Bisphenol A (BPA) is an organic plasticizer used to line metal cans for storing food and beverages as well as in the coating of metal lids for glass jars and bottles. It is also found in some plastic food containers, all disposable plastics, toys, dental devices, dental fillings, and sealants. BPA may enter the human body primarily through food and water from plastic container and through the saliva from dental sealants 1. However, recent evidence also indicates that exposure may occur through dermal contact with thermal papers used widely in cash register receipts 2.
BPA has been shown to induce various pathologic conditions such as cancers, neurological disorders, behavioral defects, and developmental anomalies 3. In addition, BPA was reported to have a significant role in cardiovascular disorders, type 2 diabetes, and liver enzyme abnormalities in a representative sample of the US population. Two studies on laboratory animals have also shown that BPA has direct adverse effects on the brain, reproductive system, and several metabolic processes 4,5.
There are several reports suggesting the reproductive toxicity of BPA in rats and mice 6,7. Accumulation of BPA in male reproductive organs has some clinical implications during fetal life, such as decrease in the efficiency of sperm production in the offspring of male mice. Some studies in rodents showed that low-dose BPA decreases testis weight associated with morphological changes, as well as sperm count and motility 8–10.
BPA may operate through several different mechanisms involving interaction with estrogen receptors (ERs) and/or by production of a minor but potent estrogenic metabolite 11. Furthermore, it was suggested that BPA causes tissue injury in the liver, kidney, brain, and other organs by the formation of reactive oxygen species, decreasing the activities of antioxidant enzymes and increasing lipid peroxidation, thereby causing oxidative stress 12,13. Some researchers 14 declared that BPA generates significantly high concentrations of malondialdehyde (MDA) in the brain and sperm of male rats by induction of oxidative damage in the brain and testes of rats.
The ER is a member of the nuclear receptor superfamily of proteins that play an important role in the regulation of testosterone synthesis. Two subtypes of ER have been cloned to date, ERα and ERβ. The ERα was the preferable subtype for BPA binding and was confined to Leydig cells, whereas ERβ was identified in the nuclei of pachytene spermatocytes at all stages, in spermatogonia as well as in Sertoli cells 15. Recently, it was shown that ERα was present in spermatids, Sertoli cells, and Leydig cells of rat testis 16.
To date, there exists controversy about the toxicity of BPA. Although FDA has labeled BPA as a safe agent 17, newly emerging data have suggested that more studies be conducted to assess the risk of BPA exposure on human health 18. This risk has especially increased in developing countries where plastic usage has increased exponentially; certain population groups such as those suffering from malnutrition may be at higher risk than other populations 19,20. Therefore, the aim of our study was to investigate the effect of the administration of BPA (50 mg/kg for 8 weeks) on the oxidant/antioxidant status and the histological structure of the testis of adult male albino rats.
Materials and methods
This study was conducted on 32 healthy adult male albino rats (4–7 months) weighing 180–200 g. The rats were obtained from the animal house of the Faculty of Medicine, Zagazig University, and maintained under controlled laboratory conditions of 12-h dark and 12-h light cycle, at 25°C. All animals were provided with water ad libitum and a standard pellet diet.
BPA (purity >99%) was purchased from Sigma–Aldrich Co. (St Louis, Missouri, USA). It was dissolved in corn oil (vehicle) and given to rats at a dose of 50 mg/kg body weight 4 for 28 days 15 by oral gavage once daily. The BPA dose was equal to the lowest dose that is commonly used to refer to environmentally relevant doses – that is, doses resulting in serum levels close to those observed in human serum 4.
The rats were divided equally into four groups: the negative control group (group I), the positive control group (group II), and two experimental groups (groups III and IV), with eight rats in each.
Group I (negative control): Group I served as the negative control, which did not receive any treatment.
Group II (positive control): Group II served as the positive control, which received a daily oral gavage of corn oil (vehicle) for 8 weeks.
Experimental group III (BPA treated): These rats received 50 mg/kg of BPA 6 days/week for 8 weeks.
Experimental group IV (recovery): The rats were given the same dose of BPA for the same period as group III and then left without treatment for another 4 weeks for follow-up.
At the time of sacrifice, all rats were anesthetized with ether inhalation. Blood samples were collected from the retro-orbital venous plexus of all animals for estimation of testosterone hormone. Each testis was dissected by abdominal incision, cleared from adhering connective tissue, and used for assessment of tissue MDA as an indicator for lipid peroxidation and glutathione (GSH) as endogenous antioxidant markers and subjected to histological and immunohistochemical studies.
Determination of tissue malondialdehyde and reduced glutathione levels
Two specimens from the testis were separated and homogenized. One of them was homogenized in PBS 50 mmol/l, pH 7.4, and the other in potassium phosphate buffer 10 mmol/l, pH 7.4, for estimation of GSH and MDA, respectively.
Lipid peroxidation was assayed by determining the level of MDA by measuring thiobarbituric reactive species using the method of Ruiz-Larrea et al. 21. The thiobarbituric acid reactive substances react with thiobarbituric acid to produce a red-colored complex that has peak absorbance at 532 nm using a Helios Alpha (UVA111615; Thermo Spectronic, Cambridge, UK).
Reduced GSH was assayed by Ellman’s method 22. The procedure is based on the reduction of Ellman’s reagent [5,5′-dithiobis(2-nitrobenzoic acid)] by –SH groups of GSH to form 2-nitro-5-mercaptobenzoic acid, which has an intense yellow color. The absorbance was measured at 412 nm and the GSH concentration was calculated by comparison with a standard curve.
Serum testosterone analysis
Blood samples were collected at a fixed time of the day to minimize the diurnal variation. The blood was collected in a special container and the serum was separated by centrifugation for 10 min and stored at −70°C until use. The serum testosterone hormone was analyzed using double-antibody radioimmunoassay method by Coat A-Count 23.
Specimens for light microscopic examination were fixed in 10% neutral formol saline. They were processed to prepare 5-μm-thick paraffin sections for H&E staining 24. Specimens for electron microscopic examination were fixed immediately in 2.5% glutaraldehyde buffer with 0.1 mol/l phosphate buffer at pH 7.4 for 2 h at 4°C and postfixed in 1% osmium tetroxide in the same buffer for 1 h at 4°C. The specimens were processed and embedded in Embded-812 resin in BEEM capsules (Polysciences, Warrington, Pennsylvania) at 60°C for 24 h. Ultrathin sections were obtained using Leica ultracut UCT, (Germany), stained with uranyl acetate and lead citrate 25, and examined with a JEOL-JEM 1010 electron microscope (JEOL, Tokyo, Japan) in the Histology Department, Faculty of Medicine, Zagazig University.
Tissues obtained from the animals of all groups were fixed in 7.4% formaldehyde for 16 h. The fixed tissues were processed and embedded in paraffin. Tissue sections were cut and dewaxed in xylene, rehydrated, and treated to block endogenous peroxidase by incubating in 3% H2O2–methanol for 15 min. After washing twice in PBS, the slides were incubated with an anti-rabbit ER antibody. After washing in (phosphate-buffered saline) PBS, sections were incubated for 30 min. with a biotinylated swin anti-rabbit immunoglobulin (Dako, Glostrup, Denmark) and then incubated with horseradish peroxidase streptavidin biotin for 20 min (Dako Corp., Carpinteria, California, USA). After two additional washes in PBS, bound antibodies were visualized using diaminobenzidine tetra-hydrochloride (Dako Corp.). Sections were then washed in distilled water and lightly counterstained with hematoxylin 15. The positive immunoreactions appeared as perinuclear brown coloration in spermatogonia, spermatocytes, and/or Leydig cells. Negative controls were processed according to the same protocol, except for the use of the primary antibody.
Five fields from three H&E-stained sections of each rat from each group were examined to measure the mean diameter and the mean germinal epithelium height of the seminiferous tubules using an image analyzer (Olympus Image J, NIH, 1.41b; Olympus, America Inc., Melville, New York, USA) at the Oral Pathology Department, Faculty of Dentistry, Ain Shams University. Rounded transversely cut tubules were selected and two diameters of each tubule, one perpendicular to the other, were measured and the average taken 26. When the sections were oblique, only the minor axis was considered for the measurements 27,28.
The mean diameter and the mean height of the germinal epithelium of the seminiferous tubules, as well as the mean blood testosterone level of the animals at the time of scarification, were measured. All values were presented as mean±SD. The differences among the groups with respect to all measured data parameters were statistically analyzed using one-way analysis of variance and the post-hoc test using SSPS program, version 17 (IBM Corporation, Somers, New York, USA). The calculations were considered significant if P value was less than 0.05.
On comparing both positive and negative control groups, no significant differences (P>0.05) were found in testicular MDA, GSH, and serum testosterone. Therefore, the negative control group was used in the statistical comparison with the other groups.
Table 1 and Histogram 1 showed that there is a highly statistically significant difference between different groups as regards testicular MDA, GSH, and serum testosterone level (P value <0.001).
Table 1 and Histogram 1 also showed that the daily oral administration of BPA resulted in significant increase in MDA, which was accompanied by a significant decrease in GSH levels in the testis after 8 weeks (30.81±0.62 and 0.92±0.15, respectively). In addition there was significant decrease in serum testosterone level, recording 101.93±0.87 when compared with the control group.
In contrast, after 2 weeks of recovery, the statistical study revealed significant improvement in the oxidative stress parameters, recording 27.83±0.87 for MDA and 1.1±0.12 for GSH when compared with the BPA group, which recorded 30.81±0.62 and 0.92±0.15, respectively. As regards the serum testosterone level of the recovery group, results showed significant increase in its level when compared with the BPA group, recording 119.62±4.78 and 101.93±0.87, respectively.
Group I (the control group)
Histological examination of the testis of positive and negative control (group I&II) showed the same structure. So, morphological results including the figures for the negative control group (I) are representive for the other (groupII). Light microscopic examination of the sections of the testes of the control group revealed that testicular parenchyma consisted of closely packed seminiferous tubules. The tubules were lined with stratified germinal epithelium and contained sperms in their lumina. They were separated by a narrow interstitium containing interstitial cells (Figs 1 and 2). The stratified germinal epithelium was formed of spermatogonia, primary spermatocytes, and spermatids. Sertoli cells with their large pale nuclei were observed between these cells. The boundary tissues between the tubules included flat myoid cells, clusters of Leydig cells, and blood capillaries (Fig. 3). Electron microscopic examination of the ultrathin sections of the testes of the same groups revealed Sertoli cells with large oval euchromatic nuclei and prominent nucleoli resting on a regular basement membrane. Spermatogonia appeared with coarse clumps of peripheral heterochromatin in their nuclei. Thin boundary tissue and primary spermatocytes were seen (Fig. 4). Spermatids had large rounded euchromatic nuclei, and their cytoplasm contained peripherally located mitochondria and rough endoplasmic reticulum (RER). One spermatid had acrosomal granules (Fig. 5). Transverse sections in different parts of the sperms revealed mid, principal, and end pieces with a central axoneme formed of nine doublets of microtubules with two central singlets. In the mid pieces, this axoneme was surrounded by nine dense bundles of fibrous sheath and mitochondrial sheath. In the principal pieces, the axoneme was surrounded by a fibrous sheath only (Fig. 6). Leydig cells had irregular nuclei with clumps of heterochromatin. Their cytoplasm contained numerous mitochondria with dilated smooth endoplasmic reticulum (SER) and many lipid droplets (Fig. 7). Immunostaining for detection of ERs showed brown coloration localized in the nuclei of spermatogonia and/or spermatocytes (Fig. 8).
Group III (the bisphenol-treated group)
Light microscopic examination of sections of the testes of the bisphenol-treated group revealed that testicular parenchyma was formed of many distorted seminiferous tubules with irregular outlines (Fig. 9). They were lined by disorganized epithelium with interepithelial vacuolations. Some tubules contained exfoliation of apoptotic cells within their lumina (Figs 10 and 11). There was an apparent decrease in the number of epithelial layers and intercellular spaces (Fig. 12). Electron microscopic examination of the ultrathin sections of the testes of the same group revealed Sertoli cells with irregular euchromatic nuclei resting on a thick basement membrane. Many spermatids were separated by variably sized spaces and had distorted and abnormally distributed mitochondria. Some of them had shrunken nuclei (Fig. 13). Almost all transverse sections in the mid pieces of sperms showed a markedly affected axoneme and distorted swollen mitochondrial sheathes (Fig. 14). Leydig cells with oval indented euchromatic nuclei, multiple electron-dense bodies, RER, and mitochondria were detected (Fig. 15). This group showed increased ER immunoreactivity (Fig. 16).
Group IV (the recovery group)
Light microscope examination of sections of the testes of the recovery group showed that they resumed nearly their normal general architecture. Closely packed seminiferous tubules had normally arranged germinal epithelium, sperms in their lumina, and were separated by narrow interstitium. Few intercellular vacuolations within the germinal epithelium were still detected (Fig. 17). Leydig cells and blood capillaries were present in the interstitium (Fig. 18). Electron microscopic examination of the ultrathin sections of the testes of the same group showed that spermatogonia, primary spermatocytes, spermatids, and Sertoli cells exhibited their normal fine structure. However, intercellular separations were still present with loss of intercellular bridge between some cells. The basement membrane was thin and regular (Figs 19 and 20). Most of the transverse sections of the mid pieces of sperms had a normal structure, except a few that showed an affected axoneme (Fig. 21). Leydig cells had oval binucleated euchromatic nuclei and peripherally arranged heterochromatin. Their cytoplasm contained numerous mitochondria, multiple variable-sized lysosomes, RER, and SER (Fig. 22). Moreover, this group revealed minimal changes in ER immunoreactivity as compared with the control group (Fig. 23).
Statistical analysis of the diameter and epithelial height of the seminiferous tubules of the bisphenol-treated group (group III) revealed a highly significant decrease as compared with the control group, whereas the recovery group showed a significant decrease in epithelial height compared with group I. In addition, there was significant improvement in both diameter and epithelial height of the seminiferous tubules of the recovery group (group IV) when compared with the bisphenol-treated group (group III) (Table 2 and Histogram 2).
Endocrine disruptors (EDs) are compounds that can interfere with and alter the homeostasis of the endocrine system. They can act at several sites, mimicking the occurrence of natural hormones, blocking their production and inhibiting or stimulating the endocrine system. EDs have long-term adverse effects on human and animal health and on their progeny. The effects also extend to the thyroid, nervous system, immune system, and metabolism in general 29.
Synthetic estrogens, also called xenoestrogens, are a diverse group of compounds in the environment that mimic the action of the natural hormone 17β-estradiol (E2) in estrogen-dependent tissues 30. Xenoestrogens are one of the most common endocrine-disrupting chemicals. Among these xenoestrogens, BPA is an estrogen-mimicking chemical that can alter the ER number and its receptor gene activity in target tissue. Recent findings have revealed that BPA is a selective estrogen modulator that can interfere with hormone synthesis and clearance as well as with other aspects of tissue metabolism 4. The extensive use of BPA-containing products results in high levels of exposure to humans worldwide 31.
The results obtained from this study showed a decrease in serum testosterone hormone levels in rats after the oral intake of BPA. This was in accordance with another study that demonstrated the inhibition of testosterone production induced by BPA. It was also associated with decreased luteinizing hormone secretion and decreased levels of steroidogenic enzyme gene 32. In addition, some studies detected a significant decrease in testicular and blood testosterone levels of different strains of male rats following prenatal and postnatal low-dose BPA exposure, and this decrement persisted even in adults. This effect was shown to be a result of decreased expression of the steroidogenic enzyme 17α-hydroxylase/17–20 lyase in Leydig cells 33.
In the present study, the recorded significant increase in MDA that was accompanied by a significant decrease in GSH in the testis of BPA-treated rats reflects a state of oxidative stress in testicular tissue. It was reported that the immediate endocrine environment of the testes has a major impact on the antioxidant status of this organ. Treatment with any chemicals that diminish the intratesticular concentration of testosterone inhibits the testicular expression of antioxidant enzymes such as GPx, SOD, and catalase 34. Also, some researchers documented that BPA generates significantly high concentrations of reactive oxygen species and resulted in the decline of the testicular antioxidant enzymes, which could increase the oxidative damage of testicular cellular membranes 35.
In the present study, examination of the bisphenol-treated group showed different degrees of seminiferous tubular degeneration. Many of them were distorted with irregular outlines and contained desquamated cells within their lumina. Some tubules were lined by the disorganized epithelium with a reduced number of epithelial layers, which was confirmed statistically. Similar results were detected by other investigators with different BPA doses and routes in rats 36 and mice 37. It was mentioned that decrease in the level of circulating estradiol and testosterone in BPA-treated rats led to atrophy of seminiferous tubules, degeneration of germ cells, and complete absence of spermatogenesis 38.
The present study revealed congestion in some blood vessels within the interstitial spaces and vacuolations within the tubules. Ultrastructurally, these vacuolations appeared as variable sized intercellular spaces separated many spermatids. These spermatids had disorganized and abnormally distributed mitochondria. Some of them had shrunken nuclei. The intercellular spaces represent progressive degenerative changes affecting cell membrane integrity secondary to oxidative stress induced by BPA. The free radicals oxygen species initiate oxidative phosphorylation reactions to cell membranes ending in disruption of the integrity of the intercellular junctional complex 1. Furthermore, it was shown that neonatal exposure with BPA affects the ectoplasmic specialization between the Sertoli cells and spermatids when the animal reaches puberty 4,39. It was also suggested that oxidative stress selectively disrupts cadherin/catenin complexes and disrupts cell–cell adhesion 40.
Ultrastructurally, the shrunken nuclei of spermatids indicated death of these germ cells. Thick boundary tissue was also recorded in our work. A significant increase in apoptosis of spermatocytes and spermatids was recorded by other investigators in EDs-exposed adult hamsters 41. It could be explained by the decreased testosterone hormone levels essential to maintain normal spermatogenesis and prevention of germ cell apoptosis in adult rats 34. In contrast, the intratesticular testosterone can be reduced by 50–60% without provoking spermatogenetic damage 42.
With regard to thick boundary tissue, some reports have shown that certain stimulants may induce myoid cells to produce more collagen and extracellular matrix, which are responsible for basal lamina thickness. Moreover, overexpression of type IV collagen that correlates with the thickened basement membrane could be related to spermatogenic dysfunction in mammals 43.
The present work revealed different degrees of sperm affection. Almost all transverse sections in the mid pieces of sperms showed a markedly disturbed axoneme and distorted swollen mitochondrial sheathes. Upregulation of ERs caused by EDs could lead to variable outcome on reproduction, such as reduced sperm quality, altered morphology, and even infertility 41. Also, Leydig cells appeared with oval binucleated euchromatic nuclei, multiple electron-dense bodies, and RER. BPA was found to exert inhibitory effects on Leydig cells. Also, it acts directly on this cell because it decreased T-cell production and suppressed aromatase gene expression and E2 biosynthesis 30. Controversially, it was mentioned that BPA treatment did not induce abnormality in serum testosterone levels and Leydig cell morphology, but altered expression levels of genes related to androgen and Leydig cells 44.
In the current study, the bisphenol-treated group (group III) showed minimal decrease in ER immunoreactivity. This result was concomitant with that of other researchers 15,45. ERs are expressed in the tissues of the male reproductive tract and play an important role in regulation of testosterone synthesis. It has been reported that BPA is an equally strong agonist for ERα as it is for ERβ. In contrast, BPA has also been reported to exhibit only agonistic activity on ERβ, whereas it has dual actions as both an agonist and antagonist of estrogen on ERα 15. Nevertheless, it is generally thought that endocrine effects of BPA on adults are few, because these effects may be transient; that is, they appear only while the chemical is present or until it is completely metabolized 45,46. The existence of ERα and ERβ in the testis suggests that estrogens directly affect germ cells during testicular development and spermatogenesis. Furthermore, the differential modulation of ERα and ERβ in the testis could be involved in the effects of BPA 15.
Stoppage of BPA in the recovery group (group IV) in the present work showed that seminiferous tubules and Leydig cells of the testis nearly retained their normal structure. The tubules appeared with normally arranged germinal epithelium and separated by narrow interstitium. Vacuolization in the germinal epithelium and intercellular separations in a few seminiferous tubules were still detected in this group. The mid pieces of sperms had a normal structure except a few of them that affected the axoneme. Similar observations were detected by other investigators 47 in BPA-treated mice and rats. They concluded that the adverse effects of BPA on rat and mouse spermiogenesis are reversible. BPA-treated males were fertile 2 months after cessation of the administration. In addition, they found no histological and ultrastructural abnormalities were found in the testis of treated rats and animals at the time of fertilization tests 39,47.
Oral routes of exposure are relevant to potential human BPA exposure because humans are primarily exposed through oral ingestion. Following this route of exposure, enterocytes and hepatocytes extensively glucuronidate BPA, leading to essentially complete first pass metabolism of BPA in the intestinal wall and liver 48,49. This results in much lower tissue levels of BPA than would occur by other routes of exposure 50. Overall, oral exposure results in much lower tissue levels of BPA than would occur by other routes of exposure. Results from studies of nonoral BPA exposures may not be applicable to ‘low-dose’ oral exposures in humans. Thus, in this study the oral route was used and preferred.
The results of the present study suggested that male exposure to BPA resulted in adverse effects on the histological structure and biochemistry of the testis. These changes resulted from decreased testosterone hormone levels and the oxidative stress induced in the testis. Furthermore, stoppage of the drug was associated with some sort of recovery. Thus, the use of BPA in different plasticizers and other industries should be limited and the erroneous handling of plastic containers should be avoided to reduce the health risks resulting from exposure to these EDs including BPA.
Conflicts of interest
There is no conflict of interest to declare.
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Keywords:© 2013 The Egyptian Journal of Histology
bisphenol A; rat; testis; ultrastructure