TOXICOLOGICAL SCIENCES 120(2), 460–474 (2011)
Advance Access publication January 25, 2011
Species-Specific Dibutyl Phthalate Fetal Testis Endocrine Disruption
Correlates with Inhibition of SREBP2-Dependent Gene Expression
Kamin J. Johnson,*,1Erin N. McDowell,* Megan P. Viereck,* and Jessie Q. Xia†
*Nemours Biomedical Research, Alfred I. duPont Hospital for Children, Wilmington, Delaware 19803; and †National Institute of Statistical Sciences,
Research Triangle Park, North Carolina 27709
1To whom correspondence should be addressed at Nemours Biomedical Research, Alfred I. duPont Hospital for Children, 1701 Rockland Road, Wilmington,
DE 19803. Fax: (302) 651-6539. E-mail: email@example.com.
Received November 29, 2010; accepted January 14, 2011
Fetal rat phthalate exposure produces a spectrum of male
reproductive tract malformations downstream of reduced Leydig
cell testosterone production, but the molecular mechanism of
phthalate perturbation of Leydig cell function is not well
understood. By bioinformatically examining fetal testis expression
microarray data sets from susceptible (rat) and resistant (mouse)
species after dibutyl phthalate (DBP) exposure, we identified
decreased expression of several metabolic pathways in both
species. However, lipid metabolism pathways transcriptionally
regulated by sterol regulatory element–binding protein (SREBP)
were inhibited in the rat but induced in the mouse, and this
differential species response corresponded with repression of the
steroidogenic pathway. In rats exposed to 100 or 500 mg/kg DBP
from gestational days (GD) 16 to 20, a correlation was observed
between GD20 testis steroidogenic inhibition and reductions of
testis cholesterol synthesis endpoints including testis total choles-
terol levels, Srebf2 gene expression, and cholesterol synthesis
pathway gene expression. SREBP2 expression was detected in all
fetal rat testis cells but was highest in Leydig cells. Quantification of
SREBP2 immunostaining showed that 500 mg/kg DBP exposure
significantly reduced SREBP2 expression in rat fetal Leydig cells
but not in seminiferous cords. By Western analysis, total rat testis
SREBP2 levels were not altered by DBP exposure. Together, these
data suggest that phthalate-induced inhibition of fetal testis
steroidogenesis is closely associated with reduced activity of several
lipid metabolism pathways and SREBP2-dependent cholesterolo-
genesis in Leydig cells.
Key Words: phthalate; developmental toxicity; prenatal;
reproductive and developmental toxicology; sterol regulatory
element–binding protein; fetal testis; endocrine disruptors;
Phthalates are esters of phthalic acid produced in high
volume and used in a variety of consumer products including
cosmetics, medical devices, building supplies, and food
packaging. Because of a noncovalent linkage to the product
matrix, phthalates leech from materials during normal use
leading to ubiquitous, daily human exposure (Koch and
Calafat, 2009; Latini, 2005). In human male infants, hypospa-
dias and undescended testes are prevalent congenital malfor-
mations (Foresta et al., 2008), and phthalate exposure is
hypothesized to contribute to these malformations (Foster,
2006). Some epidemiological data support this hypothesis
(Main et al., 2006; Swan, 2008). Pregnant rats exposed to high
levels of certain phthalate congeners during a critical late
gestational window produce male pups with a spectrum of
reproductive tract malformations such as hypospadias, retained
nipples, and undescended testes (Carruthers and Foster, 2005).
These malformations result from rapid inhibition of rat Leydig
cell testosterone and insulin like-3 production after phthalate
exposure (Thompson et al., 2004; Wilson et al., 2004).
The molecular mechanism of phthalate-induced steroido-
genic inhibition is largely unknown. Although male reproduc-
susceptibility, phthalates inhibit fetal rat Leydig cell testoster-
one production at any gestational age of active steroidogenesis
(Scott et al., 2008). Inhibition of Leydig cell testosterone
synthesis is explained at least in part by reductions in
messenger RNA (mRNA) levels of several steroidogenic genes
including Cyp11a1, Cyp17a1, Scarb1, and Star (Barlow et al.,
2003; Lehmann et al., 2004). Phthalates also reduce neutral
lipid content in fetal Leydig cells (Barlow et al., 2003;
Lehmann et al., 2004). Diminished binding of the transcription
factor CCAAT/enhancer binding protein beta to steroidogenic
gene promoters may contribute to attenuation of Leydig cell
steroidogenic gene expression after phthalate exposure (Kuhl
et al., 2007). Phthalates cause aggregation and reduction of
volume of Leydig cells (Mahood et al., 2005; Mylchreest et al.,
2002), but the total number and proliferation rate of fetal
Leydig cells appear unchanged.
In utero phthalate exposure produces species-specific
phenotypes. Although some phthalate-induced phenotypes
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such as gonocyte multinucleation are similar between mice and
rats, mice are refractory to inhibition of both fetal testosterone
production and expression of fetal testis steroidogenic genes
(Gaido et al., 2007). Thus, some phthalate-induced phenotypes
are attributed to reduced Leydig cell hormone production,
whereas others, such as gonocyte multinucleation, utilize
a different mechanism. In the gut, orally ingested phthalate
diester is quantitatively metabolized to its proximate toxicant,
phthalate monoester. However, there appears to be no
significant difference in phthalate diester metabolism or
placental transfer of the proximate toxicant to the fetus
between rats and mice (Gaido et al., 2007). In utero phthalate
exposure of marmosets perturbs normal germ cell development
but does not reduce masculinization, suggesting that the
phthalate effect on primates is similar to its effect on mice
(McKinnell et al., 2009).
transcription factors are master regulators of fatty acid and
cholesterol synthesis (Brown and Goldstein, 2009; Osborne
and Espenshade, 2009). Precursor SREBP reside in the
endoplasmic reticulum, but in response to low cholesterol or
fatty acid levels, these proteins translocate to the Golgi
complex where proteases generate small active fragments that
migrate into the nucleus and function as transcription factors.
Mammals express two SREBP homologs, SREBP1 and
SREBP2. Differential promoter usage generates two SREBP1
isoforms (SREBP1a and SREBP1c) from the Srebf1 gene
(Hua et al., 1995). In general, it is thought that SREBP1c
primarily regulates expression of fatty acid synthesis pathway
genes, SREBP2 primarily mediates expression of cholesterol
synthesis pathway genes, and SREBP1a activates expression of
genes in both pathways. Proteins in the cholesterol synthesis
pathway are highly expressed in fetal mouse Leydig cells
(Budefeld et al., 2009). Interestingly, Star and Cyp17a1
expression in cultured cells is enhanced by SREBP transcrip-
tional activity (Christenson et al., 2001; Ozbay et al., 2006;
Shea-Eaton et al., 2001). It is not yet known whether SREBP
perform these steroidogenic functions in the fetal testis.
Species-specific endocrine disruption effects highlight the
need to understand the phthalate molecular mechanism to
determine if phthalate exposure could contribute to human
reproductive malformations. In this study, we exploited species-
specific effects of phthalates by bioinformatically mining
mRNA expression profiling data to identify molecular pathways
that correlate with dibutyl phthalate (DBP)-induced endocrine
disruption. This analysis gave rise to a hypothesis that reduced
fetal Leydig cell testosterone production after exposure is
associated with attenuation of SREBP expression and activity.
MATERIALS AND METHODS
Data from several phthalate exposure studies were examined (Table 1). For
the bioinformatic pathway analysis, primary data (CEL) files were obtained
from Dr Kevin Gaido (U.S. Food and Drug Administration) from four
previously published phthalate exposure experiments of Sprague-Dawley rats
or CD1 mice (Gaido et al., 2007; Liu et al., 2005; Thompson et al., 2004).
Animal usage, phthalate exposure paradigms, and microarray data generation
for these published experiments are not described in detail here. Also included
in the bioinformatic pathway analysis was an additional expression microarray
experiment performed following a 50 mg/kg DBP (DBP50) exposure of Fischer
344 rats from gestational day (GD) 12 to 20. For the experiments examining the
affects of DBP on SREBP, DBP100 and DBP500 Sprague-Dawley rat
exposures were from GD16 to GD20, and each DBP group had a distinct
DBP Treatment Groups
Microarray and Pathway Analysis Experiments
final dose (h)
1, 3, 6, and 18
2, 4, and 8
3–4RAE230A þ Bb
RAE230A þ B
Thompson et al. (2004)
Liu et al. (2005)
Gaido et al. (2007)
Gaido et al. (2007)
SREBP expression and function experiments
final dose (h)
aValues are the number of probe sets in the entire study with an FDR q value < 0.05.
bThe RAE230A chip was employed at all time points, but the RAE230B chip was not used at 6 and 18 h time points.
PHTHALATE INHIBITION OF FETAL TESTIS SREBP2
vehicle control group. Animal usage, exposure paradigms, and molecular
techniques for both the DBP50 microarray experiment and the SREBP
experiments are detailed below.
Animals and phthalate exposure. For the DBP50 microarray experiment,
timed-pregnant Fischer 344 rats were obtained from Charles River Laboratories
(Raleigh, NC), housed in The Hamner Institutes (Durham, NC) vivarium in
polycarbonate cages containing cellulose bedding, fed NIH-07 rodent chow
(Zeigler Brothers, Gardener, PA), and provided reverse-osmosis water ad
libitum. For the studies analyzing SREBP biology, timed-pregnant Sprague-
Dawley rats were purchased from Charles River Laboratories, housed in the
Alfred I. duPont Hospital for Children vivarium in polycarbonate cages
containing pine shavings, fed LabDiet Rat Chow 5012 (PMI Nutrition
International, Brentwood, MO), and provided with tap water ad libitum. Both
vivariums are accredited by the Association for Assessment and Accreditation
of Laboratory Animal Care International, and all animal protocols were
approved by the Institutional Animal Care and Use Committee. The day of
sperm-positive identification was designated GD0.
For DBP (CAS number 84-74-2; Sigma Chemical Co., St Louis, MO)
exposures, groups of weight-randomized rats were gavaged each day of the
study between 8:00 and 10:00 A.M. with 1 ml/kg (body weight) of a DBP
solution in corn oil (Sigma Chemical Co.) or corn oil alone. DBP dose levels
between 50 and 500 mg/kg body weight were chosen to produce no significant
effect on fetal rat Leydig cell steroidogenesis (DBP50) or to produce profound
inhibition (DBP500). For comparison, average human phthalate exposure is in
the range of micrograms per kilogram per day, although certain subpopulations
may be exposed to low milligrams per kilogram per day amounts (Wittassek
et al., 2010). On the day of tissue procurement, animals were euthanized 6 h
after the final DBP dosing. Prior to euthanasia, pup anogenital distance was
measured with a stereomicroscope fitted with a 1-mm reticle. Testes were
placed in microcentrifuge tubes and immediately frozen on dry ice for
subsequent testosterone, mRNA, or protein analyses.
Testis histology. Paraffin sections were stained with hematoxylin and
eosin, and multinucleated germ cell (MNG) formation and seminiferous cord
diameters were quantified as previously described (Johnson et al., 2008).
Fetal testis cholesterol and testosterone measurements. Testis total
cholesterol was measured from two pooled testes (each from different fetuses)
per litter using a colorimetric cholesterol quantitation kit (K603-100; Biovision
Inc., Mountain View, CA) according to the manufacturer’s protocol.
When possible, two testes (each from different fetuses) per litter were pooled
for testosterone analysis. For the DBP50 expression microarray experiment,
a radioimmunoassay was performed as previously described using a Double-
Antibody-125I RIA kit (Catalog #07-189105; MP Biomedicals, Costa Mesa,
CA) (Johnson et al., 2007). For the SREBP study, testes were homogenized in
100 ll PBS, centrifuged at 15,000 3 g for 5 min, and testosterone measured in
the supernatant by the University of Virginia Center for Research in
Reproduction Ligand Assay and Analysis Lab using a radioimmunoassay kit
(Catalog #TKTT2; Siemens Medical Solutions Diagnostics, Los Angeles, CA).
DBP50 microarray. For the DBP50 exposure microarray, fetal testes were
extracted with TRIzol reagent (Invitrogen Corp., Carlsbad, CA) and total RNA
purified using an RNeasy Micro kit (Qiagen, Valencia, CA). RNA quality was
measured with a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), and
all RNA samples showed an RNA Integrity Number greater than 8. Biotin-
labeled complementary RNA probes were generated using the One Cycle
Target Labeling and Control Reagents kit (catalog number 900943; Affymetrix,
Santa Clara, CA). Probe hybridization to Affymetrix Rat Genome 230 2.0
microarrays and microarray washing and scanning were performed as
previously described (Lahousse et al., 2006). The DBP50 microarray data
have been submitted to Gene Expression Omnibus (http://www.ncbi.nlm.nih.
gov/geo/) and given accession number GSE25196.
Microarray statistical analysis. Data were analyzed statistically from five
fetal testis expression Affymetrix microarray experiments after DBP exposure
(Table 1). Although different normalization and statistical methods were used
to analyze the microarray data described in previous publications, all primary
microarray data were subjected to uniform data normalization and statistical
analysis in this study. Data were normalized using the Robust MultiChip
Average method (Irizarry et al., 2003), and raw values were log transformed.
Using Array Studio software (OmicSoft Corp., Morrisville, NC), one-way
ANOVA was used to compare overall expression across microarray data in an
experiment and was followed by Student’s t-tests to compare the expression
levels of group pairs of interest. A false discovery rate (FDR) q value < 0.05
was considered significant. Calculated fold change and FDR q values for all
microarray data sets are displayed in Supplementary files 1 and 2.
Bioinformatic pathway analysis. Two complementary methods were used
to identify biological pathways significantly altered by phthalate exposure. Both
methods determine if genes within a biological pathway are significantly enriched
(i.e., overrepresented) in gene lists identified as being altered by experimental
treatment; however, the lists of genes used as the input data and the statistical
analysis of the two methods are different. The first method, performed with
Ingenuity Pathways Analysis (IPA) version 8.7 software (Ingenuity Systems,
www.ingenuity.com), used lists of genes having statistically significant
differential expression as input data and a right-tailed Fisher exact test, whereas
the gene set enrichment analysis (GSEA) method used expression fold change
data from all genes examined as the input data and a Kolmogorov-Smirnov
running sum statistic (Subramanian et al., 2005). For IPA analysis, the pathways
queried for overrepresentation included canonical metabolic pathways, which
were derived from Kyoto Encyclopedia of Genes and Genomes (KEGG; http://
www.genome.jp/kegg/) annotations. For GSEA, lipid, carbohydrate, amino acid,
nucleotide, and energy metabolism pathways curated by KEGG were examined.
In addition, custom gene sets were queried using IPA and GSEA. These custom
gene sets included the testicular steroidogenic, fatty acid biosynthesis, and
cholesterol biosynthesis pathways and gene sets whose expression was influenced
by SREBP1 or SREBP2 transcriptional activity, as determined using the IPA
Knowledge Base (Supplementary file 3). The IPA parameters employed were the
following: (1) genes were imported in files using gene symbols as the identifier;
(2) the reference gene background was set to the genes present on the Affymetrix
microarray used in the experiment; and (3) a Benjamini-Hochberg q value < 0.05
was considered significant. GSEA was performed using the online implementa-
tion tool (version 2.0) provided by the Broad Institute (www.broadinstitute.org/
gsea/index.jsp). GSEA parameters were the following: (1) the imported data set
was collapsed to gene symbols, (2) 1000 permutations were performed using
gene set as the permutation type, (3) gene sets containing 10–500 genes were
analyzed, and (4) FDR q values < 0.05 were considered significant.
Quantitative Reverse Transcription-Polymerase Chain Reaction. Total
RNA purification, complementary DNA generation, Taqman-based PCR, and
data analysis using the delta-delta threshold cycle method were performed as
described (Barthold et al., 2008), except for performing technical duplicate
amplifications. Because DBP exposure at 500 mg/kg significantly reduced fetal
testis Gapdh expression (data not shown), target gene expression was determined
relative to Tbp expression. Fetal testis Tbp mRNA levels were not affected by
DBP exposure (data not shown). The following prevalidated Taqman assays
(Applied BiosystemsInc., Foster
Rn00568733_m1, Cyp17a1: Rn00562601_m1, Hmgcr: Rn00565598_m1, Idi1:
Rn00585526_m1, Insig1: Rn00574380_m1, Scarb1: Rn00580588_m1, Srebf2:
Rn01502638_m1, Srebf1c: Rn01495766_m1, Star: Rn00580695_m1, and Tbp:
Rn01455646_m1. To specifically quantify Srebf1a expression, no premade
Taqman assay was available; therefore, a custom assay spanning the first two
exons of Srebf1a was designed and validated by Applied Biosystems. For the
Srebf1a Taqman assay, PCR primer sequences were 5#ACGACGGAGCCATG-
GATTG and 5#AGTCACTGTCTTGGTTGTTGATGAG and the Taqman probe
sequence was 5#AAGCATGTCTTCAAATGTG
City,CA) were used—Cyp11a1:
Immunostaining and quantification of SREBP2 immunoexpression. An
immunofluorescent technique using
Following dissection, fetal testes were immersed in Bouin’s fixative for 1 h,
embedded in paraffin, and antigen retrieval performed on 5-lm deparaffinized
sections. For antigen retrieval, slides were washed for 10 min with PBS
paraffin sections wasemployed.
JOHNSON ET AL.
of cholesterol in lipid droplets while concomitantly increasing the
endoplasmic reticulum cholesterol content. Because oxysterol
derivatives of cholesterol also negatively regulate SREBP
processing and gene expression activity (Gale et al., 2009;
Radhakrishnan et al., 2007), another potential mechanism
operating is a phthalate-induced increase in oxysterol production.
Because SREBP2 is a transcription factor and reduced
steroidogenic gene expression appears to contribute to
phthalate-induced testosterone reductions, the question arises:
does SREBP2 modulate steroidogenic gene expression in
fetal Leydig cells? Although no data are available in this
cell type, SREBP can increase steroidogenic gene expression in
other cell types. Mammalian Star and Cyp17a1 promoters
contain consensus SREBP binding sites. In nonsteroidogenic
human cell lines, both SREBP1 and SREBP2 can bind to
the Star promoter and increase expression of transfected
Star promoter/reporter constructs (Christenson et al., 2001;
Shea-Eaton et al., 2001). In a human adrenocortical cell
line, mutation of the putative SREBP binding site in the
Cyp17a1 promoter and knockdown of SREBP1 both decrease
transcriptional activity of a transfected Cyp17a1 promoter
(Ozbay et al., 2006). Thus, the potential exists for SREBP
transcriptional activity to increase steroidogenic gene expres-
sion in fetal Leydig cells. Defining how SREBP2 is regulated
and its functional activity in fetal Leydig cells may provide
clues about the phthalate molecular target as well as the
signaling pathways governing fetal Leydig cell steroidogenesis.
Although some epidemiological studies suggest that phtha-
lates may perturb human Leydig cell hormone production (Main
et al., 2006; Swan, 2008), no direct evidence of human fetal
testis endocrine disruption by phthalates is available. Because
the experiments presented here did not test human tissue, these
data do not speak directly to the endocrine disruption sus-
ceptibility of human fetal testes. However, our results could
be used to help define the sensitivity of human fetal testes to
phthalate endocrine. The question of human sensitivity is
highlighted by the differential endocrine disruption observed
between mice and rats (Gaido et al., 2007). Unfortunately, the
signaling processes controlling rodent fetal Leydig cell hormone
production during the critical window of reproductive tract
programming are not known (Scott et al., 2009), which impedes
understanding of the phthalate endocrine disruption mechanism.
We show that endocrine disruption is associated with reduced
gene expression of SREBP2-dependent pathways and hypoth-
esize that this may contribute to reduced steroidogenesis. Should
an experimental system recapitulating rat sensitivity and
amenable to using human tissue become available, SREBP2-
dependent gene expression analysis could help determine
susceptibility to endocrine disruption.
Supplementary data are available online at http://toxsci.
National Institutes of Health (P20RR020173 to K.J.J.).
The authors would like to thank Duncan Wallace and Linda
Pluta of The Hamner Institutes for Health Sciences for their
expert technical help with the DBP50 study and Dr Kevin
Gaido (U.S. Food and Drug Administration) for providing
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