TOXICOLOGICAL SCIENCES 81, 60–68 (2004)
Advance Access publication May 12, 2004
Dose-Dependent Alterations in Gene Expression and Testosterone
Synthesis in the Fetal Testes of Male Rats Exposed to
Di (n-butyl) phthalate
Kim P. Lehmann, Suzanne Phillips, Madhabananda Sar, Paul M. D. Foster,1and Kevin W. Gaido,2
CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709
Received on March 19, 2004; accepted on May 5, 2004
Exposure to di (n-butyl) phthalate (DBP) in utero impairs the
development of the male rat reproductive tract. The adverse effects
are due in part to a coordinated decrease in expression of genes
involved in cholesterol transport and steroidogenesis with a resul-
mine the dose-response relationship for the effect of DBP on
steroidogenesis in fetal rat testes, pregnant Sprague-Dawley rats
received corn oil (vehicle control) or DBP (0.1, 1.0, 10, 50, 100, or
500 mg/kg/day) by gavage daily from gestation day (GD) 12 to 19.
Testes were isolated on GD 19, and changes in gene and protein
testicular testosterone concentration was determined by radio-
reductions inmRNAandproteinconcentration ofscavengerrecep-
tor, steroidogenic acute regulatory protein (StAR), cytochrome
P450 side-chain cleavage, 3b-hydroxysteroid dehydrogenase, and
cytochrome P450c17. Testicular testosterone was reduced at doses
of 50 mg/kg/day and above. Whole-testis expression of peripheral
to transport cholesterol across the mitochondrial membrane, was
upregulated following exposure to DBP at 500 mg/kg/day. By
immunocytochemistry, however, PBR protein was reduced in
interstitial cells and also expressed but not reduced in gonocytes.
Our results demonstrate a coordinate, dose-dependent reduction in
the expression of key genes and proteins involved in cholesterol
transport and steroidogenesis and a corresponding reduction in
testosterone in fetal testes following maternal exposure to DBP,
at dose levels below which adverse effects are detected in the devel-
oping male reproductive tract. Alterations in gene and protein
expression and testosterone synthesis may serve as sensitive
indicators of testicular response to DBP.
Key Words: di (n-butyl) phthalate; in utero exposure; male
reproductive development; antiandrogen; molecular mechanisms;
androgen receptor; dose response; steroidogenesis.
Di (n-butyl) phthalate (DBP) is used as a plasticizer and as a
cosmetics, latex adhesives, inks, and caulking. Because of its
widespread use, there is potential for human exposure in the
general population on a daily basis. Estimates for human expo-
sure to DBP range from 0.84 to 113 mg/kg/day (Blount et al.,
nated and does not bioaccumulate (Saillenfait et al., 1998;
Tanaka et al., 1978).
dam (100–500 mg/kg/day) can adversely affect development of
deformed epididymides, cryptorchidism, hypospadias, reduced
fertility, and Leydig cell adenoma (Mylchreest et al., 1998,
1999, 2000). The effects of DBP on the developing male repro-
ductive tract are similar, although not identical, to the effects of
antiandrogens such as flutamide and linuron (McIntyre et al.,
2000, 2001, 2002). Unlike flutamide and linuron, however,
neither DBP nor its primary metabolite monobutyl phthalate
(MBP) interacts with the androgen receptor (Foster et al.,
2001). The antiandrogenic effects of DBP are due instead to
decreased testosterone synthesis as a result of a reduction in
expression of genes involved in cholesterol transport and tes-
tosterone synthesis (Barlow et al., 2003; Shultz et al., 2001).
100, and 500 mg/kg/day), male rats exposed in utero to 100
and 500 mg DBP/kg/day showed a dose-dependent increase
in retained nipples, an indicator of reduced androgen status
during development (Mylchreest et al., 2000). Other adverse
effects such as hypospadias, absent or deformed epididymides,
vas deferens, seminal vesicles, and ventral prostate were
observed only at the highest dose level. No statistically signifi-
cant adverse effects were observed in the offspring of dams
treated with ?50 mg DBP/kg/day. We repeated the dose-
response study to examine the dose-response relationship for
the effect of DBP on gene and protein expression and testoster-
one concentration in the fetal testes. A broader range of dose
levels was selected to incorporate a dose level (0.1 mg/kg/day)
approximately equivalent to the maximum estimated level of
1Present address: National Institute of Environmental Health Sciences,
Research Triangle Park, NC 27709.
2To whom correspondence should be addressed at CIIT Centers for
Health Research, P.O. Box 12137, Research Triangle Park, NC 27709.
Fax: (919) 558-1300. E-mail: firstname.lastname@example.org.
Toxicological Sciences vol. 81 no. 1#Society of Toxicology 2004; all rights reserved.
by guest on June 1, 2013
et al., 2000; Kohn et al., 2000). Fetal testes were examined on
GD 19 based on our previous studies that showed significant
reductions in the expression of genes involved in cholesterol
transport and testosterone synthesis at this time (from GD 12–
19) following DBP treatment (Barlow et al., 2003; Shultz et al.,
fetal testicular testosterone concentration and expression of
genes and their corresponding proteins involved in cholesterol
transport and testosterone synthesis in the fetal testis at dose
levels below the levels at which adverse effects are detected.
MATERIALS AND METHODS
ResearchonGD 0,the dayspermwasdetectedinthevaginalsmear.Damswere
assigned to a treatment group by body weight randomization using Provantis
in each of the DBP-dosed groups. Animals were housed in the CIIT animal
facility, accredited by the Association for the Assessment and Accreditation
of Laboratory Animal Care, in a humidity- and temperature-controlled,
HEPA-filtered, mass air–displacement room. The room was maintained on a
12-h light/dark cycle at approximately 22 6 4?C with a relative humidity of
approximately 30–70%. Animals were identified by ear tags and cage cards and
housed individually in polycarbonate cages with Alpha-dri cellulose bedding
(Shepherd Specialty Papers, Kalamazoo, MI). Rodent diet NIH-07 (Zeigler
Brothers, Gardener, PA) and reverse-osmosis water were provided ad libitum.
and wasapprovedby the InstitutionalAnimalCareand Use Committee at CIIT.
with corn oil vehicle (Sigma Chemical Co., St. Louis, MO) or DBP (Aldrich
dose level groups, to generate samples for the testosterone radioimmunoassay
that500 mg/kg/dayproducedsignificantchangesingene expressionin the male
offspring without maternal toxicity or fetal death (Barlow and Foster, 2003;
Kohn et al., 2000).
All dams were euthanized on GD 19 by carbon dioxide asphyxiation. Fetuses
were removed by cesarean section and body weights were recorded. All fetuses
were euthanized by decapitation and then sexed by internal examination of the
reproductive organs. The right and left testes and epididymides were removed
from male fetuses and separated using a dissecting microscope with transillu-
Real-time quantitative RT-PCR.
RNA STAT-60 reagent (Tel-Test, Friendswood, TX). Subsequent reverse tran-
scription (RT) reactions, quality control for RT reactions, and quantitative PCR
reactions were performed as described previously (Barlow et al., 2003). Rat-
using Primer Express software (Applied Biosystems, Foster City, CA) with the
following parameters: low Tm5 60?C; high Tm5 64?C; optimum Tm5 62?C;
optimum length 5 20 base pairs.
Sprague-Dawley outbred rats were time-mated at Charles
Total RNA was isolated from the testes
ment group were solubilized in Laemmli buffer, and protein concentration was
for 5 min, and thenequal concentrations of protein were added to each lane on a
12% SDS–PAGE minigel (Bio-Rad Laboratories, Hercules, CA). Proteinswere
electrophoretically transferredonto polyvinylidene difluoride membranes (Bio-
Rad Laboratories). Membranes were incubated for 1 h at room temperature in
with the following primary antibodies at 4?C overnight, with the exception of
Golden, CO); P450scc (rabbit polyclonal, US Biological, Swampscott, MA);
polyclonal, provided by Dr. Dale Buchanan Hales, University of Illinois at
Chicago).Membraneswere washedinTBST for45min followedbyincubation
with secondary antibody donkey antirabbit IgG conjugated with horseradish
peroxidase (Amersham Biosciences, Piscataway, NJ). Positive bands were
detected by chemiluminescence with the ECL Plus Western Blot Detection
kit (Amersham Biosciences) after a final 1 h wash in TBST. Densitometry
was performed using FluorChem version 2.0 analysis software (Alpha Innotech
Corporation, San Leandro, CA).
Wholetestes from fourindividualfetuses per treat-
Primer Sets for Real-Time Quantitative RT-PCR Analyses
Gene Forward primerReverse primer
Probe Sequences for Real-Time Quantitative RT-PCR Analyses
DBP DOSE RESPONSE
by guest on June 1, 2013
with 500 mg DBP from the present study (as well as from a previous study,
Barlowet al., 2003)were immersion-fixed in 10% neutralbuffered formalin for
24 h and then transferred to 70% ethanol. Next, the tissues were embedded in
paraffin, sectioned at 5 mm, placed on charged slides, and stored at room tem-
perature until processed. At processing, sections were deparaffinized, treated
with 3% H2O2in water for10 min to block endogenous peroxidaseactivity,and
treated with 10% powdered nonfat milk for 20 min following 2% normal goat
serum in PBS for 10 min to reduce nonspecific staining. The sections were then
Insl3 (rabbit polyclonal IgG, 5 mg/ml) overnight at 4?C. Rabbit anti-PBR was
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); rabbit anti-
ing incubation with the primary antibodies, the slides were washed in PBS for
5min followedby incubationwitha biotinylatedsecondaryantibody,antirabbit
IgG (1:100), and then with avidin-biotin peroxidase (Vector Labs, Burlingame,
CA) for 30 min at room temperature (Sar and Welsch, 1999). The sections were
treated with liquid diamino benzidine (BioGenex) for 3 min, washed in water,
counterstained with hematoxylin, and mounted with Paramount. Antibody spe-
cificity was confirmed by excluding incubation with the secondary antibody.
per dose group, following the previously described method (Shultz et al., 2001)
using the Testosterone CT kit (ICN Pharmaceuticals, Costa Mesa, CA). Testes
were homogenized in 100 ml of PBS-Gel buffer; the homogenate was then
extracted three times with a total of 1 ml of a fresh mixture of ethylacetate
and chloroform (4:1). Extracts were dried under nitrogen and resuspended in
extraction solvent was added to standards (0.25–128 pg of steroid hormone per
nitrogen. Dextran-coated charcoal (DCC) stripped serum (25 ml) was added to
recovery tubes. Rabbit antitestosterone hormone antibody (ICN) was diluted
(1:800,000) with phosphate-buffered saline containing 0.01% g-globulin and
0.1% gelatin (PBS-Gel); 100 ml was added to each tube, gently mixed, and
incubated overnight at 4?C.125I-testosterone hormone (100 ml, 15,000 cpm)
was added, and tubes were incubated for 4 h at room temperature. The second
Cobra gamma counter (D5005, Packard Instrument Co., Downers Grove, IL).
Oil red O histochemistry.
five separate rat fetuses from different dams per treatment group, except for the
with the exception that hematoxylin was not used on sections for lipid quantita-
tion. Image-Pro Plus software (version 4.5; Media Cybernetics, Carlsbad, CA)
give the relative amount of lipid per section.
Frozen sections from GD 19 testis from four to
litter was the experimental unit. Gene expression data were analyzed by Dun-
to the control. The error term for the Dunnett’s test was generated by a one-way
ANOVA. Relative expression ratios were calculated as described previously
(Barlow et al., 2003) using the equation set forth (Pfaffl, 2001) in which effi-
ciencies for both the gene of interest and the calibrator GAPDH were used.
Analyses of relative expression ratios were considered to be statistically signifi-
cant for p 5 0.05.
Radioimmunoassay data were analyzed by Dunnett’s test comparing log10-
transformed testosterone concentrations from each nonzero dose group to the
All statistical analyses were conducted using either
control. The error term for the Dunnett’s test was generated by a two-way
day. A trend analysis was also performed after fitting the data to a one-way
ANOVA model using S-Plus. Analyses of testosterone concentrations were
considered to be statistically significant for p 5 0.05. Western blot data were
analyzed by Dunnett’s test comparing raw intensity values from each nonzero
two-way ANOVA. The two factors used in this analysis were dose and mem-
brane. Analyses of protein expression values were considered to be statistically
significant for p 5 0.05. Lipid data were analyzed by Dunnett’s test comparing
term for the Dunnett’s test was generated by a one-way ANOVA with subsam-
pling, which indicated that the subsampling and experimental errors could be
combined. Analyses of oil red O values were considered to be statistically sig-
nificant for p 5 0.05.
(0.1, 1.0, 10, 50, 100, or 500 mg/kg/day) by gavage daily from
in expression of genes involved in cholesterol transport and
(StAR), and cytochrome P450 side-chain cleavage (P450scc)
were similar, with a reduction in expression of approximately
50% for each gene at 50 mg/kg/day and 80% at 500 mg/kg/day.
SR-B1 mRNA was also significantly reduced at 1.0 mg/kg/day.
3b Hydroxysteroid dehydrogenase (3b-HSD) gene expression
tion at doses of 50 mg/kg/day and above. Cytochrome P450c17
(CYP17) was significantly reduced only at 500 mg/kg/day.
Expression of peripheral benzodiazepine receptor (PBR)
mRNA was increased in response to DBP (Fig. 1F). PBR is a
mitochondrial-membrane-spanning receptor and, like StAR, is
essential for cholesterol transport across the mitochondrial
membrane (Papadopoulos et al., 1997). Sterol regulatory
element–binding protein (SREBP) gene expression was not
altered following DBP exposure (data not shown).
Previously, we showed that in utero exposure to DBP also
resulted in the altered expression of genes involved in cell sur-
vival, including c-Kit (also known as stem cell factor receptor)
and testosterone-repressed prostate message 2 (TRPM-2;
Barlow et al., 2003; Shultz et al., 2001). We examined the
dose-response relationship for the effect of DBP on the expres-
sion of these two genes. Similar to 3b-HSD, expression of c-Kit
(Fig. 1H) was significantly reduced at 0.1 and 1.0 and further
reduced at ?50 mg/kg/day. In contrast, TRPM-2 (Fig. 1G) was
induced only at the highest dose level of DBP.
Protein expression, as determined by Western analysis, mir-
inSR-B1andStAR occurring atdoses ?50mg/kg/day(Fig.2).
P450scc protein was significantly reduced only at 500 mg/kg/
reduced at doses ?50 mg/kg/day (Fig. 1C).
LEHMANN ET AL.
by guest on June 1, 2013
Accurate quantification by Western analysis of 3b-HSD and
PBR could not be obtained. PBR was examined further by
immunocytochemistry. Staining for PBR was reduced in the
interstitial cells. PBR expression was similarly expressed in
the gonocytes of control and DBP-exposed testes (Figs. 3A
and 3B). The reduction of PBR in the testicular interstitial
cells correlates well with the reduction of StAR and other
proteins involved in cholesterol transport and steroidogenesis.
Expression of PBR in gonocytes has not been previously
reported, and the relevance of this observation remains to
be determined. In utero exposure to DBP doses of 250 and
500 mg/kg/day causedintra-abdominal
(Barlow and Foster, 2003; Mylchreest et al., 1998, 2000).
The pattern of DBP-induced cryptorchidism is similar to
the cryptorchidism that occurs with the insulin-like growth
factor 3 (Insl3) knockout mouse (Emmen et al., 2000).
In contrast, androgen receptor antagonists such as flutamide
can cause inguinal cryptorchidism (Mylchreest et al., 1999).
We examined the dose-response relationship for DBP on Insl3
to determine whether the cryptorchidism induced by DBP was
due to altered expression of this gene. Insl3 mRNA was
significantly reduced at 500 mg/kg/day DBP (Fig. 1I). By
immunohistochemistry, we showed reduced staining for
Insl3 in interstitial cells from DBP-exposed fetal testes
(Figs. 3C and 3D).
Intratesticulartestosterone wassignificantlyreduced atdoses
?50 mg/kg/day, as determined by radioimmunoassay (Fig. 4).
in a reduction in Leydig cell lipid content, as determined by oil
red O staining (Barlow et al., 2003). We examined the dose
response for this effect and found a significant reduction only
at the highest dose (Fig. 5).
expression values from DBP-exposed testes are expressed relative to control values and represent the average 6 SEM from five separate rat fetuses from
different dams per treatment group. (A) Scavenger receptor B-1 (SR-B1); (B) steroid acute regulatory protein (StAR); (C) cytochrome P450 side-chain cleavage
(P450scc); (D) CYP17; (E) 3b-hydroxysteroid dehydrogenase (3b-HSD); (F) peripheral benzodiazepine receptor (PBR); (G) testosterone-repressed prostate
message-2 (TRPM-2); (H) c-Kit; and (I) insulin-like factor 3 (Insl3). *p 5 0.05.
Real-time quantitative RT-PCR analyses of testicular mRNA collected on gestation day (GD) 19 from control and DBP-exposed fetuses. Gene
DBP DOSE RESPONSE
by guest on June 1, 2013
In this study, we demonstrate the dose-response relation-
ship for an effect of DBP on key steps in the steroidogenic
pathway and correlate changes in gene and protein expression
with a corresponding dose-dependent reduction in fetal testis
testosterone concentration. Fetal testicular testosterone was
significantly reduced at DBP doses ?50 mg/kg/day. In an
earlier study that examined 20 litters per dose group, we
established 50 mg DBP/kg/day as the no observable adverse
effect level (NOAEL) for effects on male reproductive tract
development (Mylchreest et al., 2000). Thus, while the reduc-
tion in fetal testicular testosterone production is not overtly
adverse at 50 mg DBP/kg/day, our current results indicate
that some reduction in steroidogenic gene and protein expres-
sion and fetal testicular testosterone can occur at dose levels
below those at which significant effects on reproductive
tract development can be detected. Our results establish
50 mg DBP/kg/day as a lowest observable effect level
(LOEL) and 10 mg DBP/kg/day as a no observable effect
StAR, and CYP17. (B) Average relative protein expression levels 6 SEM from four separate rat fetuses from different dams per treatment group. *P50.05
LEHMANN ET AL.
by guest on June 1, 2013
level (NOEL) for reductions in genes and proteins associated
with testosterone production together with reductions in intra-
The steroidogenic enzymes responsible for the conversion of
cholesterol to testosterone include P450scc, 3b-HSD, CYP17,
and 17b-HSD. We demonstrated previously that P450scc, 3b-
HSD, and CYP17, but not 17b-HSD, are reduced in DBP-
exposed fetal testes (Barlow et al., 2003; Shultz et al., 2001).
respond following DBP treatment with a significant decrease in
mRNA at 0.1 and 1.0 mg/kg/day. Fetal testicular testosterone
reduction in 3b-HSD mRNA at low-dose levels remains to be
Cholesterol uptake into the cell is mediated by SR-B1, also
membrane, where StAR mediates the transfer of cholesterol
from the outer to the inner mitochondrial membrane (Stocco,
2001). SR-B1 mRNA was significantly reduced at 1 mg/kg/day
and further reduced at doses ?50 mg/kg/day. SR-B1 protein
was also reduced by 20 6 10% at 1 mg/kg/day, although this
reduction was not statistically significant. SR-B1 protein was
significantly reduced at ?50 mg/kg/day.
port across the mitochondrial membrane (West et al., 2001).
and (C and D) Insl3. In control testis, interstitial cells (IC) showed strong immunostaining for PBR (A) while DBP-exposed testis (B) had little or no
immunostaining of interstitial cells. In control testis (C), interstitial cells showed increased staining for Insl3 compared to the DBP-exposed (D) testis. G,
gonocytes; magnification 3280.
Immunohistochemical staining of GD 19 testis from (A and C) control males and (B and D) DBP-exposed males (500 mg/kg/day) for (A and B) PBR
on GD 19 from control and DBP-exposed fetuses. Values are expressed
relative to control values and represent the average 6 SEM from three to four
separate rat fetuses from one to four dams per treatment group. *p 5 0.05.
Fetal testicular testosterone concentration of fetal testes collected
DBP DOSE RESPONSE
by guest on June 1, 2013
cellPBR protein levels isnotknownbutmay indicate enhanced
PBR protein turnover or another post-transcriptional event.
Alternatively, this difference may be due to differential regula-
ditions, and our results showing a decrease in PBR protein in
interstitial cells following phthalate treatment are in agreement
with a previously published study (Gazouli et al., 2002). In that
study, treatment of 12-week-old mice with 1 g/kg/day di-2-
Leydig tumor cells in culture with mono (2-ethylhexyl) phthal-
ate (MEHP), the active metabolite of DEHP, also reduced PBR
expression (Gazouli et al., 2002). Expression of PBR in fetal
gonocytes has not been previously reported. However, low
tubules has been shown, suggesting the presence of PBR in the
Sertoli and germ cell population (De Souza et al., 1985).
Steroidogenically active Leydig cells synthesize and store
fatty acids and cholesterol to help maintain steroidogenesis.
red O. (A) Control testes; (B) representative image of testes from 500 mg DBP/kg/day treatment group. (C) Image-Pro Plus software was used to quantify the
total area of the section and the area of oil red O stain to give the relative amount of lipid per section. Values are expressed relative to control values and represent
the average 6 SEM from four to five separate rat fetuses from different dams per treatment group, except for the control group that had 10 individual fetuses
from six dams. *p 5 0.05.
The effect of DBP on fetal testicular lipid. Fetal testes from control and DBP-treated male rat fetuses were collected on GD 19 and stained using oil
LEHMANN ET AL.
by guest on June 1, 2013
Androgens upregulate this process through a cascade of events
involving androgen-dependent activation of SREBP (Brown
and Goldstein, 1998; Swinnen et al., 1997, 1998). Both DBP
and flutamide, an androgen receptor–competitive antagonist,
downregulate expression of genes involved in fatty acid and
cholesterol synthesis, including long-chain-specific acyl-CoA,
acetyl-CoA carboxylase, steryl sulfatase, and low-density lipo-
protein receptor (Shultz et al., 2001). Downregulation of genes
involved in cholesterol synthesis, together with the reduction of
cholesterol import through downregulation of SR-B1, is likely
the reason for the dose-dependent decrease in Leydig cell lipid
content, as determined by oil red O staining.
During reproductive development, the fetal testes descend
from a pararenal position through the abdominal wall and into
the scrotal sac. Insl3, also known as relaxin-like factor, is
produced by Leydig cells and is essential for gubernacular
development and testicular descent from the pararenal through
the abdomen (Nef and Parada, 1999; Zimmermann et al.,
1999). Male mice deficient in Insl3 have bilateral intra-
abdominal testes (Nef and Parada, 1999; Zimmermann
et al., 1999). This form of cryptorchidism is similar to that
which occurs following in utero exposure to DBP doses of
250 and 500 mg/kg/day (Barlow and Foster, 2003; Mylchreest
et al., 1998). We have shown that Insl3 expression is sup-
pressed following exposure to DBP doses 4100 mg/kg/day,
and that the gubernaculum is underdeveloped in male rats
exposed gestationally to 500 mg/kg/day DBP (Barlow and
Foster, 2003). Our results are in keeping with a recently
published report of reduced Insl3 expression following fetal
exposure to several different phthalates, including DBP
(Wilson et al., 2004). Together, these studies suggest that
phthalate-induced cryptorchidism is due to decreased Insl3
production by the fetal Leydig cell.
Fetal testes of rats exposed in utero to DBP contain focal
regions of Leydig cell hyperplasia (Barlow and Foster, 2003;
DBP doses ?100 mg/kg/day (Mylchreest et al., 2000). Fetal
Leydig cell hyperplasia may be due in part to enhanced cell
survival since these regions contain enhanced expression of
two factors associated with cell survival, TRPM-2 and Bcl-2
(Shultz et al., 2001). We showed that the induction of TRPM-2
occurred at doses above 100 mg/kg/day, which correlates well
with the appearance of the focal lesions.
C-Kit mRNA was significantly reduced at 0.1 and
1.0 mg DBP/kg/day and further reduced at DBP doses ?50
mg/kg/day. Kit-ligand (Kitl), or stem cell factor, is produced
as both a membrane-bound form and a secreted form by the
Sertoli cell and is essential for normal gonocyte proliferation
in infertility due to germ cell loss (Feng et al., 1999; Mauduit
et al., 1999; Ohta et al., 2000). Kitl has also been shown to
influence Leydig cell steroidogenesis (Rothschild et al.,
2003), and the effect of DBP on testosterone synthesis may
be due, at least in part, to reduced stem cell factor signaling.
For several of the genes examined in this study (SR-B1,
3b-HSD, and c-Kit), we found significant reductions in
mRNA levels at DBP doses that approach maximal human
exposure levels. The biological relevance of these alterations
in gene expression at low dose levels remains to be determined,
since no statistically significant observable adverse effects on
male reproductive tract development have been identified at
DBP doses 5100 mg/kg/day, following the protocol we used
in this study (Mylchreest et al., 2000), and since fetal testicular
mRNA and corresponding proteins of these genes may be
produced in excess and small changes in their expression levels
may not significantly affect steroidogenesis. StAR transport of
cholesterol across the mitochondrial membrane is generally
considered the rate-limiting step in steroidogenesis (Stocco,
2001), and expression of StAR mRNA and protein is not
significantly altered below 50 mg/kg/day. Our results indicate
that alterations in the expression of SR-B1, c-Kit, and 3b-HSD
of adverse consequences to DBP.
Significance was not achieved at the 10 mg/kg/day dose for
the genes that had significantly altered expression at 0.1 and
1.0 mg/kg/day (SR-B1, 3b-HSD, and c-Kit). The values
obtained at the 10 mg/kg/day dose were within the expected
range of variability. Studies incorporating more litters and
additional doses may be necessary to more accurately define
the shape of the dose-response curve for these genes in the
dose range of 1–50 mg/kg/day.
In summary, we report that gestational exposure to 50 mg
DBP/kg/day results in the coordinate reduction of genes and
their corresponding proteins involved in cholesterol transport
and steroidogenesis, along with a reduction in intratesticular
testosterone. This reduction in cholesterol transport proteins,
at a dose at which no observable adverse effects on the devel-
that alterations in gene and protein expression and testosterone
synthesis are sensitive indicators of testicular response to DBP.
We thank Drs. Kamin Johnson, Li You, and Michael Shelby for their critical
review of this report; Drs. DennisHouse and Kejun Liu for their assistance with
the statistical analyses of the data; and Dr. Barbara Kuyper for her editorial
review. This study was supported by the National Institutes of Health Grant
Acton, S., Rigotti, A., Landschulz, K. T., Xu, S., Hobbs, H. H., and Krieger, M.
(1996). Identification of scavenger receptor SR-BI as a high density lipopro-
tein receptor. Science 271, 518–520.
DBP DOSE RESPONSE
by guest on June 1, 2013
Barlow, N. J., and Foster, P. M. (2003). Pathogenesis of male reproductive tract Download full-text
lesions from gestation through adulthood following in utero exposure to di
(n-butyl) phthalate. Toxicol. Pathol. 31, 397–410.
Barlow, N. J., Phillips, S. L., Wallace, D. G., Sar, M., Gaido, K. W., and
following exposure to di (n-butyl) phthalate. Toxicol. Sci. 73, 431–441.
Blount, B. C., Silva, M. J., Caudill, S. P., Needham, L. L., Pirkle, J. L.,
Sampson,E. J.,Lucier, G.W.,Jackson,R. J., andBrock,J.W. (2000).Levels
of seven urinary phthalate metabolites in a human reference population.
Environ. Health Perspect. 108, 979–982.
Brown, M. S., and Goldstein, J. L. (1998). Sterol regulatory element binding
proteins (SREBPs): Controllers of lipid synthesis and cellular uptake.
Nutr. Rev. 56, S1–S3; S54–S75.
De Souza, E. B., Anholt, R. R., Murphy, K. M., Snyder, S. H., and Kuhar, M. J.
(1985). Peripheral-type benzodiazepine receptors in endocrine organs: Auto-
radiographic localization in rat pituitary, adrenal, and testis. Endocrinology
Brinkmann, A. O. (2000). Hormonal control of gubernaculum development
like factor and androgen. Endocrinology 141, 4720–4727.
Feng, H. L., Sandlow, J. I., Sparks, A. E., Sandra, A., and Zheng, L. J. (1999).
Decreased expressionof the c-Kit receptor is associatedwith increased apop-
tosis in subfertile human testes. Fertil. Steril. 71, 85–89.
Foster, P. M., Mylchreest, E., Gaido, K. W., and Sar, M. (2001). Effects
of phthalate esters on the developing reproductive tract of male rats.
Hum. Reprod. Update 7, 231–235.
V. (2002). Effect of peroxisome proliferators on Leydig cell peripheral-type
benzodiazepine receptor gene expression, hormone-stimulated cholesterol
transport, and steroidogenesis: Role of the peroxisome proliferator-activator
receptor a. Endocrinology 143, 2571–2583.
and Needham, L. L. (2000). Human exposure estimates for phthalates.
Environ. Health Perspect. 108, A440–A442.
Mauduit, C., Hamamah, S., and Benahmed, M. (1999). Stem cell factor/c-Kit
system in spermatogenesis. Hum. Reprod. Update 5, 535–545.
McIntyre,B. S., Barlow, N. J., and Foster, P. M. D. (2001). Androgen-mediated
developmentinmaleratoffspringexposedto flutamidein utero: Permanence
and correlation of early postnatal changes in anogenital distance and nipple
Toxicol. 16, 131–139.
McIntyre,B. S.,Barlow,N.J.,Wallace,D.G.,Maness,S. C.,Gaido,K.W.,and
Foster, P. M. D. (2000). Effects of in utero exposure to linuron on androgen-
Appl. Pharmacol. 167, 87–99.
Mylchreest, E., Cattley, R. C., and Foster, P. M. D. (1998). Male reproductive
(n-butyl) phthalate: An antiandrogenic mechanism? Toxicol. Sci. 43, 47–60.
androgen-regulated male reproductive development by di (n-butyl) phthalate
during late gestation in rats is different from flutamide. Toxicol. Appl.
Pharmacol. 156, 81–95.
dependent alterations in androgen-regulated male reproductive development
Nef, S., and Parada, L. F. (1999). Cryptorchidism in mice mutant for Insl3. Nat.
Genet. 22, 295–299.
Ohta, H., Yomogida, K., Dohmae, K., and Nishimune, Y. (2000). Regulation of
and its ligand SCF. Development 127, 2125–2131.
Papadopoulos, V., Amri, H., Boujrad, N., Cascio, C., Culty, M., Garnier, M.,
diazepine receptor in cholesterol transport and steroidogenesis. Steroids 62,
Pfaffl, M. W. (2001). A new mathematical model for relative quantification in
real-time RT-PCR. Nucleic Acids Res. 29, E45.
Rothschild, G., Sottas, C. M., Kissel, H., Agosti, V., Manova, K., Hardy, M. P.,
and Besmer, P. (2003). A role for Kit receptor signaling in leydig cell
steroidogenesis. Biol. Reprod. 69, 925–932.
Saillenfait,A. M., Payan, J. P., Fabry,J. P., Beydon, D., Langonne,I., Gallissot,
F., and Sabate, J. P. (1998). Assessment of the developmental toxicity,
metabolism, and placental transfer of di (n-butyl) phthalate administered to
pregnant rats. Toxicol. Sci. 45, 212–224.
Sar, M., and Welsch, F. (1999). Differential expression of estrogen receptor-b
and estrogen receptor-a in the rat ovary. Endocrinology 140, 963–971.
Shultz, V. D., Phillips, S., Sar, M., Foster, P. M. D., and Gaido, K. W. (2001).
Altered gene profiles in fetal rat testes after in utero exposure to di (n-butyl)
phthalate. Toxicol. Sci. 64, 233–242.
Stocco, D. M. (2001). StAR protein and the regulation of steroid hormone
biosynthesis. Annu. Rev. Physiol. 63, 193–213.
diazepam-binding inhibitor/Acyl-CoA-binding protein as a sterol regulatory
element–binding protein-responsive gene. J. Biol. Chem. 273, 19938–19944.
Swinnen, J. V., Ulrix, W., Heyns, W., and Verhoeven, G. (1997). Coordinate
mechanism involving sterol regulatory element binding proteins. Proc. Natl.
Acad. Sci. USA 94, 12975–12980.
Tanaka, A., Matsumoto, A., and Yamaha, T. (1978). Biochemical studies
on phthalic esters. III. Metabolism of dibutyl phthalate (DBP) in animals.
Toxicology 9, 109–123.
West, L. A., Horvat, R. D., Roess, D. A., Barisas, B. G., Juengel, J. L., and
Niswender, G. D. (2001). Steroidogenic acute regulatory protein and
peripheral-type benzodiazepine receptor associate at the mitochondrial
membrane. Endocrinology 142, 502–505.
E. (2004). Phthalate ester–induced gubernacular lesions are associated
with reduced Insl3 gene expression in the fetal rat testis. Toxicol. Lett. 146,
Zimmermann, S., Steding, G., Emmen, J. M., Brinkmann, A. O., Nayernia, K.,
Insl3 gene causes bilateral cryptorchidism. Mol. Endocrinol. 13, 681–691.
LEHMANN ET AL.
by guest on June 1, 2013