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ARTICLES
Year : 2013  |  Volume : 26  |  Issue : 1  |  Page : 23-34

The toxic effect of phthalate on testes of albino rats and the possible protective role of curcumin


Department of Anatomy and Embryology, Faculty of Medicine, Menoufia University, Alexandria, Egypt

Date of Submission20-Feb-2013
Date of Acceptance09-Apr-2013
Date of Web Publication26-Jun-2014

Correspondence Address:
Rasha M.A. Salama
MSc, 20 Shady Abd-El Salam from Khaled Ebn El Waleed, Alexandria
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.7123/01.MMJ.0000429688.04299.73

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  Abstract 

Objectives

The aim of this study was to determine the toxic effect of di-n-butylphthalate (DBP) on the testes of developing and adult albino rats and to establish the possible protective role of curcumin.

Background

DBP is an endocrine-disrupting chemical that disrupts the growth of normal male reproductive organs of adult and developing rats after prenatal exposure.

Methods

The rats used in this study received DBP alone as well as in combination with curcumin and were subjected to biochemical (measuring the level of testosterone), histological, immunohistochemical, and morphometric (analysis of the mean diameter of the seminiferous tubules) studies.

Results

Administration of DBP to rats at a dose of 500 mg/kg dissolved in 1 ml/kg corn oil by daily gavage significantly reduced (P<0.001) the serum levels of testosterone in developing and adult rats compared with control rats. DBP-treated rats showed marked atrophy of the seminiferous tubules with apoptosis, exfoliation, and abnormal regeneration of the spermatocytes, with a highly significant decrease (P<0.001) in the mean diameter of the seminiferous tubules compared with that in the control and combined DBP and curcumin groups. DBP-induced reproductive toxicity was reversed by the addition of curcumin.

Conclusion

Exposure to DBP led to more pronounced testicular damage and impaired spermatogenesis in prepubertal and adult male rats. These changes can be prevented by addition of curcumin, which induced restoration of spermatogenesis.

Keywords: Department of Anatomy and Embryology, di-n-butylphthalate, germinal epithelium, seminiferous tubules, testis


How to cite this article:
Salama RM, Mansour FK, El-Safti F, El-Sherif NM. The toxic effect of phthalate on testes of albino rats and the possible protective role of curcumin. Menoufia Med J 2013;26:23-34

How to cite this URL:
Salama RM, Mansour FK, El-Safti F, El-Sherif NM. The toxic effect of phthalate on testes of albino rats and the possible protective role of curcumin. Menoufia Med J [serial online] 2013 [cited 2020 Feb 17];26:23-34. Available from: http://www.mmj.eg.net/text.asp?2013/26/1/23/135423


  Introduction Top


Di-n-butylphthalate (DBP) is one of the most widely studied phthalate esters; it is considered to be an endocrine-disrupting chemical that has the potential to affect male reproduction 1. It disrupts the growth of normal reproductive organs in adult rats 2. In addition, it is known to induce adverse effects in the reproductive system of male rats after prenatal exposure, as it interferes with normal development of testes and the reproductive tract when exposure occurs during gestation 3. Some of these effects are likely caused by a reduction in testicular testosterone hormone (TH) production during late gestation 4.

Humans are exposed to DBP mainly through contaminated food, personal care products, and occupational sources 5.

Curcumin has been evaluated as a potential chemoprotective agent that has intrinsic antioxidant abilities to combat oxidative damage and the reduction in TH production induced by DBP 6.

This work aimed at studying the toxic effect of DBP on the testes of developing and adult albino rats and to establish the possible protective role of curcumin.


  Methods Top


Animal protocol

This study was carried out on 72 adult albino rats of both sexes [35 females and 37 males (30 males for experimental design and seven males for mating)]. The animals were divided into two main groups:

Group I consisted of 35 pregnant rats, subdivided into three groups: group IA, subdivided into negative control, curcumin, and corn-oil subgroups; group IB (DBP group); and group IC (combined DBP and curcumin group). The male pups were sacrified 1 day or 2, 4, or 6 weeks after birth.

Group II consisted of 30 adult male albino rats, subdivided into four groups: group IIA, subdivided into negative control, curcumin, and corn-oil subgroups; group IIB (DBP group); group IIC (combined DBP and curcumin group); and group IID (recovery group).

Chemicals

DBP was administered at a dose of 500 mg/kg dissolved in 1 ml/kg corn oil by gavage daily. Curcumin, available in the form of a powder, was administrated at a dose of 200 mg/kg/day dissolved in1 ml/kg corn oil by daily gavage. Corn oil, available in the form of an oily solution, was used as a solvent for DBP and curcumin (Sigma Chemical Co., Cairo, Egypt).

Biochemical study

Blood samples were collected from the tail vein of the rat and used for the measurement of the levels of TH to determine the function of the interstitial cells of Leydig the blood was centrifuged and plasma was used for hormonal assays 6.

Histological and immunohistochemical studies

Rats were killed by decapitation; the abdomen and scrotum of each animal was opened using a pair of scissors and the testes were extracted. The testes were removed immediately, fixed in 10% formalin saline for 24 h, and then processed to obtain paraffin blocks. Sections of 4–6 μm thickness were cut using a microtome and stained with hematoxylin and eosin (H&E) stain and by immunohistochemical staining using caspase-3 and proliferating cell nuclear antigen (PCNA).

Morphometric study

Five different H&E-stained sections from five different rats were examined in each subgroup to measure the two diagonal diameters of the seminiferous tubules. This was performed at the Department of Histochemistry and Cell Biology, Alexandria University, using the Lecia Qwin 500 image analyzer computer system (UK).

Statistical analysis

The level of TH and the diameter of the seminiferous tubules in the DBP group were compared with those in the other groups using the Mann–Whitney and Kruskal–Wallis tests. The data collected were tabulated and analyzed using SPSS software (version 17.0 on an IBM compatible computer; SPSS Inc., Chicago, Illinois, USA).


  Results Top


Biochemical results

The administration of DBP highly significantly decreased serum TH levels (P<0.001) in developing and adult rats compared with the control rats. Coadministration of curcumin with DBP significantly increased serum TH levels compared with administration of DBP alone [Table 1] and [Table 2]. Serum TH levels were significantly increased (P<0.05) in 6-week-old treated rats compared with 1-day-old treated rats [Table 3].
Table 1: Statistical comparison between subgroup IAa (negative control subgroup), group IB (di-n-butylphthalate group), and group IC (combined di-n-butylphthalate and curcumin group) of group I (pregnant animals) in different sequences as regards serum testosterone hormone level

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Table 2: Statistical comparison between subgroup IIAa (negative control subgroup), group IIB (di-n-butylphthalate group), group IIC (combined DBP and curcumin group), and group IID (recovery group) of group II (adult animals) as regards serum testosterone hormone level

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Table 3: Statistical comparison between subgroup IAa (negative control subgroup), group IB (di-n-butylphthalate group), and group IC (combined di-n-butylphthalate and curcumin group) of 1-day-old rats and subgroup IAa (negative control subgroup), group IB (di-n-butylphthalate group), and group IC (combined di-n-butylphthalate and curcumin group) of 2-, 4-, and 6-week-old rats of group I (pregnant animals) in different sequences as regards serum testosterone hormone levels

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Table 4: Statistical comparison between subgroup IAa (negative control subgroup), group IB (di-n-butylphthalate group), and group IC (combined di-n-butylphthalate and curcumin group) of group I (pregnant animals) in different sequences as regards morphometry of the mean diameter of the seminiferous tubules

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Table 5: Statistical comparison between subgroup IIAa (negative control subgroup), group IIB (di-n-butylphthalate group), group IIC (combined di-n-butylphthalate group and curcumin group), and group IID (recovery group) of group II (adult animals) as regards morphometry of the mean diameter of the seminiferous tubules

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Histological and immunohistochemical results

Group I (pregnant animals)

Group IA (control group): The testes comprised many rounded or oval seminiferous tubules that increased in size with age, surrounded by well-defined basal lamina and separated from each other by interstitial tissue that decreased in amount with age. All these structures were covered by the testicular coat, which consisted of the outer tunica albuginea and the inner tunica vasculosa, consisting of large blood vessels scattered in loose connective tissue [Figure 1]a and b.
Figure 1: (a) A photomicrograph of a section of the testis of a 1-day-old albino rat of control group (subgroup IAa) showing many rounded to oval seminiferous tubules surrounded by well-defined basal lamina (arrow), separated by interstitial tissue (I) and covered by a testicular capsule. The capsule consisted of an outer layer [the tunica albuginea (TA)], which comprised connective tissue fibers and spindle-shaped cells with elongated deeply stained nuclei. Inner to it the tunica vasculosa (TV) showed large blood vessels (BV) in the loose connective tissue (H&E, ×400). (b) A photomicrograph of a section of the testis of a 2-week-old albino rat of the control group (subgroup IAa) showing a lumen starting to appear in some seminiferous tubules (L). The tubules were closely packed, separated by small amount of interstitial tissue (I) and surrounded by well-defined basal lamina (arrow). Note the decreased thickness of the testicular coat (T) (H&E, ×400). (c) A photomicrograph of a section of the testis of a 4-week-old albino rat of the control group (subgroup IAa) showing stratification and an increase in the number of cell layers of the spermatogenic epithelium in the wall of the seminiferous tubules around a well-developed lumen (L). The germ epithelium comprises spermatogonia (green arrow), primary spermatocytes (yellow arrow), and early spermatids (arrow head) near the lumen and Sertoli cells attached to the basement membrane (red arrow). The tubules were closely packed with small amounts of interstitial tissue (I) in between and were surrounded by well-defined basal lamina (black arrow; H&E, ×400). (d) A photomicrograph of a section of the testis of a 6-week-old albino rat of the control group (subgroup IAa) showing parts of two seminiferous tubules surrounded by well-defined basal lamina (two black arrows) with stratification of their lining epithelium and marked increase in the tubular cell layers comprising spermatogonia (green arrow), primary spermatocytes (yellow arrow), and early spermatids (black arrow) with the appearance of a new type of cell near the lumen (L), assumed to be late spermatids (arrow head). Sertoli cells appeared attached to the basement membrane and their nuclei were triangular in shape (blue arrow; H&E, ×1000 oil immersion).

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The lumen started to appear in the seminiferous tubules of 2-week-old rats [Figure 1]b and became well developed in 4- and 6-week-old rats [Figure 1]c and d.

The lining epithelium of the seminiferous tubules in 1-day-old rats consisted of supporting cells, which were small, rounded, or elongated in shape with oval deeply stained nuclei that were located peripherally along the basal lamina, and gonocytes, which were fewer in number, larger in size, and spherical in shape, with large lightly stained vesicular nuclei, a prominent nucleoli, and a pale stained cytoplasm that occupied the central portion of the tubules [Figure 1]a. In 2-week-old rats, two new types of cells appeared between the supporting cells: spermatogonia type A cells, which appeared along the basal lamina, had a rounded contour, were smaller in size, and had deeply stained nuclei and spermatogonia type B, which appeared near the lumen, had a rounded contour, and had nuclei that contained course chromatin clumps along the nuclear membrane. Gonocytes were rarely detected [Figure 1]b.

In 4-weeks-old rats, there was increased stratification of the lining epithelium with appearance of the primary spermatocytes, which are large cells with large round nuclei containing clumped chromatin and the early spermatid near the lumen of the tubules which was characterized by having rounded, or polygonal contour, scanty cytoplasm and fine chromatin network in their nuclei. Sertoli cells were predominant and appeared as elongated cells wedged between the spermatogenic cells. They contained basal elongated or pyramidal pale nuclei [Figure 1]c. Similar findings were observed in 6-week-old rats, with the appearance of a new type of cell near the lumen, which was assumed to be a late spermatid. Spermatids are elongated cells with deeply stained nuclei [Figure 1]d.

There was weak immunoreactivity for caspase-3 in the 1-day-old and 2-week-old rats and moderate immunoreactivity for caspase-3 in the 4- and 6-week-old rats in the germinal epithelium and interstitial cells, and the nuclei of all germ cells were positive for PCNA.

Group IB (DBP group): In the 1-day-old rats, the testes are formed of many irregular seminiferous tubules surrounded by disrupted basal lamina and separated from each other by wide interfollicular spaces with destructed interstitial tissue and clustering of Leydig cells [Figure 2]a.
Figure 2: (a) A photomicrograph of a section of the testis of a 1-day-old albino rat of the group IB [di-

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The supporting cells and gonocytes were detected within the seminiferous tubules, with appearance of sloughed germ cells near the center of some tubules. The gonocytes were increased in number as compared with that in the control group [Figure 2]a.

Immunostaining of the testes revealed a moderate immunoreactivity for caspase-3 in the germinal epithelium and interstitial cells [Figure 3]a and a negative reaction for PCNA in the nuclei of supporting cells and gonocytes [Figure 4]a.
Figure 3: (a) A photomicrograph of a section of the testis of a 1-day-old albino rat of group IB (DBP group) showing moderate immunoreactivity for caspase-3 in the germinal epithelium (black arrow) and interstitial cells (red arrow) of the seminiferous tubules (immunohistochemistry caspase-3, ×400). (b) A photomicrograph of a section of the testis of a 2-week-old albino rat of group IB [di-

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Figure 4: (a) A photomicrograph of a section of the testis of a 1-day-old albino rat of group IB (DBP group) showing seminiferous tubules with negative proliferating cell nuclear antigen (PCNA) immunostaining in the nuclei of supporting cells (red arrow) and gonocytes (blue arrow; PCNA immunostaining, ×400). (b) A photomicrograph of a section of the testis of a 2-week-old albino rat of group IB (DBP group) showing seminiferous tubules with a positive PCNA immunostaining in the nuclei of the spermatogonia type A (red arrow) and a negative PCNA immunostaining in the nuclei of the spermatogonia type B (blue arrow; PCNA immunostaining, ×400). (c) A photomicrograph of a section of the testis of a 4-week-old albino rat of group IB (DBP group) showing seminiferous tubules with positive PCNA immunostaining in the nuclei of spermatogonia (red arrow) and negative PCNA immunostaining in the nuclei of primary spermatocytes (blue arrow) and early spermatids (green arrow; PCNA immunostaining, ×400). (d) A photomicrograph of a section of the testis of a 6-week-old albino rat of group IB (DBP group) showing seminiferous tubules with positive PCNA immunostaining in the nuclei of spermatogonia (red arrow) and negative PCNA immunostaining in the nuclei of primary spermatocytes (blue arrow), early spermatids (green arrow), and late spermatids (yellow arrow; PCNA immunostaining, ×400).

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In 2-week-old rats there was loss of stratification and disorganization of the lining epithelium. Some tubules showed disruption of the basal lamina and appearance of sloughed germ cells and necrotic debris in the center of the tubules [Figure 2]b]. The testicular capsule was thickened and the tunica vasculosa contained dilated congested blood vessels.

The interfollicular spaces appeared wide and showed destructed interstitial tissue, dilated congested blood vessel [Figure 2]b], Leydig cell hyperplasia, vacuolation, and the presence of hyalinized material.

There was strong immunoreactivity for caspase-3 in the germinal epithelium and interstitial cells [Figure 3]b, a positive reaction for PCNA in the nuclei of the spermatogonia of type A, and a negative reaction for PCNA in the nuclei of the spermatogonia of type B [Figure 4]b.

In 4-week-old rats, the seminiferous tubules appeared irregular and smaller in size, with disruption of the basal lamina, and showed loss of stratification and disorganization of the lining epithelium, where some germ cells appeared destructed and separated with the appearance of empty spaces, some appeared exfoliated toward the lumen with disappearance of the early spermatids [Figure 2]c], and others detached from the basement membrane.

The interfollicular spaces showed destructed interstitial tissues in some areas and congestion with some vacuolation in other areas [Figure 2]c. The testicular capsule was similar to that in two-week-old rats.

There were apoptotic bodies exhibiting strong immunoreactivity for caspase-3 in the germinal epithelium and interstitial cells [Figure 3]c, a positive reaction for PCNA in the nuclei of spermatogonia, and a negative reaction for PCNA in the nuclei of primary spermatocytes and early spermatids [Figure 4]c.

In 6-week-old rats, the seminiferous tubules showed shrinkage and irregularity with loss of the normal arrangement and stratification of the germinal epithelium of the seminiferous tubules, appearance of many apoptotic bodies (dark shrunken pyknotic nuclei surrounded by a hollow), appearance of exfoliated germ cells in the center, and disappearance of the late spermatids. The tubules were surrounded by disrupted basal lamina and appeared crowded in some areas and widely separated in others, whereas the interfollicular spaces showed destructed interstitial tissue, dilated congested blood vessels, vacuolation, the presence of hyalinized material, and hyperplasia of the Leydig cells [Figure 2]d.

Immunostaining of the testes showed apoptotic bodies with strong immunoreactivity for caspase-3 in the germinal epithelium and interstitial cells [Figure 3]d], a positive reaction for PCNA in the nuclei of spermatogonia, and a negative reaction for PCNA in the nuclei of primary spermatocytes, early spermatids, and late spermatids [Figure 4]d.

Group IC (combined DBP and curcumin group): The seminiferous tubules of the testes of the rats in group IC were restored to their normal shape; the spermatogenic epithelium was retained to its normal arrangement and thickness with reappearance of all the germ cells and restoration of spermatogenesis, as evidenced by the improvement in the biochemical, histological, immunohistochemical, and morphometric findings.

Group II (adult animals)

Group IIA (control group): This group had normal histological and immunohistochemical findings of the testes, similar to the rats in group IA. In addition, the spermatogenic epithelium of the adult rats was arranged in several layers; the spermatogonia appeared as small cells with round dark nuclei forming the basal layer; the primary spermatocytes appeared to be the largest cells in the section with central round nuclei containing clumped chromatin in addition to spermatozoal tails in lumen [Figure 5]a.

Immunostaining of the testes revealed a negative immunoreactivity for caspase-3 in the germinal epithelium and interstitial cells [Figure 6]a and a positive reaction for PCNA in the nuclei of spermatogonia, primary spermatocytes, and sperms [Figure 7]a.
Figure 6: (a) A photomicrograph of a section of the testis of an adult albino rat of the control group (subgroup IIAa) showing negative immunoreactivity for caspase-3 in the germinal epithelium (black arrow) and interstitial cells (red arrow) of the seminiferous tubules (immunohistochemistry caspase-3,×400). (b) A photomicrograph of a section of the testis of an adult albino rat of group IIB [di-n-butylphthalate (DBP) group] showing a moderate immunoreactivity for caspase-3 in the germinal epithelium (black arrow) and interstitial cells (red arrow) of the seminiferous tubules (immunohistochemistry caspase-3, ×400). (c) A photomicrograph of a section of the testis of an adult albino rat of group IIC (combined DBP and curcumin group) showing weak immunoreactivity for caspase-3 in the germinal epithelium (black arrow) and interstitial cells (red arrow) of the seminiferous tubules (immunohistochemistry caspase-3, ×400). (d) A photomicrograph of a section of the testis of an adult albino rat of group IID (recovery group) showing moderate immunoreactivity for caspase-3 in the germinal epithelium (black arrow) and interstitial cells (red arrow) of the seminiferous tubules (immunohistochemistry caspase-3, ×400).

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Figure 7: (a) A photomicrograph of a section of the testis of an adult albino rat of the control group (subgroup IIAa) showing seminiferous tubules with positive proliferating cell nuclear antigen (PCNA) immunostaining in the nuclei of spermatogonia (red arrow), primary spermatocytes (blue arrow), and sperms (green arrow; PCNA immunostaining, ×400). (b) A photomicrograph of a section of the testis of an adult albino rat of group IIB [di-n-butylphthalate (DBP) group] showing seminiferous tubules with positive PCNA immunostaining in the nuclei of spermatogonia (red arrow) and negative PCNA immunostaining in the nuclei of primary spermatocytes (blue arrow) and sperms (green arrow; PCNA immunostaining, ×400). (c) A photomicrograph of a section of the testis of an adult albino rat of group IIC (combined DBP and curcumin group) showing seminiferous tubules with positive PCNA immunostaining in the nuclei of spermatogonia (red arrow), primary spermatocytes (blue arrow), and sperms (green arrow; PCNA immunostaining, ×400). (d) A photomicrograph of a section of the testis of an adult albino rat of group IID (recovery group) showing seminiferous tubules with positive PCNA immunostaining in the nuclei of spermatogonia (red arrow) and primary spermatocytes (blue arrow) and negative PCNA immunostaining in the nuclei of sperms (green arrow; PCNA immunostaining, ×400).

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Group IIB (DBP group): The seminiferous tubules were surrounded by disrupted basal lamina and separated from each other by wide interfollicular spaces containing dilated congested blood vessel, showing vacuolation, the presence of hyalinized material, and hyperplasia of the interstitial Leydig cells with destructed and decreased amounts of interstitial tissue. Some tubules, in which sperms were absent, appeared distorted with a small diameter and a wide lumen [Figure 5]b.

There was abnormal or reduced spermatogenesis, characterized by loss of cellular architecture, a decrease in the height of the seminiferous epithelium, a decrease in the frequency of cells (spermatogonia, spermatocytes, and spermatids), total absence of spermatozoa in some tubules, appearance of empty spaces between spermatogenic cells, and detachment of the germ cells from the basement membrane in some tubules. Many spermatocytes were exfoliated in the lumen [Figure 5]b.

The testicular capsule was thickened, and the tunica vasculosa contained dilated, congested blood vessels.

Immunostaining of the testes revealed a moderate immunoreactivity for caspase-3 in the germinal epithelium and interstitial cells [Figure 6]b, a positive reaction for PCNA in the nuclei of spermatogonia, and a negative reaction for PCNA in the nuclei of primary spermatocytes and sperms [Figure 7]b.

Group IIC (combined DBP and curcumin group): Addition of curcumin elicited significant protective effects on the structure and function of testes, making them nearly normal [Figure 5]c, [Figure 6]c, and [Figure 7]c.

Group IID (recovery group): The testicular sections of this group showed restoration of spermatogenesis, characterized by an increase in the height of the seminiferous epithelium, an increase in the frequency and stratification of the lining epithelium (spermatogonia, primary spermatocytes, and spermatids), and the appearance of spermatozoa in some tubules. Some tubules continued to show empty spaces between the germ cells, separated by wide interfollicular spaces with destructed interstitial tissue containing congested blood vessel and surrounded by a slightly thickened testicular capsule [Figure 5]d]. There was also improvement in the biochemical, immunohistochemical [Figure 6] d and [Figure 7] d, and morphometric results.

Morphometric results

The diameter of the seminiferous tubules was significantly reduced (P<0.001) in adult DBP-treated rats compared with adult rats in the control and combined DBP and curcumin groups [Table 4], as well as with the adult rats in the recovery group [Table 5].


  Discussion Top


DBP was particularly chosen as it is one of the most commonly used plasticizers; it is present in the environment and can enter organisms through various routes in utero during gestation and lactation and then affect reproductive and developmental processes of the organism and its offspring (mainly affecting male offspring), as reported by Howdeshell et al. 7. In addition, DBP is used in a variety of consumer products and can induce testicular atrophy in adults, as reported by Zhang et al. 8, who stated that DBP is a potential toxicant affecting human reproduction and development and on the basis of adult animal data showed that the male reproductive system is the main target organ of DBP toxicity.

The selection of the dose of DBP was based on previously published data of Jiang et al. 9, who found that the useful rat models of DBP for the induction of developmental abnormalities were 250, 500, 750, or 1000 mg/kg/day during late gestation.

The biochemical findings of the DBP groups confirmed that DBP is a potent antiandrogen that reduces TH in all age groups, as reported by Scott et al. 10. In addition, Borch et al. 11 and Mahood et al. 12 reported that fetal TH production was significantly inhibited by DBP at doses of 300 mg/kg/day and higher. However, Hyung et al. 13 stated that there was no significant difference in serum TH levels between DBP-treated and control animals.

Histopathologically, DBP-treated rats showed loss of stratification and disorganization of the lining epithelium of the seminiferous tubules. Some germ cells exfoliated in the lumen and detached from the basement membrane, with an increase in the number of the gonocytes in the 1-day-old rats only, disappearance of the early spermatids in the 4-week-old rats, and disappearance of the late spermatids in the 6-week-old rats. This was concordant with the findings of Barlow and Foster 3, who stated that when male rats were exposed in utero to DBP at a dose of 500 mg/kg/day during the latter half of gestation, they exhibited a characteristic suite of testicular lesions, as well as the formation of large aggregates of LC and an increase in the number of gonocytes within the seminiferous tubules. In addition, DBP induces marked testicular atrophy and Leydig cell hyperplasia in fetal male rats (PND31 and 42), and these effects were permanent.

The empty spaces in the seminiferous tubules of the testes of the rats in the DBP group might be explained by phagocytosis of the apoptotic bodies by Sertoli cells, as reported by Nishimura et al. 14. The findings in the interstitial tissues of all age groups were concordant with those of Mahood et al. 15, who explained that the occurrence of large Leydig cell aggregates may be due to an increase in focal Leydig cell proliferation or Leydig cell migration to central regions from other points of origin in the testes after exposure to DBP. In the testes of rats belonging to groups IB and IIB, the tunicae albuginea was thickened, which might be due to an increase in type I collagen synthesis, and the tunica vasculosa contained dilated congested blood vessels, which might be due to chemical mediators that were released shortly after tissue damage, causing capillaries and postcapillary venules to become dilated. The blood flow within them slowed and they became lodged with red blood corpuscles, as reported by Wheater et al. 16.

DBP induced testicular atrophy through several different mechanisms. The present study suggests that the mechanism might be hormonal, involving a reduction in TH levels, as reported by Parks et al. 17, or might involve a depletion in the level of zinc or iron, as reported by Fukuoka et al. 18, who stated that DBP can deplete testicular zinc. Zinc deficiency has been associated with increased oxidative damage to proteins and lipids, as well as with DNA damage, in rat testes.

In this study, there was weak to moderate immunoreactivity for caspase 3 in the developing rats of the control group. This was explained by Norbury et al. 19, who reported that apoptosis normally occurs during development and aging and as a homeostatic mechanism to maintain the cell population in tissues, and was supported by Yan et al. 20, who stated that during the first wave of spermatogenesis (which occurs from the first to the sixth/seventh week of postnatal life), a significant number of spermatogenic cells undergo apoptosis, and the highest rate of apoptosis occurs at 18–26 days of age to maintain an optimal Sertoli and spermatogenic cell ratio. DBP-treated rats exhibited strong immunoreactivity for caspase 3, in concordance with the explanation of Alam et al. 2, who stated that vacuolation is the earliest morphological sign of testicular injury, as observed in this study, and believed that vacuolation, followed by ultrastructural alterations in Sertoli cells induced by phthalate administration, causes detachment of spermatogenic cells from Sertoli cells, which is the first step towards cell death or apoptosis.

DBP exerts an antiproliferative effect on the different spermatogenic cells, as evidenced by the lack of PCNA immunostaining in this work. This was explained by Creasy and Foster 21 as spermatogenic arrest and is in agreement with the findings of Kelymenova et al. 22 who revealed lack of PCNA staining in DBP-exposed testes on gestational days 19–21.

The reduction in the diameter of seminiferous tubules of the rat testes might be because of severe atrophy of the tubules, low spermatogenic epithelium, and histological disruption of spermatogenesis induced by DBP. This is in concordance with the findings of Hyung et al. 13, who observed a reduction in the diameter of the seminiferous tubules after neonatal exposure to DBP, and is explained by Creasy and Foster 21, who reported that the loss of the germ cells from the seminiferous tubules of the testes could generally result in a decrease in the tubular diameter.

The observed protective effect of curcumin against DBP-induced testicular atrophy and reduced TH levels was explained by Farombi et al. 6, who stated that the protective effects of curcumin on the testes are through its intrinsic antioxidant abilities to combat oxidative damage induced by DBP by relieving the inhibition of the antioxidant enzymes and reduction in the TH levels, and/or by triggered their synthesis; they also found that coadministration of curcumin with DBP increased the TH levels by 82% compared with those in the DBP-treated animals; this is in accordance with the findings of this study.

Stoppage of DBP for 2 weeks in the adult rats led to improvement and restoration of spermatogenesis but the improvement was less than that observed on adding curcumin. This was in accordance with the findings of Alam et al. 2 who indicated that although DBP is eliminated from the body, its effects still persist in testes and cause the inability of the testes to return to normal until 30 days after treatment.

The improvement in the TH level and the diameter of the seminiferous tubules of 6-week-old rats compared with rats belonging to other developmental age groups showed that the recovery from the DBP toxicity in neonates could occur but it need longer period. These findings are in agreement with those of Walter and Talbot 23, who postulated that a damaged cell is not an inactive cell and its reaction to injury may end during recovery. The cell may return to normal but some alterations may persist.

Therefore in the present study, the observed low level of serum TH in the DBP group may be the possible cause of spermatogenic impairment; this finding is in concordance with that of Singh et al. 24, who stated that TH, secreted from the Leydig cells of the testes, is responsible for the activation and maintenance of spermatogenesis. In addition, O’Donnell et al. 25 found that TH helps in the maturation of round elongated spermatids and the protective effect of curcumin was probably mediated by an increase in the TH levels; this is in accordance with the findings of Karbalay et al. 26, who stated that one of the possible ameliorative mechanisms of curcumin is to scavenge the free radicals and thereby act as a good antioxidant; the other mechanism might be increasing serum TH levels. Moreover, Wei et al. 27 found that, in laboratory animals, curcumin has protective effect against oxidative damage induced by cisplatin, sodium arsenite, aflatoxin, ischemia-reperfusion injury, and DBP.


  Conclusion Top


Exposure to DBP led to more pronounced testicular damage in prepubertal and mature adult rats, as evidenced biochemically by a reduction in TH levels, histologically by damage induced in the testicular tissue and interference with the normal spermatogenesis process, immunohistochemically by induced apoptotic changes in the germ cells, as evidenced by caspase 3 staining, and interference with the regeneration of the germ cells, as evidenced by PCNA staining, and morphometrically by reduced seminiferous tubule diameter. These serious effects on the testes might lead to infertility. These changes could be prevented by adding curcumin, which elicited significant protective effects on the structure and function of the testes, making them nearly normal. Although stoppage of DBP for a short period of time improves the results, the improvement is less than that observed on curcumin administration.


  Recommendations Top


Exposure to DBP products during childhood and adulthood should be avoided to avoid male infertility.

Curcumin, which is orally safe and inexpensive, should be added to foods, as much as possible, to counteract the toxic effects of DBP on the testes. Additional studies with longer recovery periods should be conducted to establish methods for complete recovery from testicular damage.

Studies on the combined effect of various phthalate esters on male reproductive development should be carried out because the human male is at low risk from exposure to DBP alone and the human fetus is exposed to multiple phthalates in the womb.[27]

 
  References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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