TOXICOLOGICAL SCIENCES 60, 56 – 62 (2001)
Copyright © 2001 by the Society of Toxicology
Androgen Receptor Antagonism by the Organophosphate
Insecticide Fenitrothion
Hiroto Tamura,* ,† Susan C. Maness,† Kim Reischmann,† David C. Dorman,† L. Earl Gray,‡ and Kevin W. Gaido† ,1
*Department of Applied Biochemistry, Meijo University, Nagoya 468-8502, Japan; †CIIT Centers for Health Research, P. O. Box 12137,
Research Triangle Park, North Carolina 27709; and ‡U.S. Environmental Protection Agency,
Research Triangle Park, North Carolina 27709
Received August 17, 2000; accepted November 14, 2000
Organophosphate insecticides represent one of the most widely
used classes of pesticides with high potential for human exposure
in both rural and residential environments. We investigated the
interaction of the organophosphothioate pesticide fenitrothion
(O,O-dimethyl O-(4-nitro-m-tolyl) phosphorothioate) with the hu-
man androgen receptor (AR). Fenitrothion blocked dihydrotestos-
terone-dependent AR activity in a concentration-dependent and
competitive manner in HepG2 human hepatoma liver cells tran-
siently transfected with human AR and an AR-dependent lucif-
erase reporter gene. Schild regression analysis yielded an equilib-
rium dissociation constant value of 2.18 ⴛ 10 – 8 M. To determine
the antiandrogenic potential of fenitrothion in vivo, 7-week-old
castrated Sprague-Dawley rats were dosed once a day for 7 days
with testosterone propionate (50 ␮g/day, sc) plus gavage doses of
either corn oil vehicle or fenitrothion (15 or 30 mg/kg/day). An
additional group of rats was given testosterone propionate and
flutamide (50 mg/kg/day). Motor activity and acetylcholinesterase
activity in whole blood and brain were also assessed. Both feni-
trothion and the reference antiandrogen flutamide caused signif-
icant decreases in the ventral prostate, seminal vesicle, and levator
ani plus bulbocavernosus muscles tissue weights. In contrast,
blood acetylcholinesterase activity, a standard biomarker of or-
ganophosphate poisoning, was only inhibited at the higher dose of
fenitrothion (30 mg/kg). Our results demonstrate that fenitrothion
is a competitive AR antagonist, comparable in potency to the
pharmaceutical antiandrogen flutamide and more potent, based
on in vitro assays, than the known environmental antiandrogens
linuron and p,p’-, 2,2-bis(p-hydroxyphenyl)-1,1-dichloroethylene
(p,p’-DDE).
Key Words: androgen receptor; organophosphate pesticide; en-
docrine-active chemical; antiandrogen; HepG2 cells; Hershberger
assay; transcriptional activation.
The research described in this article has been reviewed by the National
Health and Environmental Effects Research Laboratory, U. S. Environmental
Protection Agency, and has been approved for publication. Approval does not
signify that the contents necessarily reflect the views and policies of the
Agency nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.
1
To whom all correspondence should be addressed. Fax: 919 –558 –1300.
E-mail: gaido@ciit.org.
56
Organophosphate insecticides are widely used in both agri-
cultural and landscape pest control and the potential for human
exposure to this class of compounds is significant. A profile
from 66 poison control centers in the United States in 1997
indicated that organophosphorous insecticides were involved
in more poisonings than any other single class of pesticide
(Litovitz et al., 1998). Individuals at greatest risk are those who
most frequently handle these compounds, including formula-
tors, applicators, farmers, and home gardeners. Organophos-
phate insecticides are also used extensively in the home envi-
ronment, and, as a result, young children are at increased
likelihood for exposure to these compounds (Eskenazi et al.,
1999; Landrigan et al., 1999; National Research Council,
1993). The primary toxicity associated with acute exposure to
organophosphate insecticides is cholinergic crisis resulting
from acetylcholinesterase inhibition (Pope, 1999). However,
these compounds have numerous other compound-specific
chronic effects, including delayed polyneuropathy, immuno-
toxicity, carcinogenesis, and endocrine, developmental, and
reproductive toxicity (Astroff et al., 1998; Cranmer et al.,
1978; Reuber, 1985; Sultatos, 1994).
Recent concern that chemicals in the environment are dis-
rupting normal human endocrine function leading to reproduc-
tive and developmental disorders, such as a reduction in male
fertility or an increase in female breast cancer (National Re-
search Council, 1999), has led to changes in the Food Quality
Protection and Safe Drinking Water Acts to now require the
United States Environmental Protection Agency to develop a
screening and testing program for endocrine-active chemicals.
Early efforts to identify and characterize environmental endo-
crine-active chemicals focused on xenoestrogens, which are
chemicals capable of interfering with estrogen receptor func-
tion (Chen et al., 1997; Gaido et al., 1997; Krishnan et al.,
1993; Soto et al., 1991; White et al., 1994). However, recent
publications describing environmental chemicals with antian-
drogenic activity has expanded the research effort to include
screening for chemicals capable of interfering with androgen
receptor function (Hosokawa et al., 1993; Kelce et al., 1995;
Maness et al., 1998; Wong et al., 1995).
 ANTIANDROGENIC ACTIVITY OF FENITROTHION
FIG. 1. Chemical structure of flutamide, linuron, and fenitrothion.
We investigated the interaction of the organophosphothioate
pesticide fenitrothion [O,O-dimethyl O-(4-nitro-m-tolyl) phos-
phorothioate] with the human androgen receptor. We chose to
investigate fenitrothion based on its structural similarities with
the pharmaceutical antiandrogen flutamide and the environ-
mental antiandrogenic herbicide linuron (Fig. 1). Fenitro-
thion’s androgen receptor activity was compared with its abil-
ity to inhibit acetylcholinesterase activity and alter motor
activity, standard indicators of organophosphate insecticide
toxicity. We demonstrate that fenitrothion competitively antag-
onizes androgen receptor (AR) activity in transfected cells and
causes regression of androgen-dependent tissue weights in
vivo. Inhibition of androgen receptor function in vivo occurred
at a dose of fenitrothion that did not significantly alter blood
acetylcholinesterase activity.
MATERIALS AND METHODS
Chemicals. Fenitrothion was obtained from Chem Service (West Chester,
PA). All other chemicals were obtained from Sigma Chemical Co. (St. Louis,
MO). All chemicals were ⱖ 97% pure.
Plating and transfection. Transfection experiments were performed as
previously described (Gaido et al., 1999; Maness et al., 1998). HepG2 human
hepatoma cells (ATCC, Rockville, MD) are used in our studies because of our
extensive prior experience with this cell line, the utility of the cell line for the
proposed study, the ability to compare results obtained with previously pub-
lished studies, and because of their widespread use in toxicology and hormone
receptor research. Under normal circumstances, HepG2 cells have limited
metabolic capabilities. Our experiments indicate that very little metabolism of
chemicals occurs over the 24-h treatment period of this assay (Gaido et al.,
unpublished observations).
HepG2 cells were plated in 24-well plates (Falcon Plastics, Oxnard, CA) at
a density of 10 5 cells/well in complete medium consisting of phenol red-free
Eagle’s Minimal Essential Medium (GIBCO/BRL, Grand Island, NY) supple-
mented with 10% charcoal-dextran-treated fetal bovine serum (Hyclone, Lo-
gan, UT), 2% L-glutamine, and 0.1% sodium pyruvate. Cells were transfected
with 3 plasmids: receptor plasmid pRSAR at 10 ng/well, MMTV-luc reporter
plasmid at 405 ng/well, and a constitutively expressed pCMV␤-gal plasmid
(transfection control) at 10 ng/well (Maness et al., 1998). Transfected cells
were rinsed with phosphate-buffered saline and dosed with various concentra-
tions of test chemical and dimethyl sulfoxide (vehicle control) in complete
medium. After the 24-h incubation, cells were rinsed with phosphate-buffered
saline and lysed with 65 ␮l of lysing buffer (25 mM Tris-phosphate, pH 7.8,
57
2 mM 1,2-diaminocyclohexane-N,N,N’,N’-tetraacetic acid, 10% glycerol,
0.5% Triton X-100, 2 mM dithiothreitol). Lysate was divided into two 96-well
plates for luciferase and ␤-galactosidase determination.
Luciferase activity was determined by adding 100 ␮l Luciferase Assay
Reagent (Promega, Madison, WI) to 20 ␮l of lysate per well. Luminescence
was determined immediately, using an ML3000 microtiter plate luminometer
(Dynatech Laboratories, Chantilly, VA).
␤-Galactosidase activity was determined by adding 20 ␮l ␤-galactosidase
assay reagent to 30 ␮l of lysate per well. ␤-Galactosidase assay reagent
consisted of a 4-mg/ml solution of chlorophenol red-␤-D-galactopyranoside
(CPRG) in 150 ␮l CPRG buffer (60 mM Na 2HPO 4, 40 mM NaH 2PO 4, 10 mM
KCl, 1 mM MgSO 4, 50 mM ␤-mercaptoethanol, pH 7.8). Absorbance at 570
nm was determined over a 30-min period, using a Vmax kinetic microplate
reader (Molecular Devices, Menlo Park, CA). Luciferase values obtained for
each transfected well were normalized by dividing by the associated ␤-galac-
tosidase value for that well. This normalization corrects for any variation in
transfection efficiency between wells and between experiments.
HepG2 cells lack detectable levels of endogenous steroid hormone receptors
including AR, progesterone receptor, and glucocorticoid receptor. In the ab-
sence of transfected receptor, luciferase activity remains below the level of
detection. Background activity following AR transfection averaged 5 ⫾ 1
normalized luciferase units. Values presented in this study represent the
means ⫾ SE resulting from at least 3 separate experiments with triplicate wells
for each treatment dose level. Dose-response data were analyzed using the
sigmoidal dose-response function of the graphical and statistical program
Prism (GraphPad, San Diego, CA).
Hershberger male rat assay. This study was conducted in accordance with
Federal guidelines for the care and use of laboratory animals and was approved
by the Institutional Animal Care and Use Committee at CIIT. Rats were housed
in the CIIT animal care unit, a facility accredited by the American Association
for the Accreditation of Laboratory Animal Care (AAALAC) and were kept in
a HEPA-filtered, mass air-displacement room with a 12-h light-dark cycle at
18 –26°C and relative humidity of 30 –70%. Rats had access ad libitum to
deionized water and rodent chow (NIH-07, Zeigler Brothers, Gardner, PA).
Male Sprague-Dawley rats, castrated at 4 weeks of age, were purchased
from Charles River Labs, Inc. (Raleigh, NC). Rats were weight-ranked, and a
homogeneous population (mean weight ⫾ 20%) of 32 male rats was selected
for the study. Rats were treated once a day for 7 days, beginning at 7 weeks of
age, with subcutaneous doses of testosterone propionate (50 ␮g/day in 0.2 ml
corn oil) plus gavage doses of either corn oil vehicle or 15 or 30 mg/kg/day
fenitrothion. While the acute toxicity of fenitrothion to mammals is considered
to be low, it does cause typical signs of cholinergic stimulation such as muscle
twitching, tremor, salivation, and diarrhea at a dose of 30 mg/kg/day following
14 days of exposure in rats (Kunimatsu et al., 1996). The dose of fenitrothion
(30 mg/kg/day) selected for this study was chosen to ensure that an active
concentration of fenitrothion was achieved, as determined by its ability to
inhibit cholinesterase activity, that did not cause excessive toxicity (Kunimatsu
et al., 1996). A second, lower dose (15 mg/kg/day) was selected to determine
whether antiandrogenic activity could be detected at less toxic doses. An
additional group of rats was treated with testosterone propionate plus flutamide
(50 mg/kg/day) as an antiandrogen reference control. The dose level for
flutamide was based on previously published studies (Ashby and Lefevre,
2000; Lambright et al., 2000; O’Connor et al., 1999). Each of the 4 treatment
groups consisted of 8 rats. Treatments were adjusted each day for body weight
changes.
Neurotoxicity assessment. Two h after the last dose, animals were trans-
ferred to cages containing an automated photobeam activity system to monitor
motor activity as an assessment of neurotoxicity (Dorman et al., 2000). Motor
activity was measured during ten 6-min intervals for a total of 60 min, using
an automated cage rack photobeam activity system (San Diego Instruments,
San Diego, CA). The trial was initiated by the first activity of the rat. Total
number of movements (beams broken) and number of ambulations (number of
times that more than one beam is broken in succession) was recorded. A nested
analysis of motor activity data was performed, using a repeated-measures
58 TAMURA ET AL.
FIG. 2. Fenitrothion antagonizes DHT-induced AR activity in transiently
transfected HepG2 human hepatoma cells. Cells were treated with increasing
concentrations of DHT, fenitrothion alone for detecting AR agonist activity,
and fenitrothion plus 10 –7 M DHT for detecting AR antagonist activity. Values
represent the means ⫾ SE resulting from 3 separate experiments with triplicate
wells for each treatment dose and are presented as units of luciferase activity
divided by the ␤-galactosidase control.
analysis with treatment as a grouping factor and interval as a within-subject
factor (MANOVA).
Necropsy. On the day after the last treatment, rats were anesthetized with
sodium pentobarbital. Cardiac puncture was performed to collect blood for an
acetylcholinesterase assay, and rats were euthanized by exsanguination. Fron-
tal cortex, hippocampus, and striatum were collected for the acetylcholinest-
erase assay. Liver, kidney, adrenals, ventral prostate, dorsolateral prostate,
glans penis, seminal vesicle (with coagulating glands and fluid), and levator ani
plus bulbocavernosus muscles were collected and weighed. Organ weight data
were analyzed using a regression analysis, the general-linear-models procedure
on the statistical analysis system (SAS). Post-hoc tests were conducted when
the overall analysis of variance was significant at the p ⬍ 0.05 level using the
LSMEANS procedures available on SAS.
Acetylcholinesterase activities in striatum, hippocampus, frontal cortex, and
whole blood were determined after solubilization of tissue with 1% Triton
X-100 in 0.1 M phosphate buffer (pH 8.0) for 15 min at room temperature.
Acetylthiocholine iodide (0.075 M) and 0.01 M 5,5⬘-dithio-bis(2-nitrobenzoic
acid) were used as substrate, and analysis was performed on a Roche COBAS
Fara II chemical analyzer. Protein was measured using a commercially avail-
able kit (Pierce, Rockford, IL). Acetylcholinesterase results were normalized to
total protein and expressed as change in absorbance per min. Serum testoster-
one and corticosterone were measured by radioimmunoassay using commer-
cially available kits (ICN Biomedicals, Inc., Costa Mesa, CA). Data were
analyzed by one-way analysis of variance using JMP statistical analysis
software (SAS Institute, Cary, NC).
RESULTS
We determined the in vitro interactions of fenitrothion with
the androgen receptor (AR) by measuring AR-dependent lu-
ciferase activity in HepG2 human hepatoma liver cells tran-
siently transfected with the human AR and an AR-dependent
luciferase reporter gene. Transfected HepG2 cells were treated
with fenitrothion over a concentration range from 10 – 8 to 10 –5
M in the presence or absence of a maximally activating dose of
dihydrotestosterone (DHT). Fenitrothion reduced DHT-depen-
dent AR activity in a concentration-dependent manner, thus
indicating that fenitrothion is an AR antagonist (Fig. 2). Slight
AR agonist activity was detected at the highest concentration
of fenitrothion used in this study (10 –5 M). Fenitrothion’s
agonist activity was approximately 8% of the maximally in-
ducible response with DHT.
The AR antagonist activity of fenitrothion was further char-
acterized by determining the effect of set concentrations of
fenitrothion over a complete DHT dose-response curve (Fig.
3). Fenitrothion caused a parallel displacement of the DHT
dose-response curve to the right with no concomitant depres-
sion of maximal response. These results indicate that fenitro-
thion acts as a competitive AR antagonist (Kenakin, 1993). The
equilibrium dissociation constant (K B) for the antagonist-re-
ceptor complex was determined by Schild regression (Arun-
lakshana and Schild, 1959) to be 2.18 ⫻ 10 – 8 M.
To determine the antiandrogenic potential of fenitrothion in
vivo, we performed an assay in castrated male rats following
the Hershberger protocol (Hershberger et al., 1953). This assay
was developed as a short-term, in vivo assay for androgenic
and antiandrogenic compounds, and has been recommended by
the Endocrine Disrupter Screening and Testing Advisory Com-
mittee (EDSTAC) as part of a Tier-1 screening battery for
endocrine-active chemicals (U.S. EPA, 1998).
Rats dosed with 30 mg/kg/day fenitrothion weighed 17%
less than controls at necropsy (Fig. 4A). Rats dosed with 15
mg/kg/day fenitrothion did not weigh significantly different
from control rats at necropsy. Fenitrothion at 15 mg/kg/day and
30 mg/kg/day, respectively, caused a 30% and 92% decrease in
body weight gain over the 7-day treatment period relative to
control values (Fig. 4B). The effect of fenitrothion on body
weight is a result of cholinergic stress. High-dose fenitrothion
(30 mg/kg/day) caused a 22% decrease in absolute liver weight
FIG. 3. Fenitrothion acts as a competitive reversible inhibitor of AR.
Experiments were performed as described in Figure 1, with 10 –10 to 10 –5 M
DHT either alone or in combination with 10 –7 M, 3 ⫻ 10 –7 M, 10 – 6 M
fenitrothion. Values represent the means ⫾ SE of 3 separate experiments and
are presented as percent response, with 100% activity defined as the activity
achieved with 10 –7 M DHT alone in each experiment. Inset: Schild Regression
analysis. The dose ratio (dr) is [A⬘] ⫼ [A] (Kenakin, 1993), where [A⬘] and [A]
refer to equiactive concentrations of DHT in the presence and absence of
fenitrothion, respectively.
 ANTIANDROGENIC ACTIVITY OF FENITROTHION
relative to controls, whereas flutamide treatment resulted in a
12% increase in absolute liver weight (Fig 4C). The effect of
high-dose fenitrothion on liver weight was not significant when
body weight was considered by covariance analysis (data not
shown). The effect of flutamide on liver weight remained
significant by covariance analysis with body weight. Thus, the
reduced liver weight in fenitrothion-treated rats is likely due to
the reduced animal weights, whereas, the increased liver
weights in the flutamide rats is likely to a direct effect of
59
FIG. 4. Effect of fenitrothion on an-
drogen-dependent responses in the rat.
Male Sprague-Dawley rats castrated at 4
weeks were treated once a day for 7 days
beginning at 7 weeks of age with subcu-
taneous doses of testosterone propionate
(50 ␮g/day in 0.2 ml corn oil) plus ga-
vage doses of either corn oil vehicle, 15
or 30 mg/kg/day fenitrothion, or 50 mg/
kg/day flutamide. Each treatment group
consisted of 8 rats. C, Control; F15, feni-
trothion 15 mg/kg; F30, fenitrothion 30
mg/kg; Flu, flutamide; *p ⬍ 0.05.
flutamide on the liver. Kidney weight was not affected by any
of the treatments (data not shown).
Both fenitrothion and the reference antiandrogen flutamide
caused significant decreases in ventral prostate, seminal vesi-
cle, and levator ani plus bulbocavernosus muscles tissue
weights (Figs. 4D– 4F). The reduction in androgen-dependent
tissue weights remained statistically significant (p ⬍ 0.05)
when analyzed by covariance analysis with terminal body
weight, and thus were due to a direct effect of fenitrothion and
60 TAMURA ET AL.

TABLE 1
Effect of Fenitrothion on Serum Steroid Concentration and Whole Blood and Brain Acetylcholinesterase Activity

Serum steroid (ng/ml) Acetylcholinesterase (⌬A/mg protein/min)

Treatment Testosterone Corticosterone Whole blood Striatal Hippocampal Frontal cortex

Control 0.18 ⫾ 0.03 72 ⫾ 23 4.83 ⫾ 0.44 837.5 ⫾ 57.1 84.8 ⫾ 12.3 104 ⫾ 3.5
Fen 15 0.20 ⫾ 0.03 109 ⫾ 21 3.71 ⫾ 0.21 101.1 ⫾ 6.8* 10.2 ⫾ 0.8* 23.0 ⫾ 1.3*
Fen 30 0.25 ⫾ 0.02 223 ⫾ 70 2.92 ⫾ 0.48* 61.8 ⫾ 5.5* 5.1 ⫾ 1.3* 14.7 ⫾ 1.4*
Flutamide 0.25 ⫾ 0.02 66 ⫾ 14 6.54 ⫾ 0.40* 822.1 ⫾ 57.2 89.5 ⫾ 12.9 100 ⫾ 3.5

Note. Mean ⫾ SE; *p ⬍ 0.05. Male Sprague-Dawley rats castrated at 4 weeks were treated once a day for 7 days, beginning at 7 weeks of age, with
subcutaneous doses of testosterone propionate (50 ␮g/day in 0.2 ml corn oil) plus gavage doses of either corn oil vehicle, 15 or 30 mg/kg/day fenitrothion, or
50 mg/kg/day flutamide. Fen 15, fenitrothion 15 mg/kg; fen 30, fenitrothion 30 mg/kg.

flutamide on these tissues rather than an indirect effect related DISCUSSION

to body weight (data not shown).
Serum testosterone concentrations were not altered by any of
the treatments (Table 1). A trend for increased plasma corti-
costerone concentrations, an indication of cholinergic stress
(Kunimatsu et al., 1996; Rattner et al., 1982), was evident only
in the 30-mg/kg/day fenitrothion treatment group. However,
this increase was not significantly different from control. Log
transformation of the data did not enhance significance. Feni-
trothion exposure was associated with other signs of cholin-
ergic toxicity, including excess salivation, muscle tremors, and
reduced motor activity (Fig. 5). Acetylcholinesterase activity
was significantly reduced in whole blood and brain in rats
dosed with 30-mg/kg/day fenitrothion dosage (Table 1). Brain
acetylcholinesterase activity was also significantly reduced at
15-mg/kg/day fenitrothion. Blood acetylcholinesterase activity
was not significantly reduced with 15-mg/kg/day fenitrothion
(Table 1). Blood acetylcholinesterase was significantly ele-
vated in the flutamide treatment group.
FIG. 5. Total motor activity following repeated exposure to fenitrothion or
flutamide. Motor activity was measured during ten 6-min intervals for a total
of 60 min, using an automated cage rack photobeam activity system. Values
represent mean ⫾ SE; *p ⬍ 0.05.
Fenitrothion is registered in the U.S. for use in ant and roach
baits. There are no approved domestic food or feed uses for
fenitrothion, and exposure to fenitrothion in the U.S. is mini-
mal. However, fenitrothion is used in other countries to control
pests on crops, stored grains, and cotton. Fenitrothion is also
used elsewhere in forest spraying and in public health cam-
paigns. As a result, the human health effects associated with
exposure to fenitrothion remain a concern, especially among
pesticide workers and applicators whose acute exposure to
organophosphate pesticides can sometimes occur at levels high
enough to inhibit blood acetylcholinesterase activity (Nigg and
Knaak, 2000; Ohayo-Mitoko et al., 2000; Satoh and
Hosokawa, 2000). We demonstrate that fenitrothion is a com-
petitive antagonist of the human AR and can inhibit androgen-
dependent tissue growth in vivo. Inhibition of androgen-depen-
dent tissue growth in vivo occurred with a dose of fenitrothion
(15 mg/kg) that was not associated with a significant decrease
in blood acetylcholinesterase activity, which is often used as a
biomarker for human exposure to organophosphate pesticides
(Nigg and Knaak, 2000). Thus, antiandrogenic activity in the
rat occurs at a dose level below that required for a significant
reduction in blood acetylcholinesterase activity.
Fenitrothion was included as a negative control in a recently
published study comparing the effect of antiandrogenic and
estrogenic chemicals on preputial separation (Ashby and Le-
fevre, 2000), an androgen-dependent response that can be
delayed by antiandrogen treatment (Monosson et al., 1999).
Fenitrothion (15 mg/kg/day) failed to cause a significant delay
in preputial separation (Ashby and Lefevre, 2000). However,
the results were confounded by the negative effect of fenitro-
thion on body weight, which also delayed preputial separation
(Ashby and Lefevre, 2000). As a result, the authors were
unable to identify fenitrothion as an antiandrogen. These re-
sults suggest that preputial separation may not be the best
method for detecting antiandrogens that also cause a decrease
in body weight.
The in vitro potency of fenitrothion as a competitive AR
 ANTIANDROGENIC ACTIVITY OF FENITROTHION
antagonist (K B value of 2.18 ⫻ 10 – 8 M) is comparable with the
pharmaceutical antiandrogen flutamide (K B value of 1.07 ⫻
10 – 8 M) and approximately 8- to 35-fold greater than the
environmental antiandrogens p,p’-DDE and linuron, respec-
tively (Maness et al., 1998; McIntyre et al., 2000). Based on
these results, fenitrothion represents one of the more potent
environmental AR antagonists identified to date. Fenitrothion
alone did demonstrate slight agonist activity at the highest
concentration tested (10 –5 M). We have previously reported on
the ability of some AR antagonists to demonstrate agonist
activity in vitro at high concentrations (Maness et al., 1998).
The mechanism for this switch to agonist activity at high
concentrations has not been determined.
The Hershberger assay used in this study, and proposed by
the U.S. EPA as part of their endocrine disrupter screening
program, was designed to identify agents that possess intrinsic
antiandrogenic activity (U.S. EPA, 1998). This assay has been
used for decades for screening chemicals for androgenic and
antiandrogenic activity (Dorfman, 1962) and is extremely sen-
sitive to antiandrogens, because the typical endocrine feedback
loops have been eliminated. The Hershberger assay does, how-
ever, respond to several different mechanisms of action, so it is
important to confirm the purported AR-activity with an in vitro
assay as was done here. We use the Hershberger assay to
demonstrate that fenitrothion can block androgen-dependent
tissue growth. Fenitrothion’s effects on androgen-dependent
tissue weights were comparable to those caused by treatment
with the reference antiandrogen, flutamide. Fenitrothion also
inhibited brain acetylcholinesterase activity and induced signs
of cholinergic stress. The effect, if any, of cholinergic stress on
the AR-dependent responses measured in this study remains to
be determined.
Male reproductive tract development in utero is one of the
most sensitive periods for exposure to antiandrogens, and
chemicals that have been shown to display antiandrogenic
activity in vitro and in the Hershberger in vivo assay induce
malformations in male rat offspring at lower dosage levels
following in utero exposure. Such toxicants include the herbi-
cide linuron, vinclozolin, procymidone, flutamide and p,p⬘-
DDE (Gray et al., 1999a,b; Lambright et al., 2000; McIntyre et
al., 2000; Ostby et al., 1999; You et al., 1999). When linuron
is administered during gestation, dosages as low as 12.5–25
mg/kg/day result in alterations of androgen-dependent tissues
in male rats (Lambright et al., 2000; McIntyre et al., 2000).
Similarly, administration of vinclozolin at 50 mg/kg/day dur-
ing gestation causes hypospadias, while functional alterations
(reduced AGD, areolas, retained nipples, reduced sex acces-
sory gland size) are seen at lower dosage levels, ranging from
3 to 25 mg/kg/day (Gray et al., 1999a). When administered
during sexual differentiation, procymidone induces hypospa-
dias at 50 mg/kg/day, and similar to vinclozolin, functional
alterations of androgen-dependent tissues are seen at lower
dosage levels (Ostby et al., 1999). Future studies should de-
termine the dose-responsive effect of fenitrothion on male
61
reproductive tract development following in utero exposure,
which will be more relevant for risk assessment.
Organophosphate insecticides have not been tested previ-
ously for their ability to directly interact with steroid hormone
receptors. Structural similarities between fenitrothion and other
organophosphorous compounds make it likely that additional
organophosphate insecticides will have antiandrogenic activ-
ity. Indeed, the organophosphate pesticide parathion has been
shown to inhibit DHT binding to the AR in the rat ventral
prostate (Shain et al., 1977). Preliminary experiments con-
ducted in our laboratory show that methyl parathion, as well as
other structurally related organophosphates, demonstrate anti-
androgenic activity in transiently transfected HepG2 cells (data
not shown). The high potential for human exposure and the
current concern for the effect of environmental antiandrogens
on male reproductive development indicate the need for further
study of this economically important class of compounds.
ACKNOWLEDGMENTS
We thank Joe Ostby, Melanie Struve, Carol Bobbit, Otis Lyght, Delorise
Williams, Duncan Wallace, Brian McManus, Dr. Barry McIntyre, and Dr.
Norman Barlow for their helpful suggestions and technical assistance. We
thank Dr. Paul Foster, Dr. Katrina Waters, Dr. Valerie Shultz, Dr. J. Christo-
pher Corton, Dr. Tammy Stoker, and Vickie Wilson for critical review of the
manuscript and Dr. Barbara Kuyper for editorial assistance.
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