during the time course of experiments.
1
6
made with O-water. Four microliters of sequencing grade trypsin
0.4 μg/μL in 50 mM acetic acid, 200 nmol of acetic acid) was added
(
Inhibition kinetics were performed under pseudofirst-order conditions
to each preparation, followed by 2 μL of 0.1 M ammonium bicarbonate
(200 nmol of bicarbonate) to neutralize the acetic acid. The preparations
were incubated at 37 ꢀC overnight.
Ten microliters of each digest was desalted using C18 ZipTips
(Millipore, Billerica, MA catalog number ZTC18SO96). The ZipTip
was wetted with 100% acetonitrile and activated with 0.1% trifluoro-
acetic acid. One microliter of 1% trifluoroacetic acid was added to each
(
[E] , [CBDP]). BChE active site concentration [E] in the inhibition
ꢁ11
medium was 6 ꢀ 10 mol/L, and AChE active site concentration was 5
ꢁ
11
ꢀ
10
mol/L. Enzyme active site concentrations were determined
from the catalytic activity of diluted preparations at Vmax, pH 8.0, and
ꢁ
1
27
2
5 ꢀC, taking kcat = 45,500 min for BChE with BTC as the substrate
ꢁ
1
2
8
and kcat = 400,000 min for hAChE with ATC as the substrate. The
time course of enzyme inhibition was monitored by the Aldridge sampling
method, i.e., sampleswerewithdrawn atdifferent incubationtimes(15 s to
1
0 μL aliquot of digest. Each acidified digest was loaded onto a separate
activated ZipTip, washed with 0.1% trifluoroacetic acid, and eluted with
0 μL of 60% acetonitrile/water containing 0.1% trifluoroacetic acid.
20 min) after the addition of CBDP, and the remaining enzyme activity
1
was determined. The pseudofirst-order rate constants for adduct
formation (kobs) were taken from the slopes of plots of log (residual
An aliquot of each eluant was diluted 10-fold with 50% acetonitrile/
water plus 0.3% trifluoroacetic acid. Two microliters of each dilution was
mixed with 2 μL of CHCA (alpha-cyano-4-hydroxycinnamic acid). One
microliter aliquots of the mixture were spotted onto a MALDI target
plate (384-well Opti-TOF plate, catalog number 1016491 from Applied
Biosystems, Foster City, CA) and allowed to air dry.
Samples were analyzed in a MALDI TOFTOF 4800 mass spectrom-
eter (Applied Biosystems, Foster City, CA) using both reflector
positive and reflector negative modes with delayed extraction. Six 500
laser pulses were accumulated for each measurement, over a mass range
of 2000 to 4000 amu, using a laser voltage of 3500 V in positive mode
and 3600 V in negative mode. Data collection was controlled by 4000
Series Explorer software (version 3.5). Mass calibration was made with
Cal Mix 5 (containing bradykinin, 2ꢁ9 clip; angiotensin I; Glu-
fibrinopeptide B; adrenocorticotropic hormone [ACTH], 1ꢁ17 clip;
ACTH, 18ꢁ39 clip; and ACTH, 7ꢁ38 clip, Applied Biosystems).
Crystallization of Recombinant hBChE and Generation of
the Ultimate Aged Conjugate of CBDP-Phosphorylated Hu-
29,30
activity) versus time.
Previous work suggested that initial phosphorylation of the BChE
active site serine would lead to a covalent, ring-opened CBDP-serine
1
adduct. Covalent adduct formation by CBDP may be described by the
classical scheme for the reaction of OPs with ChEs (Scheme 2). In this
scheme, EH stands for the free enzyme, I⊃ for the cyclic phosphotriester
CBDP, EH.I⊃ for the noncovalent Michaelis complex between enzyme
and CBDP, and EHI for the ring-opened CBDP phosphorylated enzyme.
K
I
= kꢁ1/k
1
is the dissociation constant of complex EH.I⊃, and k
p
is the
enzyme phosphorylation rate constant. Assuming that rapid equilibrium
conditions hold (i.e., k , k ), the observed rate for inhibition, kobs, is as
p
ꢁ1
follows:
kp½CBDPꢂ
kobs ¼
ð1Þ
KI þ ½CBDPꢂ
Analysis of kinetic data was performed using GOSA-fit, a fitting software
based on a simulated annealing algorithm (BiogLog, Toulouse, France;
http://www.bio-log.biz).
22
man BChE. Human BChE was crystallized as described previously.
The final conjugate of hBChE inhibited by CBDP was obtained by
soaking crystals for 12 h in a mother liquor solution (0.1 M MES, pH 6.5,
2.1 M ammonium sulfate) containing 1 mM CBDP (at 4 ꢀC). The
CBDP stock solution was 10 mM in DMSO. Crystals were washed for 5 s
in a cryoprotective solution (0.1 M MES buffer pH 6.5, 2.3 M
ammonium sulfate, and 20% glycerol) before being flash-cooled in
liquid nitrogen for data collection.
X-ray Data Collection and Structure Solution of the
Ultimate Aged Conjugate of CBDP-Phosphoryated Human
BChE. Diffraction data were collected at the European Synchrotron
Radiation Facility (ESRF, Grenoble, France). Data for crystalline hBChE
inhibited by CBDP were collected at 100 K, on the ID14-eh4 beamline
using λ = 0.9765 Å wavelength with an ADSC Quantum Q315r detector.
The data set was processed with XDS, and the structure was solved
with the CCP4 suite. An initial model was determined by molecular
replacement with MolRep, starting from the recombinant hBChE
structure (PDB entry 1P0I) from which all ligands and glycan chains
were removed. The initial model was refined as follows: a rigid-body
refinement, made with REFMAC5, was followed by iterative cycles of
model building with Coot and then restrained, and TLS refinement
Aging and Reactivation of CBDP-Inhibited Cholines-
terases. Reactivation and dealkylation rates for CBDP-phosphonylated
ChEs were measured using standard procedure for the investigation of
31
oxime-mediated reactivation and aging of ChEs. Enzymes were inhib-
ited with CBDP to 95% in less than 15 min, and aliquots were removed at
various times (from 15 s to 10 min) following inhibition and immediately
incubated withoxime reactivators in 0.1 M sodiumphosphate buffer atpH
8.0 at 25 ꢀC. Two different oxime reactivators were tested: 2-PAM (at 1 or
10mM) andHI-6(at0.5 mM). ChEactivity wasmeasuredasafunction of
incubation time in the presence of oxime, for up to 24 h.
Mass Spectrometry of CBDP-Inhibited BChE. hBChE was
completely inhibited by excess CBDP in the presence and absence of
32
1
8
18
18
34
O-water. For the reaction in the presence of O, 100 μL of O-water
was combined with 1 μL of 1 M ammonium bicarbonate, 5 μL of BChE
73,400 units/mL), and 1 μL of CBDP (100 mM in acetonitrile). For the
3
5
3
6
(
18
16
reaction in the absence of O, 100 μL of O-water was combined with
μL of 1 M ammonium bicarbonate, 5 μL of BChE (73,400 units/mL),
and 1 μL of CBDP (100 mM). The final reaction mixtures contained
6 μM BChE and 930 μM CBDP in 10 mM ammonium bicarbonate.
1
3
7
3
8
5
7
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Chemical Research in Toxicology
ARTICLE
37,39
was carried out with REFMAC5.
TLS groups were defined with the
Scheme 3. Reaction of CBDP with Two Cholinesterase
help of the TLS Motion Determination server (http://skuld.bmsc.
Forms in Slow Equilibrium
40
washington.edu/∼tlsmd/index.html). The bound ligand and its de-
scriptions were built using the Dundee PRODRG2.5 server including
energy minimization using GROMOS96.1 force field calculations.
’
RESULTS AND DISCUSSION
Kinetics for the Inhibition of Human Cholinesterases by
CBDP. Inhibition of BChE by CBDP did not follow simple first-
order progressive kinetics. Plots of residual activity (ln(A/A0)
versus time were biphasic, despite the fact that the basic condi-
tion for a first-order reaction was maintained, i.e., the enzyme
concentration in the reaction medium was much less than the
CBDP concentrations, and that CBDP was stable during the
course of inhibition. In addition, titration experiments with
[
CBDP] varying from 0.2 [E ] to 0.9 [E ] showed that there
0 0
was no spontaneous reactivation of inhibited enzymes.
Thus, the biphasic progressive inhibition can be regarded as the
sum of two first-order processes, a fast phase and a slow phase. It
should be noted that the first-order lines for both phases extra-
polated to ln50% activity at t = 0. Situations similar to this, where
first-order lines do not pass through the origin, were described by
30
Aldridge and Reiner. In one case, they described partial inhibi-
tion at zero-time, i.e., rapid equilibrium formation of an inhibitory
complex prior to phosphonylation. In another case, there was
41ꢁ43
ongoing inhibition during the reaction with substrate.
These
situations could be identified by the dilution of the enzyme prior
to the measurement of residual activity or by increasing the
substrate concentration during activity measurement. Neither
dilution of the inhibited enzyme prior to the measurement of
residual activity nor increasing the concentration of substrate
([BTC] from 0.25 mM to 10 mM) during the assay changed the
extrapolated ln(% activity) of BChE. These observations indicate
that neither ongoing inhibition nor rapid equilibrium binding of
inhibitor is responsible for the biphasic inhibition behavior
exhibited by CBDP. In addition, for the type of behavior
Figure 1. Time course of inhibition of hBChE by different concentra-
tions of CBDP in 50 mM sodium phosphate at pH 8.0, containing 10%
MeOH at 25 ꢀC. The solid curve represents the nonlinear regression fit
using eq 2. Insert: secondary plot used for the determination of the
0
30
41
bimolecular rate constants of inhibition (k and k ) for fast and slow
i i
described by Aldridge and Reiner and Estevez and Vilanova,
phases, according to eq 4.
the intercept on the activity axis depends on inhibitor concen-
tration. In the case of the inhibition of BChE by CBDP, first-
order lines for the slow phase extrapolated through the ordinate
ChEs by CBDP, under pseudofirst-order conditions, can be
described by the following equation:
at [E ] = 50 ( 10% regardless [CBDP]. After subtraction of the
0
slow process contribution, the first-order lines for the fast phase
ꢁ
kobs
t
0
ꢁk0
t
0
3
obs 3
½
Eꢂ ¼ ½Eꢂ e
þ ½E ꢂ e
ð2Þ
also extrapolated through the ordinate at [E ] = 50 ( 10%
t
0
0
0
whatever the [CBDP]. The sum of the two ordinate intercepts
was always equal to 100%. Both fractional activities at t may be
0
0
with kobs > k obs, and
regarded as the relative activities of two enzyme forms. Since
both fractional activities are the same, the two enzyme forms are
present in the preparation in similar amounts. Inhibition of
AChE by CBDP is also biphasic. However, in this case, the
extrapolated y-axis intercept for the fast phase is larger than the
kp ½Iꢂ
0
3
½
Etotꢂ ¼ ½Eꢂ þ ½E ꢂ , kobs ¼
,
0
0
0
K þ ½Iꢂ
I
0
0
k ½Iꢂ
p 3
koff
k
0
0
on
k
¼
, K ¼
I
, andK ¼
ð3Þ
extrapolated intercept for the slow phase. Therefore, the fast
obs
0
I
0
K þ ½Iꢂ
kon
k
I
off
0
phase form of AChE, [E ], is present in higher concentration
0
0
than the slow phase form, [E0].
By convention, we refer to kobs for the fast phase and to k for
obs
The biphasic progressive inhibition indicates that CBDP
reacts at different rates with two different active populations of
butyrylcholinesterase (E and E ) that are in slow equilibrium.
the slow phase. Figure 1 shows typical biphasic plots for the
progressive inhibition of BChE by CBDP that fit with eq 2.
0
44,45
0
Measured values for kobs and k
are calculated by nonlinear
obs
The affinity of both forms for certain inhibitors can be different,
and their reactivity can be different too. Such an inhibition
pattern has also been observed for carbamylation of BChE with
regression fit using eq 2.
For BChE, replots of kobs versus [CBDP] for both the fast and
slow phases were linear, passing through the origin (Figure 1,
insert). This indicates that for both the E and E forms of BChE,
46
0
N-methyl-N-(2-nitrophenyl) carbamoyl chloride. This situa-
tion is described in Scheme 3. Thus, the kinetics for inhibition of
the highest CBDP concentration (10 nM) remained lower than
8
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Chemical Research in Toxicology
ARTICLE
a
Table 1. Bimolecular Rate Constants (k ) for the Inhibition of Human AChE and BChE at pH 8.0 and 25 ꢀC
i
b
AChE
BChE
0
0
enzyme form
E
E
E
E
ꢁ
1
ꢁ1
6c
5
5
8
7
k
i
k
i
k
i
(M min ) 10% MeOH
>3.7 ꢀ 10
ND
(1.06 ( 0.23) ꢀ 10
(0.33 ( 0.17) ꢀ 10
(1.5 ( 0.2) ꢀ 10
ND
ND
(2.5 ( 0.7) ꢀ 10
ꢁ
1
ꢁ1
(M min ) 5% acetonitrile
ND
ꢁ1
ꢁ1
5
(M min ) 0.9% saline
ND
(0.73 ( 0.6) ꢀ 10
ND
b
a
0
E, enzyme fast forms; E enzyme slow forms; ND, not determined. Experiments were performed in triplicate (mean values ( SE). Experimental k
values for the E-form of AChE are not listed because the kinetics did not provide a second order rate constant in that instance. Estimated minimum k
value using k = 0.37 min and K , 0.1 μM.
i
i
c
ꢁ1
p
I
Scheme 4. Reaction Pathway of CBDP with Cholinesterases
0
K and K . It follows that under our experimental conditions, k
of CBDP is that CBDP is unstable in water. We found that
the hydrolysis rate of CBDP in 0.1 M sodium phosphate at
I
I
obs
0
and k reduce to
obs
ꢁ1
pH 8.0 was 0.0126 min at 25 ꢀC, i.e., t1/2 = 55 min. This agrees
with a previously reported t1/2 value of 60 min in 36 mM
0
kp
kp
0
0
kobs ¼ ½Iꢂ ¼ ki ½Iꢂ and
k
¼
½Iꢂ ¼ k ½Iꢂ ð4Þ
i 3
3
obs
0
K
I
6
KI
barbiturateꢁ8 mM potassium phosphate buffer at pH 8.0.
48
0
Saligenin-derived, six-membered cyclic phosphates (oxon) are
even moresensitiveto hydrolysis. Instability ofthesecompounds
with k and k as the bimolecular rate constants of inhibition.
Determined values of k and k are given in Table 1.
The situation was somewhat different for AChE (data not
shown). The inhibition kinetics were biphasic, and a replot of the
i
i
0
i
i
is reported to be due to a strain in the distorted benzodioxaphos-
49,50
phorin ring.
Our results (Table 1, k
phase reactivity of hBChE (form E ) with CBDP is 235 ( 110-fold
faster than the reactivity of hAChE (form E ). Though it was not
0
0
i
in 10% MeOH) show that the slow
slow phase k
versus [CBDP] was linear and passed through
obs
0
0
the origin. The linear dependence of k on [CBDP] indicates
that, as for BChE, the highest experimental CBDP concentration
5.5 μM) was far less than K . However, the fast phase of
inhibition was independent of the CBDP concentration, giving
kobs = 0.37 ( 0.15 min . Such a situation occurs if the CBDP
concentration is very much larger than K so that k reduces to
kp, i.e., the E form is saturated by CBDP even at the lowest
concentration (0.1 μM).
Determined values of k for the inhibition of AChE form E
under different solvent conditions are also given in Table 1.
Experimental values remain in the same order of magnitude
showing that the solvent effect is weak. The slight increase in k i
observed for the inhibition performed in 5% acetonitrile may
obs
0
0
possible to determine k for hAChE (form E) directly, an estimate
i
(
I
could be made from the following. The k for BChE (form E) can
p
ꢁ1
be estimated from its k , knowing from the experiments that K for
i
I
BChE must be much larger than 10 nM. Since k for BChE is 1.5 ꢀ
i
I
obs
8
ꢁ1
1
ꢁ1
1
0 M min and K . 10 nM, then k = k K for BChE . 1.5
min > 0.37 min = k for AChE. Therefore, on the one hand
I
p
i
3
I
ꢁ
ꢁ1
p
0
0
we know that k for BChE . k for AChE, and on the other hand,
p
p
i
we do not expect that K for AChE is smaller than that for BChE
I
because the active site of AChE poorly accommodates large
ligands like CBDP compared to the active site of BChE. It follows
0
that k for AChE (form E) , k for BChE, i.e., the reactivity of
i
i
reflect a specific effect of this solvent on AChE as has been
reported by several investigators, but did not reflect a change in
the mechanism of inhibition.
AChE (form E) is much lower than the reactivity for BChE (form E).
However, a lower limit value for k for AChE (fast form, E) may
be estimated from the experimental value of k
47
i
ꢁ
1
= 0.37 min and
p
6
ꢁ1
ꢁ1
The determined values of the bimolecular rate constants for
K
I
, 0.1 μM, giving k
i
. 3.7 ꢀ 10 M min . Despite the
8
the phosphonylation of both hBChE (k = 1.5 ( 0.2 ꢀ 10 and
reactivity of AChE with CBDP being lower than that of BChE, it
i
0
7
ꢁ1
ꢁ1
6
k = 2.5 ( 0.7 ꢀ 10 M min ) and hAChE (>3.7 ꢀ 10 and
appears to be larger than some nerve agents such as tabun (7.4 ꢀ
i
5
ꢁ1
ꢁ1
6
ꢁ1
ꢁ1 51
1
.06 ( 0.23 ꢀ 10 M min ) by CBDP (Table 1) are much
10 M min ).
faster than values previously reported for the inhibition of
Aging and Nonreactivatibility of CBDP-Inhibited BChE.
Previous MALDI-TOF mass spectral studies suggested that two
postinhibitory reactions occurred after the formation of the initial
BChE covalent adduct, i.e., rapid hydrolysis of the ring-opened
CBDP adduct into an o-cresyl-phospho-serine adduct with the
release of saligenin, followed by conversion of this adduct into a
cholinesterase by CBDP. Maxwell obtained a value of 1.1 ꢀ
3
ꢁ1
ꢁ1
14
1
0 M min for rat brain AChE at pH 7.4 and 37 ꢀC. Cohen
3
ꢁ1
ꢁ1
found a value of 2.1 ꢀ 10 M min for recombinant human
4
ꢁ1
ꢁ1
AChE and 2 ꢀ 10 M min for human BChE at pH 8.0 and
2
0
2
7 ꢀC. The major difference between those experiments and
1
ours is that those authors used CBDP stock solutions that were
phospho-serine adduct plus o-cresol (Scheme 4). Both deal-
made in aqueous buffer. The problem with aqueous solutions
kylation steps can be considered aging steps.
8
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Chemical Research in Toxicology
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18
Figure 2. MALDI TOF mass spectra of the active site tryptic peptide from reactions of BChE with CBDP in the presence and absence of O-water.
þ1
Reactions of BChE with CBDP were conducted as described in the Materials and Methods section. Masses of interest are shown: 2928 amu [M þ H]
ꢁ
1
for the unlabeled active site peptide SVTLFGESAGAASVSLHLLSPGSHSLFTR in positive mode; 3006 amu [M ꢁ H] for the phosphorylated active
16
ꢁ1
18
site peptide in the presence of O-water in negative mode; 3008 amu [M ꢁ H] for the phosphorylated active site peptide in the presence of O-water
ꢁ1
18
16
in negative mode; 3096 amu [M ꢁ H] for the o-cresyl-phosphorylated active site peptide in the presence of O-water and O-water in negative
mode. Limited mass ranges around the masses of interest are shown to emphasize the isotopic splittings and peak resolution, to illustrate the relative
intensities of the members of the isotopic families, and to avoid interference from other peptides with greater signal intensities. Panels A, E, and I are from
18
16
the reaction of BChE with CBDP in the presence of O-water; panels B, F, and J are from the reaction of BChE with CBDP in the presence of O-water;
panels C, G, and K are from an incubation of BChE in O-medium without CBDP; and panels D, H, and L are from an incubation of BChE in O-
18
16
1
8
medium without CBDP. A mass shift of þ2 from the incorporation of O-water is seen only in panel E for the phosphorylated active site peptide of mass
18
3
008, which demonstrates that the PꢁO bond was broken when the cresylphosphate adduct aged to form phospho-serine. In contrast, O was not
introduced during the formation of the cresylphosphate adduct (mass 3096 in panels I and J), which means this adduct resulted from cleavage of the
OꢁC bond.
1
It is generally accepted that oximes can reactivate the initial,
covalent adduct of organophosphate inhibited cholinesterases,
but that after aging, oxime reactivation is ineffective. In order to
determine how fast the CBDP aging reactions occur, we mea-
sured oxime-mediated reactivation of CBDP-inhibited BChE
and AChE.
reaction between CBDP and tyrosine. The dealkylation reac-
tions that follow the initial adduct formation involve hydrolysis of
1
8
the PꢁOꢁC linkage. The question to be addressed by the O-
mass spectral experiments is whether the PꢁO bond or the OꢁC
bond is cleaved during the course of these dealkylation reactions.
1
8
If the PꢁO bond is cleaved in a medium containing O-water,
18
Attempts to reactivate both CBDP-phosphonylated AChE
and BChE by oximes 2-PAM and HI-6 failed. Experiments were
performed in triplicate. This strongly suggests that aging is very
rapid after inhibition by CBDP. The kinetics indicated that loss of
reactivatibility occurs in less than 15 s following inhibition. Aging
then an OH will be added to the phosphorus; but if the OꢁC
18
bond is cleaved, then the OH will be added to the carbon, and
16
18
the original O will remain on the phosphorus. Exchange of O
16
for O can be discerned by a 2 amu increase in mass of the
phospho(organophospho)-peptide adduct. MALDI TOF mass
spectral analysis of tryptic digests from BChE reacted with CBDP
5
2
19
of R-chymotrypsin and fly AChE inhibited by CBDP was also
found to be very rapid. These enzymes were not reactivatable even
with strong nucleophilic compounds. A previous mass spectro-
metry study of human albumin phosphonylated by CBDP in-
dicated that a rapid, water-mediated loss of saligenin occurred after
18
16
in the presence O-water or in the presence of O-water is
shown in Figure 2 (panels A, B, E, F, I, and J). The results are
compared to tryptic digests of BChE incubated under the same
conditions without CBDP (Figure 2; panels C, D, G, H, K, and
L). Limited mass ranges are presented in order to illustrate the
isotopic splitting for the masses of interest and to reduce
interference from more intense signals. Spectra in panels A, B,
C, and D show the mass of the unlabeled active site peptide from
BChE, SVTLFGESAGAASVSLHLLSPGSHSLFTR, taken in
positive reflector mode. The theoretical, protonated [M þ
1
phosphonylation. This reaction occurred despite the fact that
53
albumin adducts of other organophosphates do not age at all.
A mass spectral study of the human BChE-CBDP adduct indicated
that two consecutive postphosphorylation reactions occur: a rapid
loss of saligenin was followed by a loss of the cresyl moiety, leading
1
to a phospho-serine adduct. Our kinetic results confirm the
þ1
rapidity of the first postinhibitory reactions of CBDP-phosphony-
lated BChE and CBDP-phosphonylated AChE to form aged
adducts, and illustrate the difficulties in reactivating such aged
enzymes.
H] , monoisotopic mass of this peptide is 2928.52 amu
(obtained with the assistance of the MS-Product algorithm from
Protein Prospector at http://prospector.ucsf.edu/prospector).
Spectra in panels E, F, G, and H show the mass of the phospho
adduct of the active site peptide taken in negative reflector mode.
The theoretical, deprotonated [M ꢁ H] , monoisotopic mass
of this peptide, is 3006.52 amu in negative mode. Spectra in
panels I, J, K, and L show the mass of the cresyl-phospho adduct
of the active site peptide taken in negative reflector mode. The
Mass Spectrometry. Following the formation of the initial
ꢁ
1
reaction of CBDP with BChE, there are two adduct modifica-
1
tions (Scheme 4). The initial ring opened adduct is not seen
with BChE, but its presence can be inferred by analogy with the
reactions of other OP with BChE and from a similar nucleophilic
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Chemical Research in Toxicology
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theoretical, deprotonated [M ꢁ H]ꢁ1, monoisotopic mass of this
peptide is 3096.52 amu. Negative mode spectra of the phospho-
adducts showed better resolution and better signal-to-noise than
spectra in the positive mode, despite the fact that the signal
intensities were greater in the positive mode.
Table 2. Data Collection and Refinement Statistics
Data Collection
phospho-hBChE
(PDB code 2y1k)
data
space group
The unlabeled active site peptide produced by digestion in
1
8
16
either O- or O-media gave identical spectra with a mono-
I422
þ1
isotopic, [M þ H] mass of 2928.27 ( 0.08 amu (Figure 2,
unit cell axes, a = b, c (Å)
no. of reflections
153.77, 127.60
243 040
18
panels C and D), indicating that spontaneous exchange of
O
into the peptide did not occur. The 1 amu isotopic splitting and
the isotopic pattern of this family of peaks are consistent with a
mass of 2928 amu (the isotopic pattern was checked with the
MS-Isotope algorithm from Protein Prospector at http://pro-
spector.ucsf.edu/prospector). Weak, unresolved spectra in the
vicinity of 2928 amu were seen in the digests from the CBDP-
containing samples (Figure 2, panels A and B), despite the fact
that CBDP had inhibited all of the BChE activity. The unresolved
character of these spectra indicates that these masses arose in the
MALDI TOF mass spectrometer from fragmentation in the flight
path after the reflector.
unique reflections
25 339
a
resolution range (Å)
40ꢁ2.5 (2.6ꢁ2.5)
94.8 (97.4)
8.9 (49.0)
26.0 (4.8)
6.7 (7.0)
completeness (%)
b
R
merge (%)
I/σ (I)
redundancy
Refinement Statistics
c
d
R-factor (R-free )
no. of atoms
protein
18.3 (24.6)
4180
250
The cresyl-phospho adducts from the CBDP reactions con-
18
16
solvent
ducted in the presence of either O- or O-media gave identical
ꢁ
1
others
154
spectra with a monoisotopic, [M ꢁ H] mass of 3096.15 ( 0.05
amu (Figure 2, panels I and J), indicating that oxygen from the
medium was not added to the phosphorus during hydrolysis of
saligenin. This in turn indicates that OꢁC bond fission accom-
panied this hydrolysis. This sort of dealkylation is characteristic
2
mean B-factor (Å )
36.0
rms from ideality
bond length (Å)
angles (deg)
0.018
1.954
0.151
5
4
of aging in BChE. Aging is assisted by groups in the pi-cation
3
chiral (Å )
5
5,56
binding pocket of the active site
bound into this pocket.
and is specific for the ligand
a
b
Values in brackets refer to the highest resolution shell. Rmerge = (Σ|I ꢁ
2
3,24,57,58
This suggests that for the initial,
ÆIæ|)/ΣI, where I is the observed intensity and ÆIæ is the average intensity
obtained from multiple observations of symmetry related reflections
ring-opened adduct, the saligenin moiety is bound to the pi-
cation binding pocket. The 1 amu isotopic splitting and the
isotopic pattern of this family of peaks support the argument that
these signals are a genuine reflection of a monoisotopic 3096 amu
mass. Absence of signal at 3096 amu from the digests of BChE
incubated without CBDP (Figure 2, panels K and L) indicates
that the 3096 signal is a characteristic of the CBDP reaction and is
not an adventitious mass occurring in the BChE tryptic digest.
The phospho adduct from the reaction of BChE with CBDP in
c
after rejections. R-factor = Σ |F ꢁ |F /Σ|F |, F and F are observed
o
c
o
o
c
d
and calculated structure factors, respectively. The R-free set uses 5% of
randomly chosen reflections.
positive peak is observed (above 15σ) at covalent bonding distance
of the catalytic Ser198 confirming the presence of the phosphorus
atom. The structure was unambiguously refined as a phospho-serine
adduct, indicating the departure of both the o-cresyl and saligenin
substituents after 12 h of incubation (Figure 3).
In the structure of the BChE phosphoserine adduct, the
phosphorus atom of the phosphate adduct is at 1.8 Å from
1
8
the presence O-water gave a mass of 3008.23 amu, whereas the
1
6
same reaction in O-water resulted in a mass of 3006.09
Figure 2, panels E and F). Since the theoretical mass for the
phosphorylated active site peptide in negative mode is 3006.51
(
Ser198Oγ. The oxygen O of the phosphate adduct is negatively
2
18
amu, the 3008.23 amu mass from the O-reaction indicates that
one oxygen atom from the medium was added to the phosphorus
during hydrolytic release of the cresyl. This in turn indicates that
the PꢁO bond was broken during this hydrolysis. PꢁO bond
breakage is consistent with the cresyl moiety being located in the
acyl binding pocket where the classical aging type of assistance
for fission of the OꢁC bond is unavailable. The 1 amu isotopic
splittings and the isotopic patterns of these two families of peaks
support the argument that these signals are genuine reflections of
monoisotopic masses at 3006 and 3008 amu. The absence of
signal in the 3006ꢁ3008 amu region from the digests from BChE
incubated without CBDP (Figure 2, panels G and H) indicates
that the 3006 and 3008 amu signals are characteristic of the
CBDP reaction and are not adventitious masses occurring in the
BChE tryptic digest.
charged and forms a salt bridge with the catalytic histidine
His438Nε2 (2.9 Å). The oxygen Oδ is stabilized by H-bonding
with atoms from residues forming the oxyanion hole, Gly116N
(2.7 Å), Gly117N (2.6 Å), and Ala199N (2.7 Å). O is oriented
3
toward the acyl-binding pocket and interacts with water molecule
w100 (3.3 Å) stabilized by H-bond interaction with the main
chain oxygen of the Leu286 (3.1 Å). The choline-binding pocket
is occupied by water molecules (w125 and w103) at equal
distance of the Trp82 and the oxygen O of CBDP adduct
2
(approximately 4 Å). This crystal structure confirmed the
formation of a phospho-serine adduct resulting from two con-
secutive postphosphorylation reactions of the ring opened
CBDP adduct in the hBChE active site.
This is the first time that a phosphoserine has been observed as
the final conjugate from the reaction of BChE with an OP.
A phosphoserine adduct resulting from dealkylation of human
AChE inhibited by the phosphoramidate mipafox was already
X-ray Structure of Phospho-hBChE. The structure of CBDP-
inhibited hBChE after 12 h of soaking was solved at 2.5 Å
resolution. Data and refinement statistics are shown in Table 2.
In the experimental |F | ꢁ |F | electron density map, a strong
59
reported. Evidence for this adduct was based on mass spectro-
metry and immuno-precipitation of trypsin digests with
o
c
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Chemical Research in Toxicology
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Figure 3. Active site view of the phospho-serine-hBChE conjugate. Key residues and the OP adduct are displayed as sticks, with carbon atoms in green,
oxygen atoms in red, nitrogen atoms in blue, and the phosphorus atom in orange. Hydrogen bonds are represented as red dashed lines. The F
map is contoured at 3σ.
o
ꢁ F
c
omit
monoclonal antibodies to phosphoserine. Our previous studies
with phosphoramidates (tabun and tabun derivatives) showed
that for phosphoramidates, displacement of isopropyl amine
groups results from acid hydrolysis during sample preparation
reaction involves the hydrolysis of the OꢁC bond in the
PꢁOꢁC linkage between the phosphorus and the saligenin
moiety, which is indicated by the observation that oxygen from
the medium is not added to the phosphorus.
23
for mass spectrometry. Thus, although mass spectrometry alone
does not provide absolute evidence for nonartifactual formation of
phosphoserine adduct, immuno-precipitation of trypsin digests
rules out possible artifactual acid-induced formation of this adduct.
In our study, X-ray analysis of the CBDP-aged BChE adduct
provides direct and 3D evidence that the phosphoserine adduct
does not result from sample preparation but results from postpho-
sphorylation chemical events. Interestingly, both CBDP-phos-
phorylated human BChE and mouse AChE (Carletti et al., in
preparation) lead to phosphoserine adducts, while only mipafox-
inhibited AChE leads to phosphoserine adduct. With BChE, the
mipafox-aged adduct is a monoisopropylphosphoroamido
Fission of the OꢁC bond is characteristic of enzyme assisted
54
aging for phosphylated BChE adducts. Aging involves phos-
phorus ligands located in the pi-cation binding site (i.e., near
23,57,58
Trp82 in hBChE and Trp87 in hAChE).
This argues that the saligenin moiety probably occupied the
choline-binding site in the initial ring-opened form of the CBDP
adduct, suggesting in turn that the o-cresyl substituent occupied
the acyl-binding pocket of hBChE.
52
On the basis of chemical analyses, Toia and Casida proposed
a similar dealkylation process for chymotrypsin and trypsin
inhibited by saligenin cyclic phosphonates and phosphates. In
addition to the release of saligenin from phosphorus into the
medium, they proposed that a portion of the saligenin released
from phosphorus was trapped by the active site histidine.
No evidence for a similar trapping of the released saligenin was
seen for either AChE or BChE adducts of CBDP.
6
0
adduct.
Concluding Remarks. Kinetics and Mechanism. With k values
i
7
8
ꢁ1
ꢁ1
of 10 ꢁ10 M min , CBDP is one of the most potent OP
inhibitors yet found for BChE. Comparable reactivities are seen
8
ꢁ1
ꢁ1 61
8
with cyclosarin (3.8 ꢀ 10 M min ), FP-biotin (1.6 ꢀ 10
The second reaction involves hydrolysis of the PꢁO bond
between the phosphorus and the cresyl moiety. Cleavage of the
PꢁO bond is indicated because oxygen from the solvent is added
to the phosphorus during this reaction. Fission of the PꢁO bond
ꢁ
1
ꢁ1 62
7
7
ꢁ1
ꢁ1 62
M
(
min ), diazoxon (7.7 ꢀ 10 M min ), and soman
ꢁ1 ꢁ1 63
9
5.1 ꢀ 10 M min ). Only chlorpyrifos-oxon (1.7 ꢀ 10
ꢁ
1
ꢁ1 64
8
ꢁ1
ꢁ1 63
M
min ) and MEPQ (6.3 ꢀ 10 M min ) are
appreciably more reactive. Thus, BChE should be a sensitive
marker of low level exposure to CBDP.
is consistent with either a dissociative (S 1) hydrolysis of
N
6
5
phosphoesters or a nucleophilic (S 2) hydrolysis. An SN2
N
The ultimate product of the reaction of CBDP with BChE is a
unique phosphopeptide not previously reported for the reaction
of OP with BChE. This phosphopeptide is the result of two
consecutive, postphosphorylation reactions (aging), starting
from the CBDP ring-opened adduct (see Scheme 4). The first
was earlier suggested as one of two alternative mechanisms for
66
aging of diethylphosphoryl-BChE, but it was later discarded in
favor of a dealkylation-based mechanism because no incorpora-
18
tion of O-water to phosphorus was found by mass spectro-
metry analysis. However, structural analysis of the aging of some
8
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Chemical Research in Toxicology
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Scheme 5. Proposed Reaction Mechanism of CBDP with
Cholinesterases, Leading to the Phospho Serine Adduct
chromatography to give 15ꢁ30% pure BChE) yields a phosphory-
lated peptic peptide that is specific for the active-site sequence of
BChE (manuscript submitted for publication). This peptide is
readily detectible by MALDI TOF TOF mass spectrometry, and
its identity has been confirmed by mass spectral fragmentation
analysis. It provides a unique biomarker for the reaction of CBDP
with BChE. We have used this peptide marker to address the
question of whether there is sufficient cresyl phosphate in aircraft
cabin air to cause detectible adduct formation in passengers. This
question is at the heart of the debate between expert bodies on the
extent of potential exposure to and the toxic potency of cresyl
phosphate. To date, we have found that about half of the
passengers tested carried evidence for exposure to TOCP despite
the fact that none of the subjects was exposed to high levels of
engine gases (i.e., a fume event) and that none reported toxic
symptoms. Since only a subgroup of passengers experience aero-
toxic symptoms after a fume event, it can be concluded that some
individuals are hyper-sensitive to the toxicants in the engine fumes.
Evidence for TOCP toxicant in a substantial fraction of passengers
exposed to normal cabin air provides ample reason to suspect that
the level of TOCP in aircraft air (however small it may be) is a
potential threat to sensitive passengers exposed to fume events.
The high reactivity of BChE toward CBDP also argues that it
should play an important role in natural defenses against toxicity
from low dose exposure to TOCP by scavenging CBDP from the
bloodstream. Variation in the reactivity of BChE or cytochrome
6
8
P450 enzymes due to naturally occurring mutations or diseases
analogues of the nerve agent tabun suggested an S 2 based
may contribute to the explanation for why only selected passen-
gers and aircraft workers fall victim to aerotoxic syndrome.
The initial neurological symptoms associated with aerotoxic
syndrome are similar to symptoms associated with organophos-
phate inhibition of AChE. Though AChE is less reactive than
BChE with CBDP, the reactivity of AChE with CBDP is similar
N
6
7
mechanism. An S 2 mechanism requires that the nucleophile
N
(
in this case water) approach the phosphorus from the face of the
tetrahedral phosphate structure that is opposite from the group
to be displaced. This face is accessible to water after the departure
of saligenin substituent in the previous step. The dissociative
mechanism requires that the leaving group (in this case the
cresyl) detach from the phosphorus and depart to make room for
the replacing group (in this case water) to attach. At this point,
the data do not allow one to discriminate between an S 1 or S 2
6
ꢁ1
to the reactivity with potent OPs such as paraoxon (k ≈ 10 M
i
ꢁ
1
7
ꢁ1
ꢁ1 69
min ) or (ꢁ)-tabun (k ≈ 2.7 ꢀ 10 M min ), suggesting
i
that human exposure to high doses of CBDP should cause the
typical symptomatology of OP poisoning. Neurotoxicity that
persists for years suggests targets in addition to AChE.
N
N
mechanism. The complete inhibition mechanism by CBDP up to
the formation of the phosphoserine is summarized in Scheme 5.
Aerotoxic Syndrome and Toxicity of CBDP. Aerotoxic syndrome
is speculated to be asociated with exposure to tri-ortho-cresylphos-
phate, but this association has not been proven. After exposure to
TOCP, the CBDP generated in vivo can react with tissue enzymes
such as liver carboxylesterase and neuropathy target esterase
Origin of Aerotoxic Syndrome and Organophosphate-In-
duced Delayed Neuropathy. Aerotoxic syndrome is assumed to
2
be caused by exposure to low doses of TOCP. Organophos-
phate-induced delayed neuropathy (OPIDN) is known to be
10,70ꢁ72
caused by exposure to high doses of TOCP.
High doses
of TOCP result in paralysis of the legs, whereas low doses do not
cause delayed paralysis. The processes leading to OPIDN and the
long-term symptoms of aerotoxic syndrome are probably related.
A long-standing candidate for the target of CBDP responsible for
the induction of OPIDN is neuropathy target esterase (NTE), a
membrane bound phospholipase that catalyzes the deacylation of
membrane-phophatidylcholine to soluble glycerophosphocho-
(
NTE), and with blood bioscavengers such as albumin and BChE
(note that CBDP does not react with paraoxonase 1, the naturally
occurring plasma phosphotriesterase; Masson et al., unpublished
results). Detection of low dose exposure to CBDP is critical to
ongoing investigations into aerotoxic syndrome. It is suspected that
aerotoxic syndrome is caused by internalization of an engine oil
additive (TOCP) that gives rise to CBDP in vivo. Epidemiological
studies into aerotoxic syndrome are currently ham-strung by the
absence of a direct, quantitative, in vivo measure for exposure to
TOCP. Phosphorylated BChE, as a biomarker for TOCP/CBDP
exposure, is an appealing candidate to fill this vacancy. The unique
nature of this adduct will reduce the incidence of false positive
identifications when screening populations.
Use of a phosphorylated protein as a biomarker for aerotoxic
syndrome entails the potential for conflict with other phosphorylated
proteins, of which there are many. However, ongoing work in our
laboratory has demonstrated that phosphorylated BChE (partially
purified from serum by anion exchange and procainamide affinity
73ꢁ75
line and fatty acids.
Low doses of TOCP inhibit neuropathy
target esterase, though they do not cause neuropathy. A role for
NTE in delayed neuropathy is supported by the finding that
human genetic variants of NTE have motor neuron disease
7
6,77
characterized by spastic paraplegia and distal muscle wasting.
Proposed noncholinergic mechanisms of OP neurotoxicity in-
clude activation of glutamatergic neurons and oxidative damage
7
8
from reactive oxygen species and from peroxynitrite radicals.
The recent discovery that OP can covalently modify amino acid
residues other than the traditionally accepted active site serines of
serine esterases/proteases has fueled interest in nontraditional
targets for OP. The covalent modification of tyrosine residues by
8
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Chemical Research in Toxicology
ARTICLE
OP on nonenzymatic proteins such as serum albumin and
81
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1
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term neurological symptoms associated with aerotoxic syndrome
and organophosphate-induced delayed neuropathy.
(
(
cyclization reactions envolved in the metabolism of tri-o-cresyl phos-
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’
AUTHOR INFORMATION
Corresponding Author
(8) De Nola, G., Kibby, J., and Mazurek, W. (2008) Determination
of ortho-cresyl phosphate isomers of tricresyl phosphate used in aircraft
turbina engine oils by gas chromatography and mass spectrometry.
J. Chromatogr., A 1200, 211–216.
*E-mail: pmasson@unmc.edu.
Funding Sources
This work was supported by U.S. Army Medical Research and
Materiel Command [W81XWH-07-20034 to O.L.]; National
Institutes of Health [U01 NS058056 to O.L. and P30CA36727
to Eppley Cancer Center]. Mass spectra were obtained with the
support of the Mass Spectrometry and Proteomics core facility at
the University of Nebraska Medical Center. We are grateful to
the ESRF for beam-time under long-term projects MX498,
MX609, and MX722 (IBS BAG) and MX551 and MX 666
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jet oils. Environ. Res. 89, 146–164.
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6
(
5
(
(radiation-damage BAG), and the ESRF staff for providing
tion mechanism for soman (pinacolylmethylphosphonofluoridate) in
mice. Biochem. Pharmacol. 33, 3807–3811.
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efficient help during data collection. Financial support by the
CEA, the CNRS, and the UJF is acknowledged, as well as a grant
to M.W. and F.N. from the Agence Nationale de la Recherche
(
ANR; project number ANR-09-BLAN-0192-04) and to M.W.
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’
ACKNOWLEDGMENT
6
We thank Dr. D. Lenz (USAMRICD, Aberdeen PG, MD,
(
USA) for the gift of CBDP and Dr. D. Genkin (Pharmsynthez,
Saint Petersburg, Russia) for the gift of HI-6 dichloride.
benzodioxa-phosphorin-2-oxide (CBDP) on organophosphate poison-
ing and its therapy. Arch. Toxicol. 42, 207–216.
(17) Cohen, O., Kronman, C., Raveh, L., Mazor, O., Ordentlich, A.,
’
ABBREVIATIONS
and Shafferman, A. (2006) Comparison of polyethylene glycol-conju-
gated recombinant human acetylcholinesterase and serum human
butyrylcholinesterase as bioscavengers of organophosphate compounds.
Mol. Pharmacol. 70, 1121–1131.
AChE, acetylcholinesterase (EC 3.1.1.7); ATC, acetylthiocho-
line; BChE, butyrylcholinesterase (EC. 3.1.1.8); BSA, bovine
serum albumin; BTC, butyrylthiocholine; CBDP, 2-(o-cresyl)-
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HI-6, 1-[[[4-(aminocarbonyl) pyridinio] methoxy] methyl]-2-
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hAChE, human AChE; hBChE, human BChE; MALDI-TOF,
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