Enantioselectivity in estrogenic potential and uptake of bifenthrin

Article abstract of DOI:10.1021/es070220d

Despite the fact that the biological processes of chiral compounds are enantioselective, the endocrine disruption activity and uptake of chiral contaminants with respect to enantioselectivity has so far received limited research. In this study, the estrogenic potential and uptake of the enantiomers of a newer pyrethroid insecticide, bifenthrin (BF), were investigated. Significant differences in estrogenic potential were observed between the two enantiomers in the in vitro human breast carcinoma MCF-7 cell proliferation assay (i.e., the E-SCREEN assay) and the in vivo aquatic vertebrate vitellogenin enzyme-linked immunosorbent assay (ELISA). In the E-SCREEN assay, the relative proliferative effect ratios of 1S-cis-BF and 1R-cis-BF were 74.2% and 20.9%, respectively, and the relative proliferative potency ratios were 10% and 1%, respectively. The cell proliferation induced by the two BF enantiomers may be through the classical estrogen response pathway via the estrogen receptor (ER), as the proliferation induced by the enantiomers could be completely blocked when combined with 10^-6 mol/L of the ER antagonist ICI 182,780. Measurement of vitellogenin induction in Japanese medaka (Oryzias latipes) showed that, at an exposure level of 10 ng/mL, the response to 1S-cis-BF was about 123 times greater than that to the R enantiomer. Significant selectivity also occurred in the uptake of BF enantiomers in the liver and other tissues of J. medaka. These results together suggest that assessment of the environmental safety of chiral insecticides should consider enantioselectivity in acute and chronic ecotoxicities such as endocrine disruption.

Full text of DOI:10.1021/es070220d

Environ. Sci. Technol. 2007, 41, 6124-6128
EDCs are xenoestrogens, which mimic the activity of en-
Enantioselectivity in Estrogenic
Potential and Uptake of Bifenthrin
dogenous estrogens such as 17â-estradiol (E2), and may
influence reproductive development, senescence, and car-
cinogenesis in humans and wildlife (3, 4).
There are several methods for investigating xenoestrogens,
including in vivo assays and in vitro assays. In vivo assays
include the uterotrophic assay, the vaginal cornification assay,
and the vitellogenin induction assay. In vitro assays include
ligand binding assay, recombinant receptor-receptor assay,
yeast estrogen screening assay, and cell proliferation assay
(3, 5-9). The human breast carcinoma MCF-7 cell prolifera-
tion assay (i.e., the E-SCREEN assay) is an in vitro assay for
testing the estrogenic potential using the proliferative effect
of estrogens on their target cells, i.e., the estrogen-dependent
MCF-7 human breast carcinoma cell line, as an end point
(10, 11). On the other hand, vitellogenin (VTG), a phospho-
lipoprotein and yolk protein precursor, is normally produced
in the liver of adult female oviparous vertebrates such as fish
(12, 13). While little or no vitellogenin can be detected in the
liver of normal male fish, when they are exposed to
xenoestrogens, the VTG gene is activated and vitellogenin is
synthesized in the liver (12). The vitellogenin enzyme-linked
immunosorbent assay (ELISA) has been developed based on
this interaction and proved to be a simple and sensitive in
vivo test to assess environmental estrogens (12, 13). VTG
ELISA assay using Japanese medaka (Oryzias latipes) is an
especially preferred method because J. medaka is a popular
laboratory model fish with high sensitivity to hormone
administration (12, 14, 15).
Synthetic pyrethroids (SPs) are among the most widely
used insecticides. With the restriction on the use of orga-
nophosphorus insecticides, the usage of pyrethroids is
expected to further increase (16). However, studies are starting
to show that pyrethroids possess hormone agonist or
antagonist activities. For example, fenvalerate, sumithrin,
cypermethrin, and deltamethrin displayed significant es-
trogenicity in MCF-7 human breast carcinoma cell line and
Ishikawa Var-I human endometrial cancer cell line, while
fenvalerate and d-trans-allethrin significantly antagonized
the action of progesterone in T47D human breast carcinoma
cell line (4, 8, 17). Bifenthrin (BF), λ-cyhalothrin, and
fenvalerate also induced thyroid hormone regulation dys-
function in rats (18, 19).
†
L U M E I W A N G , † , ‡ W E I P I N G L I U , * ,
C A I X I A Y A N G , † Z H I Y A N P A N , †
J I A N Y I N G G A N , § C H A O X U , ‡
M E I R O N G Z H A O , † A N D
D A N I E L S C H L E N K §
Research Center of Environmental Science, Zhejiang
University of Technology, Hangzhou 310032, China;
Institute of Environmental Science, Zhejiang University,
Hangzhou 310027, China; and Department of Environmental
Sciences, University of California, Riverside, California 92521
Despite the fact that the biological processes of chiral
compounds are enantioselective, the endocrine disruption
activity and uptake of chiral contaminants with respect
to enantioselectivity has so far received limited research.
In this study, the estrogenic potential and uptake of the
enantiomers of a newer pyrethroid insecticide, bifenthrin
(BF), were investigated. Significant differences in estrogenic
potential were observed between the two enantiomers
in the in vitro human breast carcinoma MCF-7 cell proliferation
assay (i.e., the E-SCREEN assay) and the in vivo aquatic
vertebrate vitellogenin enzyme-linked immunosorbent assay
(ELISA). In the E-SCREEN assay, the relative proliferative
effect ratios of 1S-cis-BF and 1R-cis-BF were 74.2% and
20.9%, respectively, and the relative proliferative potency
ratios were 10% and 1%, respectively. The cell proliferation
induced by the two BF enantiomers may be through the
classical estrogen response pathway via the estrogen receptor
(ER), as the proliferation induced by the enantiomers
could be completely blocked when combined with 10-6
mol/L of the ER antagonist ICI 182,780. Measurement of
vitellogenin induction in Japanese medaka (Oryzias latipes)
showed that, at an exposure level of 10 ng/mL, the
response to 1S-cis-BF was about 123 times greater than
that to the R enantiomer. Significant selectivity also occurred
in the uptake of BF enantiomers in the liver and other
tissues of J. medaka. These results together suggest that
assessment of the environmental safety of chiral
insecticides should consider enantioselectivity in acute
and chronic ecotoxicities such as endocrine disruption.
Pyrethroids contain 1-3 asymmetric positions, making
them a family of chiral insecticides with 1-4 pairs of
enantiomers. Several recent studies focused on the occur-
rence of enantioselectivity of chiral pesticides, including
pyrethroids, in acute aquatic toxicity (20-25), and environ-
mental fate (16, 26-30). However, so far little attention has
been given to enantioselectivity in chronic toxicities such as
endocrine disruption activity (31-33). The purpose of this
study was to evaluate enantioselectivity in estrogenic po-
tential of pyrethroids using BF as a model compound.
Bifenthrin is a newer pyrethroid insecticide and has wide-
spread use in both agricultural and urban environments.
Introduction
The adverse effects of man-made endocrine-disrupting
chemicals (EDCs) on both humans and wildlife have recently
become an issue of great concern (1). Endocrine disrupting
chemicals are defined as “exogenous agents that interfere
with the synthesis, secretion, transport, binding, action, or
elimination of natural hormones in the body, which are
responsible for the maintenance of homeostasis, reproduc-
tion, development, and/or behavior” (2). The most studied
Materials and Methods
Chemicals and Preparation of Bifenthrin Enantiomers. An
analytical standard of racemic (Z)-cis-BF [99.5%, 2-methyl-
biphenyl-3-ylmethyl-(Z)-(1RS)-cis-3-(2-chloro-3,3,3-trifluo-
roprop-1-enyl)-2,2-dimethylcyclopropane carboxylate] was
purchased from Sigma (St. Louis, MO) and used for the MCF-7
cell proliferation assay and the medaka VTG ELISA. The [14C]
phenyl-labeled BF (radioactive purity > 98%; radioactive
specific activity 75.48 kBq/mol) was obtained from FMC
(Princeton, NJ) and used for the uptake test in J. medaka.
* Corresponding author. Tel.: +86-571-8832-0666. Fax: +86-571-
8832-0884. E-mail: wliu@zjut.edu.cn.
† Zhejiang University of Technology.
‡ Zhejiang University.
§ University of California.
9
6124 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 17, 2007
10.1021/es070220d CCC: $37.00
2007 American Chemical Society
Published on Web 07/20/2007

FIGURE 1. Enantiomers of (Z)-cis-bifenthrin (BF) (* indicates chiral position).
The stereoconfiguration of BF is given in Figure 1. 17â-
estradiol (E2; purity > 98%) was purchased from Sigma and
used as the positive control. ICI 182,780 (Tocris, Avonmouth,
U.K.), an estrogen receptor (ER) antagonist, was used to test
whether the cell proliferation was through the ER pathway.
For the cell proliferation assay, all tested chemicals were
initially dissolved in ethanol, with the final solvent concen-
tration at <0.1% (v/v). Charcoal-dextran treated fetal bovine
serum (CDFBS) was purchased from HyClone (Logan, UT).
Other chemicals or solvents used in this study were of cell
culture or high-performance liquid chromatography (HPLC)
or analytical grade.
The enantiomers of [14C] phenyl-labeled and nonlabeled
(Z)-cis-BF were resolved on a Jasco LC-2000 series HPLC
system (Jasco, Tokyo, Japan) equipped with a PU-2089
intelligent quaternary gradient pump, a mobile-phase vacuum
degasser, an AS-1559 autosampler with a 100 µL loop, a CO-
2060 column temperature-control compartment, a UV-2075
ultraviolet-visible (UV) detector, a variable-wavelength CD-
2095 circular dichroism (CD) detector, and a LC-Net II/ ADC
data collector. The chromatographic conditions were similar
to the methods reported in a previous study (22). The
detection wavelength of CD detector was 230 nm. Chro-
matographic data were acquired and processed with the
ChromPass software (Jasco, Tokyo, Japan). The resolved
enantiomers were individually collected at the HPLC outlet,
evaporated to dryness, and used in the following bioassays.
Concentrations of the enantiomers were determined on an
Agilent 6890N gas chromatograph (GC) equipped with an
electron capture detector (ECD), assuming the same response
factor for enantiomers as for the racemate of (Z)-cis-BF.
Experimental Fish. The Japanese medaka (Oryzias latipes)
(2.5-3.5 cm in total body length) used in this study were
purchased from a local supplier. All new fish were observed
daily for 10 days for signs of illness and maturity. The healthy
adult male fish were kept in glass tanks under a summer
photoperiod (14 h light/10 h dark) at 25 ( 1 °C. The
dechlorinated water was used and changed three times per
week. The water was aerated by continuous air-bubbling.
Fish were fed brine shrimp eggs (Red Sun Aquaculture,
Tianjin, China) twice a day.
MCF-7 Cells and Cell Proliferation Assay. MCF-7 cells,
purchased from the Cell Bank of the Chinese Academy of
Science, Shanghai, China (the original source is American
Type Culture Collection, ATCC, Manassas, VA), were grown
in the seeding medium (Eagle’s minimum essential medium
(MEM) with phenol red, supplemented with 100 U/mL of
penicillin, 100 µg/mL of streptomycin, 0.1 mM of nonessential
amino acids, 4 mM of L-glutamine, 0.1 mM of sodium
pyruvate, 10 µg/mL of insulin, and 10% of fetal bovine serum)
at 37 °C with 5% CO2 in a humidified Thermo model 3111
forma series II water-jacketed CO2 incubator (Thermo
Electron, Marietta, OH). The overall procedures for the test
were based on previously described methods with some
modifications (8, 10, 11). Briefly, MCF-7 cells (1 000 cells/
well) were seeded into 96-well plates (Costar, Cambridge,
MA) and were allowed to attach for 24 h. Then the medium
was replaced with the experimental medium (phenol red-
free MEM containing 5% CDFBS) for 2 days to improve the
sensitivity of MCF-7 cells to estrogen. After that, the medium
was changed to the dosing medium (the experimental
medium along with a range of concentration of test com-
pounds). After 3 days, the dosing medium was refreshed.
Again 3 days later, cell proliferation was terminated by
removing the medium from the wells, adding a thiazolyl blue
(Amresco, Solon, OH) solution (5 mg/mL of phosphate-
buffered saline, PBS). After 4 h, the solution was removed
and 150 µL of dimethyl sulfoxide (DMSO) per well was added.
After 10 min of shaking with a micromixer, the absorbance
was measured at 490 nm with a Bio-Rad model 680 microplate
reader (Bio-Rad Laboratories, Hercules, CA). Results were
calculated as the ratio of each exposure group over the vehicle
control (0.1% ethanol, 1-fold) and as the mean ( standard
deviation (SD) of five independent measurements performed
in four replicates. The results were analyzed using t-test with
Origin (OriginLab, Northampton, MA). The estrogenicity of
BF enantiomers was evaluated (a) by assessing the prolifera-
tive effect (PE), which is the ratio between the highest cell
yield obtained with the test compound and that with the
hormone-free negative control; (b) by determining the relative
proliferative effect (RPE), which is 100 times the ratio between
the highest cell yield obtained with the xenoestrogens and
with E2; and (c) by measuring the relative proliferative potency
(RPP), which is the ratio between the minimal concentration
of E2 needed for maximal cell yield and the minimal dose of
the test xenoestrogen needed to achieve its maximal cell
yield effect. RPE is calculated as the ratio (PE-1) of the test
xenoestrogen over (PE-1) of E2, multiplied by 100. Thus, RPE
indicates whether the chemical being tested induces a
proliferation quantitatively similar to the one obtained with
E2. That is, RPE of 100 suggests a full agonist, while RPE
significantly lower than 100 is a partial agonist (10). The cell
proliferation induced by 17â-estradiol (E2) was included as
a positive control to validate the cell line. 1 × 10-12 to 1 ×
10-8 mol/L of E2 induced pronounced proliferation (p < 0.01)
of MCF-7 cell, and the maximum proliferation was 4.57-fold
relative to the negative control (0.1% ethanol) at 1 × 10-10
mol/L (Figure 2). Therefore, 1 ×10-10 mol/L of E2 was selected
as the positive control in the cell proliferation tests by the
two enantiomers of BF. The proliferation induced by E2 was
completely inhibited by addition of 1 × 10-6 mol/L of ICI
182,780, a pure antiestrogen (Figure 2). The solvent ethanol
was also tested for its ability to affect MCF-7 cell proliferation
and was found to have neither estrogenic nor inhibitory
activity (data not shown).
Measurement of Liver Vitellogenin Level. Bifenthrin
enantiomers were dissolved in ethanol (1.0 mg/mL) and
diluted to 10 ng/mL in rearing water immediately prior to
use. Adult male medaka fish were exposed to the above testing
solutions under static conditions for 10 d. The vitellogenin
synthesis level in livers of medaka was quantified using the
medaka vitellogenin ELISA kit (Biosense Laboratories, Bergen,
Norway). The fish were kept on ice for 1-2 min, and the
livers were extracted, weighed, and then homogenized in
250 µL of the ice-cold dilution buffer included in the ELISA
kit. The homogenized liver samples were immediately
centrifuged (8000×g, 10 min, 4 °C) and the supernatant was
stored at -80 °C until use. The samples were measured within
9
VOL. 41, NO. 17, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 6125

FIGURE 2. Proliferation of MCF-7 cells grown in 5% CDFBS-
supplemented medium exposed to E2 and combined with 10-6 mol/L
of ICI 182,780. Results are presented as mean ( SD of five
independent assays (** indicates p < 0.01 by t-test).
FIGURE 3. HPLC chromatogram showing enantiomeric separation
of (Z)-cis-bifenthrin on a Sumichiral OA-2500-I column (detection
wavelength 230 nm).
1 week according to the manufacturer’s protocol. Briefly, the
samples were thawed on ice, and then the dilutions of
standard and samples were prepared. Dilution buffer
(100 µL) was added to the nonspecific binding (NSB) wells,
and diluted standards and liver samples (100 µL) were added
to the remaining wells, followed by incubation at room
temperature for 1 h. After washing three times with 200 µL
of washing buffer per well, 100 µL of the diluted detecting
antibody was added to all wells and incubated at room
temperature for 1 h. Subsequently, the plates were washed
with 200 µL of washing buffer per well for three times, and
100 µL of the diluted horseradish peroxidase (HRP)-labeled
secondary antibody was added to all wells and incubated at
room temperature for 1 h. After washing the plates five times
with 200 µL of washing buffer per well, 100 µL of o-
phenylenediamine (OPD) peroxidase substrate solution was
added to all wells and incubated in the dark at room
temperature for 30 min. A 50 µL aliquot of 2 M H2SO4 per well
was used to stop the reaction. After 5 min, the absorbance
was read at 490 nm with a Bio-Rad model 680 microplate
reader. The data were analyzed using the Origin software
according to the methods provided by the manufacturer of
the test kit, and the t-test was used to indicate the significant
differences (n ) 5).
FIGURE 4. Proliferation of MCF-7 cells grown in 5% CDFBS-
supplemented medium exposed to enantiomers of (Z)-cis-bifenthrin
and combined with 10-6 mol/L of ICI 182,780. Results are presented
as mean ( SD of five independent assays (* indicates p < 0.05 and
** indicates p < 0.01 by t-test).
Uptake of Bifenthrin Enantiomers by J. Medaka. 14C-
labeled BF enantiomers were dissolved in acetone (1.0 mg/
L) and diluted to 10 ng/mL in rearing water before use. Half
of the adult male medaka fish were exposed to the above
testing solution of 1R-cis-bifenthrin, and the other half of
the fish were exposed to that of 1S-cis-BF under static
conditions for 4 d, respectively. At 1, 2, 3, and 4 d after the
exposure was begun, the fish were rinsed with deionized
water, from which the livers were extracted. The liver tissue
and the remaining whole fish were then dried at room
temperature and combusted on an OX500 biological pxidizer
(R.J. Harvey, Hillsdale, NJ). A LS 5000TD liquid scintillation
counter (LSC; Beckman, Fullerton, CA) was used to measure
the released 14C activity, from which the uptake of BF
enantiomers was determined (34).
TABLE 1. Estrogenic Effect of Enantiomers of Bifenthrin (BF)
Measured by the E-SCREEN Assay
compound concentrationa (mol/L)
PEb
RPEc (%) RPPd (%)
E2
1 × 10-10
1 × 10-9
1 × 10-8
4.49
3.59
1.73
100
74.2
20.9
100
10
1
1S-cis-BF
1R-cis-BF
a The lowest concentration needed for maximal cell proliferation.
b The proliferative effect (PE) calculated as the ratio of the maximal cell
yield of the test compound to the cell yield of the negative control.c The
relative proliferative effect (RPE) calculated as the ratio (PE-1) of the
test chemical over (PE-1) of E2 (×100). d The relative proliferative potency
(RPP) calculated as the ratio between E2 and xenoestrogen doses needed
to produce maximal cell proliferation (×100).
Results and Discussion
effect at 230 nm on the CD detector. The purity of the
enantiomers was determined to be >99% as analyzed by
GC-ECD.
Enantioselectivity in in vitro E-SCREEN Assay. The dose-
response relationships of cell proliferation induced by the
BF enantiomers and the combined effects with 10-6 mol/L
of ICI 182,780 are shown in Figure 4. From Table 1, PE values
of 1S-cis-BF and 1R-cis-BF were 3.59 and 1.73, respectively.
In comparison with the PE of E2 synchronously (4.49), RPE
Enantiomer Separation and Isolation. Baseline separation
of (Z)-cis-BF enantiomers was achieved at a reduced flow
rate of the mobile phase at 0.4 mL/min (Figure 3). From the
previous studies (22, 23) and Figure 3, it may be concluded
that the 1R-cis-BF is dextral (+) at 675 nm and sinistral (-)
at 365 nm and has a negative (-) Cotton effect at 230 nm on
the CD detector, while the 1S-cis-BF is sinistral (-) at 675 nm
and dextral (+) at 365 nm and has a positive (+) Cotton
9
6126 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 17, 2007

was determined to be 74.2% for 1S-cis-BF and 20.9% for 1R-
cis-BF. In addition, by comparing with the concentration
needed for maximal cell proliferation of E2 (1 × 10-10 mol/L),
RPP was calculated to be 10% for 1S-cis-BF and 1% for 1R-
cis-BF. Therefore, the estrogenic potential of 1S-cis-BF was
greater than that of 1R-cis-BF. The proliferation induced by
BF enantiomers was completely blocked by 10-6 mol/L of
ICI 182,780 (Figure 4). These results suggest that BF enan-
tiomers acted through the classical estrogen response
pathway via the estrogen receptor (4, 8). Bifenthrin having
the structural components similar to estrogen metabolites,
may have contributed to its estrogenic response (4). On the
other hand, the open phenyl group of BF could undergo
hydroxylation via a cytochrome P450 monooxygenase-
mediated conversion and generate metabolic byproducts in
situ by the MCF-7 cells. These metabolic byproducts are
possible EDCs. The enantioselectivity of BF in inducing
estrogenic activity may be attributed to the different ste-
reoconfigurations of BF enantiomers when interacting with
enzymes or other naturally occurring chiral biomolecules.
FIGURE 5. Uptake of the enantiomers of bifenthrin in Japanese
medaka (n ) 5). 1S-BF in liver and 1R-BF in liver indicated the
uptake of 1S-cis bifenthrin and 1R-cis bifenthrin in the fish liver
tissues, respectively. 1S-BF in fish and 1R-BF in fish indicated the
uptake of 1S-cis-bifenthrin and 1R-cis-bifenthrin in the fish tissues
excluding liver, respectively.
Enantioselectivity in in vivo Vitellogenin ELISA Assay.
No fish mortality was observed during the liver vitellogenin
synthesis induced by BF enantiomers under the test condi-
tions. The liver vitellogenin level of the adult male medaka
fish placed in BF-free dechlorinated water (containing 0.001%
solvent ethanol) for 10 d was the negative control and was
below the detection limit as measured with the medaka
vitellogenin ELISA kit. Both 1S-cis-BF and 1R-cis-BF showed
the ability to induce the production of VTG. However, there
was a drastic difference between the two enantiomers in
their relative estrogenic potential for inducing VTG produc-
tion. The average level of VTG in the adult male medaka
exposed to 1S-cis-BF for 10 d was 1532 ng/mg, while that
with exposure to 1R-cis-BF was only 12.45 ng/mg. Therefore,
at the given exposure level (10 ng/mL), the response to 1S-
cis-BF was about 123 times greater than that to the R
enantiomer based on medaka vitellogenin induction. As with
the MCF-7 cells, the VTG expression effects observed by the
BF enantiomers may be due to the in situ formation of
metabolic intermediates.
TABLE 2. Uptake of the Enantiomers of Bifenthrin in Japanese
Medaka (n ) 5)
day
R/S (fish)a
R/S (liver)b
liver/fish (R)c
liver/fish (S)d
1
2
3
4
3.7
2.0
1.9
3.0
5.0
4.0
4.6
5.2
12.8
10.1
10.5
9.5
9.6
5.1
4.3
5.5
a R/S (fish)-ratio of 1R-cis-bifenthrin to 1S-cis-bifenthrin in fish tissues
excluding the liver. b R/S (liver)-ratio of 1R-cis-bifenthrin to 1S-cis-
bifenthrin in the liver tissue of J. medaka. c Liver/fish (R)-ratio of 1R-
cis-bifenthrin in the liver to that in the other tissues. d Liver/fish (S)-
ratio of 1S-cis-bifenthrin in the liver to that in the other tissues.
Several recent studies addressed the occurrence of
enantioselectivity in aquatic toxicity and biodegradation of
BF and other pyrethroids. For instance, Liu et al. (22) found
that 1R-cis-BF was 18 and 22 times more active than the
1S-cis enantiomer in causing acute toxicity to aquatic
invertebrates Ceriodaphnia dubia and Daphnia magna,
respectively. The 1S-cis enantiomer of BF was found to be
preferentially degraded in sediments, and the deviation in
enantiomer ratio from the original value increased with the
age of the sediments (20, 21). In this study, both the in vitro
mammalian cell proliferation assay and the in vivo aquatic
vertebrate vitellogenin synthesis assay showed clear enan-
tioselectivity between BF enantiomers in inciting estrogenic
activity. In fact, as 1S-cis-BF uptake into the liver of J. medaka
was relatively less but the induction of the liver vitellogenin
synthesis was substantially greater than that of the 1R
enantiomer, the absolute estrogenic potential of 1S-cis-BF
could be much stronger than that of its R counterpart
compared to what was shown through the in vivo ELISA test.
Results from this study suggest that enantiomers of BF
possess estrogenic properties and that the estrogenic activity
is enantioselective. This is the first report on the enanti-
oselectivity in endocrine-disruption activity of synthetic
pyrethroids. Further in vitro and in vivo studies are needed
to better understand the effects of such xenoestrogens on
the endocrine system in both humans and wildlife. It must
also be noted that while 1R-cis-bifenthrin has higher acute
aquatic toxicity, 1S-cis-BF displays higher estrogenic poten-
tial. Therefore, a general rule with regard to the direction of
enantioselectivity may not exist between acute and chronic
toxicities. Studies so far suggest that monitoring of the
racemate concentration will give an inadequate or misleading
basis for assessing the environmental risk of chiral pesticides
Previous studies showed enantioselectivity in estrogenic
activity of the enantiomers of o,p′-Dichloro-Diphenyl-
Trichloroethane (DDT), suggesting that the R-o,p′-DDT was
a more active estrogen mimic in rats (31, 32). Hoekstra et al.
(33) used a yeast-based assay to assess the enantiomer-
specific transcriptional activity of o,p′-DDT with the human
estrogen receptor (hER) and found that the R enantiomer
was the active estrogen mimic, whereas the hER activity of
S-o,p′-DDT was negligible. DDT and analogs have been
banned for use in most parts of the world for several decades.
This study suggests that enantioselectivity in endocrine
disruption may also occur among chiral pesticides that have
current use.
Enantioselectivity in Uptake in J. Medaka. Significant
differences were noted in the uptake of BF enantiomers by
J. medaka (Figure 5). The uptake of BF in the liver was
generally greater than that in the other tissues. For example,
the ratios of 1R-cis-BF in the liver to that in the other tissues
were 12.8, 10.1, 10.5, and 9.5 after 1, 2, 3, and 4 d exposure,
respectively. The ratios of the 1S enantiomer in the liver to
that in the other tissues were 9.6, 5.1, 4.3, and 5.5 after 1, 2,
3, and 4 d exposure, respectively (Table 2). Under the same
conditions, uptake of 1R-cis-BF in both the liver and the
other tissues was consistently greater than that of 1S-cis-BF.
For example, the ratios of 1R-cis-BF to 1S-cis-BF in liver tissues
were 5.0, 4.0, 4.6, and 5.2 after 1, 2, 3, and 4 d exposure,
respectively. The ratios of 1R-cis-BF to 1S-cis-BF in the other
fish tissues were 3.7, 2.0, 1.9, and 3.0 after 1, 2, 3, and 4 d
exposure, respectively (Table 2).
9
VOL. 41, NO. 17, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 6127

screening. Anal. Bioanal. Chem. 2004, 378, 621-633.
(14) Iwamatsu, T. Convenient method for sex reversal in a freshwater
teleost, the medaka. J. Exp. Zool. 1999, 283, 210-214.
(15) Chikae, M.; Ikeda, R.; Hasan, Q.; Morita, Y.; Tamiya, E. Effect
of alkylphenols on adult male medaka: plasma vitellogenin
goes up to the level of estrous female. Environ. Toxicol.
Pharmacol. 2003, 15, 33-36.
(16) Qin, S. J.; Budd, R.; Bondarenko, S.; Liu, W. P.; Gan, J. Y.
Enantioselective degradation and chiral stability of pyrethroids
in soil and sediment. J. Agric. Food Chem. 2006, 54, 5040-5045.
(17) Garey, J.; Wolff, M. S. Estrogenic and antiprogestagenic activities
of pyrethroid insecticides. Biochem. Biophys. Res. Commun.
1998, 251, 855-859.
(18) Akhtar, N.; Kayani, S. A.; Ahmad, M. M.; Shahab, M. Insecticide-
induced changes in secretory activity of the thyroid gland in
rats. J. Appl. Toxicol. 1996, 16, 397-400.
(19) Kaul, P. P.; Rastogi, A.; Hans, R. K.; Seth, T. D.; Seth, P. K.; Srimal,
R. C. Fenvalerate-induced alterations in circulatory thyroid
hormones and calcium stores in rat brain. Toxicol. Lett. 1996,
89, 29-33.
(20). Given the widespread use of pyrethroids, a more
comprehensive understanding of the significance of enan-
tioselectivity in both acute and chronic ecotoxicities is
imperative for improving risk assessment and regulation of
these pesticides (20). Development of enantiomer-enriched
pesticide products should consider the selection of the
enantiomer with a high potency toward the target organisms
but a low adverse effect to nontarget species.
Acknowledgments
This study was supported by the National Basic Research
Program of China (2002CB410800), the program for Cangjiang
Scholars and Innovative Research Team in University (No.
IRT 0653), and the national natural science foundation of
China for international cooperation of distinguished young
scholars program awarded to J.G. and W.L. (No. 20628708).
We thank FMC for providing 14C-labeled bifenthrin.
(20) Liu, W. P.; Gan, J. Y.; Schlenk, D.; Jury, W. A. Enantioselectivity
in environmental safety of current chiral insecticides. Proc. Natl.
Acad. Sci. U.S.A. 2005, 102, 701-706.
(21) Liu, W. P; Gan, J. Y.; Lee, S. J.; Werner, I. Isomer selectivity in
aquatic toxicity and biodegradation of bifenthrin and per-
methrin. Environ. Toxicol. Chem. 2005, 24, 1861-1866.
(22) Liu, W. P.; Gan, J. J; Qin, S. J. Separation and aquatic toxicity
of enantiomers of synthetic pyrethroid insecticides. Chirality
2005, 17, S127-S133.
(23) Liu, T. L.; Wang, Y. S.; Yen, J. H. Separation of bifenthrin
enantiomers by chiral HPLC and determination of their toxicity
to aquatic organism. J. Food Drug Anal. 2005, 13, 357-360.
(24) Liu, W. P.; Lin, K. D.; Gan, J. Y. Separation and aquatic toxicity
of enantiomers of the organophosphorus insecticide trichlo-
ronate. Chirality 2006, 18, 713-716.
(25) Lin, K. D.; Zhou, S. S.; Xu, C.; Liu, W. P. Enantiomeric resolution
and biotoxicity of methamidophos. J. Agric. Food Chem. 2006,
54, 8134-8138.
(26) Garrison, A. W.; Schmitt, P.; Martens, D.; Kettrup, A. Enantio-
meric selectivity in the environmental degradation of dichlo-
rprop as determined by high-performance capillary electro-
phoresis. Environ. Sci. Technol. 1996, 30, 2449-2455.
(27) Lewis, D. L; Garrison, A. W.; Wommack, K. E.; Whittemore, A.;
Steudler, P.; Melillo, J. Influence of environmental changes on
degradation of chiral pollutants in soils. Nature 1999, 401, 898-
901.
(28) Buser, H. R.; Mu¨ller, M. D.; Poiger, T.; Balmer, M. E. Environ-
mental behavior of the chiral acetamide pesticide metalaxyl:
Enantioselective degradation and chiral stability in soil. Environ.
Sci. Technol. 2002, 36, 221-226.
Literature Cited
(1) Colborn, T.; vom Saal, F. S.; Soto, A. M. Developmental effects
of endocrine-disrupting chemicals in wildlife and humans.
Environ. Health Perspect. 1993, 101, 378-384.
(2) International workshop on endocrine disruptors, 1997; http://
epa.gov/endocrine/Pubs/smithrep.pdf (accessed Jan 23-24,
1997).
(3) Gillesby, B. E.; Zacharewski, T. R. Exoestrogens: Mechanisms
of action and strategies for identification and assessment.
Environ. Toxicol. Chem. 1998, 17, 3-14.
(4) Go, V.; Garey, J.; Wolff, M. S.; Pogo, B. G. T. Estrogenic potential
of certain pyrethroid compounds in the MCF-7 human breast
carcinoma cell line. Environ. Health Perspect. 1999, 107, 173-
177.
(5) Korach, K. S.; McLachlan, J. A. Techniques for detection of
estrogenicity. Environ. Health Perspect. 1995, 103, 5-8.
(6) Andersen, H. R.; Andersson, A. M.; Arnold, S. F.; Autrup, H.;
Barfoed, M.; Beresford, N. A.; Bjerregaard, P.; Christiansen, L.
B.; Gissel, B.; Hummel, R.; Jørgensen, E. B.; Korsgaard, B.; Guevel,
R. L.; Leffers, H.; McLachlan, J.; Møller, A.; Nielsen, J. B.; Olea,
N.; Oles-Karasko, A.; Pakdel, F.; Pedersen, K. L.; Perez, P.;
Skakkeboek, N. E.; Sonnenschein, C.; Soto, A. M.; Sumpter, J.
P.; Thorpe, S. M.; Grandjean, P. Comparison of short-term
estrogenicity tests for identification of hormone-disrupting
chemicals. Environ. Health Perspect. 1999, 107, 89-108.
(7) Andersen, H. R.; Vinggaard, A. M.; Rasmussen, T. H.; Gjer-
mandsen, I. M.; Bonefeld-Jørgensen, E. C. Effects of currently
used pesticides in assays for estrogenicity, androgenicity, and
aromatase activity in vitro. Toxicol. Appl. Pharmacol. 2002, 179,
1-12.
(8) Chen, H. Y.; Xiao, J. G.; Hu, G.; Zhou, J. W.; Xiao, H.; Wang, X.
R. Estrogenicity of organophosphorus and pyrethroid pesticides.
J. Toxicol. Environ. Health, Part A 2002, 65, 1419-1435.
(9) Xie, L.; Sapozhnikova, Y.; Bawardi, O.; Schlenk, D. Evaluation
of wetland and tertiary wastewater treatments for estrogenicity
using in vivo and in vitro assays. Arch. Environ. Contam. Toxicol.
2004, 48, 81-86.
(10) Soto, A. M.; Sonnenschein, C.; Chung, K. L.; Fernandez, M. F.;
Olea, N.; Serrano, F. O. The E-SREEN assay as a tool to identify
estrogens: an update on estrogenic environmental pollutants.
Environ. Health Perspect. 1995, 103, 113-122.
(11) Payne, J.; Jones, C.; Lakhani, S.; Kortenkamp, A. Improving the
reproducibility of the MCF-7 cell proliferation assay for the
detection of xenoestrogens. Sci. Total Environ. 2000, 248, 51-
62.
(12) Nishi, K.; Chikae, M.; Hatano, Y.; Mizukami, H.; Yamashita, M.;
Sakakibara, R.; Tamiya, E. Development and application of a
monoclonal antibody-based sandwich ELISA for quantification
of Japanese medaka (Oryzias latipes) vitellogenin. Comp.
Biochem. Physiol., C 2002, 132, 161-169.
(29) Wedyan, M.; Preston, M. R. Isomer-selective adsorption of amino
acids by components of natural sediments. Environ. Sci. Technol.
2005, 39, 2115-2119.
(30) Garrison, A. W. Probing the enantioselectivity of chiral pesticides.
Environ. Sci. Technol. 2006, 40, 16-23.
(31) McBlain, W. A.; Lewin, V.; Wolfe, F. H. Differing estrogenic
activities for the enantiomers of o,p′-DDT in immature female
rats. Can. J. Physiol. Pharmacol. 1976, 54, 629-632.
(32) McBlain, W. A. The levo enantiomer of o,p′-DDT inhibits the
biding of 17â-estradiol to the estrogen receptor. Life Sci. 1987,
40, 215-221.
(33) Hoekstra, P. F.; Burnison, B. K.; Neheli, T.; Muir, D. C. G.
Enantiomer-specific activity of o,p′-DDT with the human
estrogen receptor. Toxicol. Lett. 2001, 125, 75-81.
(34) Yang, W. C.; Gan, J. Y.; Hunter, W.; Spurlock, F. Effect of
suspended solids on bioavailability of pyrethroid insecticides.
Environ. Toxicol. Chem. 2006, 25, 1585-1591.
Received for review January 28, 2007. Revised manuscript
received June 5, 2007. Accepted June 15, 2007.
(13) Nilsen, B. M.; Berg, K.; Eidem, J. K.; Kristiansen, S. I.; Brion, F.;
Porcher, J. M.; Goksøyr, A. Development of quantitative vitel-
logenin-ELISAs for fish test species used in endocrine disruptor
ES070220D
9
6128 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 17, 2007