T. Matsushita et al. / Chemosphere 261 (2020) 127743
3
from Fujifilm Wako Pure Chemical Corporation.
each solution was assayed by LC on a 100 mm ꢄ 2.1 mm Cortecs
UPLC HILIC column (1.6- m particle size, Waters Corporation,
m
2.2. Anti-acetylcholinesterase activity assay
Milford, MA, USA). The mobile phase was a binary gradient of
100 mM ammonium formate in Milli-Q water (solvent A) and 100%
An in vitro anti-AChE activity assay was conducted with
with some modification as follows. Phosphate buffer (1 mM, pH
7.4), prepared by dissolving Na2HPO4 and NaH2PO4 in Milli-Q water,
was supplemented with NaCl at a concentration of 150 mM.
Hereafter, this buffer is referred to as the assay buffer. A working
solution of AChE was prepared by adding AChE (240 units/L) to the
acetonitrile (solvent B) at a flow rate of 200 mL/min as follows:
begin with 5% B (v/v), increase linearly to 60% B over a period of
0.75 min, hold at that percentage for 0.25 min, decrease linearly to
30% B over a period of 0.25 min, hold at that percentage for 1 min,
and then decrease linearly to 5% B over a period of 3.75 min. The
mass spectrometer was operated in electrospray-ionization mode
(positive) with a spray voltage of 3.2 kV. The temperatures of the
capillary heater and the electrospray-ionization-probe heater were
220 and 450 ꢁC, respectively. The flow rates of the sheath gas,
auxiliary gas, and sweep gas were 50, 15, and 0 units, respectively.
The S-lens ratio frequency level was set to 57. The concentration of
Ch was quantified in selected-ion-monitoring mode (m/z 104.1070)
at a resolution of 70,000. Detection limit of Ch was 5 nM.
assay buffer, and a working solution of ACh (120
by adding ACh to the assay buffer.
mM) was prepared
Each sample solution was diluted to the desired concentration
with the assay buffer, and then 285 L of the diluted sample was
poured into at least three wells of an ice-cooled 96-well microplate.
m
Each sample was supplemented with 7.5 mL of the AChE working
solution and then preincubated at 37 ꢁC for 30 min, during which
inhibition of the AChE by compounds in the sample was expected to
occur. After the preincubation, the 96-well plates were cooled on
The concentrations of insecticides and oxons were measured
with the same apparatus used to measured Ch concentration. A 5-
m
L sample of each solution was assayed by LC on a 50 mm ꢄ 2.1 mm
ice, and then each sample was supplemented with 7.5
mL of the ACh
Hypersil Gold column (1.9- m particle size, Thermo Fisher Scien-
m
working solution on ice and then incubated at 37 ꢁC for 120 min,
during which time release of Ch from ACh by uninhibited AChE was
tific). The mobile phase was a binary gradient of 2 mM ammonium
formate in Milli-Q water (solvent A) and 100% methanol (solvent B)
expected to occur. After the incubation, an aliquot (200
mL) of each
at a flow rate of 200 mL/min as follows: 1% B (v/v) for 1.5 min, in-
sample was mixed with 200 L of acetonitrile to inactivate the
m
crease linearly to 60% B over a period of 1 min, increase linearly to
99% B over a period of 5.5 min, hold at that percentage for 1.5 min,
decrease linearly to 1% B over a period of 0.5 min, and then hold at
that percentage for 2.0 min. The mass spectrometer was operated
in electrospray-ionization mode (positive) with a spray voltage of
3.2 kV. The temperatures of the capillary heater and the
electrospray-ionization-probe heater were 220 and 450 ꢁC,
respectively. The flow rates of the sheath gas, auxiliary gas, and
sweep gas were 50, 15, and 0 units, respectively. The S-lens ratio
frequency level was set to 57. The concentrations of malathion,
malathion-oxon, methidathion, and methidathion-oxon were
quantified in selected-ion-monitoring mode (m/z 331.0433,
315.0662, 302.9691, and 286.9920, respectively) at a resolution of
70,000. Detection limits of malathion, malathion-oxon, methida-
AChE. The concentration of Ch in the final solution was measured
by liquid chromatographyemass spectrometry (LC-MS). A control
(prepared with the assay buffer instead of the diluted sample) and a
blank (prepared with the assay buffer instead of both the diluted
sample and the AChE working solution) were also subjected to the
assay procedure.
Anti-AChE activity by each sample was calculated by means of
½Chꢃcontrol ꢂ ½Chꢃsample
½Chꢃcontrol ꢂ ½Chꢃblank
Anti ꢂ AChE activity ¼
(1)
where [Ch]control, [Ch]sample, and [Ch]blank are the Ch concentrations
in the control, the sample, and the blank, respectively.
thion, and methidathion-oxon were 3, 2, 0.3, and
respectively.
2 nM,
2.3. Batch chlorination experiments
3. Results and discussion
Malathion and methidathion were separately dissolved in
10 mM phosphate buffer (pH 7.0) at concentrations of 28 and
3.1. Comparison of anti-acetylcholinesterase activities of
insecticides and oxons
230 mM, respectively, in glass vials. For chlorination, these solutions
were supplemented with sodium hypochlorite at mol-Cl2/mol-C
ratios of 1 and 3 for malathion and methidathion, respectively, so
that chlorine was present throughout the procedure; and the
supplemented samples were magnetically stirred for 5 min. Then
the vials were tightly sealed with screw caps and Parafilm to pre-
vent volatilization and allowed to stand at 20 ꢁC in the dark for
168 h. During this period, aliquots were withdrawn at pre-
determined intervals, quenched with sodium sulfite to remove
residual chlorine, and subjected to the anti-AChE activity assay
(after 200-fold dilution with the assay buffer). In addition, the
concentrations of the parent insecticides and their oxons in the
aliquots were determined by LC-MS.
First, we assayed the anti-AChE activities of chemical standards
of the four parent insecticides and their respective oxons (Fig. 1).
Among the insecticides, only malathion showed relatively strong
anti-AChE activity; methidathion and diazinon showed only slight
activity, and dimethoate showed no activity at all in the tested
concentration range. In contrast, all the oxons showed dose-
dependent anti-AChE activity, and in all cases the oxons were
more active than the respective parent insecticides. To compare the
activities of the compounds, we calculated median half maximal
inhibitory concentrations (IC50) by using logistic regression analysis
(Table 1). The IC50 values of the parent insecticides were more than
3 orders of magnitude higher than those of their respective oxons,
indicating that the oxons were >1000 times as active as the parent
insecticides. This tendency is consistent with published data ob-
2.4. Quantification of the concentrations of choline, parent
insecticides, and oxon
ꢁ
The concentration of Ch was measured with
quadrupole-orbitrap mass spectrometer (Q-Exactive, Thermo
Fisher Scientific, Inc., Waltham, MA, USA) coupled with an LC sys-
tem (UltiMate 3000, Thermo Fisher Scientific). A 5-mL sample of
a hybrid
ꢀ
ꢁ
different among literature probably due to the differences in
experimental conditions as shown in Table S1. The activities of the