|Year : 2014 | Volume
| Issue : 1 | Page : 51-56
Protective effects of resveratrol against di-n buthyl phthalate induced toxicity in ductus epididymis and ductus deferens in rats
Erhan Sahin1, Celal Ilgaz2, Deniz Erdogan2, Gülnur Take2, Güleser Göktas2
1 Histology and Embryology Department, Eskisehir Osmangazi Universty, Medicine School, Turkey
2 Histology and Embryology Department, Gazi Universty Medicine School, Turkey
|Date of Submission||13-May-2013|
|Date of Decision||08-Jul-2013|
|Date of Acceptance||11-Nov-2013|
|Date of Web Publication||16-Jan-2014|
Histology and Embryology Department, Eskisehir Osmangazi Universty, Medicine School
Source of Support: None, Conflict of Interest: None
Objective: This study aimed to observe the possible protective effects of resveratrol (RSV) against damage induced by di-n-butylphthalate (DBP), on the ductus epididymis and deferens in rats.
Materials and Methods: Six groups of rats were used in the experiment: Group 1: Control group; Group 2: Solvent (carboxymethylcellulose (CMC), 10ml/kg); Group 3: 500 mg/kg/day DBP; Group 4: 500 mg/kg/day DBP+20 mg/kg/day RSV; Group 5: 1000 mg/kg/day DBP; Group 6: 1000mg/kg/day DBP + 20 mg/kg/day RSV. Groups were treated by gavage for 30 days. Immunohistochemical, electronmicroscopic and histomorphometric examinations were carried out in the epididymis and deferens.
Results: In the ductus epididymis and deferens mitochondrial crystolysis, exfoliation of the stereocilia and openings in lateral surface increased with DBP dosage, but these structures were recovered with RSV. DBP reduced the epithelial height of epididymis and vas deferens. Lumen dilatation was observed in both tissues. These disorders may lead to dysfunction of epithelial absorption. In the TUNEL examinations in both tissues, there were no apoptotic cells or apoptotic bodies.
Conclusion: In conclusion, DBP administration caused structural degeneration in the epididymis and deferens, parallel to dose evaluation and RSV can reverse these changes with its protective effects.
Keywords: Di n buthyl phthalate, Ductus deferens, Ductus epididymis, Resveratrol
|How to cite this article:|
Sahin E, Ilgaz C, Erdogan D, Take G, Göktas G. Protective effects of resveratrol against di-n buthyl phthalate induced toxicity in ductus epididymis and ductus deferens in rats. Indian J Pharmacol 2014;46:51-6
|How to cite this URL:|
Sahin E, Ilgaz C, Erdogan D, Take G, Göktas G. Protective effects of resveratrol against di-n buthyl phthalate induced toxicity in ductus epididymis and ductus deferens in rats. Indian J Pharmacol [serial online] 2014 [cited 2020 Aug 4];46:51-6. Available from: http://www.ijp-online.com/text.asp?2014/46/1/51/125167
| » Introduction|| |
An increasing number of reports show that human semen quality has decreased. However the incidence of male genital tract abnormalities and infertility has increased in last 50 years.  Exposure to environmental factors, such as chemical pollutants, which are introduced and spread by human activity, have been suggested as a potential risk for abnormalities and infertility.  There is an increasing awareness that chemicals can adversely affect human semen quality. Many environmental agents have been investigated for their associations with male reproductive function, including endocrine disrupters, pesticides,  heavy metals, , compounds associated with plastics (bisphenols A and phthalates  ) and organochlorine compounds. 
Endocrine disrupters are substance or substance mixtures that change the function and development of the endocrine system. Phthalates are the primary chemical endocrine disrupters. The widespread presence and intensive use of phthalates have encouraged studies on the toxic effects of these chemicals. ,
Di-n-butylphthalate, which causes changes in the function and development of the endocrine system, is also a ubiquitous environmental pollutant and used for plastic coating and in the cosmetic industry.  Similar to other phthalates, DBP is not covalently bound to the plastic matrix and therefore, they leach out, migrate or evaporate from plastics into the environment, food or other materials.  In general, exposure to humans occurs primarily through contaminated food and water, which may have been in contact with plastic, adhesives or other packing materials that contain DBP. ,
In recent years, it has become evident that DBP has toxic effects on the male reproductive system. , These toxic effects are hypospadias, cryptorchidism, and testicular atrophy (which is present at birth), low sperm counts, sexual dysfunction, and testicular germ cell cancer, which manifests in young adulthood. , Evidence suggests that DBP is toxic throughout all phases of development. , DBP and its metabolites inhibit the synthesis of testosterone through the inhibition of cholesterol transport, and the synthesis of gene products related to testosterone synthesis. ,
Recent studies suggest that the polyphenolic compounds contained in fruits and vegetables are very important for the prevention and reduction of many diseases. RSV is a polyphenol that is synthesized by plants to fight fungus and traumatic injury. It is found in about 70 plants, particularly in red wine and grapes, pineapple and peanuts. , RSV has many biological and pharmacological properties, such as antioxidant effects, intrinsic antioxidant capacity and induces the expression of many antioxidants. RSV also exhibits a broad range of biological activities, including anti-inflammatory, anti-viral and anti-tumoral properties. ,
Some recent in vivo studies in animal models demonstrated that a long-term application of RSV increases the amount of testosterone and gonadotropins in the blood and enhances sperm production by stimulating the hypothalamic-pituitary-gonadal axis without inducing adverse effects. ,
After formation in the seminiferous tubules, sperm are non-motile, and they cannot fertilize ova. However, after the sperm have been in the epididymis for 18 to 24 h, they develop the capacity for motility. The epididymis and ductus deferens are known to play an important role in providing a microenvironment for sperm maturation and sperm storage. The epithelial cells of the epididymis and ductus deferens synthesize and secrete numerous proteins for the microenvironment and male reproduction activities, including the initiation of sperm maturation, sperm-oocyte recognition and the acrosome reaction, directly or indirectly. No studies have explored the protective effect of RSV on the epididymis and ductus deferens after exposure to DBP; therefore this study aimed to observe the possible protective effects of RSV against the damage of an endocrine disruptor, DBP, on the epididymis and ductus deferens.
| » Materials and Methods|| |
Drugs and Chemicals
DBP (purity of 99.8%), RSV, corn oil, carboxymethyl cellulose (CMC) were purchased from Sigma Chemical Co. (St. Louis MO, USA). ApopTag® plus peroxidase ύn situ apoptosis detection kit purchased from Chemicon (Chemicon International, USA). Liquid DAB-Plus Substrate Kit purchased from In Vitro Gen (Camarillo, USA).
Healthy male pre-pubertal (25 days old) Wistar rats were obtained from the Gazi University, Laboratory Animal Breeding and Experimental Research Center animal experimental protocols were performed in accordance with guidelines issued by the Local Institutional Committee for the Ethical Use of Animals of Gazi University. The animals were housed in individual polycarbonate cages at a temperature-controlled room (21-24°C) and humidity of 30-40% under 12 h light/12 h dark cycle and animals were fed with standard diet and water ad libitum throughout the experiment. All animals were acclimatized for a minimum period of 1 week prior to the beginning of the study.
Experimental Protocol and Treatment
The animals were randomly divided into six experimental groups (n = 6 animal, in each group).
Applications were made by oral gavage between 08.30 a.m and 11.30 a.m for 30 days. RSV 20mg/kg/day was prepared freshly by dissolving in 10ml/kg CMC. DBP 500 and 1000mg was prepared freshly in 1mL/kg corn oil. The dose of phthalate was based on several previous studies that reported observation of adverse effects on reproductive system at a dosage of 500-1000 mg/kg/day. ,,, CMC was prepared freshly (0.9%). The doses were adjusted for recorded body weight changes during the study. After a 30 day treatment, the rats were intraperitoneally injected with 0.08mL of a 1:8 mixture of 2% xylazine (Rompun; Bayer) and 2.5% ketamine hydrochloride (Ketalar; Parke-Davis) for anesthesia. Rats were sacrificed by decapitation. The epididymises and ductus deferenses were removed immediately, and washed with sodium phosphate buffer (pH 7.4). Tissues were prepared for electron and light microscopic investigations.
Electron Microscopic Studies
Tissues of all groups were fixed in phosphate-buffer containing 2.5% glutaraldehyde (Sigma-Aldrich Co.) for 2-3 h, post-fixed in 1% osmium tetraoxide (Sigma-Aldrich Co.) and dehydrated in a series of graded alcohols (50, 60, 70, 80, 90, 96 and 100% ethanol). After passing through propylene oxide (Sigma-Aldrich Co.), the specimens were embedded in Araldite CY 212 (Ciba-Geigy), (2-dodecen-1-yl)succinic anhydride (Sigma-Aldrich Co.), benzyldimethyl amine (Poly Sciences Inc.) and dibutylphtalate (Sigma-Aldrich Co.). The semi-thin sections were stained with toluidine blue (Sigma-Aldrich Co.) and examined with a photomicroscope (BH2 Olympus, Japan). After the selection of appropriate specimens, thin sections were cut and stained with uranyl acetate (ProSciTech) and lead citrate (Sigma-Aldrich Co.). They were examined by an electron microscope (Carl Zeiss EM 900, Germany).
The TUNNEL Assay
Apoptosis in the epididymis and vas deferens was demonstrated in situ by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) staining assay. In order to determine the number of apoptotic and oncotic SGCs, sections were stained with in situ cell death detection kit (Apop Tag Plus Peroxidase In Situ Apoptosis Detection Kit, S7101, Lot: 25050483, Chemicon, Temecula, CA, USA). Slices were incubated with 20 μg/ml proteinase K. Washing with PBS was performed in every stage. Endogenous peroxidase activity was blocked with 3% H 2 O 2 . After washing with PBS, sections were incubated with equilibration buffer and TdT enzyme (77 μl reaction buffer + 33 μl TdT enzyme mix, 1 μl TdT enzyme) at 37°C. Working strength stop/wash buffer (1:10) was applied at room temperature and slices were incubated with anti-digoxigenin conjugate for 30 min. After washing with PBS the sections were stained with 3'3 diaminobenzydine components to detect TUNEL positive cells and then counter stained with methyl green. The sections were examined with a photomicroscope (BH2 Olympus, Japan).
Histological and Morphometric Analysis
Paraffin embedding, 4-5 μm sectioning, mounting and hematoxylin and eosin staining were completed according to standard techniques. The slides were visualized and the images were captured digitally by using a photo-light microscope (DM 4000 B, Leica, Wetzlar, Germany) attached to a camera (DFC280 Plus, Leica, Wetzlar, Germany). Morphometric analysis was performed on for all animals (n = 6 per treatment group) with using image analyzing software (Leica Q Win V3 Plus Image). Epithelial height, lumen and tubule total diameter were measured in epididymis and ductus deferens at 6 area for 6 experimental subjects per group.
Statistical analysis is carried out by using SPSS statistical software, version 16.0.for Windows (SPSS Inc., Chicago, IL, USA). All data were tested for normal distribution (Shapiro-Wilks) for defining whether the results should be analyzed parametrically or non-parametrically. P < 0.05 was regarded as statistically significant for all statistical analysis.
| » Results|| |
Electron Microscopy Results
In the control group, stereocilia at the apical cell surface and cell-cell adhesion units were normal. Basal interdigitations were obvious at the basal surface of the epithelium. In Group 2, cell-cell adhesion units were normal. There was a general presence of crystolysis in the mitochondria. On the apical surface of the cell, the arrangement of the stereocilia had an abnormal histological appearance. The stereocilia were mostly lost and some fused to create groups. In Group 3, the ultrastructure was more degenerative than Group 2. The stereocilia were mostly lost and some were fused. The epithelial cells were arranged by lighter and darker cytoplasm. The mitochondrial crystolysis was common in the epithelium, especially in the darker cytoplasm cells. Small and large vacuoles attract darker cytoplasm cells. There were openings in the cell-cell adhesion units in some areas of the epithelium. In Group 4, the ultrastructure was normal in comparison to Groups 2 and 3. While some cells showed mitotic cell divisions, some darker cytoplasm cells showed increased mitochondrial crystolysis. Cell-cell adhesion units were normal. In Group 5, the ultrastructure was the same as Group 3. A marked loss of the stereocilia and cytoplasmic particles was observed on the surface of the epithelium. Apoptotic bodies were detected in some areas. Mitochondrial degenerations, crystolysis and swelling were prominent. Some darker cytoplasm cells had a heterochromatic nucleus. Compared to Group 5, the stereocilia and cytoplasmic particles in Group 6 were lower. The appearance of cytoplasm and cell-cell adhesion units was similar to the control group [Figure 1].
In the control group, stereocilia and cell-cell adhesion units were normal. In some areas, mitochondrial degenerations, crystolysis and swelling were prominent. In Group 2, the ultrastructure was similar to the control group. However, in some areas stereocilia were lost and some were thrown into the lumen. In Group 3, the epithelial cells were arranged by lighter and darker cytoplasm. Mitochondrial degenerations were prominent in lighter and darker cytoplasm cells. The ultrastructure of the stereocilia was degenerated. The conjunction units on the sides of the cells were normal. In Group 4, the ultrastructure was similar to the control group. In Group 5, the characteristics of the surface epithelium were noted missing. The epithelial cells were arranged lighter and darker cytoplasm. Mitochondrial degeneration and crystolysis were prominent. When Group 6 was compared to Group 5, in terms of cytoplasmic structure, the ultrastructure of Group 6 was similar to the control group, but apical surface of the epithelium was noted missing [Figure 2].
TUNEL results in ductus epididymis and ductus deferens
In the TUNEL examinations, in both tissues, there were no apoptotic cells or apoptotic bodies [Figure 3] and [Figure 4].
|Figure 4: TUNEL examination of ductus deferens in Control, CMC, 500 DBP, 500 DBP + Resveratrol, 1000 DBP and 1000 DBP + Resveratrol. E: Epithelium, *: Connective tissue, S: Stereocilia. Scale bar: 50 Ām|
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Epithelial height, lumen diameter, and tube diameter were statistically evaluated in the epididymis. Additionally, epithelial height, muscle thickness and diameter of the vas deferens were evaluated in the ductus deferens.
The epithelial height of the control group was significantly higher than Groups 3 and 5 ( a P < 0.01). The epithelial height of Group 3 was significantly lower than Group 4 ( b P < 0.01). Moreover, the epithelial height of Group 5 was significantly lower than Group 6 ( b P < 0.01). The lumen diameter of the control group was significantly lower than the Groups 3 and 5 ( c P < 0.01). The lumen diameter of the group 4 was significantly higher than group 3 ( g P < 0.01) and the lumen diameter of the group 6 was significantly higher than group 5 ( h P < 0.01) [Table 1].
|Table 1: Morphometric analysis of ductus epididymis sections in Control, CMC, 500 DBP, 500 DBP+RSV, 1000 DBP and 1000 DBP+RSV groups. Results are expressed as mean ± SEM|
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The epithelial height of the control group was significantly higher than that of Groups 3 and 5 ( e P < 0.01). The epithelial height of Group 3 was significantly lower than Group 4. ( F P < 0.01) Additionally, the epithelial height of Group 5 was significantly lower than Group 6( Y P < 0.01). The diameter of the control group was significantly higher than that of Groups 3 and 5 ( m P < 0.01). The diameter in Group 4 was significantly higher than Group 3 ( n P < 0.01)[Table 2].
|Table 2: Morphometric analysis of ductus deferens sections in Control, CMC, 500 DBP, 500 DBP + RSV, 1000 DBP and 1000 DBP+RSV groups. Results are expressed as mean ± SEM.|
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| » Discussion|| |
Until now, studies related to DBP were carried out on adult rats during pregnancy and the lactation period, , but this study focuses on the degenerative effects of DBP during adolescence until youth to observe developmental toxicities. Kohn et al., have shown that 95% of people are exposed to 10 μg DBP per a day. In particular, women between the ages of 20-40 are exposed to DBP at a much higher increment than the other gender and other ages.  Zhou et al., in their study of adult rats, reported that 100, 250 and 500 mg/kg/day doses of DBP create a dose-dependent toxicity in the epididymis. In addition, they demonstrated in that epididymal weight, alpha-glucosidase, and glutathione peroxidase enzyme activity significantly decreased in a group that was administered 500 mg/kg/day of DBP. As a result of their study, they determined that DBP caused oxidative stress in the epididymis of adult rats. 
The current study investigated the effects of increasing the DBP dose on the epididymis and ductus deferens in rats, ranging in age from adolescent to youth. Parallel to the findings of Zhou et al., this study determined that an increase in the DBP dose creates a dose-dependent toxicity in the epididymis and ductus deferens. It was observed that an increased DBP dose reduced the epithelial height, and increased the lumen diameter of the epididymis.
Zhu et al., showed that diisobutyl phthalate (DIBP), used in place of DBP in plastics, in pre-pubertal (21-day-old) Sprague-Dawley rats, caused apoptosis in spermatogenic cells, disruption of vimentin filaments in the Sertoli cells More Details and testicular atrophy. 
In contrast to Zhu et al., this study revealed that after the investigation of DBP apoptotic effect with the TUNEL method at the ductus epididymis and ductus deferens, DBP not increase apoptosis in these two organs. Wellerson et al., investigated the effects of 100mg/kg/day DBP exposure to rat from pregnancy to lactation. Fetal testes of the DBP treated group showed Leydig-cell clusters, presence of multinucleated germinative cells, and an increase of the interstitial component. The study revealed that DBP treatment did not markedly affect relative proportions of epithelial, stromal or luminal compartments in the epididymis; sperm counts in the testis and epididymis; sperm transit time; or sperm morphology and motility in rats. However, the current study observed degenerative changes in the testis, epididymis and sperm in these organs. 
Zhang et al., reported that exposure to 250 mg/kg/day DBP causes testicular atrophy, underdeveloped or absent epididymis, undescended testes, obvious decline of epididymal sperm parameters and a decrease of organ/body weight ratio of the epididymis and prostate.  In the present study, in groups that were administered 500mg and 1000mg/kg DBP by oral gavage, sperm head abnormalities and settlement disorders were observed in the epididymis. This finding, parallel to the previous studies, reveals that DBP administration affects the myogenesis and spermatogenesis stages in the testis. Thus, it can be concluded that sperm defects in the lumen of the epididymis may be associated with testis toxicity. In the present study, it was also observed that DBP dose-dependent degeneration of stereocilia at the epididymis and ductus deferens may lead to abnormality of sperm preservation and supply.
After reviewing the literature, it has been found that the potential effects of DBP on the development of reproductive system of embryos of pregnant rats have been examined in most studies. There is almost no study carried out about infants, pre-puberty and adults. No study has been encountered about the effect of DBP on testis and the potential protective effect of RSV in these conditions. Our study differs from other studies in that it examines the potential effects that can arise after exposure to DBP particularly from puberty to adolescence.
The literature reveals that RSV has many protective or therapeutic properties on many organs. Thus, in this study, RSV was selected to be a protective agent against the adverse effects of DBP. As a result of the evaluations that were assessed from apoptosis with TUNEL examinations and research with electron microscopy reveal that DBP induced toxicity was more effective on epithelium and stereocilia than connective tissue. It may lead to abnormality of sperm preservation and supply.DBP reduced the epithelial height of epididymis and vas deferens. Lumen dilatation was observed in both tissues. These disorders may lead to disfunction of epithelial absorption. The epithelial cells of the epididymis and ductus deferens synthesize and secrete numerous proteins for the micro-environment and male reproduction activities, including the initiation of sperm maturation, sperm-oocyte recognition and the acrosome reaction and RSV is highly protective on epithelium with low doses of DBP and therefore RSV may use safely as an alternative therapeutic agent in low doses of DBP. However in high doses of DBP, RSV is efficient too but inadequate. Through these findings, in addition to the fact that RSV is a protective antioxidant against DBP, it is concluded that the agent-applied DBP in increasing doses should be adjusted to the dose level of applied RSV .
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]