Reproductive endocrine disorders and sexual dysfunction are common in men with epilepsy [
]. In addition, these disorders have been frequently associated with long-term treatments with antiepileptic drugs (AED) [
]. CBZ is a first-line AED also utilized for treatment of psychiatric disorders such as bipolar affective disorder [
], alcohol withdrawal syndrome [
] and neuropathic pain [
Various clinical and experimental studies involving the effects of CBZ and other AED on male reproduction have been carried out. They have mainly shown hormonal changes and seminal alterations during adult phase, including reduced sperm motility, sperm morphological alterations and decrease in the sperm concentration [
]. However, studies have not investigated the occurrence of damage to the prostate in the phase of sexual maturation when the CBZ is chronically administered from the pre-pubertal phase.
The reproductive hormone status differs noticeably in humans, according to the phase considered, i.e., in childhood, adolescence and adulthood. Thus, endocrine changes due to long-term CBZ-treatments may cause a negative impact on pubertal development and fertility both in boys and young men [
]. Damage on the seminiferous epithelium, testicular interstitial oedema, reductions of testosterone levels and increase of estradiol levels were also observed in rats treated with CBZ since weaning [
The onset and maintenance of prostatic development are androgen-dependent phenomena [
]. However, in rodents, the prostatic development is also sensitive to other hormones such as estrogen and prolactin [
]. The prostate secretion is important for normal male reproductive function as well as sperm survival within the female reproductive tract during fertilization [
The responsive morphological alterations of the prostate to androgen deprivation caused by diabetes [
], chronic alcoholism [
], nicotine [
] and castration [
] have been shown. In addition, as the chronic administration of CBZ is a common schedule utilized for treatment of children and adolescents [
] and can provoke hormonal alterations as well as damage on fertility [
]. In addition, given that there are not studies investigating the extension of the prostatic damage, caused by CBZ chronic administration, during the sexual maturation, we assumed that this theme is an essential subject to be investigated. Thus, considering that the maturation of the hypothalamic-pituitary-gonadal (HPG) axis in pre-puberty and puberty is more susceptible to a cytotoxic agent [
] and also, taking into account that recently we observed estrogen and testosterone level alterations in CBZ-treated rats from the weaning [
], we proposed to evaluate the side effects of CBZ on the ventral prostate of rats in different phases of sexual development; for this goal, the CBZ was administered to rats from the pre-puberty until the adulthood and the histopathological, morphometric and stereological analyses of their prostate ventral lobe were carried out.
In the prostate, death and cell proliferation are hormone regulated events. During the periods of normal growth of prostate, androgens are essential for stroma and epithelial cell differentiation throughout branching morphogenesis and ductal canalization [
]. At the age of sexual maturity, the secretory activity of the epithelium and the differentiation of smooth muscle are also maintained by androgens [
]. In the prostate, luminal and basal epithelium as well as stroma and smooth muscle cells express ARs at sexual development and hence are capable of mediating androgen's actions [
]. Thus, in response to androgens, cells of the prostate interact in a autocrine-paracrine way, influencing various aspects of the growth of this gland in normal and diseased states [
]. In fact, differential responsiveness of the prostate to androgens has been observed in rat at different phases of the sexual development [
]. On the other hand, CBZ treatment can cause alterations in the weight of seminal vesicle and prostate gland which are age-dependent; this probably occurs due to the higher responsiveness of the prostate to testosterone during the pre-puberty and adulthood than during the puberty [
]. During the pubertal period, testosterone clearance may be increase and this could account for the apparent reduction in prostate sensitivity during the pubertal period. It is possible that the alterations in prostate response reported are inherent to the tissue themselves and are dependent on their own spontaneous maturation independent of other hormonal events [
Puberty in the male rat is a complex process that involves maturational changes in the hypothalamus, pituitary, testes, and secondary sexual organs and in their interrelationships. During the course of sexual maturation, the negative feedback control systems for the gonadotropins become less responsive to testosterone while the testes become more responsive to LH. In the immature rat, testosterone can potentiate the effect of GnRH on pituitary LH release; this response is lost with sexual maturation. The responsiveness of the prostate to testosterone is also altered with age due proportion of testosterone and androstendione secreted by the testes. Experiments designed to prevent or mimic the transition in testicular steroid secretion suggest that it may be a critical component of sexual maturation in the male rat. An increase of androstendione appears to be capable of delaying the maturation of the LH negative feedback system, the prostate gland, and the GnRH self-priming effect [
]. Moreover, the metabolism of testosterone and/or alterations androgen receptor activity could be responsible for these sensibility differences [
]. In addition, estrogens also play a physiologic role in the prostate development with regard to programming stromal cells and directing early morphogenic events [
The significant reductions of ventral prostate weight and volume observed in CBZ93 rats probably occurred due to the CBZ-induced hormonal alteration. These data are in accordance with our previous study [
]; in this former study, a decrease in testosterone plasma levels and an increase in the estrogen level were respectively observed in 63 and 93 day-old rats which were also CBZ-treated from the weaning. In addition, the decrease in the weight of androgen-dependent organs as the ventral prostate is consistent with the decrease in serum levels of testosterone [
]. Besides, both androgen and estrogen regulate AR at the level of mRNA and protein. Then, abnormalities in the AR signaling pathway have also been linked to male reproductive alterations [
]. Moreover, post-transcriptional regulation through stability of the mRNA product is believed to be a major mechanism of androgens' effects [
]. In the current work, the CBZ treatment caused decrease in the AR reactivity of the 93-day old rat prostate; this event was confirmed by the optical density of nuclear reactivity. Prins and Birch [
] observed that neonatal chronic estrogen exposure resulted in an immediate and sustained decrease in AR protein levels in the developing and adult rat ventral prostate that in turn led to its abnormal growth and decreased secretory capacity.
In this present study, an increase of the estrogen plasma levels during puberty phase was observed in CBZ-treated rats. In addition, the estrogen plasma levels remained high in the adulthood and this phenomenon probably corroborated the reduction of the morphometric and stereological parameters evaluated. In fact, a decrease in the glandular acini length as well as in the glandular lumen, glandular epithelium and fibromuscular stroma volumes were observed in the CBZ93 group. These phenomena resulted in a reduction of the ventral prostate weight and volume in rats of this group. Additionally, we suggested that the reduction of glandular epithelium surface was a consequence of the diminution of the glandular epithelium volume. Thus, although CBZ chronic treatment since weaning has induced significant prostatic alterations observed in adult rats, the harm was started from the pre-puberty phase.
Similar to androgens, circulating levels of estradiol are high during the fetal and early neonatal life in both humans and rodent models [
] and there is compelling evidence that the developing prostate gland is particularly sensitive to these estrogens. Although the natural role for estrogens during prostatic development is unclear, it has been proposed that excessive estrogenization during prostatic development may contribute to the high incidence of benign prostatic hyperplasia and prostatic carcinoma [
]. Following neonatal exposure to high-dose estradiol, both epithelial and stromal cell proliferation and differentiation are markedly disturbed leading to defects that persist throughout the lifespan of the animal [
]. Neonatal estrogen exposure interrupts intercellular communication and blocks certain epithelial cells within the rat prostate from entering a normal differentiation pathway, beyond that the activational response to androgens during adulthood is permanently blunted in estrogenized rats [
] and this effect is mediated, in part, through an immediate and permanent reduction in prostatic AR expression [
]. Furthermore, the temporal expression patterns and quantitative levels of several other members of the steroid receptor superfamily are deregulated by early exposure to high doses of estradiol [
Besides, the possible direct effects of CBZ should be also considered as this drug is highly lipid-soluble [
]. Furthermore, some drugs alter the prostatic stroma-epithelial communication and this occurrence may affect the expression or distribution of growth factors, steroid hormones, and their respective receptors [
]. These phenomena corroborate the possible occurrence of a specific effect of the CBZ on ventral prostate, independently from the hormonal testosterone and estradiol level alterations caused by this drug.
Although structural alterations of the prostatic tissue have not been noted during the histopathological analysis, we could observe the presence of degranulated mast cells in fibromuscular stroma, both in the CBZ43 and CBZ63 rats.
There are mast cells in almost all organic systems, including lungs, skin, heart, and gastrointestinal tract. Depending on the organic system, differentiation state, stage of maturation as well as pathological conditions, mast cells can be heterogeneous in terms of their phenotype, functional properties, and local distribution [
]. In the ventral prostate, a quantitative analysis revealed a correlation between the numerical density of mast cells and the age of the rat, since their frequency increases during pre-puberty, remaining constant in the puberty and adult phases [
]. Mast cells participate in inflammatory reactions, angiogenesis, extracellular matrix reabsorption [
], fibrosis and tissue reconstruction [
]. Activated mast cells contain several inflammatory mediators, such as histamine, serotonin, cytokines, leukotrienos, prostaglandins, chemotatic substances, platelet-activing factor and potent vasodilators molecules [
]. These mastocitary products may be activated by different stimuli such as chemical substances, drugs, free radicals, estradiol and radiation [
]. Tobacco smoke, for example, can directly activate mast cells and to act releasing mediators such as histamine and tryptase, causing degranulation of mast cells [
The hormonal status may also induce activation and degranulation of mast cells. Estrogens can induce rat prostate inflammation [
] and then, mast cells can undergo degranulation [
]. Many experimental studies showed that steroid sexual hormones can influence the immune response and allergy development, in which estrogen is able to enhance humoral immune response and antibody synthesis, whereas androgen seems to exhibit inhibitory effects [
]. Therefore, the occurrence of activated and degranulated mast cells located in the fibromuscular stroma, of the CBZ63 group might be caused by alterations of the testosterone and estradiol levels.
Since changes of the testosterone or estradiol plasma levels this age were not at 43 days of age, the presence of activated and degranulated mast cells in the prostatic stroma of CBZ43 rats cannot be only explained based on hormonal alterations. Thus, in the our experiment, the frequent observation of activated and degranulated mast cells located in the fibromuscular stroma of the CBZ43 and CBZ63 groups again suggests that a direct effect of the CBZ on ventral prostate may have occurred, inducing possible transitory inflammatory process. In fact, mast cells granules containing preformed tumor necrosis factor α and releasing this cytokine from mast cells is important for the initiation of an inflammatory response [
Some experiments indicate that mast cell infiltration can enhance carcinogenesis [
]. Mast cells contribute to the development of skin cancer in K14-HPV16 transgenic mouse by proteases releasing, such as tryptase and chymase and stimulating angiogenesis [
In addition, the presence of degranulated mast cells in the prostatic stroma of CBZ63 rats was followed by increase of the macrophages number. This increase of macrophages might partly be due to the cytokines released from infiltrating mast cells. Macrophages have shown clearly to aid in both the initiation and progression of experimental cancers [
]. Similar increases in mast cells and macrophages were observed in rats exposure to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine in cooked meat at high temperatures [
]; then, the inflammatory response to PhIP may help explain the tissue-specific and prostate lobe-specific carcinogenesis in the rat prostate induced by a long term dietary carcinogen [
]. Moreover, after castration, an increase of the mast cells can be observed in the rat prostate; besides, the number of ED-1-immunostained macrophages was also markedly increased in the epithelium and in the stroma of the rat ventral prostate [
In the present study, stereological and histomorphometric analyses revealed that CBZ chronic treatment since weaning causes, in the adult phase, decreases in: the volume of prostatic components, glandular epithelium surface and acini lenght. As a result, these alterations may have probably provoked reductions of the ventral prostate weight and volume in response to the CBZ direct effect. Besides, testosterone and estradiol plasma level alterations caused by CBZ can also indirectly act on the ventral prostate, influencing its maintenance and development. In addition, the presence of activated mast cells during pre-puberty and puberty phases may indicate a possible transitory inflammatory process, suggesting direct effect of the CBZ on ventral prostate.
In addition, we must remember that inflammatory reactions of the male genitourinary tract can affect male infertility in different degrees. In fact, high levels of cytokines secreted by mast cells can play a role in the decrease of the sperm function and sperm-egg interaction and this phenomenon have been widely neglected so far. Moreover, cytokines does not act only as key mediators of inflammation, but may also play important roles in the carcinogenesis and cancer progression [
]. Moreover, mast cells have been used as markers for risk stratification in invasive breast cancers [
]; then, it is possible that they can also function as cellular marker in other neoplasm, such as prostate tumor.
SUO performed histological procedures, morphometric and stereological evaluation, histological analysis numerical, densities of mast cells and macrophages and hormonal levels determination. Immunohistochemistry and AR semi-quantitative analysis were carried out by WRS. SUO and WRS performed statistical analysis of the data. SUO, FKO and SMM conceived the study and drafted the manuscript. All authors read and approved the final manuscript.