Journal of Geoscience and Environment Protection, 2014, 2, 167-172
Published Online April 2014 in SciRes. http://www.scirp.org/journal/gep
How to cite this paper: Huang, Y.J. et al. (2014) Combined Effects of Dibutyl Phthalate (DBP) and Benzo(a)Pyrene on Fertil-
ity in Male Rats. Journal of Geoscience and Environment Protection, 2, 167-172. http://dx.doi.org/10.4236/gep.2014.22023
Combined Effects of Dibutyl Phthalate
(DBP) and Benzo(a)Pyrene on
Fertility in Male Rats
Yujing Huang, Ji’an Chen, Weiqun Shu*
Department of Environmental Hygiene, College of Preventive Medicine, Third Military Medical University,
Received Dec emb er 2013
Our previous studies reveal ed the polycyclic aromatic hydrocarbon and phthalic acid esters were
the major organic pollutants in the Jialing Rive r and Yangtze River in the Three Gorges area, and
they might cause the toxicity in male fertility when combined. Thus we used di-n-butyl phthalate
(DBP) and Benzo(a)pyrene (Bap) as their representatives respectively to explore their effects on
the spermatogenesis in male rats. Male Sprague Dawley rats were randomly divided into 4 groups
and respectively exposed to corn oil, Bap (5 mg/kg/ d), DBP (2 50 mg/kg/d), and combined doses
of Bap (5 mg/kg/d) and DBP (250 mg/kg/d) for 90 days. We observed a significant increase in the
stillbirth rate after Bap and combined treatments, while the mean area of seminiferous tubules
was reduced after Bap, DBP and combined treatments. Bap and combined treatment had a sup-
pressing effect on meiosis in germ cells, which reduced the haploid contents and the ratio between
haploid and diploid but increased the tetraploid and diploid contents and the ratio between hap-
loid and tetraploid. These effects were more obvious in the combined group. Furthermore, the ex-
pression of a number of proteins was ch anged , of which was associated with the oxidative stress
and cAMP/PKA signaling pathway. Our results suggest that Bap has significant toxic effects on
male fertility, while the combined treatment of Bap and DBP has more toxic effects.
Di-n-Butyl Phthalate; Benz o(a)Pyrene; Male Reproductive; Protein Expression; Tubules Structure;
Di-n-butyl phthalate (DBP), a representative of phthalate acid esters (PAEs) that have been used in a wide va-
riety of industrial and common household products, can induce the atrophy in the testes and seminiferous tu-
bules, reproductive tract malformation, the change in enzyme activity in testicular cells, hormone reduction,
Y. J. Huang et al.
spermatogenic cell apoptosis and sperm counts reduction (Kavlock et al., 2002; Shultz et al., 2001; M. S. Alam
et al., 2010a; M. S. Alam et al., 2010b). Benzo(a)pyrene (Bap), a prototype of polycyclic aromatic hydrocarbon
(PAH) that is released into the environment by industrial emission, has attracted worldwide attention as its toxic
effect on male reproduction. It has been demonstrated that Bap can cause seminiferous tubules malformation,
change the epididymis function and enzyme activity in testicular cells, induce DNA damage and synthesis inhi-
bition in spermatogenic cells, and decrease serum testosterone level and the amount of spermatogenic cells and
sperms (Zhu et al., 2010; Sipinen et al., 2010; Inyang et al., 2003; Mohammad Shah Alam et al., 2010; Ramesh
et al., 2008).
In our previous study, PAHs and PAEs are the most organic pollutants in the Jialing river and Yangtze river
in Three Gorges area (Chen et al., 2008). The organic extracts from Jialing River in Chongqing can cause pa-
thological damage and peroxidation response to testis (Cui et al., 2010). When we chose DBP and Bap as model
compounds, we found that in vivo exposure to DBP and Bap, either separately or in combination, resulted in
adverse effects on the reproductive system of male rats, including disturbing the secretion of T, influencing the
production and morphology of sperm and damaging the seminiferous tubule, which may induced through oxida-
tive stress from their ROS-metabolites (Qing, 2009; QIU et al., 2009; Huang et al., 2010; Chen et al., 2011).
And the adverse effects in rats exposed to the DBP and Bap are unexpected and elusive, which suggesting a fur-
ther investigation is needed to explore the complex mechanism of action of these organic pollutions in greater
In this study, we investigated whether the reproductive toxicity of Bap and DBP can affect the male fertility
and explored the possible mechanisms. Our study provides strong evidences for the toxic effects of Bap and
DBP on male fertility.
2.1. Animals and Treatment
Male Sprague Dawley (SD) rats (n = 48, 4 to 5 weeks of age) were provided by the Laboratory Animal Center
of Third Military Medical University (TMMU, China). This study was approved by the Third Military Medical
University Institutional Animal Care and Use Committee. All rats were housed in polycarbonate cages with chip
hardwood, allowed to access to food and filtered tap water ad libitum and maintained at 20˚C - 22˚C and 50% -
70% humidity with controlled lighting (12 h light/12 h darkness).
DBP (Cat. No. 84-74-2, 99% purity), Bap (Cat. No. 50-32-8, 96% purity) and corn oil (Cat. No. 8001-30-7)
were purchased from Sigma Aldrich (St. Louis, MO). After one week acclimatization period, male SD rats were
randomly assigned into 4 groups (12 per group) and administrated corn oil (Control group), 5 mg/kg Bap (Bap
group), 250 mg/kg DBP (DBP group), combination of 5 mg/kg Bap and 250 mg/kg DBP (Combined group)
each day at the same time via gavage for 90 days. After treatment, 8 rats in each group were anesthetized deeply
with 20% urethane and sacrificed to collect the testes. Other rats (n = 4) were mated with health female SD rats
(22 - 24 weeks, 1:1 randomly matched). The day on which the vaginal plug was observed was set as the 1st day
of pregnancy. Pregnant rats were anesthetized with 20% urethane and sacrificed on the 20th day of pregnancy.
The fetuses were separated to examine the total stillbirth rate (including absorptive fetus).
2.2. Examination of Histopatological Testes
The left testes from each group were fixed in Bouin’s fixative at room temperature for 72 h. Following infiltrat-
ing and embedding with paraffin wax, serial sections (4 μm) were cut from the middle of each testis preparation.
Sections were stained with haematoxylin and eosin (HE), observed and evaluated on a light microscope (BX51)
equipped photomicroscope (DP72; OLYMPUS) which coupled with a computerized morphometric planimetry
system (DP72-BSW; OLYMPUS) to facilitate the measurement of tubule area and area percentage (volume
percentage) occupied by seminiferous tubules. The percentage of seminiferous tubules was computed according
to Ramesh (Ramesh et al., 2008).
2.3. Germ Cell Preparation
Right testes without albuginea were washed and decapsulated by phosphate buffer saline (PBS) (Zhongshan
golden bridge Biotechnology Co., Ltd., Beijing, China). Seminiferous tubules were separated with pipette,
Y. J. Huang et al.
washed and sedimentated in PBS and discarded supernatant. Separated seminiferous tubule fragments were in-
cubated in PBS containing 1 mg/ml type IV collagenase (C5138, sigma) for 20 min at 37˚C, washed and pipet-
ted in PBS three times. After each wash, the supernatant was discarded. The pellet were incubated in 0.25%
trypsogen (SH30042.01, HyClone, Thermo Scientific, Pittsburgh, PA) for 10 min at 37˚C, then mixed with
equal volume of DMEM/F12 (SH30023.01, HyClone) medium containing 10% bovine calf serum (Sijiqing Bi-
ological Engineering Materials Co., Ltd., Hangzhou, China). The samples were centrifuged at 100 × g for 3 min
to remove the supernatant, and then washed in PBS three times. The pellet was composed of the mixture of germ
cells and the purity was assessed by the staini ng of vimentin that is expressed in other cells (including sertoli
cells, interstitial cells and peritubular cells) of testes but not in the germ cells.
2.4. Flow Cytometric Analysis (FCM) of DNA Content in Isolated Germ Cells
The mixture of germ cells was fixed in 70% ethanol and washed twice in PBS. Then incubated with 1 mg/ml
RNase for 30 min at 37˚C and stained by propidium iodide (PI) for 30 min at 4˚C. The fluorescent signals of
PI-stained cells were recorded using a flow cytometer (FACSCalibur; BD Immunocytometry Systems, San Di-
ego, CA) and then analyzed by CellQuest (BD Immunocytometry Systems). According to DNA content, germ
cells were divided into 4 phases , including haploid (1C) that is round and elongated spermatids, diploid (2C)
that is spermatogonia, tetraploid (4C) that is primary spermatocytes, and S-phase in which the cells can actively
synthesize DNA. The germ cell ratios among haploid, diploid and tetraploid were determined by 1C:4C, 1C:2C,
2.5. Two-Dimensional Electrophoresis (2-DE) and the Identification of Differential
Proteins in Isolated Germ Cells by Proteomics Method
The germ cells mixture was suspended in cell lysate (8 M urea (GibcoBRL), 4% CHAPS (Calbiochem), 40 mM
Tris base (GibcoBRL), 1 mM PMSF (Pierce Chemicals, Rockford, IL), 2 mM EDTA (GibcoBRL), 10 mM DDT
(Promega, Madison, WI)) and sonicated for 5 min on ice by a ultrasonic vibrator (JY92-II; Scientz, Ningbo,
China) and centrifuged at 12,000 × g for 20 min. The protein concentration in supernatant was measured by
Bradford method and the samples were stored at −80˚C.
The 2-DE was carried out using pre-cast IPG strip (pH 3-10; 7 cm) gels (three parallel samples per group) on
an IPGphor unit (IPGphor, Amersham Biosciences, Piscataway, NJ), followed by the second dimension using
pre-cast polyacrylamide gel (Ettan DALT II Gel, Amersham Biosciences) on a vertical electrophoresis system
(Ettan DALT SIX, Amersham Biosciences) following user’s manual (Amersham Biosciences). The gels were
stained with the Plus One Silver Staining Kit (Amersham Biosciences) using the Multi-Processor (Amersham
Biosciences) and scanned and analyzed with the ImageMaster 5.00 analysis software (Amersham Biosciences).
For the identification of protein spots by mass spectrometry, the protein spots were cut into 1.5 mm cubes,
faded in a 50% CAN (Fisher), digested with trypsin (Promega) and analyzed by using delayed extraction ma-
trix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer system (Bruker Ul-
traflex). Monoisotopic peptide masses obtained from MALDI-TOF were queried against the entries for protein
databases in NCBI using a protein search program, Mascot (Matrix Science Ltd., London).
3. Results and Discussion
3.1. The Effect of DBP and Bap on Rat Growth
Compared with control rats, the rats after Bap and DBP treatments did not show the change in weekly weight
gains (P ≥ 0.05), and the organ coefficients of heart, spleen, kidney, liver, epididymis and testis were not
changed (P ≥ 0.05).
3.2. The Effect of DBP and Bap on Rat Fertility, Histological Changes and Cell Cycle in
There was no difference in the pregnancy rate among different groups (P ≥ 0.05). The stillbirth rate (including
absorptive fetus) was significantly increased after Bap treatment and the combined treatment of Bap and DBP
compared to control (P < 0.05). The atrophy in the seminiferous tubules was observed in all groups treated with
Y. J. Huang et al.
Bap, DBP, and both (P < 0.05). The atrophy in the Bap group was lighter than that in the DBP and combined
groups (P, 0.05B). However, there was no significant difference in the percentage of seminiferous tubules in tes-
tis among four groups (P ≥ 0.05). Compared with the control group, the proportion of different germ cell popu-
lations and their ratios were not changed after treatments with DBP and Bap, while in the combined group, the
haploid content and the ratio of 1C:4C was significantly reduced (P < 0.05) but the tetraploid contents and the
ratio of 4C:2C were significantly increased, even compared with the Bap group (P < 0.05). These data indicate
that the combined treatment with Bap and DBP may induce a suppressing effect on the meiosis in germ cells in
3.3. The Changes in Total Protein Expression
2-DE was used to separate the proteins, and the results from ImageMaster 2D Platinum 5.0 analysis screened 35
different proteins between control and treated groups. 21 proteins were identif ied by mass spectrometric analysis
and 4 proteins were identified as Serum albumin.
Bap, DBP and their combined exposure could decreases SOD and GSH-Px activities and increase CAT activ-
ity, which indicates an oxidative stress state in the testis (Chen et al., 2011). In this study, the expression of
many proteins which associated with oxidative stress was changed in the germ cells after treatments with Bap
and DBP and may associate with their reproductive toxicology. PKA C-beta and tubulin beta-5, which would be
associated with germ cell structure, meiosis process and male fertility (Burton et al., 2006; Fujimori et al., 2012),
were up-regulated after treatment with Bap. HnRNP DL, which acted as a transcriptional regulator and was as-
sociated with cell cycle (Akagi et al., 2000), also varied when exposed to Bap. Both of them are associated with
the structural defects, the suppression on meiosis process and adverse effects on male fertility. All these proteins
and others, including Mitofilin, Rho GTPase activating protein 11A and MST4, are associated with cAMP/PKA
signal pathway, which may be activated by the oxidative stress (Barlow et al., 2006). Other oxidative stress as-
sociated proteins like serotransferrin, ECE-2, retinoid binding protein, endoplasmin, hemopexin and ALDH2
were also associated with Bap and DBP exposure (Klipper et al., 2010). Suggesting that the exposure of Bap and
DBP may disturb the oxidant/antioxidant balance and active the cAMP signal pathway in germ cells (Arafa et al.,
2009; Mayati et al., 2012). Mitofilin, endoplasmin and MST4 are also associated with apoptotic process. The
change of their expression in germ cells those are exposed to Bap may be associated with the atrophy in the se-
miniferous tubules and structural defects. The structural damage in the Bap group included blood-testis barrier
disruption, which can be the reason of enhanced expression of tubulin beta and albumin (Liao et al., 2006;
Christensen et al., 1985).
Our data demonstrated that in vivo exposure to Bap and DBP resulted in directly adverse effects on germ cells
and spermatogenesis, which may be associated with oxidant/antioxidant balance and activation of the cAMP/
PKA signaling pathway, and the reproductive toxicity of combined exposure is stronger than individual expo-
Acknowledgem ent s
This work was supported by a Key Project of National Na tur al Science Foundation of China (No. 30630056)
and the Natural Science Foundation of China (Grant No. 81072262).
Akagi, T., Kamei, D., Tsuchiya, N., Nishina, Y., Horiguchi, H., Matsui, M. et al. (2000). Molecular Characterization of a
Mouse Heterogeneous Nuclear Ribonucleoprotein D-Like Protein JKTBP and Its Tissue-Specific Expression. Gene, 2 45,
267-273. http://dx.doi.org/10.1016/S0378-11 19 (00) 0003 2 -9
Ala m, M. S., Andrina, B. B., Tay, T. W., Tsunekawa, N., Kanai, Y., & Kurohmaru, M. (2010). Single Administration of
Di(n-butyl) Phthalate Delays Spermatogenesis in Prepubertal Rats. Tissue and Cell, 42, 129-13 5.
Ala m, M. S., Ohsako, S., Matsuwaki, T., Zhu, X. B., Tsunekawa, N., Kanai, Y. et al. (2010a). Induction of Spermatogenic
Cell Apoptosis in Prepubertal Rat Testes Irrespective of Testicular Steroidogenesis: A Possible Estrogenic Effect of
Y. J. Huang et al.
Di(n-butyl) Phthalate. Reproduction, 139 , 427-43 7. http://dx.doi.org/10.1530/REP-09-02 26
Ala m, M. S., Ohsako, S., Tay, T. W., Tsunekawa, N., Kanai, Y., & Kurohmaru, M. (2010 b ). Di(n-butyl) Phthalate Induces
Vimentin Filaments Disruption in Rat Sertoli Cells: A Possible Relation with Spermatogenic Cell Apoptosis. Anatomy
Histology and Embryology, 39, 186-193. http://dx.doi.org/10.1111/j.1439-0264. 2010 .00 99 3.x
Arafa, H. M., Aly, H. A., Abd-Ellah, M. F., & El-Refaey, H. M. (2009). Hesperidin Attenuates Benzo[Alpha] Pyrene-In-
duced Testicular Toxicity in Rats via Regulation of Oxidant/Antioxidant Balance. Toxicology and Industrial Health, 25,
Barlow, C. A., Shukla, A., Mossman, B. T., & Lounsbury, K. M. (2006). Oxidant-Mediated cAMP Response Element Bind-
ing Protein Activation: Calcium Regulation and Role in Apoptosis of Lung Epithelial Cells. American Journal of Respi-
ratory Cell and Molecular Biology, 34, 7-14. http://dx.doi.org/10.1165/rcmb.2005-0153 OC
Burton, K. A., McDermott, D. A., Wilkes, D., Poulsen, M. N., Nolan, M. A., Goldstein, M. et al. (2006). Haploinsufficiency
at the Protein Kinase A RI Alpha Gene Locus Leads to Fertility Defects in Male Mice and Men. Molecular Endocrinology,
20, 2504-2513. http://dx.doi.org/10.1210/me.2006-0060
Chen, J. A., Luo, J., Qiu, Z., Xu, C., Huang, Y., Jin, Y. H. et al. (2008). PCDDs/PCDFs and PCBs in Water Samples from
the Three Gorge Reservoir. Chemosphere, 70, 1545 -1551 . http://dx.doi.org/10.1016/j.chemosphere.2007.08.063
Chen, X., An, H., Ao, L., Sun, L., Liu, W., Zhou, Z. et al. (2011 ). The Combined Toxicity of Dibutyl Phthalate and Ben-
zo(a)Pyrene on the Reproductive System of Male Sprague Dawley Rats in V ivo. Journal of Hazardous Materials, 186,
Christ ensen , A. K., Komorowski, T. E., Wilson, B., Ma, S. F., & Stevens 3rd, R. W. (1985). The Distribution of Serum Al-
bumin in the Rat Testis, Studied by Electron Microscope Immunocytochemistry on Ultrathin Frozen Sections. Endocri-
nology, 116 , 1983-199 6. http://dx.doi.org/10.1210/endo-116-5-1983
Cui, Z., Liu, J., Li, P., & Cao, J. (2010). Male Reproductive and Behavior Toxicity in Rats after Subchronic Exposure to Or-
ganic Extracts from Jialing River of Chongqing, China. Birth Defects Research. Part B, Developmental and Reproductive
Toxicology, 89, 34-42.
Fujimori, C., Ogiwara, K., Hagiwara, A., & Takahashi, T. (2012). New Evidence for the Involvement of Prostaglandin Re-
ceptor EP4b in Ovulation of the Medaka, Oryzias Lati pes. Molecular and Cellular Endocrinology, 362, 76-84.
Huang, Y., Chen, J.-A., & Sh u, W. (2010). Subchronic Toxic Effects of Exposure in Combination to Dibutyl Phthalate and
Benzo[a]Pyrene on Spermatogenesis in Male Rats. Journal of Environmental Health, 27, 215-218.
Inyang, F., Ramesh, A., Kopsombut, P., Niaz, M. S., Hood, D. B., Nyanda, A. M. et al. (2003). Disruption of Testicular Ste-
roidogenesis and Epididymal Function by Inhaled Benzo(a)Pyrene . Reproductive Toxicology, 17, 527 -537.
Kavlock, R., Boekelheide, K., Chapin, R., Cunningham, M., Faustman, E., Foster, P. et al. (2002). NTP Center for the Eval-
uation of Risks to Human Reproduction: Phthalates Expert Panel Report on the Reproductive and Developmental Toxicity
of Di-n-butyl Phthalat e. Reproductive Toxicology, 16, 489 -527. http://dx.doi.org/10.1016/S0890-6238(02 )00 033 -3
Klipper, E., Levit, A., Mastich, Y., Berisha, B., Schams, D., & Meidan, R. (2010 ). Induction of Endothelin-2 Expression by
Luteinizing Hormone and Hypoxia: Possible Role in Bovine Corpus Luteum Formation. Endocrinology, 151, 1914-1922.
Liao , X., Terada, N., Ohno, N., Li, Z., Fujii, Y., Baba, T. et al. (2006). Immunohistochemical Study of Serum Albumin in
Normal and Cadmium-Treated Mouse Testis Organs by “in Vivo Cryotechnique”. Histology and Histopathology, 21, 35-
Mayati, A., Levoin, N., Paris, H., N’Di aye , M., Courtois, A., Uriac, P. et al. (2012 ). Induction of Intracellular Calcium Con-
centration by Environmental Benzo(a)Pyrene Involves a Beta2-Adrenergic Receptor/Adenylyl Cyclase/Epac-1/Inositol
1,4,5-Trisphosphate Pathway in Endothelial Cells. The Journal of Biological Chemistry, 287, 4041-40 5 2.
Qing, Z. (20 09). Effects of Di-n-butyl Phthalate on Testicular Insl3 mRNA Expression in Pubertal Male Rats. Carcinogene-
sis, Teratogenesis and Mutagenesis, 21, 0161-01 64 .
Qiu, Z.-Q., Shu , W.-Q., Chen, J., Luo, J.-H., Yang, L., Zheng, Y.-K. et al. (2009). Combined Effect of Di-n-butyl Phthalate
and Benzo(a) Pyrene on Vimentin and α-Tubulin in Rat Sertoli Cells. Carcinogenesis, Teratogenesis and Mutagenesis, 21,
Rame s h, A., Inyang, F., Lunstra, D. D., Niaz, M. S. , Kopsombut, P., Jones, K. M. et al. (2008). Alteration of Fertility End-
points in Adult Male F-344 Rats by Subchronic Exposure to Inhaled Benzo(a) Pyr ene. Experimental and Toxicologic Pa-
thology, 60, 269-280. http://dx.doi.org/10.1016/j.etp.2008.02.010
Shultz, V. D., Phillips, S., Sar, M., Foster, P. M., & Ga i d o, K. W. (2001). Altered Gene Profiles in Fetal Rat Testes after in
Utero Exposure to Di(n-butyl) Phthalate. Toxicological Sciences, 64, 23 3-242. http://dx.doi.org/10.1093/toxsci/64.2.233
Y. J. Huang et al.
Sipinen, V., Laubenthal, J., Baumgartner, A., Cemeli, E., Linschooten, J. O., Godschalk, R. W. et al. (2010 ). In Vitro Evalu-
ation of Baseline and Induced DNA Damage in Human Sperm Exposed to Benzo[a] Pyrene or Its Metabolite Ben-
zo[a] Pyre ne -7 ,8 -di ol -9,10-epoxide, Using the Comet Assay. Mutagenesis, 25, 417-425.
Zhu, X. B., Tay, T. W., Andriana, B. B., Alam, M. S., Choi, E. K., Tsunekawa, N. et al. (2010). Effects of Di -iso-butyl
Phthalate on Testes of Prepubertal Rats and Mice. Okajimas Folia Anatomica Japonica, 86, 129-136.