Download article in press

G Model
RTX-6420;
No. of Pages 8
ARTICLE IN PRESS
Reproductive Toxicology xxx (2010) xxx–xxx
Contents lists available at ScienceDirect
Reproductive Toxicology
journal homepage: www.elsevier.com/locate/reprotox
Combined effects of two environmental endocrine disruptors nonyl phenol and
di-n-butyl phthalate on rat Sertoli cells in vitro
Dongmei Li a,b , Yang Hu a,b , Xiahong Shen a,b , Xinjue Dai a,b , Xiaodong Han a,b,∗
a
b
Immunology and Reproduction Biology Laboratory, Medical School, Nanjing University, Nanjing, Jiangsu 210093, China
Jiangsu Key Laboratory of Molecular Medicine, Nanjing University, Nanjing, Jiangsu 210093, China
a r t i c l e
i n f o
Article history:
Received 19 January 2010
Received in revised form 17 May 2010
Accepted 16 June 2010
Available online xxx
Keywords:
Nonyl phenol
Di-n-butyl phthalate
Sertoli cell
Combined effect
a b s t r a c t
In this study, our purpose is to analyze combined effects of nonyl phenol (NP) and di-n-butyl phthalate
(DBP) for rat testicular Sertoli cell toxicity in vitro. Sertoli cells were isolated, purified, cultured, and identified with FSHR fluorescence staining. Then the purified Sertoli cells were treated with different doses of
NP, DBP or NP + DBP. Although we did not find dramatic morphological changes, cell viability decreased
significantly at high-dose NP and their mixture. The following Annexin V-PI staining demonstrated that
DBP alone did not show apoptosis induction, the combination effect on apoptosis induction was due to
NP, in addition, nucleus of Sertoli cell showed apoptosis morphological changes. In addition, increased
LDH leakage was also observed in high-dose mixture. According to the above phenomena, we inferred
that the combined effect of the two substances on Sertoli cell toxicity had an additive effect, and the
induction of apoptosis may play an important role.
© 2010 Published by Elsevier Inc.
1. Introduction
Decline in human and wildlife reproductive health, which is
mainly caused by environmental pollution, is receiving more and
more attention from the public and the scientific community.
So many chemicals as environmental endocrine disruptors have
experimentally demonstrated to be able to affect endocrine processes to a great extent. Among them, nonyl phenol (NP) and
di-n-butyl phthalate (DBP) are commonly found in water environment [1,2]. It has been observed that they are seriously threatening
reproductive health of human and animals.
NP which has weak estrogenic activity is a primary degradation
product of nonylphenol ethoxylate (NPEO), a major group of multipurpose nonionic surface active agents [3,4]. It has been found in
preliminary studies that NP can interfere with reproduction in fish,
reptiles, and mammals, induce the cell death in gonads, and change
other reproductive parameters [3,5,6]. DBP belongs to phthalates
which are man-made chemicals widely used in industry and commerce. It has been shown that DBP and its metabolite mono-n-butyl
phthalate can cause such anti-androgenic effects as decreased
anogenital distance (AGD), cryptorchidism, decreased testosterone
levels, decreased sperm production, and infertility [7–9].
∗ Corresponding author at: Immunology and Reproduction Biology Laboratory,
Medical School, Nanjing University, 22 HanKou Road, Nanjing, Jiangsu 210093,
China. Tel.: +86 25 83686497; fax: +86 25 83686497.
E-mail address: [email protected] (X. Han).
In general, level of human exposure to a single environmental
endocrine disruptor is low, and it has much less potent than natural hormones. However, humans are often exposed to a mixture of
these chemicals and combined effects should be considered to evaluate the human exposure risk. Therefore, Kortenkamp [10] claimed
that epidemiology should focus on the mixed effects of multiple
endocrine disrupters rather than a single endocrine disrupter. Since
the above two compounds have effects on male reproductive system, it can be concluded that testis is one of the target organs of NP
and DBP. They are widely and quickly consumed. As a result, they
accumulate in the environment at an increasing level, and often
appear together. Various channels are available for a mixture of NP
and DBP to reach humans. Therefore, it is necessary to explore the
combined effect of NP and DBP on male reproduction.
A series of studies on combined effects of mixture focused
on the combinations of estrogenic, thyroid-disrupting, and antiandrogenic chemicals at low doses [11–16]. Existing data showed
that if they were present synchronously, combined effects may
result from the same endocrine disruption. However, there are
few studies on the combined effects of a weak estrogenic chemical
and an anti-androgenic chemical on reproductive toxicity in Sertoli
cell in vitro. The two compounds are often present simultaneously
and ranked the top two in the water environment. For example, in
China, an analysis revealed that levels of NP and DBP in raw water
from Yellow River were as high as 0.47 ␮mol/L and 0.021 ␮mol/L,
respectively. After water treatment, water content of the residents
was still 0.086 ␮mol/L and 0.008 ␮mol/L, respectively [2]. In Spain
and Canada, it was also found that concentration of NP and DBP
0890-6238/$ – see front matter © 2010 Published by Elsevier Inc.
doi:10.1016/j.reprotox.2010.06.003
Please cite this article in press as: Li D, et al. Combined effects of two environmental endocrine disruptors nonyl phenol and di-n-butyl phthalate
on rat Sertoli cells in vitro. Reprod Toxicol (2010), doi:10.1016/j.reprotox.2010.06.003
G Model
RTX-6420;
No. of Pages 8
ARTICLE IN PRESS
2
D. Li et al. / Reproductive Toxicology xxx (2010) xxx–xxx
existence was of the order of magnitude ␮g/L in test samples from
a number of water treatment plants [1,17]. In selected tissues from
ewes and their lambs which were grazed on pastures fertilized with
sewage sludge or with inorganic fertilizer, NP and phthalate also
existed together in muscle and liver as well as fat tissue in treatment
groups and there was also significant accumulation [18].
Sertoli cells exert important functions in supporting and nourishing germ cells as well as the constitution of blood–testicle
barrier. Onset of spermatogenesis and the eventual production of a
sufficient sperm number to insure fertility depend on Sertoli cells.
Therefore, specific impairment of Sertoli cells will produce a parallel dysfunction in sperm production. In our laboratory, long-term
research data on the Sertoli cell exposure to NP revealed that NP
can induce oxidative stress and cytotoxicity in testicular Sertoli cell
[19] and induce endoplasmic reticulum stress which may plays an
important role in the induction of apoptosis [20]. Expression of 41
proteins among 63 protein spots identified by proteomic approach
was altered in rat Sertoli cells after treated with low NP concentrations, similar to environmental conditions, for 24 h. Further analysis
by Western blot found these proteins are mainly involved in the
response of Sertoli cells to programmed cell death [21]. Additionally, study on the membrane dynamics of Sertoli cells indicated
cellular membranes represented a plausible target for NP-induced
cytotoxicity [22]. The study of Wang et al. [23] demonstrated that
impairment of spermatogenesis caused by DBP should be partly due
to the suppression of androgen binding protein (ABP) and inhibin
(INH) alpha biosynthesis in Sertoli cells, and Sertoli cells should be
one of the major toxic targets. All the above studies confirmed that
Sertoli cells were the targets of the two chemicals [24]. Damage to
Sertoli cells may lead to impairment of male reproduction.
Many related studies share an assumption that environmental
endocrine disruptors disturb endocrine through hormone receptor, but some other studies have confirmed that this is not the
case. Thus, it is particularly important to understand the mechanism of these compounds from a different perspective. The purpose
of this study is to investigate combined effects in vitro of the aforementioned two environmental endocrine disruptors on primary rat
Sertoli cells. It is elucidated whether a mixture of the two endocrine
disrupters interacts in an additive, a synergistic, or an antagonistic
way.
2. Materials and methods
2.1. Chemicals and reagents
NP (4-nonyl phenol, C15 H24 O, CAS: 25154-52-3) with 98% analytical standard
was from Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan). DBP (di-n-butyl phthalate, C16 H22 O4 , CAS: 84-74-2) was purchased from Sigma–Aldrich Inc. (St. Louis,
MO, USA). Dulbecco’s modified Eagle’s medium-Ham’s F-12 medium (DMEM-F12
medium), collagenase I, trypsin, C8H17N2O4Sna (HEPES sodium salt), penicillin,
streptomycin sulfate, Hoechst 33342, fluorescein diacetate (FDA), propidium iodide
(PI) were purchased from Sigma–Aldrich Inc. (St. Louis, MO, USA). Anti-Follicle
stimulating hormone receptor (FSHR) antibody and donkey anti-goat IgG-CY3
were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).
Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies,
Inc. (Kumamoto, Japan). Annexin V-PI apoptosis assay kit was purchased from
Nanjing KeyGen Biotech. Co. Ltd. (Nanjing, Jiangsu, China). 2-(4-Amidinophenyl)-6indolecarbamidine dihydrochloride (DAPI) was purchased from Beyotime Institute
of Biotechnology (Nantong, Jiangsu, Chain). CytoTox-ONETM Homogeneous Membrane Integrity Assay was obtained from Promega Corporation (Madison, MI, USA).
NP and DBP were dissolved in 99% pure dimethylsulfoxid (DMSO, no. S 26740
916) from Schuchardt (Hohenbrunn, Germany) into stock solutions of 20 mM and
200 mM, respectively.
2.2. Primary culture of rat testicular Sertoli cells
Sprague–Dawley rats were purchased from Nanjing Medical University and
kept in accordance with NIH Guide for the Care and Use of Laboratory Animals.
The method in which primary Sertoli cell cultures were prepared from 3-week-old
Sprague–Dawley rats by sequential enzymatic treatment have been used routinely
in our laboratory as previously described [25]. Testes were aseptically removed,
decapsulated, and washed twice, and tubules were carefully separated. The loosened tissues were transferred into 50 ml plastic tubes and sequentially digested
in 0.25% trypsin in a rocking incubator (32 ◦ C, 210 rpm, 30 min), followed by 0.1%
collagenase I (34 ◦ C, 150 rpm, 45 min). The homogenate was filtered through a 100mesh stainless steel filter and the cell suspension was centrifuged at 200 × g for
5 min. Cells were washed twice in DMEM-F12 medium supplemented with 5% FBS.
Isolated cells were plated on tissue culture dishes (10 mm in diameter, Greiner BioOne GmbH, Frickenhausen, Germany) at a density of 1.5 × 106 cells/ml in DMEM-F12
supplemented with 4 mM glutamine, 15 mM Hepes, 6 mM l-(1)-lactic hemicalcium
salthydrate, 1 mM sodium pyruvate, antibiotics (final concentrations: penicillin,
100 IU/ml; streptomycin sulfate, 100 ␮g/ml) and 5% fetal bovine serum (FBS). Cells
were maintained in a humidified atmosphere of 95% air/5% CO2 (v/v) at 37 ◦ C for
48 h. Sertoli cells attached to the bottom of dishes with only tiny dendrites protruding, but most of germ cells suspended in the medium and can be removed
by changing the medium. Another 48 h later, the medium was changed again for
second purification until Sertoli cells grow quickly to form a monolayer in new
medium.
Cell purity was determined by immune fluorescence of Anti-FSHR antibody.
After purification, cells were fixed with methanol for 5 min, washed three times in
PBS. Then cells were incubated at 37 ◦ C for 2 h with the primary anti-FSHR antibody.
To block nonspecific binding, the primary antibody was diluted 1:100 with 1% BSA in
PBS. Following incubation with the primary antibody, the cells were then incubated
at 37 ◦ C for 1 h with CY3-conjugated secondary antibody diluted in 1% BSA–PBS.
Nuclear was stained with DAPI. The cells were washed and observed under the fluorescent microscopy (Nikon, Chiyoda-ku, Tokyo, Japan) with an appropriate barrier
filter set.
2.3. Cell viability and toxicity assay
Cell viability assays were performed using CCK-8. The purified cells were
digested with trypsin and plated in 96-well plates at 1 × 104 cells per well and cultured in the serum-free growth medium for 24 h. Then, Sertoli cells were exposed to
vehicle (Control) or different concentrations of NP (N1 (0.1 ␮mol/L), N2 (1 ␮mol/L),
N3 (10 ␮mol/L)), DBP (D1 (1 ␮mol/L), D2 (10 ␮mol/L), D3 (100 ␮mol/L)) or NP + DBP
(N1 + D1, N2 + D2, N3 + D3). At the indicated time points, the cell numbers in sextuple wells were measured on an automated microplate reader (Bio-Rad, Japan)
as the absorbance (A) (450 nm) of reduced WST-8 (2-(2-methoxy-4-nitrophenyl)3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt). Cell
viability was calculated as follows:
cell viability (%) =
A(treatment) − A(blank)
× 100%
A(Control) − A(blank)
2.4. Staining with FDA and PI for morphologic evaluation
A rapid, simultaneous double-staining procedure with fluorescein diacetate
(FDA) and propidium iodide (PI) was used for morphologic evaluation [26]. This
assay is based on the simultaneous determination of viable and dead cells with the
detection of intracellular lipase activity by FDA and of plasma membrane integrity
by PI, respectively. The purified Sertoli cells were transplanted into 96-well plates at
1 × 104 cells per well and exposed to vehicle (Control) or different concentrations of
NP (N3 (10 ␮mol/L)), DBP (D3 (10 ␮mol/L)) or NP + DBP (N3 + D3) for 24 h, cells in tripartite wells were stained with 5 ␮g/ml PI and 4 ␮g/ml FDA for 5 min and observed
under the fluorescent microscopy with an appropriate barrier filter set.
2.5. Flow cytometric assay
Annexin V-FITC/PI staining combined with flow cytometry was used to quantitatively determine the percentage of cells undergoing apoptosis and necrosis. The
purified cells were digested with trypsin and plated in 6-well plates at 1 × 106 cells
per well and treated with vehicle (Control) or NP (N3 (10 ␮mol/L)), DBP (D3
(10 ␮mol/L)) or NP + DBP (N3 + D3) for 48 h and then harvested. Then Sertoli cells
in tripartite were stained with a saturating concentration of Annexin V-FITC and PI
in Assay Buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2 , pH 7.4) at a concentration of 1 × 106 cells/ml in the dark for 20 min. Cells were pelleted and analyzed
using a FACScan flow cytometer (Becton-Dickson, Immunocytometry System, San
Jose, CA) immediately after staining.
2.6. In situ labeling of apoptotic cells
A DNA dye, Hoechst 33342 was used for examining nuclear morphology. The
purified Sertoli cells were transplanted into 96-well plates at 1 × 104 cells per well
and exposed to vehicle (Control) or NP (N3 (10 ␮mol/L)), DBP (D3 (10 ␮mol/L)) or
NP + DBP (N3 + D3) for 48 h, cells in tripartite wells were stained with Hoechst 33342
according to the protocol of the kit. The result of staining was visualized under a
fluorescent microscope that was excited at a wavelength of 350 nm and measured
at 460 nm.
Please cite this article in press as: Li D, et al. Combined effects of two environmental endocrine disruptors nonyl phenol and di-n-butyl phthalate
on rat Sertoli cells in vitro. Reprod Toxicol (2010), doi:10.1016/j.reprotox.2010.06.003
G Model
RTX-6420;
No. of Pages 8
ARTICLE IN PRESS
D. Li et al. / Reproductive Toxicology xxx (2010) xxx–xxx
3
2.7. Membrane integrity assay
Membrane integrity assay is determined by a rapid, fluorescent measure of the
release of lactate dehydrogenase (LDH) from cells with a damaged membrane using
CytoTox-ONETM Homogeneous Membrane Integrity Assay kit. In brief, Sertoli cells
were digested with trypsin and plated in 96-well plates at 1 × 104 cells per well, then
Sertoli cells in sextuple wells were exposed to vehicle (Control) or different concentrations of NP (N1, N2, N3), DBP (D1, D2, D3) or NP + DBP (N1 + D1, N2 + D2, N3 + D3)
for 24 h or 48 h. According to the protocol of the kit, the LDH activity was measured
in culture supernatants (S) and in the remaining cells (C) after lysis using an automated microplate reader with excitation/emission wavelengths at 560/590 nm. The
percentage of LDH leakage was calculated as follows:
leakage (%) =
S − blank
× 100%
S − blank + C − blank
2.8. Statistical analysis
Three replicates of each exposure were performed. Data are presented as the
mean ± standard deviation (SD). Significance of the difference from the respective
controls for each experimental test condition was assessed with one-way analysis
of variance (ANOVA). Statistical significance was determined by Dunnett t test, and
p < 0.05 was considered significant.
3.1. Sertoli cell identification
Fig. 1. Immunofluorescence photomicrographs of Sertoli cells dyed with FSHR with
nuclei stained by DAPI. Sertoli cells were positive immunostaining of the FSHR,
showing red, and nuclei showed blue fluorescence. Sertoli cells were those expressing FSHR, purity of Sertoli cells was >95%. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of the article.)
FSH plays an important role to maintain Sertoli cell functions
to ensure the maintenance of qualitatively and quantitatively normal spermatogenesis. FSH action on Sertoli cells is mediated by a
specific receptor, the FSH receptor (FSHR). In spermatogenic epithelium, FSHR specifically expresses in the membrane of Sertoli cell.
Using anti-FSHR primary antibody and CY3-conjugated secondary
antibody, Sertoli cells were immunopositive and emerged in the red
fluorescence, nuclei showed a blue fluorescence (Fig. 1). All these
primary cells expressed FSHR, indicating that they were Sertoli
cells, and purity of Sertoli cells is >95%.
3. Results
Fig. 2. Effect of NP or/and DBP on the viability of cultured Sertoli cells. Sertoli cells were treated with NP (N1 (0.1 ␮mol/L), N2 (1 ␮mol/L), N3 (10 ␮mol/L)), DBP (D1 (1 ␮mol/L),
D2 (10 ␮mol/L), D3 (100 ␮mol/L)) or NP + DBP (N1 + D1, N2 + D2, N3 + D3) for 6 h, 12 h, 24 h and 48 h, respectively, and the viability of cells was determined by CCK-8. Data
were presented as mean ± SD. Statistically different from the control is marked with asterisk (*P < 0.05, **P < 0.01).
Please cite this article in press as: Li D, et al. Combined effects of two environmental endocrine disruptors nonyl phenol and di-n-butyl phthalate
on rat Sertoli cells in vitro. Reprod Toxicol (2010), doi:10.1016/j.reprotox.2010.06.003
G Model
RTX-6420;
4
No. of Pages 8
ARTICLE IN PRESS
D. Li et al. / Reproductive Toxicology xxx (2010) xxx–xxx
Fig. 3. Observation of morphological changes of Sertoli cells after treatment. (A) Phase-contrast microphotographs of Sertoli cells. After being incubated with NP (N1
(0.1 ␮mol/L), N2 (1 ␮mol/L), N3 (10 ␮mol/L)), DBP (D1 (1 ␮mol/L), D2 (10 ␮mol/L), D3 (100 ␮mol/L)) or NP + DBP (N1 + D1, N2 + D2, N3 + D3) for 48 h, Sertoli cells were
photographed under phase-contrast microscope. Bar = 100 ␮m. (B) Fluorescent photomicrographs of Sertoli cells dyed with FDA/PI. Cells in the control and N3, D3, N3 + D3
for 48 h were stained using FDA and PI staining. From micrographs, no remarkably morphological changes were observed.
Please cite this article in press as: Li D, et al. Combined effects of two environmental endocrine disruptors nonyl phenol and di-n-butyl phthalate
on rat Sertoli cells in vitro. Reprod Toxicol (2010), doi:10.1016/j.reprotox.2010.06.003
G Model
RTX-6420;
No. of Pages 8
ARTICLE IN PRESS
D. Li et al. / Reproductive Toxicology xxx (2010) xxx–xxx
5
Fig. 3. (Continued).
3.2. Effect on cell viability
The result of cell viability assay was presented in histogram
in Fig. 2. As can be seen, after 6 h treatment, Sertoli cell viability
showed significant increase between control and N1, N2, D1, D2, D3,
respectively (p < 0.05 or p < 0.01), and showed significant decrease
between control and N3, N3 + D3, respectively (p < 0.01). However,
there were no significant variations after 12 h treatment. With the
exposure time increasing to 24 h, cell viability in N3 and N3 + D3
declined significantly (p < 0.05 or p < 0.01). With the exposure time
increasing to 48 h, in addition to N3 and N3 + D3, cell viability in
N2 + D2 also declined significantly (p < 0.05).
Annexin V-FITC staining. The result of flow cytometry analysis
revealed that the proportion of apoptotic cells was significantly
increased at N3 and N3 + D3 groups (Fig. 4A). Although the proportion of apoptotic cells in D3 had a trend to increase, there was
no statistical difference. In order to further verify that two compounds induced apoptosis and necrosis of Sertoli cells, nuclear
staining with Hoechst 33342 at 48 h demonstrated that such significant morphological changes in nuclear as chromatin condensation
with bright blue, lobulated, debris, or edge set were observed in N3,
D3, or N3 + D3 compared with the normal nuclear morphology of
control cells (Fig. 4B). (For interpretation of the references to color
in this sentence, the reader is referred to the web version of the
article.)
3.3. Morphological changes of Sertoli cells
3.5. LDH leakage for membrane integrity assay
Under the inverted phase-contrast microscope, Sertoli cells
showed wide columnar or irregular-shaped with thick processes.
No obvious morphological changes of Sertoli cells induced by NP,
DBP, NP + DBP could be observed at 24 h (not shown) or 48 h
(Fig. 3A). Cell survival or death was assessed by observing fluorescence photomicrographs of Sertoli cells stained with FDA and
PI. In Fig. 3B, we showed photomicrographs of the effects on Sertoli
cells in N3, D3, N3 + D3 for 48 h. The fluorogenic substrate lipase
is cleaved only in viable Sertoli cells to form the green fluorescence. (For interpretation of the references to color in this sentence,
the reader is referred to the web version of the article.) These
cells remained viable when cultured with medium alone (Control).
The PI, a high-affinity red fluorescent DNA staining, is only able to
pass through the compromised membranes of dead cells. At 24 h
or 48 h, cells exposed to NP, DBP, NP + DBP did not exhibit significant changes by morphologic observation under the fluorescent
microscope.
3.4. Examination of NP, DBP, or NP + DBP-induced apoptosis and
necrosis
We first determined whether apoptotic events were induced
in Sertoli cells after exposure to NP, DBP, NP + DBP for 48 h using
Plasma membrane integrity was assessed by monitoring LDH
leakage into the extracellular medium (Fig. 5). After 24 h treatment,
LDH leakage in N3 + D3 increased significantly (p < 0.05). With the
exposure time prolonging to 48 h, N3 + D3 still resulted to significant increase of membrane permeability (p < 0.01).
4. Discussion
Morphological and functional characteristics of Sertoli cells
determine that they are important in nutrition, support, and regulation for germ cell in spermatogenesis. Some chemical poisons
can specifically act on Sertoli cells at different stages during the
development of germ cells, and as a result, lead to reproductive hazards. FSH and testosterone are two major signaling molecules in the
control of spermatogenesis. FSHR specifically expresses on the cell
membrane of Sertoli cells. Therefore, we use specific immunofluorescence expression of FSHR on Sertoli cell membrane to identify
cultured cell purity, and it was verified that this method was specific, intuitive, and simple.
In preliminary study, we determined half maximal effective concentration (EC50 ) of two compounds added in Sertoli cells for 24 h
using CCK-8 kit, respectively. Results indicated that EC50 of NP
Please cite this article in press as: Li D, et al. Combined effects of two environmental endocrine disruptors nonyl phenol and di-n-butyl phthalate
on rat Sertoli cells in vitro. Reprod Toxicol (2010), doi:10.1016/j.reprotox.2010.06.003
G Model
RTX-6420;
6
No. of Pages 8
ARTICLE IN PRESS
D. Li et al. / Reproductive Toxicology xxx (2010) xxx–xxx
Fig. 4. Detection of apoptosis and necrosis. (A) Annexin V-FITC and PI staining for the detection of apoptosis. After exposure 48 h, Sertoli cells were collected for Annexin
V-FITC and PI staining followed by flow cytometry analysis. The upper were flow cytometric plots. The lower was flow cytometric analysis result. Data are presented as
mean ± SD, n = 3. Statistically different from the control is marked (*P < 0.05, **P < 0.01). (B) Fluorescent photomicrographs of Sertoli cells dyed with Hoechst 33342. Cells in
the control and N3, D3, N3 + D3 for 48 h were stained using Hoechst 33342 staining. Condensed and fragmented nuclei were marked with white arrows.
Please cite this article in press as: Li D, et al. Combined effects of two environmental endocrine disruptors nonyl phenol and di-n-butyl phthalate
on rat Sertoli cells in vitro. Reprod Toxicol (2010), doi:10.1016/j.reprotox.2010.06.003
G Model
RTX-6420;
No. of Pages 8
ARTICLE IN PRESS
D. Li et al. / Reproductive Toxicology xxx (2010) xxx–xxx
Fig. 5. Effect of NP or/and DBP on LDH leakage of cultured Sertoli cells. Sertoli
cells were treated with NP (N1 (0.1 ␮mol/L), N2 (1 ␮mol/L), N3 (10 ␮mol/L)), DBP
(D1 (1 ␮mol/L), D2 (10 ␮mol/L), D3 (100 ␮mol/L)) or NP + DBP (N1 + D1, N2 + D2,
N3 + D3) for 24 h and 48 h, respectively, and the viability of cells was determined
using CytoTox-ONETM Homogeneous Membrane Integrity Assay kit. Data were presented as mean ± SD. Statistical different from the control was marked with asterisk
(**P < 0.01).
was 16.5 ␮mol/L and EC50 of DBP was about 12 mmol/L. In this
study, although it seemed that the highest concentration of NP
was 10 ␮mol/L, about 1/2 EC50 of NP, and was relatively high compared with expected environmental exposure, this concentration
is much lower than that in other studies on NP toxicity (30 ␮mol/L)
[19,20,22]. Other concentrations of NP were low, similar to environmental conditions. DBP concentration ranged from 1 ␮mol/L
to 100 ␮mol/L and the highest concentration of DBP applied in
this experiment was about 1/100 EC50 of DBP. Hallmark et al. [27]
showed that DBP was not effective in testicular Leydig cells after
up to millimolar concentrations in vitro. Therefore, in this study,
one of the purposes that we determined the highest dose of DBP at
100 ␮mol/L was to show whether DBP was effective in Sertoli cells,
and the other purpose was to observe if DBP at 100 ␮mol/L would
increase or decrease the cytotoxicity of NP in vitro.
It was found that after 6 h of exposure, 0.1 ␮mol/L NP, 1 ␮mol/L
NP, and three DBP treatment groups all showed significantly
increased cell viability. Low- and medium-dose combination
groups showed the same trend despite there was no significant
difference compared with the control group. At the high dose,
10 ␮mol/L NP inhibited Sertoli cell viability and showed certain
toxicity. The high-dose combination group further inhibited cell
viability. The results suggested that NP and DBP might have hormesis, which showed stimulating effect at low doses but at high
concentrations showed inhibitory effects in a short period. After
12 h exposure cell viability had no difference in each group compared with the control. We suggested that cells were in a state
7
of compensatory to some extent, indicating that Sertoli cells have
certain capacity of resistance against foreign chemicals. Exposed
to 24 h, 10 ␮mol/L NP still significantly inhibited cell viability. This
result was different from Gong and Han’s [19]. In their experiments,
it showed that 10 ␮mol/L NP did not induce the decline of cell viability. We considered that CCK-8 was of higher sensitivity than MTT.
At 24 h, 100 ␮mol/L DBP showed no influence to cell viability, and
high-dose combination group continued to show a trend same as
NP. Compared with the control group, such inhibition was much
severer. We speculated that there was an additive effect in the presence of NP and DBP. At 48 h, medium-dose combination group also
showed a visible decline.
Annexin V-FITC-PI double-staining flow cytometry revealed
that 10 ␮mol/L NP, 100 ␮mol/L DBP and their mixture can induce
cell apoptosis and increase the portion of cell death. Hoechst 33342
fluorescent staining of the nucleus changes for 48 h. Significant
changes in nuclear morphology with apoptosis character were
found in high-dose NP, DBP and their mixture. Results indicated
that NP and their mixture might impair cell function by inducing
apoptosis. Gong found that 20 ␮mol/L and 30 ␮mol/L NP treatment
led to remarkably increased apoptosis in Sertoli cells, while NP can
induce ER-stress in Sertoli cells, and thus Sertoli cell apoptosis could
be induced by ER-stress [20]. In addition, it was found that Fas/FasL
system, caspase-3 activation, and mitochondrial depolarization all
were involved in NP-induced thymocyte apoptosis [28,29]. DBP
related researches basically revolved around prepubertal exposure
to focus on the interference of gonadal development, including the
embryonic stage. Most recently, it was found that DBP could induce
spermatogenic cell apoptosis in prepubertal rats [30]. In Naarala
and Korpi’s study, both doses (500 ␮mol/L and 1 mmol/L) of DBP
increased apoptosis of murine macrophages in a short term exposure by two- and fourfold, though the result was not significant in
statistics [31]. Similarly, we did not find 100 ␮mol/L DBP increased
apoptosis of Sertoli cells after a 24 h or 48 h exposure with a statistically significant result. Therefore, the present results indicated
that DBP alone did not show apoptosis induction, the combination
effect on apoptosis induction was due to NP, and the combination
had an additive effect. As for the mechanisms of the mixture leading
to cell apoptosis, we need further study.
Previous studies have found cell membrane was the target
attacked by NP [22]. Two studies in vitro indicated that phthalates disturbed the energy transfer chain existing between germ
cells and Sertoli cells to lead to adverse changes in cell polarization, which affects membrane proteins [32,33]. In recent studies,
it was shown that the metabolite and main toxicants of DBP and
MBP induced damage to tight junctions between adjacent Sertoli
cells at doses of 150 ␮mol/L and 600 ␮mol/L. Thus, cell membrane
integrity was also the object we observed. In this experiment, in
24 h, LDH leakage increased significantly only in high-dose mixture group. When the exposure time was prolonged to 48 h, only
high-dose mixture induced LDH leakage to increase significantly. It
is interesting that we did not find that LDH leakage significantly
increased in 10 ␮mol/L NP group or 10 ␮mol/L DBP. We speculated that there was an additive effect in the presence of NP and
DBP.
In conclusion, additive effects were observed in the mixture of
NP and DBP on Sertoli cell. In this study, the evaluation method was
based on the principle of concentration addition, which, we think,
was a valid tool for assessing mixture effects of both similarly and
dissimilarly acting compounds in this in vitro model. A mixture of
NP and DBP can induce destruction of cell membrane integrity and
increase membrane permeability so that the amount of NP and DBP
into the cells increased. Although DBP showed no influence to cell
viability, the combined effect of the two substances on Sertoli cell
toxicity increased, showing a certain additive effects. Furthermore,
the induction of apoptosis may play an important role.
Please cite this article in press as: Li D, et al. Combined effects of two environmental endocrine disruptors nonyl phenol and di-n-butyl phthalate
on rat Sertoli cells in vitro. Reprod Toxicol (2010), doi:10.1016/j.reprotox.2010.06.003
G Model
RTX-6420;
No. of Pages 8
8
ARTICLE IN PRESS
D. Li et al. / Reproductive Toxicology xxx (2010) xxx–xxx
Conflict of interest statement
There is no conflict of interest.
Acknowledgements
The work was supported by National Natural Science Foundation of China (30800115 and 30970530), Foundation for
Junior Faculty in Doctoral Program, Ministry of Education, China
(200802841006), and Doctoral Program of Higher Specialized
Research Fund, Ministry of Education, China (20090091110048).
The authors wish to thank Dr. Yi Gong and Xuping Pan for their
help in the experiment.
References
[1] Zafra-Gómez A, Ballesteros O, Navalón A, Vílchez JL. Determination of some
endocrine disrupter chemicals in urban wastewater samples using liquid
chromatography–mass spectrometry. Microchemical Journal 2008;88:87–94.
[2] Zhao Y, Guo D. Analysis to the content of di-n-butyl phthalate, nonylphenol and
bisphenol A in the water of Yellow River. Environmental Monitoring in China
2007;23:19–21.
[3] Han XD, Tu ZG, Gong Y, Shen SN, Wang XY, Kang LN, et al. The toxic effects of
nonylphenol on the reproductive system of male rats. Reproductive Toxicology
2004;19:215–21.
[4] Tanaka JN, Grizzle JM. Effects of nonylphenol on the gonadal differentiation of the hermaphroditic fish, Riulus marmoratu. Aquatic Toxicology
2002;57:117–25.
[5] Cardinali M, Maradonna F, Olivotto I, Bortoluzzi G, Mosconi G, Polzonetti-Magni
AM, et al. Temporary impairment of reproduction in freshwater teleost exposed
to nonylphenol. Reproductive Toxicology 2004;18:597–604.
[6] Nagao T, Wada K, Marumo H, Yoshimura S, Ono H. Reproductive effects of
nonylphenol in rats after gavage administration: a two-generation study.
Reproductive Toxicology 2001;15:293–315.
[7] Fisher JS, Macpherson S, Marchetti N, Sharpe RM. Human “testicular dysgenesis syndrome”: a possible model using in-utero exposure of the rat to dibutyl
phthalate. Human Reproduction 2003;18:1383–94.
[8] Foster PM. Disruption of reproductive development in male rat offspring following in utero exposure to phthalate esters. International Journal of Andrology
2006;29:140–7.
[9] McKee RH, Butala JH, David RM, Gans G. NTP center for the evaluation of risks to
human reproduction reports on phthalates: addressing the data gaps. Reproductive Toxicology 2004;18:1–22.
[10] Kortenkamp A. Low dose mixture effects of endocrine disrupters: implications for risk assessment and epidemiology. International Journal of Andrology
2008;31:233–40.
[11] Brian JV, Harris CA, Scholze M, Backhaus T, Booy P, Lamoree M, et al. Accurate prediction of the response of freshwater fish to a mixture of estrogenic
chemicals. Environmental Health Perspectives 2005;113:721–8.
[12] Crofton KM, Craft ES, Hedge JM, Gennings C, Simmons JE, Carchman RA,
et al. Thyroid-hormone-disrupting chemicals: evidence for dose-dependent
additivity or synergism. Environmental Health Perspectives 2005;113:
1549–54.
[13] Rajapakse N, Silva E, Kortenkamp A. Combining xenoestrogens at levels below
no-observed-effect-concentrations dramatically enhances steroid hormone
action. Environmental Health Perspectives 2002;110:917–21.
[14] Silva E, Rajapakse N, Kortenkamp A. Something from “nothing”-eight
weak oestrogenic chemicals combined at concentrations below NOECs produce significant mixture effects. Environmental Science and Technology
2002;36:1751–6.
[15] Tinwell H, Ashby J. Sensitivity of the immature rat uterotrophic assay to mixtures of estrogens. Environmental Health Perspectives 2004;112:575–82.
[16] Hass U, Scholze M, Christiansen S, Dalgaard M, Vinggaard AM, Axelstad M,
et al. Combined exposure to anti-androgens exacerbates disruption of sexual
differentiation in the rat. Environmental Health Perspectives 2007;115(Suppl.
1):122–8.
[17] Fernandez MP, Ikonomou MG, Buchanan I. An assessment of estrogenic organic
contaminants in Canadian wastewaters. Science of the Total Environment
2007;373:250–69.
[18] Rhind SM, Kyle CE, Telfer G, Duff El, Smith A. Alkyl phenols and diethylhexyl
phthalate in tissues of sheep grazing pastures fertilized with sewage sludge or
inorganic fertilizer. Environmental Health Perspectives 2005;113:447–53.
[19] Gong Y, Han XD. Nonylphenol-induced oxidative stress and cytotoxicity in
testicular Sertoli cells. Reproductive Toxicology 2006;22:623–30.
[20] Gong Y, Wu J, Huang Y, Shen S, Han X. Nonylphenol induces apoptosis in
rat testicular Sertoli cells via endoplasmic reticulum stress. Toxicology Letters
2009;186:84–95.
[21] Wu J, Wang F, Gong Y, Li D, Sha J, Huang X, et al. Proteomic analysis of changes
induced by nonylphenol in Sprague–Dawley rat Sertoli cells. Chemical Research
in Toxicology 2009;22:668–75.
[22] Gong Y, Pan XP, Huang YF, Gao ZS, Yu HX, Han XD. NP-induced biophysical and
biochemical alterations of rat testicular Sertoli cell membranes related to disturbed intracellular Ca(2+) homeostasis. Toxicology Letters 2008;183:10–20.
[23] Wang YB, Song L, Zhu ZP, Chen JF, Wang XR. Effects of dibutyl phthalate on
Sertoli cells of rat testis. Zhonghua Yu Fang Yi Xue Za Zhi 2005;39:179–81.
[24] Monsees TK, Franz M, Gebhardt S, Winterstein U, Schill WB, Hayatpour J. Sertoli
cells as a target for reproductive hazards. Andrologia 2000;32:239–46.
[25] Li DM, Liu Q, Gong Y, Huang YF, Han XD. Cytotoxicity and oxidative stress
study in cultured rat Sertoli cells with methyl tert-butyl ether (MTBE) exposure.
Reproductive Toxicology 2009;27:170–6.
[26] Jones KH, Senft JA. An improved method to determine cell viability by
simultaneous staining with fluorescein diacetate-propidium iodide. Journal of
Histochemistry and Cytochemistry 1985;33:77–9.
[27] Hallmark N, Walker M, McKinnell C, Mahood IK, Scott H, Bayne R, et al. Effects of
monobutyl and di(n-butyl) phthalate in vitro on steroidogenesis and leydig cell
aggregation in fetal testis explants from the rat: comparison with effects in vivo
in the fetal rat and neonatal marmoset and in vitro in the human. Environmental
Health Perspectives 2007;115:390–6.
[28] Kim SK, Kim BK, Shim JH, Gil JE, Yoon YD, Kim JH. Nonylphenol and octylphenolinduced apoptosis in human embryonic stem cells is related to fasfas ligand
pathway. Toxicological Sciences 2006;94:310–21.
[29] Yao GH, Yang LS, Hu YL, Liang J, Liang JF, Hou YY. Nonylphenol- induced thymocyte apoptosis involved caspase-3 activation and mitochondrial depolarization.
Molecular Immunology 2006;43:915–26.
[30] Alam S, Ohsako S, Matsuwaki T, Bo Zhu X, Tsunekawa N, Kanai Y, et al. Induction of spermatogenic cell apoptosis in prepubertal rat testes irrespective of
testicular steroidogenesis: a possible estrogenic effect of di(n-butyl) phthalate.
Reproduction 2009;(November) [Epub ahead of print].
[31] Naarala J, Korpi A. Cell death and production of reactive oxygen species by
murine macrophages after short term exposure to phthalates. Toxicology Letters 2009;188:157–60.
[32] Foster PMD, Foster JR, Cook MW. Changes in ultrastructure and cytochemical
localization of zinc in rat testis following the administration of di-n-pentyl
phthalate. Toxicology and Applied Pharmacology 1982;63:120–32.
[33] Li LH. In vitro culture method. In: Ding XC, Jiang XZ, Gu ZW, editors. Male
reproductive toxicology. Beijing: Chinese Population Publishing House; 1997.
p. 78–82.
Please cite this article in press as: Li D, et al. Combined effects of two environmental endocrine disruptors nonyl phenol and di-n-butyl phthalate
on rat Sertoli cells in vitro. Reprod Toxicol (2010), doi:10.1016/j.reprotox.2010.06.003