Stereoselective biotransformation of permethrin to estrogenic metabolites in fish

Article abstract of DOI:10.1021/tx100167x

This study investigated the stereoselective biotransformation and resulting estrogenic activity of the pyrethroid insecticide, permethrin (PM). Results of both in vivo (male Japanese medaka, vitellogenin (VTG) protein in plasma) and in vitro (primary rainbow trout hepatocyte VTG-mRNA expression) assays indicated stereoselective estrogenic activity of PM. 1S-cis-PM was observed to have significantly higher activity (P ≤ 0.05) than the 1R-cis enantiomer in both in vivo and in vitro evaluations. All enantiomers of PM were oxidized to a 4′-hydoxy PM (4OH PM) metabolite and underwent esterase cleavage to 3-phenoxybenzyl alcohol (3-PBOH) and 3-(4′-hydroxyphenoxy)-benzyl alcohol) (3,4′-PBOH). Racemic 4OH PM as well as 3-PBOH, and 3,4′-PBOH possessed significant (P ≤ 0.05) estrogenicity. 1S-trans-PM underwent esterase cleavage more extensively than the corresponding 1R-trans-PM. Inhibition studies with ketoconazole confirmed cytochrome P450-catalyzed hydroxylation as well as esterase cleavage of PM for all stereoisomers. These studies indicated stereoselectivity in the estrogenic activity of PM resulting from stereoselective biotransformation of the parent compound to more estrogenic metabolites.

Full text of DOI:10.1021/tx100167x

1568
Chem. Res. Toxicol. 2010, 23, 1568–1575
Stereoselective Biotransformation of Permethrin to Estrogenic
Metabolites in Fish
Mae Grace Nillos,†,‡ Sarah Chajkowski,§ John M. Rimoldi,§ Jay Gan,† Ramon Lavado,† and
Daniel Schlenk*,†
Department of EnVironmental Sciences, UniVersity of California, RiVerside, California 92521, Department of
Chemistry, UniVersity of the Philippines, Miag-ao, Iloilo 5023, Philippines, and Department of Medicinal
Chemistry, School of Pharmacy, UniVersity of Mississippi, Mississippi 38677
ReceiVed May 15, 2010
This study investigated the stereoselective biotransformation and resulting estrogenic activity of the
pyrethroid insecticide, permethrin (PM). Results of both in ViVo (male Japanese medaka, vitellogenin
(VTG) protein in plasma) and in Vitro (primary rainbow trout hepatocyte VTG-mRNA expression) assays
indicated stereoselective estrogenic activity of PM. 1S-cis-PM was observed to have significantly higher
activity (P e 0.05) than the 1R-cis enantiomer in both in ViVo and in Vitro evaluations. All enantiomers
of PM were oxidized to a 4′-hydoxy PM (4OH PM) metabolite and underwent esterase cleavage to
3-phenoxybenzyl alcohol (3-PBOH) and 3-(4′-hydroxyphenoxy)-benzyl alcohol) (3,4′-PBOH). Racemic
4OH PM as well as 3-PBOH, and 3,4′-PBOH possessed significant (P e 0.05) estrogenicity. 1S-trans-
PM underwent esterase cleavage more extensively than the corresponding 1R-trans-PM. Inhibition studies
with ketoconazole confirmed cytochrome P450-catalyzed hydroxylation as well as esterase cleavage of
PM for all stereoisomers. These studies indicated stereoselectivity in the estrogenic activity of PM resulting
from stereoselective biotransformation of the parent compound to more estrogenic metabolites.
Recent investigations reported stereoselective aquatic toxicity
and environmental degradation of pyrethroids, including permethrin
(PM), bifenthrin, λ-cyhalothrin, and cypermethrin (11, 12). Dif-
ferences in estrogenic potential were observed between bifenthrin
enantiomers in the MCF-7 human carcinoma cell proliferation
assay, and in ViVo in Japanese medaka (Oryzias latipes) (13).
Induction of estrogen-responsive genes by PM enantiomers was
recently reported in larval and adult zebrafish (Danio rerio) (14).
In addition, PM metabolites 3-phenoxybenzyl alcohol (3-PBOH)
and 3-(4′-hydroxyphenoxy)-benzyl alcohol (3,4′-PBOH) have been
shown to mimic the interaction of 17-ꢀ-estradiol (E2) with estrogen
receptors (15, 16), while 3-phenoxybenzoic acid (3-PBCOOH) was
found to be antiestrogenic (15).
Introduction
Pyrethroids are a group of current-use insecticides extensively
used in agriculture, nurseries, and various urban and household
pest control, as less-toxic alternatives to the more acutely toxic
organophosphorus insecticides that have been banned or re-
stricted (1). Despite their hydrophobic characteristics and low
aqueous solubility (2), pyrethroids have been shown to mobilize
via surface runoff or erosion into streams, creeks, and estuaries,
and can eventually be deposited into sediments (3). Widespread
use of pyrethroids has resulted in their detection in surface
streams at many locations in California at levels that are acutely
toxic to aquatic invertebrates (4).
The acute toxicity of pyrethroid insecticides occurs through
altering the kinetics of the voltage-gated sodium channels of
neurons, which is reportedly similar across species (5). The lower
acute toxicity to birds and mammals, however, has been attributed
to their rapid metabolism and excretion from the body (6). Early
reports indicate that pyrethroid metabolism and elimination are
substantially lower in fish and qualitatively different from that
reported in mammals (7, 8). Thus, pyrethroids are highly acutely
toxic to fish and most other aquatic organisms (5).
Evidence of stereoselective metabolism of some pyrethroids has
been reported in rats (17). In carp and rainbow trout liver
microsomes, trans-PM was more susceptible to microsomal
esterases than cis-PM (18). Previous studies also showed significant
species differences in PM diastereoisomer metabolism (7, 17). For
instance, in contrast to rats, limited hydrolysis of cis- and trans-
PM was observed in trout (Oncorhynchus mykiss). Instead, both
PM stereoisomers were readily converted to a hydroxylated
derivative (7, 8). These previous studies only evaluated diastero-
meric mixtures and did not examine specific enantiomers.
Because of the presence of multiple asymmetric carbons in
their structures, most pyrethroids consist of 4-8 optical isomers.
The significance of the overall molecular shape in the mode of
action of pyrethroids is evident in the stereospecificity of its
insecticidal action (9). The stereospecificity also extends to other
pyrethroids with a chiral center at the alcohol moiety of the
carbon bearing the hydroxyl group (10).
Since little is known regarding the stereoselectivity of
biotransformation or estrogenic activity of pyrethroids in fish.
This study investigated the stereoselective metabolism and
resulting estrogenic activity of PM (Figure 1). Permethrin
stereoisomers were investigated for estrogenic activity in Vitro,
using transcriptional measurements of the egg-yolk precursor,
vitellogenin, in rainbow trout primary hepatocyte cultures and
in ViVo using vitellogenin protein measurements in male
Japanese medaka. To determine whether trout were capable of
stereoselective activation of PM to more estrogenic metabolites,
in Vitro biotransformation studies were carried out using trout
* To whom correspondence should be addressed. Tel: 951-827-2018.
Fax: 951-827-3993. E-mail: dschlenk@ucr.edu.
† University of California, Riverside.
‡ University of the Philippines.
§ University of Mississippi.
10.1021/tx100167x  2010 American Chemical Society
Published on Web 09/13/2010

StereoselectiVe Biotransformation of Permethrin
Chem. Res. Toxicol., Vol. 23, No. 10, 2010 1569
Figure 1. Structure of permethrin enantiomers. (A) trans-Permethrin and (B) cis-Permethrin.
3-(2, 2-Dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic Acid.
To a solution of permethrin (50 mg, 0.128 mmol), in 5 mL of a
CH2Cl2/CH3OH (9:1, v/v) mixture was added a 0.5 N methanolic
solution (5 mL) of NaOH. After 5 min of stirring, the solution
became cloudy, and a precipitate formed. The mixture was stirred
at room temperature for 15 h until all of the ester was consumed.
The reaction was monitored by TLC for the disappearance of the
permethrin. The solvent was removed by rotary evaporation, and
the residue was diluted with water. The aqueous solution was
extracted with diethyl ether to remove the water insoluble benzyl
alcohol and to remove any remaining unreacted ester. The aqueous
phase was cooled, acidified to pH 2-3 with 6 N HCl, and extracted
with CH2Cl2. The combined organic layers were dried with Na2SO4,
and the solvent was removed to afford acid as an isomeric mixture
as an off-white solid (23.5 mg, 87.5% yield). Rf ) 0.18 (25:75
hexanes/chloroform); MS (ESI-) m/z: [ion] (rel. int. %) 206.6 [M]-
(100), 208.6 [M + 2]- (70.8), 210.6 [M + 4]- (12.6).
1,1′-Carbonylbis(3-methylimidazolium) Triflate (CBMIT).
Synthesis of CBMIT was performed as previously reported (39).
4-OH-c, t-Permethrin. A solution of CBMIT (0.0462 mmol)
was transferred via syringe into a suspension of 3-(2,2-dichlorovi-
nyl)-2,2-dimethylcyclopropanecarboxylic acid (0.0478 mmol, 10
mg) in nitromethane (90 µL). After 5 min, a solution of (4-(3-
(hydroxymethyl)phenoxy)phenol (0.0462 mmol, 10 mg) in anhy-
drous THF (185 µL) was added via syringe. The reaction was
quenched with water (0.5 mL) after 17 h, and the mixture was
extracted into diethyl ether, washed successively with 5% aqueous
Na2CO3 and brine, and dried over Na2SO4. The organic layer was
concentrated by rotary evaporation to yield the crude product,
purified by silica gel column chromatography (20:80 ethyl acetate/
hexanes). This afforded 4-OH-permethrin as a mixture of cis- and
trans-isomers. Colorless oil (4.2 mg, 22.3% yield). Rf ) 0.40 (20:
80 ethyl acetate/hexanes); MS (ESI+) m/z: [ion] (rel. int. %)
[MNa]+ 429.2 (100), 431.2 [MNa + 2]+(62.2). 1H NMR was
compared to previously reported data (40) to verify product as a
mixture of cis- and trans-isomers.
hepatic microsomes. Results indicated stereoselective oxidation
and hydrolysis of PM to estrogenic metabolites, which may
cause the induction of vitellogenin in male fish.
Materials and Methods
Chemicals. Analytical standards of racemic PM (40:60 cis/trans),
PM (97% purity; 3-phenoxybenzyl(1RS)-cis,trans-3-(2,2-dichlo-
rovinyl)-2,2-dimethylcyclopropanecarboxylate), and cis-bifenthrin
(97% purity; 2-methylbiphenyl-3-ylmethyl-(1RS)-cis-3-(2-chloro-
3,3,3-trifluoro-1-propenyl)-2,2-dimethylcyclopanecarboxylate) were
obtained from FMC (Philadelphia, PA). Pure standards of PM
enantiomers (enantiomeric and chemical purity >99%) were
provided by U.S. EPA. 3-Phenoxybenzyl alcohol (3-PBOH; 97%
purity) was purchased from Sigma-Aldrich (St. Louis, MO).
Ketoconazole ((()-cis-1-acetyl-4-(4-[(2-[2,4-dichlorophenyl]-2-[1H-
imidazol-1-ylmethyl]-1,3-dioxolan-4-yl)-methoxy]phenyl)pipera-
zine) and 3-(4-methoxy phenoxy)-benzaldehyde (97% purity) were
purchased from Sigma-Aldrich (St. Louis, MO). All other reagents
used in this study were of analytical or HPLC grade.
Metabolite Synthesis. 3-(4′-Hydroxyphenoxy)benzyl Alcohol.
3-(4′-Hydroxyphenoxy)benzyl alcohol (3,4′-PBOH), >99% purity,
was synthesized using the following procedure. The Ag2O oxidation
(19) of commercially available 3-(4-methoxyphenoxy)benzaldehyde
afforded 3-(4-methoxyphenoxy)benzoic acid in 80% yield, which
was deprotected and subsequently reduced according the standard
method (20) yielding the desired metabolite 3-(4 (hydroxymeth-
yl)phenoxy)phenol.
Silver I oxide (3.94 mmol) was prepared by the dropwise addition
of NaOH (8.75 mmol) in 3.88 mL of H2O to a boiling solution of
1.49 g of AgNO3 (8.77 mmol) in 11.66 mL of H2O. The precipitated
dark brown Ag2O was filtered, washed successively with H2O, acetone,
and ether. A suspension of freshly prepared Ag2O (0.5 g, 2.15 mmol)
and 10% NaOH (10 mL) in 10 mL of water was vigorously stir-
red and was added to 3-(4-methoxyphenoxy) benzaldehyde (0.250 g,
1.09 mmol) in small portions over a 30 min period, keeping the
temperature below 30 °C. The mixture was stirred for 1 additional
hour, and the silver was removed with a Celite pad and washed with
water. The filtrate was acidified with concentrated HCl, and the
precipitate was collected and washed with water to afford 210 mg of
3-(4-methoxyphenoxy) benzoic acid as a white solid (80% yield). 1H
NMR-for the carboxylic acid: (acetone-d6, 500 MHz) δ 4.00 (s, 3H,
OCH3), 7.15 (d, 2H, J ) 9.0 Hz, ArH), 7.25 (d, 2H, J ) 9.0 Hz,
ArH), 7.40 (d, 1H, J ) 10.2 Hz, ArH), 7.65 (t, 1H, J ) 8.0 and 15.5
Hz, ArH), 7.73 (s, 1H, ArH), 7.93 (d, 1H, J ) 8.0 Hz, ArH). 13C
NMR (acetone-d6, 125 MHz) δ 53.7, 113.8, 116.3, 119.9, 120.6, 122.3,
128.7, 131.0, 148.1, 155.3, 157.7, 164.9.
Stereoisomer Preparation and Analysis. Stereoisomers of PM
were resolved on an Agilent 1100 Series HPLC (Wilmington, DE,
USA) equipped with an online laser polarimeter detector (PDR-
Chiral, Lake Park, FL, USA) (21). Stereoisomer resolution and
isolation were carried out at room temperature on a Chiralcel OJ
column (250 mm × 4.0 mm, cellulose tris-(4-methyl benzoate) on
a silica gel substrate (Daicel Chemical Industries, Tokyo, Japan)
using hexane/isopropanol (96%:4% v/v) as the mobile phase. The
flow rate of the mobile phase was 0.8 mL/min. The UV detection
wavelength was set at 230 nm for all analyses. The specific rotation
of the resolved stereoisomers was determined at 675 nm and using
a 50 mm cell path. The rotation sign (+ or -) was directly indicated
by a positive or negative peak on the polarimeter. Pure stereoisomers

1570 Chem. Res. Toxicol., Vol. 23, No. 10, 2010
Nillos et al.
used for estrogenic activity and metabolic transformation experi-
ments were manually collected at the HPLC outlet and evaporated
to dryness under a stream of pure nitrogen, followed by re-
dissolution in acetone (carrier solvent). The purity of the derived
stereoisomers was checked with re-analysis on HPLC and was found
to be >99% in all cases.
Organisms. Juvenile rainbow trout (Oncorhynchus mykiss) (15
( 2 g; 15 ( 3 cm) were purchased from Jess Ranch Fishing Lakes
(Apple Valley, CA) and were held in a flow-through living-stream
system (Toledo, OH, USA) with carbon-filtered dechlorinated
municipal water at 13-15 °C. The fish were fed daily with
commercial fish food (Nelson and Sons Silvercup, Murray, UT).
Gonadal morphology was not observed in any of the animals
indicating juvenile animals without endogenous expression of
vitellogenin (22).
Adult male Japanese medaka, >90 day old posthatch (18.8 (
1.3 mm; 64.8 ( 14.7 mg), were obtained from an ongoing culture
at the University of California, Riverside. The fish were kept in
glass aquaria in a chemical free room with the temperature
maintained at 25 ( 2 °C and a light cycle of 16:8 h of light/dark
photoperiods. Dechlorinated water was used for all stock cultures
and experiments. Water quality parameters were constantly moni-
tored, and fish were fed live Artemia sp. nauplii ad libitum twice
daily. Fish were not fed during exposure.
Primary Hepatocyte Culture Preparation and Exposure.
Primary cultures of rainbow trout hepatocytes were isolated by
enzymatic digestion with trypsin followed by mechanical disag-
gregation and gradient centrifugation with Percoll, as described
previously (23). Following hepatocyte isolation, the cells were
seeded in 48-well plates with a density of 1 × 106 cells/well and
allowed to settle for 2 h prior to treatment with PM enantiomers
and metabolites. Each treatment had three replicates as well as
positive (17-ꢀ estradiol) controls and solvent controls (1% acetone).
Cells were incubated for 24 h at 18 °C and then resuspended in
PBS buffer, centrifuged at 5,200g for 5 min, and the pellets washed
twice with PBS. Cells used for PCR were immediately processed
for total mRNA extraction.
To ascertain that exposure concentrations used in this study were
not cytotoxic, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium) test was conducted for acetone (carrier solvent),
PM, PM stereoisomers, PM metabolites, and 17-ꢀ estradiol (positive
control), prior to the vitellogenin-mRNA (VTG-mRNA) expression
assay. Cell viability was determined by the MTT reduction assay
adapted from Mosmann (24). Briefly, the isolated hepatocytes were
seeded in 96-well plates with a density of 1 × 106 cells/well in
100-µL volumes. The cells were exposed to racemic PM, cis/trans
PM, racemic 4OH PM, and the PM metabolites at 5, 10, 25, 50,
and 100 µM for 24 h at 18 °C. MTT solution (5 mg/mL in sterile
PBS) was added (20 µL per in each well) 4 h prior to exposure
termination. The water-insoluble purple formazan crystals formed
during incubation were then dissolved in dimethyl sulfoxide/ethanol
(1:1 in v/v). The amount of formazan formed was determined by
measuring the absorbance at 595 nm using an ELISA plate reader,
and well imperfections were corrected by the measure of the
absorbance at 650 nm. Cell viability was determined as a percentage
of the control value.
In Vitro Measurement of Vitellogenin (VTG) mRNA Expres-
sion in Rainbow Trout Hepatocytes. Vitellogenin (VTG) mRNA
was measured by quantitative PCR (qPCR), used previously in our
laboratory (23). Total mRNA was extracted from cells using
QIAshredder and RNeasy Mini RNA Extraction Kit (Qiagen,
Valencia, CA) and quantified by qPCR using iScript One-step RT-
PCR Kit with SYBR Green from Bio-RAd (Hercules, CA).
Thermocycling parameters were as follows: 10 min at 50 °C (cDNA
synthesis); 5 min at 95 °C (reverse transcriptase inactivation); and
40 cycles of 10 s at 95 °C and 30 s at 56 °C. A melting curve
analysis was carried out between 60 and 95 °C following the
amplification reaction to ensure gene specificity. To correct for
variations in mRNA and cDNA quantity and quality, all VTG values
were normalized against ꢀ-actin. Fluorescence data were collected
at the end of each cycle every 0.1 °C, and the Ct value was
determined to be in the linear phase of amplification. All real-time
reactions were done in iCycler-MyIQ Single Color Real-Time PCR
Detection System (Bio-Rad), and data analysis was done using IQ5
(Bio-Rad).
In ViWo Measurement of VTG Protein Expression in Medaka.
An initial range finding test was performed to estimate the range
of concentrations at which VTG production occurs in male fish
without lethality. The 96 h-LC50 of PM to adult Japanese medaka
was 39.5 µg/L. Upon the basis of this value and other studies
evaluating the estrogenic activity of permethrin and bifenthrin in
fish (13, 14), the exposure concentration that was used for the in
ViVo exposure was 10 µg/L, and the duration of exposure was 8
days. The exposure protocol was based on standard methods with
slight modification (25). Adult male Japanese medaka were held
in 500-mL glass jars (3 fish per jar, three replicates per treatment)
under static conditions for 8 days. On the last day of exposure, the
fish were anesthetized with 1 g/L tricaine methane sulfonate (MS-
222), and the livers were excised and pooled from each individual
replicate and frozen at -80 °C until VTG analysis. The VTG protein
level in the liver was quantified by an enzyme-linked immunosor-
bent assay (ELISA) using commercially available kits for Japanese
medaka (BiosenseTM, Bergen, Norway). The VTG concentrations
were normalized to the total protein in the liver homogenate
determined with the Coumassie blue protein assay (Pierce Bio-
technology, Rockford, IL). The limit of detection for the assay was
0.1 ng/µg protein. VTG production was compared between PM
enantiomers to determine enantioselectivity.
Microsomal Biotransformation. Microsomal fractions were
prepared from livers dissected from 5 rainbow trout and pooled
(26). The excised liver was weighed and rinsed with ice cold 1.15%
KCl and homogenized in 1:5 w/v cold homogenization buffer (100
mM KH2PO4/K2HPO4 buffer at pH 7.4, containing 100 mM KCl,
1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM phenyl-
methyllsulfonylfluoride (PMSF), 1 mM dithiothreitol (DTT), and
0.1 mM 1,10-phenanthroline). The homogenate was centrifuged at
1500g for 15 min, the fatty layer removed, and the obtained
supernatant centrifuged at 12,000g for 20 min. The 12,000-g
supernatant was further centrifuged at 100,000g for 60 min to obtain
microsomal fractions. Microsomal pellets were resuspended in a
small volume of homogenization buffer containing 20% w/v
glycerol. Protein concentrations were measured by the Bradford
method, using the Coomasie Plus-200 Protein Assay reagent (Pierce,
Rockford, IL, USA) and the bovine serum albumin (BSA) standard.
Hepatic microsomal activity was determined on the basis of the
method by Godin et al. (27) and Glickman and Lech (7) with some
modifications. A 250-µL reaction volume containing 250 µg of
microsomal protein, 100 µM substrate (racemate and enantiomers),
1 mM NADPH, and 100 mM Tris-HCl buffer (pH 7.4) was
incubated at 25 °C for 60 min. Previous studies indicated that this
was in the linear range for catalytic activity (7). The reaction was
quenched by the addition of equal volumes of ice-cold acetonitrile
following the addition of internal standards (20 µM cis-bifenthrin
and 10 µM 3-(4-methoxyphenoxy)-benzaldehyde). Following cen-
trifugation, the supernatant was transferred to HPLC glass vials
and analyzed for metabolic products by HPLC/UV at λ ) 230 nm.
Negative controls omitted NADPH or included boiled microsomal
protein. Cytochrome P450 inhibition experiments were conducted
with coincubation with 500 µM ketoconazole added immediately
after NADPH addition and incubated for 5 min prior to the addition
of individual PM enantiomers.
Reverse-phase HPLC-UV analysis of the products was based on
the method of Choi et al. (28). Analysis was performed on an
Agilent 1100 Series HPLC (Wilmington, DE, USA) equipped with
a reverse-phase C-18 HPLC column (Dionex Acclaim 120; 4.6 ×
250 mm, 5 µm). The mobile phase consisted of solvents A (90%
acetonitrile and 10% water) and B (100% water adjusted to pH 1.7
with 85% phosphoric acid). The analytes were eluted using the
following gradient program: 0 min (50% A; 50% B), 6 min (75%
A; 25% B); 7 min (100%A; 0% B); 11 min (100% A; 0% B); 12
min (50% A; 50%B); and 20 min (50% A; 50% B) at a flow rate

StereoselectiVe Biotransformation of Permethrin
Chem. Res. Toxicol., Vol. 23, No. 10, 2010 1571
of 1 mL/min. Analytes were detected at 230 nm and quantified
using external standards.
Identification of the hydroxylated derivative was conducted on
Waters ACQUITY Ultra Performance Liquid Chromatography
(UPLC) System (Waters, Milford, MA, USA) using an Acquity
UPLC BEH C18 column (100 mm × 2.1 mm, i.d., 1.7 µm particle
size) from Waters (Milford, MA, USA). Mass spectrometric
detection was carried out using an ACQUITY TQD tandem
quadrupole mass spectrometer (Waters, Milford, MA, USA). The
mobile phase consisted of ammonium acetate (solvent A) and
methanol (solvent B). The gradient conditions were 0 min (80%A;
20% B); 0.5 min (50%A; 50%B); 9 min (0% A; 100% B); 10 min
(0% A; 100% B); 11 min (80%A; 20% B); and 15 min (80% A;
20%B) at a flow rate of 0.2 mL/min. The ESI parameters were
capillary voltage of 3.0 kV, source temperature of 120 °C, and
desolvation temperature of 350 °C. The cone and desolvation gas
flows were 10 L/h and 600 L/h, respectively, and were obtained
from an in-house nitrogen source. Data acquisition was performed
using MassLynx 4.0 software with the QuanLynx program (Waters).
The analysis was done in negative mode, and the data were acquired
in full-scan at m/z 70-450. The accurate molecular mass measure-
ment was further confirmed at UCR Mass Spectrometry Facility
(Riverside, CA, USA) using Agilent 6210 ESI-TOF (Agilent Corp,
Santa Clara, CA, USA), operated in negative ESI mode. Quantifica-
tion was made using external standards.
Figure 2. Vitellogenin protein concentrations in the livers of adult male
Japanese medaka (Oryzias latipes) following 8 days of exposure to
permethrin (PM) enantiomers (10 ug/L) and expression of vitellogenin-
mRNA in primary rainbow trout (Oncorhynchus mykiss) hepatocytes
exposed to 50 µM permethrin. Values are the mean ( SEM; n ) 3 for
medaka; and n ) 9 for hepatocytes. * Indicates significant difference
from the control (p e 005).
Data Analysis. Data were presented as the mean ( standard
error of the mean (SEM). Statistical differences between treatments
(p < 0.05) were determined using analysis of variance (ANOVA).
Prior to performing ANOVA, the experimental data were checked
for the homogeneity of variance using Bartlett’s test. Treatment
differences were assessed with Bonferroni’s Multiple Comparison
Test (GraphPad Prism v.4.03; GraphPad Software Inc., San Diego,
CA, USA).
1.2-, and 1.4-fold higher VTG-mRNA expression than that with
1R-cis-, 1S-trans-, and 1R-trans-PM, respectively.
In Vitro Estrogenic Activity of Permethrin Metabolites.
To determine whether fish are capable of stereoselectively
activating PM to more estrogenic compounds, PM metabolites
previously reported as estrogenic in mammalian cell lines (4OH
PM, 3-PBOH, and 3,4′-PBOH) were investigated for their
estrogenic activity in the primary trout hepatocyte assay. A
concentration-related increase in VTG-mRNA expression level
was observed for 4OH PM and 3,4′-PBOH (Figure 3). In
contrast, significant VTG-mRNA expression was only observed
at 10 µM for 3-PBOH. VTG-mRNA expression observed after
treatment with 50 µM 3,4′-PBOH was 3-fold higher than the
1S-cis-PM enantiomer at the same concentration (Figures 2 and
3).
In Vitro Biotransformation of Permethrin Stereoisomers.
Stereoselective formation of three metabolites was observed
following the incubation of PM in rainbow trout liver mi-
crosomes (Table 1). trans-PM underwent ester clevage more
extensively than cis-PM. However, the hydroxylated derivative
of the intact ester was the predominant metabolite that was
produced (Table 1). In addition, cis-PM diastereomers were
observed to be more susceptible to hydroxylation, forming more
than twice the amount of hydroxylated product (M4) than trans-
PM diastereomers (Table 1).
Identification of the hydroxylated product (M4) was ac-
complished by accurate-mass measurements on LC-MS (TOF)
and LC-MS/MS (TQD) (Figure 4A and B). In all samples, the
[M - 1]- ion was the most abundant and gave the characteristic
two-chlorine isotope cluster (m/z 405:407 at 1:0.7 ratio),
consistent with the expected molecular mass (m/z 405) and
structure of the hydroxylated parent compound. The retention
times were consistent with those of the parent stereoisomers
(trans eluted before cis on the C18 column), further suggesting
that the stereochemistry was likely conserved during the
hydroxylation process.
Of the four stereoisomers, 1S-cis-PM had the highest
NADPH-dependent metabolic conversion with the hydroxylated
metabolite (M4) constituting 99% of the total metabolite profile.
Formation of M4 from 1S-cis-PM was approximately 25 times
higher than the formation from 1R-cis-PM, indicating a sig-
nificant stereoselectivity toward the 1S-cis-PM for the hydroxy-
Results
Chiral Separation and Identification. Permethrin consists
of two diastereomers (cis- and trans-) and four stereoisomers
(1R-cis-, 1S-cis-, 1R-trans-, and 1S-trans-) (Figure 1). Optimal
resolution and isolation of PM stereoisomers was achieved on
a Chiralcel OJ column using hexane/isopropanol (96:4% v/v)
as the mobile phase. Assignment of absolute configurations to
the resolved peaks was made by comparing chromatograms with
enantiopure standards. Elution order for the PM stereoisomers
was 1S-(+)-cis, 1R-(-)-cis, 1S-(+)-trans, and 1R-(-)-trans
(12, 41).
In ViWo Estrogenic Activity of Permethrin Stereo-
isomers. To determine stereoselectivity for estrogenic activity,
vitellogenin (VTG) production in adult male Japanese medaka
livers was compared following 8 days of exposure to sublethal
concentrations of PM stereoisomers (Figure 2). All PM stere-
oisomers showed the ability to induce the production of VTG
at levels significantly higher than that in controls (p e 0.05).
Statistically significant differences (p e 0.05) in the relative
estrogenic potential between enantiomers were also observed,
with 1S-cis- and 1S-trans-PM eliciting, respectively, 2.5 and
1.3 times greater responses than their respective corresponding
antipodes.
In Vitro Estrogenic Activity of Permethrin Stereo-
isomers. Treatment of juvenile rainbow trout primary hepatocyte
cultures with individual PM stereoisomers resulted in significant
increases in VTG-mRNA expression in the primary hepatocytes
cultures (p e 0.05) (Figures 2 and 3). The 1S-cis-PM treatment
resulted in approximately 2-fold higher VTG-mRNA expression
levels compared to that with 1R-cis-PM. The same trend was
observed for the trans-PM enantiomers, although the difference
was not statistically significant (p > 0.05) (Figure 2). Between
the 4 stereoisomers, treatment with 1S-cis-PM resulted in 2-,

1572 Chem. Res. Toxicol., Vol. 23, No. 10, 2010
Nillos et al.
Figure 3. Expression of vitellogenin-mRNA (VTG-mRNA) in primary rainbow trout (Oncorhynchus mykiss) hepatocytes following treatment with
4-hydroxy PM, 3-phenoxybenzyl alcohol, and 3-(4-hydroxy-3-phenoxy)-benzyl alcohol. * Indicates significant difference from the control (p e
0.01). VTG-mRNA expression in the positive control (E2; 3.6 × 10-2 µM) ) 142.5 ( 5.1-fold relative to the solvent control.
Table 1. Stereoselective Biotransformation of Permethrin in
Trout (Oncorhynchus mykiss) Liver Microsome and the
Effects of the CYP Inhibitor Ketoconazole on
Biotransformation
in conversion was observed presumably due to the diminished
formation of M4.
Discussion
+ ketoconazole
The adverse effect of endocrine disrupting chemicals (EDCs)
in aquatic environments has become a major issue in environ-
mental research and policy (29). Estrogenic agents have received
the most attention primarily because of the relatively simple
methods of measuring exposure and bioavailability in oviparous
aquatic vertebrates in which estrogens activate the hepatic
synthesis of vitellogenin (VTG). VTG is the major precursor
in egg-yolk proteins, which provides energy reserves for
embryonic development. 17ꢀ-Estradiol (E2) is the feminization
hormone of female fish and stimulates the liver to produce VTG
(30). The male fish liver also has the receptor for E2, which
may be stimulated by estrogens initiating the transcription and
subsequent translation of VTG. Thus, VTG induction in adult
male and juvenile fish has been used as a biomarker to assess
exposure to estrogenic contaminants in aquatic environments
(29, 30).
substrate metabolitesa
- NADPH
1S-cis-PM
+ NADPH
(500 µM)
M1
M2
M3
M4
BDL
4.28 ( 1.31
BDL
3.27 ( 0.15
0.64 ( 0.35
BDL
0.39 ( 0.23
1.55 ( 0.89
BDL
BDL
0.91 ( 0.67
BDL
711.77 ( 24.25
53.93 ( 2.56
% conversion 3.92 ( 1.76
1R-cis-PM
M1
M2
M3
M4
BDL
4.45 ( 0.09
BDL
1.99 ( 0.44
11.95 ( 0.19
BDL
BDL
4.16 ( 0.17
BDL
BDL
1.38 ( 0.58
BDL
28.09 ( 0.04
10.40 ( 1.46
% conversion 6.79 ( 3.03
1S-trans-PM
0.72 ( 0.64 21.54 ( 2.15
M1
M2
M3
M4
0.80 ( 0.01
4.05 ( 0.08
BDL
BDL
6.44 ( 1.94
46.65 ( 0.79 51.86 ( 0.93
16.49 ( 0.47 22.65 ( 0.43
BDL
57.01 ( 0.67
13.74 ( 0.90
% conversion 5.00 ( 0.78
In this study, all PM stereoisomers showed the ability to
induce the production of VTG at levels significantly higher than
the control. A 123-fold difference in VTG induction in adult
male medaka was observed among cis-bifenthrin enantiomers
(13). In the present study, enantioselectivity was observed for
cis-PM enantiomers but not for trans-PM enantiomers. In the
MCF-7 cell proliferation assay, a 3.6 times higher proliferative
effect was observed for 1S-cis-bifenthrin relative to the 1R-cis
enantiomer (13). In addition, stereoselective induction of the
hepatic estrogen responsive gene transcription in male adult
zebrafish (Danio rerio) was observed following a 2-day
exposure to PM enantiomers (14). Expression of VTG1 and
VTG2 in zebrafish was 2.6 and 1.8 times higher, respectively,
than its antipode following exposure to (-)-trans-PM.
1R-trans-PM
M1
M2
M3
M4
BDL
5.72 ( 0.33
BDL
4.57 ( 2.20
10.71 ( 0.36
ND
0.86 ( 0.17
4.36 ( 0.33
BDL
BDL
2.51 ( 2.26
BDL
27.55 ( 0.78
7.36 ( 2.09
% conversion 0.45 ( 0.18
a M1, 3,4′-PBOH; M2, 3-PBOH; M3, 3-PBCOOH; M4, OH-PM; Data
are presented as the mean ( SEM (n ) 3) in pmol/min/mg protein;
BDL, below the detection limit (<0.3 pmol/min/mg protein); M4 is
estimated from the calibration curve generated from the parent
compound standards.
lation of PM. In contrast, the NADPH-catalyzed cleavage of
the cis-PM enantiomers was enantioselective for 1R-cis-PM
(Table 1).
In addition to the in ViVo medaka response, the present study
also utilized juvenile rainbow trout primary hepatocyte cultures
in evaluating the enantiomer-specific estrogenic potential of PM.
Significant increases in VTG-mRNA expression was observed
in the primary hepatocytes cultures (p e 0.05) following a 24-h
treatment with PM stereoisomers. Results from both in ViVo
and in Vitro studies indicated stereoselective estrogenic activity
with the 1S enantiomer of both cis- and trans-PM. The
magnitude of enantioselectivity observed in the present study
was comparable to those reported in the livers of zebrafish (14).
The phenolic functionality enhances binding to the estrogen
receptor, suggesting that phenolic metabolites of PM may be
1S-trans-PM was observed to be the most susceptible to ester
cleavage among the PM stereoisomers. Although the trans-PM
stereoisomers were observed to be more susceptible to ester
cleavage than the cis-stereoisomers, hydroxylation in trout liver
microsomes was also greater than ester cleavage. Between the
two trans-PM enantiomers, 1S-trans-PM was more extensively
hydroxylated than 1R-trans-PM (Table 1).
In general, metabolite formation and enantiomer conversion
were significantly inhibited with ketoconazole when individual
enantiomers were coincubated with ketoconazole. The greatest
inhibition was observed with 1S-cis-PM where a 98% reduction

StereoselectiVe Biotransformation of Permethrin
Chem. Res. Toxicol., Vol. 23, No. 10, 2010 1573
Figure 4. HPLC trace and (A) LC-TQD-MS/MS and (B) LC-TOF-MS spectra of the major metabolite from 1S-cis-PM biotransformation in trout
liver microsome. Most probable structure of the metabolite is indicated; the major metabolite collected from 1S-trans-PM biotransformation is
identical to the figure shown here.
more effective estrogen mimics than either 3-PBOH or the
parent PM stereoisomers (31). In Vitro estrogenic activity was
observed with 4-OH PM, 3-PBOH, and 3,4′-PBOH (15, 16).
Consistent with these studies, VTG-mRNA expression following
3,4′-PBOH treatment was dramatically higher than the 1S-cis-
PM enantiomer at the same exposure concentration. While
concentration-dependent increases were observed with 3,4′-
PBOH and 4OH PM, VTG-mRNA expression was observed
only at the 10 µM exposure concentration for 3-PBOH and not
at higher concentrations. One potential explanation may be the
subsequent oxidation of 3-PBOH to the antiestrogenic 3-PB-
COOH (15). Oxidation of 3-PBOH to 3-PBCOOH was observed
in the present study at high substrate concentrations and also
in a previous study with human hepatic microsomes, where one
or more isoforms of alcohol dehydrogenase (ADH) was shown
to catalyze 3-PBOH oxidation to 3-PBCOOH (28). ADH has
also been reported in primary hepatocyte culture from rainbow
trout (32).
Although metabolites from ester cleavage were formed after
microsomal incubations with each stereoisomer, the predominant
metabolite formed from the most estrogenic stereoisomers (1-
S) was the 4′-hydroxylated PM (4OH PM). The relatively high
acute toxicity of PM to many fish species is likely due to reduced
clearance brought about by the significantly lower esterase
activity in fish compared to mammals. In mammals and fish,
trans-Permethrin has been observed to be qualitatively more
susceptible to hepatic microsomal esterases than cis-PM (18).
However, in contrast to mammals where hydrolytic cleavage is
the major detoxification pathway, very little quantitative hy-
drolysis of PM diastereomers has been observed in fish (7).

1574 Chem. Res. Toxicol., Vol. 23, No. 10, 2010
Nillos et al.
Figure 5. Proposed biotransformation pathway of permethrin in fish ((i) ester hydrolysis; (ii) CYP-catalyzed hydroxylation; (iii) glucuronide/sulfate
conjugation); * indicates a chiral center.
Consistent with these results, ester cleavage does not appear to
be the major biotransformation pathway for the stereoisomers
of PM in trout liver microsomes but rather oxidation.
of cis-bifenthrin (cis-BF) enantiomers in medaka liver, showing
greater uptake of 1R-cis-BF over 1S-cis-BF, which may be
attributed to either the preferential uptake of the 1R-cis-
enantiomer or the stereoselective metabolism of 1S-cis-bifentrhin
(13). Taken together, the differences in the metabolic pathway
between PM stereoisomers may contribute to stereoselective
reproductive toxicity not just in fish (Figures 3-5) but quite
possibly in humans as well (35).
Previous studies with trout liver microsomes indicated a
strong preference for hydroxylation at the 4′ position of the
alcohol moiety of trans-PM as opposed to other sites (7, 18). It
is likely that stereoselective cleavage of the hydroxylated ester
occurred in the present investigation, as evidenced by the greater
than 2-fold higher levels of 3,4′-PBOH produced from trans-
PM over cis-PM stereoisomers indicating hydroxylation at the
4′-position of the alcohol moiety of the parent PM compound
(7, 18). The significant inhibition of 4OH PM formation by
ketoconazole is consistent with a cytochrome P450 (CYP)
catalyzed hydroxylation reaction. In addition, the formation of
the ester cleavage products was also diminished by ketoconazole.
CYP has been shown to catalyze NADPH-dependent cleavage
of bulky esters (33). Since ketoconazole is a mechanism-based
inhibitor for CYP, inhibition of microsomal carboxylesterases
is unlikely. Stereoselectivity of CYP has not been previously
evaluated in piscine CYPs but is clearly evident in mammals
particularly with CYP2 family members (33). CYP2C19 had
the highest clearance of PM in studies with heterologously
expressed human CYPs (34). Additional studies should help
identify the isoforms responsible for each reaction in rainbow
trout and help to better understand the potential for mixture
interactions with other CYP inhibitors (PBO) and substrates.
Commercially available PM is usually a mixed product
containing cis and trans isomers at respective ratios of 40:60
or 25:75. cis-PM is reportedly more acutely toxic to mammalian
species than the trans isomer, with 1R-cis-PM having equal
insecticidal toxicity but greater mammalian toxicity than 1R-
trans-PM (35, 36). Recent environmental degradation studies
on PM stereoisomers showed significant enrichment of the 1S-
cis-PM in field soils and sediments due to enhanced mineraliza-
tion of the 1R-enantiomer of both cis- and trans-PM (12). Thus,
the stereoisomer with the higher estrogenic potential was more
persistent and may potentially move into the aquatic environ-
ment. Typical PM concentrations in surface water average in
the low ng/L concentrations with rare occurrences in low µg/L
in runoff events (37). With approximate BCFs of 2800 (38),
accumulation to body burdens capable of eliciting estrogenic
responses in fish is plausible and deserves further study.
The results of the present study showed that the insecticidally
inactive 1S-cis- and 1S-trans- stereoisomers of PM are mainly
responsible for the estrogenic potency of cis- and trans-PM,
respectively. In addition, our results also show significant
differences in metabolic pathways between PM stereoisomers
in fish, which may contribute to the differences in the estrogenic
activity between stereoisomers.
In the present study, consistently greater biotransformation
of the 1S-cis- and 1S-trans-PM was observed over that of their
corresponding antipodes. In addition, enantiomer-specific hy-
droxylation (1S-cis-PM) and esterase (1S-trans-PM) activities
were also observed. Recent studies reported similar observations
of enhanced oxidative biotransformation, consistent with the
clearance of the 1S enantiomers of PM diastereomers in human
liver microsomes (28, 34). In contrast, in rat/mouse hepatic
microsome hydrolysis predominates (35). Although stereose-
lective biotransformation of PM has not been studied previously
in fish, significant stereoselectivity was observed in the uptake
Acknowledgment. We acknowledge the University of Cali-
fornia Toxic Substances Research and Training Program (UC-
TOXICS) for the support of M.G.N. This study was partially
supported by USDA-National Research Initiatives Grant 2005-
35107-16189.

StereoselectiVe Biotransformation of Permethrin
Chem. Res. Toxicol., Vol. 23, No. 10, 2010 1575
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