STEREOSELECTIVE DEGRADATION OF ALPHA-CYPERMETHRIN
59
G1314BVWD were equipped. The mobile phase was a mixture of 97%
n-hexane and 3% isopropanol with a flow rate of 0.5 ml/min. Chromato-
graphic separation was conducted at 20 °C and UV detection at 230 nm.
Methods of Experiments
A stock solution of α-CP was dried under a stream of nitrogen and dis-
solved with alcohol, and the volume of alcohol added to each incubate
was less than 1.0% v/v. The final incubation concentration of α-CP was
changed from 10 μM to 100 μM (rac-α-CP) or 5 μM to 40 μM (enantiomer
monomers of α-CP) to obtain the Michaelis constants and the optimum
concentration of enantiomers’ degradation. Substrate-depletion studies
in vitro were performed by incubation of α-CP in rat liver microsomes
with 1 mg microsomal protein in 50 mM Tris–HCl buffer (pH 7.4) with
Fig. 1. The chemical structure of α-CP enantiomers.
2
5.0 mM MgCl . All reaction mixtures were preincubated in a heated water
bath at 37 °C for 5 min before the addition of NADPH with a concentra-
tion of 1 mg/ml (the final reaction volume was 1.0 ml). After incubation
in a water bath (37 °C) for 10 min, the reactions were terminated by
adding 4 ml of ice-cold ethyl acetate and the sample was whirled for
information and explore the stereoselective behaviors of
α-CP, herein we investigated the stereoselective degradation
of α-CP enantiomers and the interaction of two enantiomers
in vitro utilizing rat liver microsomes that mainly contained
cytochrome P450s for xenobiotics metabolism. This research
will provide a more thorough understanding of chiral pyre-
throid pesticides and could contribute to the risk assessment
of α-CP.
5
min. After centrifugation at 4000 rpm for 5 min, the supernatant was con-
veyed and aqueous solution was extracted with another 4 ml ethyl acetate
again. The two-time supernatant was collected after the second centrifu-
gation to a clean tube and evaporated to dryness under a stream of nitro-
gen at 35 °C, then the residue was reconstituted in 200 μl of isopropanol
and filtered through a 0.22-μm filter for HPLC analysis.
MATERIALS AND METHODS
Chemicals and Reagents
Rac-α-CP standard (95%) was obtained from the Institute for the Control
According to the Michaelis constants, 10 μM of rac-α-CP and 5 μM of
(ꢀ)-(1S-cis-αR)-CP and (+)-(1R-cis-αS)-CP were incubated in a water bath
(
37 °C) for 0–40 min under the same condition as the kinetic assays. As-
says to assess the microsomal hydrolysis of the α-CP were conducted
as described above but in the absence of NADPH.
To assess α-CP enantiomers recovery, the rat liver microsomes were
inactivated by hot water (100 °C) before incubation. All of the assays were
carried out in microsomes by measuring the remaining concentration of
parent compounds.
of Agrochemicals, Ministry of Agriculture (Beijing, China). Optically pure
(
ꢀ)- (1S-cis-αR)-CP and (+)-(1R-cis-αS)-CP were separated by high-
performance liquid chromatography (HPLC) with a cellulose-tris (3,
-dimethylphenylcarbamate)-based chiral stationary phase (CDMPC-
5
CSP). A stock solution of rac-α-CP and enantiomer monomers of α-CP
standard were prepared in n-hexane and stored at 4 °C. Working
standard solutions were obtained by dilutions of the stock solution in
n-hexane. Water was purified by a Milli-Q system (Millipore, Bedford,
MA). β-Nicotinamideadenine dinucleotide phosphate (NADPH) was
purchased from Sigma-Aldrich (St. Louis, MO). All the mobile phase
reagents were chromatographically pure from Sinopharm Chemical
Reagent (Beijing, China), filtered through a 0.45-μm filter.
Data Analysis
The degradation of rac-α-CP appeared to follow a first-order kinetic
reaction, and the degradation rate constants were derived from “C versus
t” plots by curve fitting through Origin8.0 software for the experiment.
The starting point was the maximum concentration. The elimination,
t
1/2, was determined by the following:
Preparation of Rat Liver Microsomes
ln2
k
Adult male Sprague–Dawley rats (200–250 g) were provided by the
Vital River Laboratory Animal Technology Company (Beijing, China),
housed in solid-bottom cages with hardwood chips, and acclimatized
t1=2 ¼
(1)
Where k is speed constant which is obtained from Eq. (2) fitted by
(
1 week) in a humidity- and temperature-controlled room with a 12-h
regression analysis
light/dark cycle before use. The rat liver microsomes were prepared as
C ¼ C0eꢀ
kt
(2)
17
described in the literature . The rat liver was quickly removed after the
rats were anesthetized and placed in an ice-cold 1.15% KCl solution. Tis-
sue was minced with scissors after being washed with 1.15% KCl solution
to remove blood. After draining the 1.15% KCl solution, individual liver
was homogenized in an ice-cold SET solution (1 mM ethylen-ediamine
tetra-acetic acid [EDTA] and 50 mM Tris–HCl, pH7.4). The homogenate
was centrifuged at 9700 rpm for 20 min at 4 °C and the pellet was
discarded. The supernatant was centrifuged at 33,000 rpm for 60 min at
The enantiomer fraction (EF) was used as a measure of the
stereoselectivity of the α-CP enantiomers in vitro:
ðþÞ
EF ¼ peak areas of the
(3)
½
ðþÞ þ ðꢀÞꢁ
where (+) and (ꢀ) are the first and second eluting enantiomers deter-
5
mined by the polarimeter in reference. A racemic EF = 0.50, whereas
4
°C. The supernatant (cytosol) was decanted. The pellet was washed
preferential degradation of the (+) or (ꢀ) yields EF < 0.50 and > 0.50,
with 50 mM Tris–HCl and the homogenate was centrifuged at
respectively.
3
5
3,000 rpm for 60 min at 4 °C again. The pellet was resuspended in
0 mM Tris–HCl buffer (pH 7.4) containing 20% glycerol. This procedure
Nonlinear regression of substrate concentration versus reaction veloc-
ity curves were analyzed using Origin8.0 software by fitting experimental
data to the Michaelis–Menten equation. The degradation of the α-CP en-
antiomers by rat liver microsomes was fitted to Eq. (4), and the K
max values were calculated by the following equation:
was used for the preparation of microsomes from Sprague–Dawley rats
male, n = 6). The protein concentration was determined by the method
(
m
and
1
8
of Bradford with bovine serum albumin (BSA) as the standard, and
V
microsomes were stored at ꢀ80 °C until use.
VmaxꢂS
V ¼
(4)
Chromatographic Condition
Chromatography was performed using an Agilent (Palo Alto, CA) 1200
series HPLC equipped with a cellulose-tris- (3, 5-dimethylphenylcarbamate)
Km þ S
m
V, S, Vmax, and K represent the velocity of metabolism, substrate con-
(
CDMPC)-based chiral stationary phase to separate enantiomers. G1311A
centration, maximum velocity of metabolism, and Michaelis constant,
respectively.
pump, G1322A degasser, G1328A injector, a 20-ml sample loop, and
Chirality DOI 10.1002/chir
Yan, Jin
