TOXICOLOGICAL SCIENCES 122(1), 45–51 (2011)
Advance Access publication May 2, 2011
Upregulation of Clusterin in Prostate and DNA Damage in Spermatozoa
from Bisphenol A–Treated Rats and Formation of DNA Adducts in
Cultured Human Prostatic Cells
Silvio De Flora,*,1Rosanna T. Micale,* Sebastiano La Maestra,* Alberto Izzotti,* Francesco D’Agostini,* Anna Camoirano,*
Serena A. Davoli,† Maria Giovanna Troglio,† Federica Rizzi,† Pierpaola Davalli,‡ and Saverio Bettuzzi†
*Department of Health Sciences, University of Genoa, I-16132 Genoa, Italy; †Department of Experimental Medicine, University of Parma, I-43100 Parma, Italy;
and ‡Department of Biomedical Sciences, University of Modena-Reggio Emilia, I-41125 Modena, Italy
1To whom correspondence should be addressed. Fax: þ39-010-3538504. E-mail: email@example.com.
Received January 13, 2011; accepted April 18, 2011
Among endocrine disruptors, the xenoestrogen bisphenol A
(BPA) deserves particular attention due to widespread human
exposure. Besides hormonal effects, BPA has been suspected to be
involved in breast and prostate carcinogenesis, which share
similar estrogen-related mechanisms. We previously demon-
strated that administration of BPA to female mice results in the
formation of DNA adducts and proteome alterations in the
mammary tissue. Here, we evaluated the ability of BPA, given
with drinking water, to induce a variety of biomarker alterations
in male Sprague-Dawley rats. In addition, we investigated the
formation of DNA adducts in human prostate cell lines. In BPA-
treated rats, no DNA damage occurred in surrogate cells
including peripheral blood lymphocytes and bone marrow
erythrocytes, where no increase of single-strand DNA breaks
was detectable by comet assay and the frequency of micro-
nucleated cells was unaffected by BPA. Liver cells were positive at
transferase dUTP nick end labeling assay, which detects both
single-strand and double-strand breaks and early stage apoptosis.
BPA upregulated clusterin expression in atrophic prostate
epithelial cells and induced lipid peroxidation and DNA
fragmentation in spermatozoa. Significant levels of DNA adducts
were formed in prostate cell lines treated either with high-dose
BPA for 24 h or low-dose BPA for 2 months. The BPA-related
increase of DNA adducts was more pronounced in PNT1a
nontumorigenic epithelial cells than in PC3 metastatic carcinoma
cells. On the whole, these experimental findings support mecha-
nistically the hypothesis that BPA may play a role in prostate
carcinogenesis and may, potentially, affect the quality of sperm.
Key Words: bisphenol A; DNA adducts; clusterin; Sprague-
Dawley rats; prostate; spermatozoa.
The term endocrine disruptors (EDs) was coined 20 years
ago to identify a large number of structurally diverse chemicals
sharing the ability to disrupt the endocrine system of both
humans and wild animals (Colborn et al., 1993; Wingspread
Consensus Statement, 1992). Among them, estrogen-mimicking
compounds or xenoestrogens are of particular concern be-
cause estrogens play important roles not only in the regulation
of the physiological homeostasis but also because they are
involved in the pathogenesis of various diseases including
cancers of the genital system (Soto and Sonnenschein, 2010).
Bisphenol A (BPA), 2,2-bis-4-hydroxyphenyl propane, is
a prototype of xenoestrogenic ED. Due to its high production
volume and widespread human exposure, BPA has received
outstanding attention and has raised concern in the public
opinion (Vandenberg et al., 2009). BPA is a monomer in the
manufacture of epoxy resins, polycarbonate plastics, and flame
retardants and is used as a dental sealant and for coating water
pipe walls, food packaging, and plastic bottles (Staples et al.,
1998). It is also a contaminant of food and water, which are the
major sources of human exposure (Staples et al., 1998;
Vandenberg et al., 2009).
BPA has been investigated for genotoxicity in a variety of
test systems, both in vitro and in vivo, but the results are
controversial (Izzotti et al., 2009; Vandenberg et al., 2009). It
has been shown to bind DNA in acellular systems, in which
this ED reacts with DNA after metabolic activation (Atkinson
and Roy, 1995a; Edmonds et al., 2004; Izzotti et al., 2009); in
cultured mammalian cells (Tsutsui et al., 1998); and in the liver
of both rats (Atkinson and Roy, 1995b) and mice (Izzotti et al.,
2009) treated in vivo. Moreover, we recently demonstrated that
administration of BPA with drinking water results in the
formation of DNA adducts and proteome alterations in the
mammary tissue of mice (Izzotti et al., 2009, 2010). These
results deserve attention also because BPA accumulates in the
mammary cells of rodents after oral intake (Yoo et al., 2001).
Interestingly, exposure to estrogens throughout life is a major
risk factor for breast cancer (Pike et al., 1993), and the
chemical structure of BPA resembles that of diethylstylbestrol
(DES), a well known carcinogen in humans (International
Agency for Research on Cancer, 1979). BPA can induce
mammary carcinomas in prenatally exposed rats, and the early
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exposure to this ED sensitizes the mammary gland to
carcinogenic insults experienced later in life (Keri et al.,
2007; Vandenberg et al., 2009).
Intriguingly, estrogens play a physiological role also during
prostate development, growth, and differentiation (Carruba,
2007). Aromatization of androgens to estrogens may be
involved in prostate carcinogenesis (Bosland, 2000; Carruba,
2007; Ellem et al., 2004), and depurinating estrogen-DNA
adducts could serve as potential biomarkers to predict the risk
of prostate cancer (Yang et al., 2009). Thus, estrogens might be
involved in the etiology of prostate cancer and breast cancer
with similar mechanisms (Yang et al., 2009). These types of
cancers share many similarities, such as risk factors, bio-
molecular determinants, geographical distribution, and natural
history (Carruba, 2007; Cavalieri and Rogan, 2006).
These premises prompted us to evaluate a variety of
biomarkers in cells of male rats receiving BPA with drinking
water. The investigated biomarkers included histological,
immunohistochemical, and Western blot analyses of prostates
for the detection of clusterin (CLU), whose gene has been
cloned and identified as one of the most highly induced genes
during the involution of the rat prostate gland following either
surgical castration (Bettuzzi et al., 1989) or treatment with the
anti-androgen finasteride (Astancolle et al., 2000). Besides
many growth factors and hormones, CLU expression is under
the control of estrogens (Filippi et al., 2002). In addition, we
analyzed spermatozoa for the levels of reactive oxygen species
(ROS) and of malondialdehyde (MDA) and other thiobarbituric
acid reactive substances (TBARS) and for the assessment of
DNA fragmentation by using the sperm chromatin dispersion
(SCD) test (Ferna `ndez et al., 2003). Liver cells were analyzed
by terminal deoxynucleotidyl transferase dUTP nick end
labeling (TUNEL). Cytogenetical damage was evaluated in
bone marrow erythrocytes, and single-strand DNA breaks
(SSBs) were measured in peripheral blood lymphocytes. In
addition, we evaluated in vitro the ability of BPA to form DNA
adducts in two human prostatic cell lines. PNT1a cells are
nontumorigenic epithelial cells, immortalized through stable
transfection with the SV40 large T antigen (Berthon et al.,
1997), whereas PC3 cells are androgen-independent prostate
cancer cells originated from bone metastasis of prostatic
carcinoma (Kaighn et al., 1979).
MATERIALS AND METHODS
Rats. Sixteen male adult Sprague-Dawley rats, weighing on an average
250 g, were purchased from Harlan Italy (San Pietro al Natisone, Udine, Italy).
The rats were housed in Makrolon cages on sawdust bedding and maintained
on standard rodent chow (Teklad 2018, Harlan Italy) and tap water ad libitum.
The animal room had a temperature of 23 ± 2?C, a relative humidity of 55%,
and a 12-h day/night cycle. Housing and treatment of rats were in accordance
with our national and institutional guidelines.
Treatment of rats. After 10 days of acclimatization, eight rats were treated
with BPA (Sigma Chemical Co., St Louis, MO), dissolved in absolute ethanol
(25 mg/ml, wt/v) and then diluted in tap water to yield a calculated daily intake
of 200 mg BPA/kg body weight. This dilution corresponded to a concentration
of ethanol in drinking water of 4% (vol/vol). BPA was administered daily via
drinking water for 10 consecutive days. Eight control rats received 4% ethanol
in drinking water for the same period of time. At the end of the treatment, the
body weights of control rats were 282.0 ± 16.0 g versus 274 ± 20.1 of BPA-
Sacrifice of rats and collection of biological samples. After overnight
starving, all rats were anesthetized with diethyl ether and killed by cervical
dislocation. Blood samples were collected from the lateral tail vein for isolating
lymphocytes to be analyzed by comet assay. The livers were collected, and
fragments were fixed in formalin and embedded in paraffin for TUNEL
analyses. Immediately after surgical excision, prostates were weighed and
divided into two parts. One was fixed in formalin for histological and
immunohistochemical analyses, whereas the other was frozen at ?80?C for
protein extraction and Western blot analyses. The left femurs were removed,
and bone marrow smears were prepared by means of a paintbrush for
cytogenetical analyses. The testes were removed, and mature spermatozoa were
collected from epididymal heads and used for evaluating DNA fragmentation
and concentrations of ROS and TBARS.
Histological, immunohistochemical, and Western blot analyses of
prostates. Five micrometer sections of paraffin-embedded prostates were
stained with hematoxylin and eosin for evaluating prostate histology. For
immunohistochemical analyses, formalin-fixed prostate fragments were trans-
ferred to 70% ethanol and then embedded in paraffin. Sections (5 lm) were cut
and mounted onto slides, which were hydrated with xylene and graded alcohol
and equilibrated in phosphate-buffered saline (PBS). Antigen retrieval was
performed with 10mM sodium citrate, pH 6.0, using a microwave for 3 3 5
min at 700 W. Endogenous peroxidase was quenched with 3% H2O2in H2O.
A specific binding was blocked with swine serum diluted 1:10 in 1% bovine
serum albumin (BSA) in PBS. Immunostaining was performed using mono-
clonal antibodies anti-rat CLU (Millipore Corporate, Billerica, MA), diluted
1:50 in 1% BSA in PBS and incubated for 1 h at room temperature. From the
secondary antibody to the chromogen reaction, the Universal LSABþSystem-
HRP kit (DakoCytomation, Milan, Italy) was used. Negative controls were
prepared by excluding the primary monoclonal antibodies from the reaction.
Counterstaining was performed with hematoxylin, and cover slips were
mounted with Eukitt (O. Kindler GmbH & Co., Freiburg, Germany).
For protein extraction and Western blot analyses, lysates from frozen mouse
prostates were homogenized in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid buffer (50mM Tris-HCl, pH 7.4, 150mM NaCl, 0.25% Na deoxycholate,
1% NP-40, 50 lg/ml DNase, and 50 lg/ml RNase), supplemented with
Complete protease inhibitor according to manufacturer’s instruction (Roche
Diagnostics Corporation, Milan, Italy), and heated at 100?C in SDS–
polyacrlyamide gel electrophoresis loading buffer. The equivalent of 50 lg
of total protein was loaded on each lane and resolved by electrophoresis on
10% polyacrylamide gel and blotted onto a polyvinylidene fluoride membrane
(Millipore Corporate, Billerica, MA). Membranes were incubated in Western
Blocking Reagent (Roche Diagnostics, Mannheim, Germany) diluted 1:10 in
Tris-buffered saline (TBS) for 1 h at room temperature, then either in anti-
b-CLU primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA), diluted
1:3000, kept overnight at þ 4?C, or in anti-b-actin (Millipore Corporate,
Billerica, MA), diluted 1:4000, for 1 h at room temperature. After 4 3 10 min
washes in 0.1% Tween-20 in TBS, the membranes were incubated with
secondary antibodies conjugated with peroxidase, anti-goat (PIERCE, Rock-
ford, IL) diluted 1:25000 for b-CLU and anti-mouse (Sigma-Aldrich) diluted
1:5000 in blocking solution for 1 h at room temperature. Blots were washed
4 3 15 min in Tween-20 in TBS. Immunoreactive bands were detected with the
Chemiluminescence Blotting Substrate (Roche Diagnostics Corporation).
Analysis of spermatozoa for ROS, TBARS, and SCD. Spermatozoa
suspensions from each rat sample were assayed for ROS by using the
membrane-permeable lipophilic fluorochrome 2#,7#-dichlorofluoresceindiace-
tate (DCF-DA). The cell suspensions were incubated with 1 lm DCF-DA for
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30 min at 37?C, and the whole populations were selected by forward scatter and
side scatter parameters. The emission signals, due to highly fluorescent ROS-
induced DCF, were collected in the green fluorescence channel (530 ± 20 nm).
Concentrations of TBARS in spermatozoa were measured in each rat sample
as described by Ohkawa et al. (1979). The results were expressed as nanomoles
MDA equivalent per milligrams protein.
The SCD test was performed as described by Ferna `ndez et al. (2003). This
method is based on the principle that spermatozoa with fragmented DNA fail to
produce the characteristic halo of dispersed DNA loops that is observed in
sperm with nonfragmented DNA, following acid denaturation and removal of
nuclear protein. The results were expressed as nuclear spreading factor, which
is the ratio between the halo of chromatin dispersion and the nucleus, calculated
by using the ImageJ software (National Institutes of Health). Low values of
nuclear spreading factor are indicative of the occurrence of DNA damage.
A total of 4304 cells from the eight untreated rats (538 cells per rat) and of 4496
cells from the eight BPA-treated rats (562 cells per rat) were analyzed.
Analysis of liver cells by TUNEL method. Apoptosis was evaluated by
TUNEL method by using the TACS XL Blue Label in Situ Apoptosis Detection
kit (Trevigen, Gaithersburg, MD) following the manufacturer’s directions. The
slides were scored at a magnification of 3400, and 1000 cells per slide were
examined. The results were expressed as percentage of TUNEL-positive cells.
Cytogenetical analysis of bone marrow erythrocytes. Bone marrow slides
were fixed in methanol and stained with hematoxylin and eosin. The frequency
of micronucleated (MN) polychromatic erythrocytes (PCE) was evaluated by
scoring 1000 cells per slide.
SSBs in lymphocytes. SSBs in peripheral blood lymphocytes were
evaluated by single-cell gel electrophoresis (SCGE), or comet assay, performed
in alkaline environment (Tice et al., 2000). Images of at least 100 randomly
selected nuclei were acquired from each rat and analyzed on an automated
imaging system (CASP or Comet Assay Software Project, http://www.cas-
p.sourceforge.net). SSBs were quantified in terms of tail moment, which is the
product of tail length and the fraction of total DNA in the tail.
Cell cultures. Two human prostatic cell lines were used, PNT1a cells and
PC3 cells (American Type Culture Collection, Manassas, VA). PNT1a cells
were cultivated in RPM1 1640 medium (Sigma, Inc., St Louis, MO),
supplemented with 10% fetal bovine serum (FBS) and 2mM L-glutamine.
PC3 cells were cultivated in Dulbecco’s modified Eagle’s medium Ham’s F12
medium (Sigma), supplemented with 7% FBS and 2mM L-glutamine. The cells
were incubated at 37?C in 5% CO2atmosphere.
Treatment of cells. PNT1a and PC3 cells were seeded in six T75 flasks
(5 3 106cells per flask). Twenty-four hours after seeding, the cells were treated
with BPA, previously dissolved in ethanol (concentration of the stock solution
was 0.2 M) at a final concentration corresponding to the IC50(200lM for PNT1a
and 250lM for PC3). During the treatment period, cells were maintained in
media depleted of steroids and phenol red (Sigma) and were supplemented with
FBS treated with charcoal/dextran (Hyclone Laboratories, Inc., Logan, UT).
After an additional 24 h, the medium was removed and the cells were detached
with trypsin. Cell suspensions were pooled in such a way to have approximately
5 3 106cells per tube in triplicate. The cells were centrifuged at 1200 3 g for 5
min. After removal of the supernatant, the pellet was frozen at ?80?C. In other
experiments, PNT1a cells were treated at a final concentration of 1nM, and after
2 months of treatment, the cells were processed as described above. Control cells
were incubated with the medium containing 0.1% ethanol.
DNA extraction and detection of DNA adducts. DNA was extracted from
control cells and BPA-treated cells by means of a commercially available kit
using phenol-free reagents (Genelute DNA Miniprep kit, Sigma). The 260/280
absorbance ratio was > 1.70 and < 1.85 in all samples, as determined by fiber
optic spectrophotometry (NanoDrop Technologies, Wilmington, DE), thus
showing the lack of protein and RNA contamination. Bulky lipophilic DNA
adducts were enriched by nuclease P1 digestion and detected by
postlabeling, as described previously (Izzotti et al., 1999). DNA adducts,
quantified by calculating the ratio between cpm detected in DNA adducts
and cpm in normal nucleotides. Their levels were expressed as adducts per 108
32P-imaging (InstantImager, Packard, Meriden, CT), were
Statistical analysis. All quantitative results were expressed as means ± SD
either of the results obtained within the mice composing each experimental
group for in vivo data or of replicates within each cell line and treatment for
in vitro data. The overall statistical significance was evaluated by ANOVA,
followed by Student’s t-test for paired data. The significance of the relative
increase of DNA adduct levels within each line treated with BPA was evaluated
by Student’s t-test comparing the difference between the means before and after
treatment and the relative weighed SD.
Rat Prostate Biomarkers
The average weight of prostates was not significantly
different in control rats (0.36 ± 0.03 g; prostate weight per
animal weight ratio 0.76 ± 0.07 3 10?3) and in rats receiving
BPA with drinking water for 10 days (0.36 ± 0.05 g, prostate
weight per animal weight ratio 0.79 ± 0.13 3 10?3).
The histological analysis of prostate sections, examples of
which are shown in Figure 1 (A and B), provided evidence for
a normal appearance of the prostate from control rats (Fig. 1A),
whereas the samples from BPA-treated rats were extensively
atrophic. In addition, extravasation of lymphocytes was
observed in some samples (Fig. 1B, arrows).
The immunohistochemical analysis of CLU protein in prostate
sections showed that, in samples from control rats, the protein is
well detectable in epithelial cells, with cytoplasmic and apical
localization (Fig. 1C). In contrast, the signal was more intense in
tion of clusterin (C, D) in prostate sections from Sprague-Dawley rats, either
untreated (A, C) or receiving BPA (200 mg/kg body weight) with drinking
water for 10 consecutive days (B, D). The arrows in B show extravasation of
lymphocytes. The asterisks show the cytoplasmic and apical localization of
CLU in control rats (C) and positivity for CLU in the whole cytoplasm and
sometimes in the nucleus of cells in atrophic areas of BPA-treated rats (D).
Histological appearance (A, B) and immunohistochemical detec-
BPA ALTERATIONS IN PROSTATE AND SPERM CELLS
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the atrophic areas of BPA-treated rats, staining the whole
cytoplasm and sometimes the nucleus (Fig. 1D). However, when
prostate homogenates were assayed by Western blot analysis for
CLU and actin (Fig. 2), the CLU/actin densitometric ratio was
not significantly different in control rats (1.02 ± 0.30) and BPA-
treated rats (1.11 ± 0.25).
Rat Spermatozoa Biomarkers
The results relative to the analysis of ROS, TBARS, and
SCD in mature spermatozoa are summarized in Table 1. ROS
concentrations were higher, but not to a significant extent, in
spermatozoa from BPA-treated rats, whereas BPA treatment
resulted in a significant 3.4-fold increase of TBARS.
Moreover, BPA caused a significant 2.1-fold decrease of the
nuclear spreading factor, evaluated by SCD, which provides
evidence for an increased BPA-related fragility of spermatozoa.
Figure 3 shows the dispersion of SCD data relative to the 538
cells per rat analyzed from untreated rats and the 562 cells per
rat from BPA-treated rats. These data show at a glance the
homogeneity of results within each one of the two experimental
groups and the sharp difference between controls and BPA-
TUNEL Assay in Liver Cells
As shown in Table 1, the frequency of TUNEL-positive cells
was increased 3.8-fold in the liver of BPA-treated rats as
compared with control rats.
Cytogenetical Analyses in Bone Marrow Erythrocytes
The frequency of MN PCE was similar in the bone marrow
of control rats and BPA-treated rats (Table 1).
Comet Assay in Peripheral Blood Lymphocytes
The comet assay did not show any significant difference in
the intensity of SSBs from untreated rats and BPA-treated rats
Formation of DNA Adducts in Cultured Human Prostatic
Table 2 summarizes the results relative to the measurement,
in triplicate32P postlabeling analyses, of bulky DNA adducts
in two human prostatic cell lines, either untreated or treated
with BPA at two different doses and exposure times. The
background levels of DNA adducts were slightly higher in PC3
metastatic carcinoma cells than in PNT1a nontumorigenic
epithelial cells, but not to a significant extent. Both types of
cells responded to treatment for 24 h to a high dose of BPA
homogenates from eight control rats and 8 BPA-treated rats. CLU bands (40
kDa) have been normalized by actin expression for quantitative analysis.
Detection of CLU and actin by Western blot analysis in prostate
Intermediate Biomarkers in Various Cells of Controls and
End point (unit) Controls BPA
ROS (DCF-DA emission/lg protein)
TBARS (nmol MDA/lg protein)
SCD (nuclear spreading factor)
TUNEL-positive cells (%)
Bone marrow erythrocytes
MN PCE (&)
Peripheral blood lymphocytes
SSB (tail moment at comet assay)
1.4 ± 0.30
1.7 ± 1.98
1.4 ± 0.83
1.8 ± 0.41
5.7 ± 3.73**
0.7 ± 0.18***
0.4 ± 0.52 1.5 ± 1.31*
0.5 ± 0.76 0.6 ± 0.74
1.6 ± 0.74 1.2 ± 0.43
Note. The data are means ± SD within each experimental group.
*p < 0.05.
**p < 0.01, significant increase as compared with controls.
***p < 0.05, significant decrease as compared with controls.
samples from BPA-treated rats.
SCD data among 4304 samples from eight control rats and 4496
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(250lM and 200lM, respectively) with a significant formation
of DNA adducts. Two major spots were located in the lower
left and lower right parts of the chromatographic area, whereas
one minor adduct was located in the upper left area. Formation
of DNA adducts was remarkable, with a 4.2-fold increase over
controls in PNT1a cells and a 2.7-fold increase over controls in
PC3 cells. The difference in the BPA-related increase of DNA
adduct levels in the two cell lines approached the statistical
significance threshold (p ¼ 0.08). When PNT1a cells were
exposed for 2 months to a much lower dose of BPA (1nM), the
increase of DNA adducts was borderline to statistical
significance (Table 2).
The results of the present study provide evidence that BPA is
able to form DNA adducts in cultured human prostatic cells
and to induce a variety of alterations in cells of rats receiving
this ED with drinking water.
The observed formation of DNA adducts in PNT1a and PC3
cells after a short (24 h) treatment with high-dose BPA and, to
a lesser extent, in PNT1a cells after a long-lasting (2 months)
treatment with a much lower BPA dose indicates that these
human prostatic cells possess the metabolic machinery needed
to activate this ED. Recently, formation of DNA adducts has
been reported to occur in human prostate carcinoma cells
(LNCaP line) after treatment with the polycyclic aromatic
hydrocarbon benzo(a)pyrene (Hruba ´ et al., 2010), a well
Both in humans and in experimental animals, BPA is
metabolized to its glucuronide and hydroxylated derivatives,
mainly 3-hydroxy-BPA (3-OH-BPA or BPA catechol), which
is then oxidized to its ortho-quinone, i.e., BPA-3,4-quinone
(BPAQ) (Atkinson and Roy, 1995b; Edmonds et al., 2004). It
is noteworthy that oxidation of catechols to semiquinones and
quinones is a mechanism of tumor initiation for endogenous
estrogens as well as for synthetic estrogens such as DES
(Cavalieri and Rogan, 2006). The fact that the BPA-related
increase of DNA adducts was greater in nontumorigenic
prostate cells than in cancer cells, with a difference that was
close to the statistical significance threshold, is consistent with
the notion that, in general, metabolic activation of procarci-
nogens and formation of DNA adducts are less pronounced in
cancer cells as compared with the corresponding healthy tissue
(van Schooten et al., 1990).
In rats receiving BPA with drinking water for 10 consecutive
days, we did not observe any DNA damage in surrogate cells.
These included peripheral blood lymphocytes, where no increase
of SSB levels was detected by SCGE, and bone marrow
erythrocytes, where the frequency of MN PCE was not increased
after BPA treatment. Conversely, a significant increase of
TUNEL-positive cells occurred in liver cells from BPA-treated
rats. The liver is the major organ of BPA metabolization, where
this ED has been shown to form DNA adducts in both mice
(Izzotti et al., 2009) and rats (Atkinson and Roy, 1995b) and to
induce generation of ROS in male rats, even at low doses
(Bindhumol et al., 2003). The TUNEL assay detects both SSBs
and double-strand DNA breaks as well as early stage apoptosis.
The specificity of this assay for the detection of apoptosis in liver
tissue has been questioned (Sta ¨helin et al., 1998). Clearly,
evaluation of apoptosis in liver cells of BPA-treated rats would
have required additional methodological approaches, but this end
point was not the primary goal of the present study. Nevertheless,
the results obtained are likely to be interpreted as the
consequence of liver cell death in BPA-treated rats, in which
hepatotoxicity has been ascribed to binding of BPAQ with DNA
(Atkinson and Roy, 1995b).
The most remarkable alterations produced by BPA in vivo,
among those investigated, affected possible target cells, such as
prostate cells and spermatozoa. In the prostate, alterations of
CLU protein were not so intense to be detected by Western blot
analysis of whole organ homogenates. However, an accumu-
lation of CLU was well evident in epithelial cells affected by
BPA-related atrophy, in agreement with our previous conclu-
sion that CLU overexpression is associated with atrophy of rat
prostate epithelial cells (Marinelli et al., 1994). Increased levels
of CLU are typical of cells doomed to die by apoptosis
(Caccamo et al., 2004, 2005). The CLU gene has been found to
express, through mechanisms that are not completely un-
derstood, a complex protein profile composed of extracellularly
secreted CLU (sCLU) and intracellular nucleus-targeted CLU
(nCLU). sCLU is probably cytoprotective, whereas nCLU is
clearly proapoptotic (Caccamo et al., 2004, 2005). It is now
believed that the balance between sCLU and nCLU may drive
the fate of the cell (Bettuzzi and Rizzi, 2009), to such an extent
that plasmatic sCLU might be proposed as a marker of prostate
cancer (Girard et al., 2010). In the present study, the CLU
signal was more intense in the atrophic prostate epithelium of
BPA-treated rats and affected the whole cytoplasm and
sometimes the nucleus. It is noteworthy that, after neonatal
exposure to BPA, prostates from aged rats exhibited an
increased incidence and score of prostate intraepithelial
neoplasia as compared with controls (Prins et al., 2011).
Levels of Bulky DNA Adducts in Human Prostatic Cells
nucleotides (means ± SD)
PNT1a cellsPC3 cells
BPA (200–250nM) for 24 h
BPA (1nM) for 2 months
1.9 ± 0.25
8.0 ± 3.26**
3.4 ± 0.72*
2.3 ± 0.27
6.3 ± 0.90***
Note. NT, not tested.
*P ¼ 0.08.
**P < 0.05.
***P < 0.01, as compared with the corresponding controls.
BPA ALTERATIONS IN PROSTATE AND SPERM CELLS
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Administration of BPA to rats resulted in evident alterations
of spermatozoa. Although the increase in ROS levels was not
statistically significant, concentrations of lipid peroxidation
products, in the form of TBARS, were considerably elevated in
spermatozoa from BPA-treated rats. Moreover, an enhanced
fragility of spermatozoa was documented by a significant
decrease of SCD. The male germ line is one of the most
sensitive tissues to the damaging effects of toxicants (Bonde,
2010), and BPA has been shown to affect the reproduction of
male rodents (Chitra et al., 2003). In previous studies, the oral
treatment of male rats with BPA for 44–45 days resulted in
disorganization, distortion, and degeneration of late spermatids
(Takahashi and Oishi, 2001) and in a reduction of epididymal
sperm motility and sperm count, with increased levels of H2O2
and lipid peroxidation products, accompanied by loss of
superoxide dismutase, catalase, glutathione reductase, and
glutathione peroxidase (Chitra et al., 2003). Administration
of BPA to rats with drinking water for 1 week increased the
production of ROS in sperm cells, an effect that was inhibited
by the antiodidant N-acetylcysteine (Minamiyama et al., 2010).
In humans, one study in a workplace in China showed inverse
relationships between urinary BPA levels and sperm concen-
tration, count, vitality, and motility (Li et al., 2011). However,
in another study in men attending an infertility clinic in the
United States, the association between urinary BPA and sperm
concentration, motility, and morphology was less evident
(Meeker et al., 2010).
The results observed in experimental test systems with high
doses of compounds cannot be automatically transferred to the
human situation. Nevertheless, the BPA-related formation of
DNA adducts in the mammary tissue of female mice (Izzotti
et al., 2009, 2010) and the alterations detected in prostate cells
and spermatozoa of male rats, together with the evidence that
BPA forms DNA adducts in cultured human prostatic cells, as
shown here, deserve attention. They suggest, as postulated with
natural estrogens (Bosland, 2000; Cavalieri and Rogan, 2006;
Carruba, 2007; Ellem et al., 2004; Yang et al., 2009), that the
estrogen-mimicking compound BPA may be involved in
mammary carcinogenesis and prostate carcinogenesis with
similar mechanisms. Moreover, the DNA damage observed in
spermatozoa of rats treated with this ubiquitous ED may be
a mechanism contributing to explain the epidemiological
finding that the quality and quantity of human sperm has
decreased during the last six decades (Maffini et al., 2006; Soto
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