Stereoselective metabolism of fenoxaprop-ethyl and its chiral metabolite fenoxaprop in rabbits

Article abstract of DOI:10.1002/chir.21009

The stereoselective metabolism of the enantiomers of fenoxaprop-ethyl (FE) and its primary chiral metabolite fenoxaprop (FA) in rabbits in vivo and in vitro was studied based on a validated chiral high-performance liquid chromatography method. The information of in vivo metabolism was obtained by intravenous administration of racemic FE, racemic FA, and optically pure (-)-(S)-FE and (+)-(R)-FE separately. The results showed that FE degraded very fast to the metabolite FA, which was then metabolized in a stereoselective way in vivo: (-)-(S)-FA degraded faster in plasma, heart, lung, liver, kidney, and bile than its antipode. Moreover, a conversion of (-)-(S)-FA to (+)-(R)-FA in plasma was found after injection of optically pure (-)-(S)- and (+)-(R)-FE separately. Either enantiomers were not detected in brain, spleen, muscle, and fat. Plasma concentration-time curves were best described by an open three-compartment model, and the toxicokinetic parameters of the two enantiomers were significantly different. Different metabolism behaviors were observed in the degradations of FE and FA in the plasma and liver microsomes in vitro, which were helpful for understanding the stereoselective mechanism. This work suggested the stereoselective behaviors of chiral pollutants, and their chiral metabolites in environment should be taken into account for an accurate risk assessment.

Full text of DOI:10.1002/chir.21009

CHIRALITY 23:897–903 (2011)
Stereoselective Metabolism of Fenoxaprop-Ethyl and Its Chiral
Metabolite Fenoxaprop in Rabbits
1
2
2
2
2
2*
2*
YANFENG ZHANG, XUEFENG LI, ZHIGANG SHEN, XINYUAN XU, PING ZHANG, PENG WANG, AND ZHIQIANG ZHOU
1
Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China
2
Department of Applied Chemistry, China Agricultural University, Beijing, China
ABSTRACT
The stereoselective metabolism of the enantiomers of fenoxaprop-ethyl (FE)
and its primary chiral metabolite fenoxaprop (FA) in rabbits in vivo and in vitro was studied
based on a validated chiral high-performance liquid chromatography method. The information
of in vivo metabolism was obtained by intravenous administration of racemic FE, racemic FA,
and optically pure (2)-(S)-FE and (1)-(R)-FE separately. The results showed that FE degraded
very fast to the metabolite FA, which was then metabolized in a stereoselective way in vivo:
(
2)-(S)-FA degraded faster in plasma, heart, lung, liver, kidney, and bile than its antipode.
Moreover, a conversion of (2)-(S)-FA to (1)-(R)-FA in plasma was found after injection of
optically pure (2)-(S)- and (1)-(R)-FE separately. Either enantiomers were not detected in
brain, spleen, muscle, and fat. Plasma concentration–time curves were best described by an
open three-compartment model, and the toxicokinetic parameters of the two enantiomers were
significantly different. Different metabolism behaviors were observed in the degradations of FE
and FA in the plasma and liver microsomes in vitro, which were helpful for understanding
the stereoselective mechanism. This work suggested the stereoselective behaviors of chiral
pollutants, and their chiral metabolites in environment should be taken into account for an
accurate risk assessment. Chirality 23:897–903, 2011. VV C 2011 Wiley-Liss, Inc.
KEY WORDS: stereoselectivity; chiral analysis; fenoxaprop-ethyl; fenoxaprop; in vivo; in vitro
INTRODUCTION
This study was conducted to evaluate the stereoselectivity
of FE and FA in rabbit. First, this research was conducted to
get the features of the FE and FA enantiomers in rabbit
plasma and tissues after in vivo exposure and obtain a better
understanding of the biological fate of each enantiomer in
rabbit. Second, the behavior of FE and FA in plasma and
liver microsomes from rabbit was conducted to assess the in
vitro stereoselective metabolism of FE and FA. All the infor-
mation may be used in construction of a physiologically
based toxicokinetic model for FE and FA in the adult male
rabbit and may have some implication for the environmental
and ecological risks assessment for chiral pesticides.
Fenoxaprop-ethyl (FE, Fig. 1), (6)-ethyl 2-[4-[(6-chloro-2-
benzoxazolyl)oxy]phenoxy]propanoate, a selective aryloxy-
phenoxypropionate herbicide, is registered for postemer-
gence control of various grass weeds in dicotyledonous crops
1
as well as in grains. FE is not persistent in the environment
and easily undergoes ester cleavage to its corresponding acid
fenoxaprop (FA, Fig. 1), 2-[4-(6-chloro-2-benzoxazolyloxy)
phenoxy] propionic acid, which possesses herbicidal activity.
FE (or FA) has an asymmetrically substituted C-atom and
thus consists of a pair of enantiomers that are (2)-(S)
2
and (1)-(R), but the herbicidal activity originates from the
(
1)-(R)-enantiomer.
Stereoselective metabolism of chiral pesticides has been3
MATERIALS AND METHODS
Chemicals and Reagents
observed in various animals in vivo and in vitro. Yang et al.
demonstrated that mice and quail have different enantiose-
lective biotransformation of a-Hexachlorocyclohexane (a-
HCH), and enantiomer fraction (EF) trends in brain tissues
of both animals were quite different from those in other tis-
sues. Wang et al. reported that the degradation of the (1)-
theta-cypermethrin was much faster than that of its antipode
in plasma, heart, liver, kidney, and fat after i.v. administration
Racemic fenoxaprop-ethyl (rac-FE, 98.0%) and (1)-(R)-fenoxaprop-
ethyl (R-FE, 98.0%) were kindly provided by Institute for Control of
Agrichemicals, Ministry of Agriculture of China. Racemic fenoxaprop
rac-FA, 97.5%) was obtained from the hydrolysis of their corresponding
parent compounds. (2)-(S)-Fenoxaprop-ethyl (S-FE, 99.0%) was collected
manually on an Agilent high-performance liquid chromatography
HPLC) with a chiral column (250 mm 3 4.6 mm (i.d.)). b-Nicotinamide
adenine dinucleotide phosphate (NADPH) was purchased from Sigma-
Aldrich (St Louis, MO). Hexane, 2-propanol (HPLC grade), and trifluoro-
acetic acid (TFA) were from Yili Fine Chemicals (Beijing, China). Water
(
4
(
5
of racemic TCYM in rats. Zhu et al. elaborated that the in
vivo degradation rate of tebuconazole enantiomers was differ-
ent. Warner et al. demonstrated that chiral polychlorinated
biphenyls were biotransformed enantioselectively by mam-
malian cytochrome P-450 isozymes to form hydroxylated
metabolites in vitro. Such stereoselective degradation in
organisms has mainly been attributed to uptake, distribution,
metabolism, and excretion, but other biological processes,
including selective protein binding and tissue transport, can
Contract grant sponsor: National Natural Science Foundation of China;
Contract grant number: 20707038.
Correspondence to: Peng Wang, China Agricultural University, Beijing,
China. E-mail; wangpeng@cau.edu.cn or Zhiqiang Zhou, China Agricultural
University, Beijing, China. E-mail: zqzhou@cau.edu.cn
Received for publication 7 December 2010; Accepted 29 June 2011
DOI: 10.1002/chir.21009
Published online 20 September 2011 in Wiley Online Library
(wileyonlinelibrary.com).
*
6
–11
also influence enantiomer compositions in biota.
VV C 2011 Wiley-Liss, Inc.

8
98
ZHANG ET AL.
Fig. 1. Chemical structures of fenoxaprop-ethyl and its primary metabolite fenoxaprop. Chiral center is denoted by an asterisk (*).
was purified by a Milli-Q system. All other chemicals and solvents were
of analytical grade and purchased from commercial sources.
In Vitro Incubation Procedure
Substrate-depletion assays in plasma. The degradations of rac-
FE, (2)-(S)-FE, (1)-(R)-FE, and rac-FA in rabbit plasma in vitro were
performed to investigate the stereoselectivity and the chiral conversion
of the enantiomers. Plasma was obtained by centrifugating the blood
that was collected from the untreated rabbits at 4000 rpm for 10 min at
The procedure of preparation of rabbit hepatic microsomes from rab-
bits (Japanese white rabbit, male, Approximately 2 kg, provided by the
Experimental Animal Research Institute of China Agriculture University)
12
followed the method of Zhang et al. and the microsomes were immedi-
ately stored at 2808C. Hepatic microsomal activity was determined
according to the method of Bradford with BSA as the standard.
4
8C. A 10% (v/v) plasma solution was prepared (diluted by Tris-HCl
buffer, pH 7.4) and preincubated at 378C for 5 min, and then the individ-
ual substrate (rac-FE, (2)-(S)-FE, (1)-(R)-FE, and rac-FA) prepared in
alcohol was added into each 1-ml plasma incubation forming a final con-
centration of 30 lM and incubated at 378C. Samples were collected at 0,
5, 15, 30, and 60 min, and 5 ml of ice-cold ethyl acetate was added imme-
diately to terminate the enzyme–substrate reaction. One set of similar
incubations without substrate was served as control. The extraction pro-
cedure was the same as mentioned above.
Degradation Kinetic and Toxicokinetic Assays
Drug administration and sample collection. Male Japanese white
rabbits (ꢀ2 kg) were fasted for 12 h before the experiment. Racemic FE
or FA was dissolved in alcohol (50%, w/v), diluted to final concentrations
by normal saline, and then administered at 30 mg/kg body weight by in-
travenous (i.v.) injection in the ear vein. Blood samples were collected
from the heart of treated rabbits into heparinized tubes at 0.5, 1, 1.5, 2,
Substrate-depletion assays in microsomes. The stereoselective
degradations of rac-FE, (2)-(S)-FE, (1)-(R)-FE, and rac-FA in rabbit
liver microsomes in vitro were also conducted. Each incubation (1.0 mg
protein per milliliter, adjusted by Tris-HCl buffer of pH 7.4) was added
with the substrate in alcohol to a concentration of 30 lM and preincu-
bated at 378C for 5 min. The reaction was regenerated with 100 ll of
NADPH to a final concentration of 1.0 mM (final total reaction volume
3
, 4, 8, and 12 h after exposure; each data point is the mean of six repli-
cates. Blood was centrifuged at 4000 rpm for 10 min, and plasma was
transferred into a new test tube. After all blood samples collection, the
rabbits were killed at 12 h after being anesthetized. The heart, kidney,
liver, lung, fat, muscle, spleen, bile, and brain samples were collected
from each subject. Plasma and tissue samples were stored at 2208C for
further treatment. To determine the chiral conversion of the two FE
enantiomers in the rabbit, (2)-(S)- and (1)-(R)-FE were also adminis-
tered separately at 15 mg/kg body weight in the same manner as men-
tioned above.
1
.0 ml). Samples were taken out after incubation at 378C for 0, 5, 15, 30,
and 60 min, respectively, and 5 ml of ice-cold ethyl acetate was added to
terminate the reaction. One set of similar incubations without substrate
was served as control. The extraction procedure was the same as men-
tioned above.
Enzyme kinetic experiments in liver microsomes. The stereose-
lective disappearance of rac-FE or rac-FA in the rabbit liver microsomes in
a series of concentrations 5, 15, 30, 60, 120, 240, and 300 lM was con-
ducted to assess the relative contributions of enzymes such as esterases
and CYP450s responsible for FE and FA metabolism based on the rela-
tionship of stereoselective disappearance and substrate concentrations.
The incubating time was 30 min, and other procedures were the same as
those in substrate-depletion assays in microsomes.
Extraction procedure. After being thawed, 1 ml of the rabbit
plasma or 1 g of homogenized tissue matrix was transferred into a 15-ml
polypropylene centrifuge tube, acidified with 100 ll of 1 M HCl, and
13
added with 5 ml of ethyl acetate. After vortex for 5 min and centrifuga-
tion at 4000 rpm for 5 min, the clear organic supernatant was decanted
into a test tube. The extraction was repeated with another 5 ml of ethyl
acetate. The organic phase was combined and evaporated to dryness
under a stream of nitrogen at 408C. To get rid of fat, the residue was dis-
solved in 1 ml of acetonitrile and partitioned thrice with 2 ml of n-hexane,
and then the acetonitrile phase was evaporated to dryness under a
stream of nitrogen. The residue was reconstituted in 200 ll of 2-propanol
and passed through a 0.22-lm syringe filter. A 20-ll aliquot was injected
into the HPLC.
14
Data analysis. Michaelis–Menten rate constants for FE and FA dis-
appearance were calculated using linear and nonlinear regression as ap-
plicable with Origin 7.5 software yielding the maximum velocity (Vmax
)
and affinity constant (K
m
), using the following Eq. 2:
Vmax 3 S
V ¼
;
ð2Þ
Km þ S
Data analysis. EFs were used to denote stereoselectivity, defined
by Eq. 1.
where V, S, Vmax, and Km represent the rate of metabolism,
substrate concentration, maximum rate of metabolism, and
Michaelis constant, respectively. The metabolic efficiency
ðꢁÞ
EF ¼ Peak areas of the
;
ð1Þ
½
ðꢁÞ þ ðþÞꢂ
was estimated by calculating intrinsic clearance (CL ) by
int
the following Eq. 3:
where (2) and (1) are the concentrations of the first eluted (2)-(S)-
enantiomer and the second eluted (1)-(R)-enantiomer. A racemic
standard had EF of 0.5, whereas preferential degradation of one of the
enantiomers made EF under or over 0.5. Statistical differences between
treatments (P < 0.05) were determined using a paired t-test.
Individual toxicokinetic parameters of FE and FA enantiomers were
determined using standard compartmental analysis methods and
calculated with the Drug and Statistics software (Section of Quantitative
Pharmacology, Chinese Pharmacological Society).
Vmax
CLint ¼
:
ð3Þ
Km
Apparatus and Chromatographic Conditions
Analytes were quantified using an Agilent 1200 series HPLC system
(Agilent Technology) equipped with a G1322A degasser, a G1311A quat
pump, a G1329A ALS, a G1314B VWD, and Agilent Chemstation software.
Chirality DOI 10.1002/chir

METABOLISM OF FENOXAPROP-ETHYL AND FENOXAPROP
899
Fig. 2. Representative HPLC chromatograms of plasma and tissues. (a) Untreated rabbit plasma; (b) untreated plasma spiked with rac-FE (10.0 lg/ml) and
rac-FA (10.0 lg/ml); (c) plasma at 0.5 h after i.v. treatment with rac-FE at 30 mg/kg body weight; (d) plasma at 0.5 h after i.v. treatment with (2)-(S)-FE at 15
mg/kg body weight; (e) plasma at 0.5 h after i.v. treatment with (1)-(R)-FE at 15 mg/kg body weight; (f) heart, (g) lung, (h) liver, (i) kidney, and tissue samples
collected from a rabbit at 12 h after i.v. treatment with rac-FE at 30 mg/kg body weight. Peaks: 1, (2)-(S)-FE; 2, (1)-(R)-FE; 3, (2)-(S)-FA; 4, (1)-(R)-FA. [Color
figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Enantiomer analysis was performed on an amylose-tri-(3,5-dimethylphenyl-
carbamate)-based column (250 mm 3 4.6 mm (i.d.), provided by the
Department of Applied Chemistry, China Agricultural University, Beijing).
The chromatographic separation was conducted at 208C and monitored
at 230 nm with the mobile phase containing n-hexane–isopropanol–
trifluoroacetic acid (95:5:0.1, v/v/v) at a flow rate of 1.0 ml/min.
signal-to-noise (S/N) ratio of 3. The limit of quantification (LOQ) was
defined as the lowest concentration in the calibration curve with accepta-
ble precision and accuracy.
RESULTS AND DISSCUSSION
Method Validation
Method Validation
The two pairs of enantiomers: (2)-(S)-FE, (1)-(R)-FE,
(
2)-(S)-FA, and (1)-(R)-FA, were baseline separated under
Blank plasma was spiked with rac-FE and rac-FA working standard
solutions to generate calibration samples ranging from 0.10 to 200lg/ml
for the single enantiomer. Calibration samples were prepared as
described earlier. Calibration curves were generated by plotting the con-
centration of each enantiomer in the spiked samples versus the peak
area of each enantiomer. The within-day precision was determined in six
replicates at concentrations of 1.0, 10.0, and 50.0 lg/ml on the same
day, whereas the inter-day precision was evaluated in six replicates at
the aforementioned concentrations on 6 different days. The standard
deviation (SD) and the relative standard deviation (RSD 5 SD/mean)
were calculated over the entire calibration range. The recoveries were
estimated by the peak area ratio of the extracted analytes with an
equivalent amount of the standard solution in pure solvents. The limit of
detection (LOD) was considered to be the concentration that produced a
the optimized condition shown in Figure 2b. Linear calibra-
tion curves were obtained over the concentration range of
0.10–200 lg/ml for (2)-(S)-FE (y 5 220.92x 2 41.72, R 5
0
(
(
for each enantiomer ranged from 3 to 16% (SD) and precision
from 3 to 11% (RSD). Recoveries of each enantiomer
fortified at 1.0, 5.0, and 50.0 lg/ml ranged from 88% 6 3% to
2
2
.998), (1)-(R)-FE (y 5 214.03x 2 43.18, R 5 0.998), (2)-
2
S)-FA (y 5 232.21x 2 93.39, R 5 0.992), and (1)-(R)-FA
2
y 5 223.21x 2 95.14, R 5 0.993). The accuracy of the assay
1
08% 6 8%. The LOD and LOQ in plasma were 0.03 and
0.1 lg/ml, respectively. The data showed that the method
was satisfying.
Chirality DOI 10.1002/chir

9
00
ZHANG ET AL.
Fig. 3. Plasma concentration–time curves and EFs of FA enantiomers in rabbit following (a) rac-FE administration at 30 mg/kg body weight; (b) rac-FA admin-
istration at 30 mg/kg body weight; (c) (2)-(S)-FE administration at 15 mg/kg body weight; and (d) (1)-(R)-FE administration at 15 mg/kg body weight.
Degradation Kinetic and Toxicokinetic Analysis in Plasma
found in plasma after administration of (1)-(R)-FE (Figs. 2e
and 3d). The results clearly indicated that there was chiral
conversion from (2)-(S)-FE or (2)-(S)-FA to (1)-(R)-FA but
no conversion from (1)-(R)-FE or (1)-(R)-FA to (2)-(S)-FA.
Chiral conversion of FA in plasma was unidirectional
and may play an important role in the enantioenrichment of
Racemic parent compound FE was not detected in rabbit
plasma after administration of rac-FE in 30 mg/kg body
weight, which indicated that rac-FE was hydrolyzed rapidly
to its carboxylated acid FA by esterase. The concentration of
produced (1)-(R)-FA was much higher than its antipode
even in the first time point, and the primary metabolite FA
underwent further degradation. The plasma concentration–
time curves of (2)-(S)- and (1)-(R)-FA are shown in
Figure 3a, and the degradations followed first-order kinetics.
(
1)-(R)-FA. Chiral conversion of FA in plasma was likely the
result of biotic interactions because the conversion rate was
very fast. Stereoselective protein binding may also lead to
1
5,16
the differences.
Stereochemical aspects of FA toxicokinetics were eval-
uated after single intravenous dose. Plasma concentration–
time curves of (2)-(S)-FA and (1)-(R)-FA were best
described by an open three-compartment model. Compart-
mental toxicokinetic analysis and paired t-test results showed
that differences between the principal toxicokinetic parame-
ters of the two enantiomers were significant (Table 1).
Plasma concentration–time curves of (2)-(S)-FA and (1)-
(
2)-(S)-FA was preferentially degradated (Fig. 2c), and it
was not detected at the 12th hour, but (1)-(R)-FA still
showed a high level of 18.92 mg/kg. The T1/2 of (2)-(S)-FA
and (1)-(R)-FA was 1.10 and 6.30 h, respectively. The EFs
decreased gradually far from 0.5 showing a prominent enan-
tioselective residue of R-enantiomer.
Plasma concentration–time curves of (2)-(S)- and (1)-(R)-
FA after i.v. administration of 30 mg/kg of rac-FA are shown
in Figure 3b. The degradations also followed first-order
kinetics and a similar enantioselectivity with preferential
elimination of (2)-(S)-FA. The T1/2 of (2)-(S)-FA and
(
R)-FA after i.v. administration of rac-FA were also best
described by an open three-compartment model, and the dif-
ferences between the principal toxicokinetic parameters of
the two enantiomers were significant (Table 1), indicating
that distribution or elimination of FA was stereoselective.
The different toxicokinetic parameters for the two enantiom-
ers were used for elucidating the mechanisms underlying
the stereoselectivity of FA metabolism in vivo.
(
1)-(R)-FA was 0.92 and 3.15 h, respectively. The concentra-
tions of (2)-S and (1)-(R)-FA were 0.60 and 30.89 mg/kg,
respectively, at the time point of 12 h, and the EF was 0.02.
To clarify the mechanism of the enantioselectivity, the
metabolisms of optically pure (2)-(S)-FE and (1)-(R)-FE in
rabbits were studied. The (1)-(R)-FA was detected in rabbit
plasma after injecting the single (2)-(S)-FE, and its concen-
tration was significantly higher than that of its antipode at
each time point (Figs. 2d and 3c). The EFs were from 0.090
to 0.019 during assays. However, the (2)-(S)-FA was not
Analytes in Rabbit Tissues
The data of the final residue in tissues such as heart, liver,
kidney, lung, and bile at 12 h after i.v. administration of the
rac-FE are shown in Table 2. In accord with that in plasma,
Chirality DOI 10.1002/chir

METABOLISM OF FENOXAPROP-ETHYL AND FENOXAPROP
901
TABLE 1. Toxicokinetic parameters of FA enantiomers after
i.v. administration with rac-FE and rac-FA of 30 mg/kg body
weight in rabbit
lung > liver. These results indicated that there was substan-
tial stereoselectivity on degradation or distribution of FA
enantiomers in rabbits.
The (1)-(R)-FA could be detected in rabbit heart, liver,
kidney, bile, and lung after injecting the single (2)-(S)-FE,
but (2)-(S)-FA nearly disappeared. However, the (2)-(S)-FA
was not found in these tissues after administration of (1)-
Administration with
Administration with
rac-FE
rac-FA
Toxicokinetic
Parameters
(2)-(S)-FA (1)-(R)-FA (2)-(S)-FA (1)-(R)-FA
(
R)-FE, whereas (1)-(R)-FA was still detected. The results
t
t
t
1/2a (h)
1/2b (h)
1/2g (h)
0.060
0.883
69.315
0.215
0.773
11.929
10.230
0.033
3.080
69.315
0.197
0.023
6.216
0.126
0.734
36.015
0.123
0.352
7.555
23.851
1.576
9.342
9.345
0.093
0.012
showed that FA underwent a unidirectional metabolic inver-
sion of the (2)-(S)-FA to its antipode with no other change
in vivo.
There were several possible factors involved in the stereo-
selective behaviors of FA enantiomers in vivo. The first factor
was chiral inversion of the (2)-(S)-FA in plasma. The second
factor was likely stereoselective distribution of (2)-(S)- and
V1 (L/kg)
CL (L/h/kg)
t
AUC (0-t)
mg/L*h)
AUC (0-1)
mg/L*h)
MRT (0-t) (h)
MRT (0-1) (h)
1/2z (h)
6.517
807.424
436.083
(
15.418
580.813
28.674
1097.899
(
1)-(R)-FA in tissues. In this study, the concentration of FE
(
was very high in the lung because of lung first-pass effect,
and the stereoselective distribution may be happened when
FE was transported to the other tissues. Stereoselective
2.642
11.538
4.498
9.505
2.371
7.197
4.292
8.819
16
metabolism and excretion would also result in stereoselec-
tive behavior of the chiral chemicals in animals.
parent compound was not detected in the tissues except for
the lung. (2)-(S)-FA degraded faster than its antipode in the
heart, lung, liver, kidney, and bile, and both enantiomers
were undetected in brain, fat, spleen, and muscle. In particu-
lar, in the lung, the concentration of (2)-(S)-FE and (1)-(R)-
FE was 189.8 and 192.8 lg/g, respectively, and there was no
obvious stereoselectivity with EF of 0.496; the concentration
of the (2)-(S)-FA was a little lower than that of the (1)-(R)-
FA with EFs of 0.415. Typical chromatograms for analytes in
heart, liver, kidney, and lung tissues after i.v. administration
of the rac-FE are shown in Figure 2. The (1)-enantiomer
concentrations were in the following order at 12 h: kidney >
bile > heart > lung > liver. For the (2)-enantiomer, the
order was as follows: lung > kidney > bile > heart > lung >
liver. These results indicated that there was substantial
stereoselectivity on degradation or distribution of FA enan-
tiomers in rabbit.
After i.v. administration of the rac-FA, correlative data of
the final residue at 12 h are shown in Table 2. The results
of administration with the rac-FA were similar to those of
administration with the rac-FE. (2)-(S)-FA was observed to
have significantly lower concentration (P < 0.01) than its an-
tipode in the heart, lung, liver, kidney, and bile, and both
enantiomers were undetected in brain, fat, spleen, and mus-
cle. The (1)-enantiomer concentrations were in the following
order: kidney > bile > heart > lung > liver, whereas the
In Vitro Kinetic Degradation in Plasma and Liver
Microsomes
Concentration–time curves of (2)-(S)- and (1)-(R)-FE
after incubation of rac-FE at 30 lM in rabbit plasma are
shown in Figure 4. The biotransformation of FE to FA was
stereoselective in favor of the (2)-(S)-FE, but at a much
slower rate. Furthermore, the enantiospecific disappearance
of the (2)-(S)-FE was certified indirectly by the preferential
generation of (2)-(S)-FA. The t1/2 of (2)-(S)- and (1)-(R)-FE
was 64.8 and 91.2 min, respectively, and the EFs decreased
with time from 0.492 to 0.409. At the same time, the EFs of
the metabolite FA decreased with time from 0.864 to 0.561. A
t-test between the EFs of FE and EF 5 0.5 yielded a P value
of 0.002. Enantioselectivity existed in the degradation of FE.
In contrast to the result of incubation of rac-FE, the elimina-
tion of (2)-(S)- and (1)-(R)-FA after incubation of rac-FA at
30 lM in plasma was very slow with EFs from 0.500 to 0.509.
Further analysis using a paired t-test indicated that there was
no statistical significance (P 5 0.117). There was no enantio-
selectivity in the degradation of FA. These data may be
important to elucidate that the difference between two FE
enantiomers was attributed to the stereoselective elimination
of FE in plasma. Results of the incubation with single (2)-
(S)- and (1)-(R)-FE at 30 lM in plasma indicated that there
was no chiral conversion from (2)-(S)-FE or (2)-(S)-FA to
(
2)-enantiomer as followed: lung > kidney > bile > heart >
TABLE 2. Concentrations and EFs in plasma and tissues at 12 h following i.v. administration with rac-FE and rac-FA at 30 mg/kg
a
body weight in rabbit
i.v. administration with FE
i.v. administration with FA
Plasma and
tissues
(2)-(S)-FA (mg/kg)
(1)-(R)-FA (mg/kg)
EF
(2)-(S)-FA (mg/kg)
(1)-(R)-FA (mg/kg)
EF
b
Plasma
Kidney
Bile
Lung
Heart
Liver
ND
18.92 6 3.71
81.16 6 9 10
35.65 6 5.39
7.72 6 0.95
4.95 6 0.69
2 77 6 0.57
0.60 60.03
0 81 6 0.08
1.97 6 0.33
9.946 1.10
0.89 6 0.11
0 88 6 0.56
30.89 6 4.27
84.30 6 8 49
44.27 6 4.22
18.31 6 4.06
7.91 1 1.29
6.62 1 0.77
0.019 6 0.004
0.010 6 0.001
0.043 6 0.011
0.352 6 0.019
0.101 6 0.008
0.117 6 0.010
1.68 6 0.21
1.26 6 0.12
5.47 6 0.16
0.20 6 0.06
0.31 6 0.02
0.020 6 0.005
0.034 6 0.012
0.415 6 0.023
0.039 6 0.012
0.101 6 0.012
a
Values represent the mean 6 SD (n 5 6).
Not detected.
b
Chirality DOI 10.1002/chir

9
02
ZHANG ET AL.
Fig. 4. Concentration–time curves of FE enantiomers or FA enantiomers with incubation of (a) rac-FE (10 lg/ml) and (b) rac-FA (10 lg/ml) in 10% plasma,
c) rac-FE (30 lM) and (d) rac-FA (30 lM) in liver microsomes.
(
(
1)-(R)-FA and vice versa. This suggested that plasma was
by FE and FA. Metabolic rate constants (Table 3) of FE were
calculated using linear regression, and Michaelis–Menten
plot is shown in Figure 5a. The parameters did not differ sig-
nificantly between the enantiomers, and the inhibition of
microsomal enzymes was nonstereoselective. For rac-FA,
metabolic rate constants (Table 3) were calculated using
nonlinear regression, and Michaelis–Menten plot is shown
in Figure 5b. The two enantiomers had identical enzyme ki-
netic characteristics, and no significant stereoselectivity was
found in the inhibition by FA.
not the major site for the chiral conversion.
The possible metabolisms of FE and FA in liver were
examined by the degradation of rac-FE, rac-FA, (2)-(S)-FE,
and (1)-(R)-FE in liver microsomes in vitro. The metabolism
of rac-FE at 30 lM in liver microsomes showed that (2)-(S)-
and (1)-(R)-FE were metabolized very quickly to FA and
became undetectable after incubation. The concentrations of
the metabolite FA enantiomers were approximately equal at
the period of incubation with EFs from 0.504 to 0.523, reveal-
ing that the degradation of FA in liver microsomes was none-
nantioselective (Fig. 4). The metabolism of rac-FA in hepatic
microsomes was also investigated at 30 lM. There was not
significant enantiomer enrichment between the dispositions
of the (2)-(S)- and (1)-(R)-FA with EFs from 0.506 to 0.521,
also suggesting that the metabolism of FA in liver micro-
somes was nonenantioselective (Fig. 4). The observation was
similar to the nonstereoselective FA elimination in plasma in
vitro, but not consistent with the stereoselective toxicoki-
netics of FA in vivo. Such nonenantioselective metabolism
could not account for the dramatic difference observed in
rabbit liver and revealed that the metabolism in liver would
not have led to the observed enantioenrichment of (1)-(R)-
FA in liver tissues. Chiral inversion of FA enantiomers
was not observed in microsomes with the incubation with
single (2)-(S)- and (1)-(R)-FE at 30 lM in rabbit liver
microsomes.
(2)-(S)-FA degraded faster than its antipode in the plasma,
heart, lung, liver, kidney, and bile in vivo, while there was
nonstereoselectivity in 10% plasma and microsomes incuba-
tion in vitro except that (2)-(S)-FE was stereoselectively
eliminated. The fact that in vivo and in vitro results revealed
1
7–20
significant differences was observed in previous studies.
Several reasons have been offered to explain the in vitro and
in vivo discrepancy. It may be due to enantioselectivity in
some of these downstream processes, and there was no
TABLE 3. Metabolic rate constants of FE and FA in rabbit
a
liver microsomes
V
max (lmol /min/mg
CLint (ml/min/mg
protein)
Substrate
protein)
m
K (lM)
(
(
(
(
2)-(S)FE
1)-(R)-FE
2)-(S)-FA
1)-(R)-FA
1674.3 6 628.9
1021.1 6 709.1
8.6 6 1.3
51409.8 6 8867.9
31431.4 6 9276.6
225.3 6 70.0
32.6 6 9.8
32.5 6 92
38.4 6 4.9
45.1 6 10.9
Enzyme Kinetic Analysis of Liver Microsomes
7.9 6 1.0
175.2 6 50.1
An in vitro enzyme kinetics study was conducted to inves-
tigate the stereoselective inhibition of microsomal enzymes
a
Values represent the mean 6 SD (n 5 6).
Chirality DOI 10.1002/chir

METABOLISM OF FENOXAPROP-ETHYL AND FENOXAPROP
903
Fig. 5. Degradation rate of (2)-(S)-FE and (1)-(R)-FE (left), (2)-(S)-FA and (1)-(R)-FA (right) in rabbit liver microsomes after 30-min incubation of rac-FE
and rac-FA.
4
5
21
significant metabolism or enantioenrichment induced by the
initial plasma attack. Alternatively, selective uptake and trans-
port across tissues or selective protein binding may also be
granule cells and Ca -uptake in rat cerebellum. Toxicol Lett 2005;
56:391–400.
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8
9
. Hoekstra PF, Burnison BK, Neheli T, Muir DCG. Enantiomer-specific
activity of o,p -DDT with the human estrogen receptor. Toxicol Lett
0
2
1,22
responsible for the enantiomer enrichment of FA.
How-
2
001;125:75–81.
ever, it should be noted that a variety of other potentially
enantioselective enzyme activities may exist in vivo in rab-
bits, which may also act upon FA enantioselectively.
. Wong CS. Environmental fate processes and biochemical transformations
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CONCLUSIONS
The chiral HPLC method described in this investigation
was validated for the study of the stereoselective metabolism
of the FE and FA enantiomers in rabbits. The in vivo study
showed that the degradation and the toxicokinetic behavior
of FA were stereoselective in rabbits, and (2)-(S)-enantiomer
was preferentially eliminated in plasma and tissues compared
with its antipode. The in vitro investigation of the stereose-
lective metabolisms of the enantiomers in plasma and liver
microsomes demonstrated that there was no significant ste-
reoselectivity was noted in the metabolism except the stereo-
selective degradation of the (2)-(S)-FE by plasma. The data
for the enantioselective behaviors in the degradation of the
enantiomers may have some implication for the environmen-
tal and ecological risk assessment for chiral pesticides or
their chiral metabolites.
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Chirality DOI 10.1002/chir