The results showed that cimetidine causes significant reductions in the perimeter of epithelium and area of the smooth muscle layer of vas deferens. The reduction in the epithelial perimeter could be due to death of epithelial cells which was confirmed by the presence of nuclei with condensed chromatin and also labeled by TUNEL, mainly in the basal cells. According to Paniagua et al., the basal cells are able to differentiate into the different columnar cells, playing a role in the epithelial turnover. Therefore, the significant reduction in the epithelial perimeter may be due, at least in part, to a possible interference of cimetidine on the basal epithelial cells, leading to a total decrease in the cellular population. The mechanism by which cimetidine induces epithelial cell death in the vas deferens should be further investigated. It is known that cimetidine inhibits ARs in different tissues that require androgens [17–19]. However, studies have demonstrated that androgens are essential for the structural and functional maintenance of the vas deferens. In castrate mice, Hamilton et al. demonstrated alterations in the epithelial cells of vas deferens such as absence of smooth endoplasmic reticulum, reduction in the size of the Golgi apparatus and decreased number of mitochondria. In mammals, including rodents , ARs have been detected in the epithelial cells of vas deferens [38, 39]. According to Zhou et al., the basal cells show a more accentuated AR immunoexpression in comparison to the other epithelial cells. Moreover, alterations in the basal cells of rat vas deferens caused by suppression of testosterone levels confirm that these cells are dependent of androgen . Therefore, the morphological alterations observed in the epithelial layer of CMTG animals such as: reduction of the epithelial perimeter, presence of pyknotic nuclei, TUNEL-positive nuclei and intraepithelial vacuoles may be related to a possible antiandrogenic effect of cimetidine on the epithelial cells.
The smooth muscular layer was also affected by cimetidine treatment since muscle cell death, confirmed by TUNEL and TEM, and a significant reduction of muscular area was detected in CMTG. The reduction in the muscular layer is related to the muscle cells death by apoptosis. Additionally to the TUNEL method, the presence of ultrastructural features typical of apoptosis such as the presence of peripheral condensed chromatin in the nuclei, nuclear fragments and cellular shrinkage , indicates that these cells undergo apoptosis. In previous studies, cimetidine has been demonstrated to exert a harmful effect on peritubular myoid cells, [21, 24] and also on the smooth muscle cells of testicular blood vessels , leading to apoptosis in these cells. Thus, in the present study, the presence of muscle cell death and muscular layer atrophy in the vas deferens confirm the susceptibility of smooth muscle cells to cimetidine treatment. However, the cellular way by which cimetidine induces smooth muscle cell death needs to be clarified. It is possible that the atrophy of vas deferens muscular layer is related to the cimetidine antagonist effect on histamine H2 receptors since histamine exerts an inhibitory effect, via H2 receptors, on the vas deferens contraction [6, 7]. The activation of H2 receptors by histamine inhibits the muscle contraction response due to a decrease in the Ca2+ influx necessary for contraction. However, this effect (muscle relaxing) is reduced or totally antagonized by cimetidine [8, 42], leading to increased intracellular calcium influx. The excess of calcium ions seems to be one of the main routes involved in the induction of programmed cell death [43, 44]. It has been demonstrated that simvastatin induces apoptosis in the smooth muscle cells due to increased Ca2+ levels and, subsequently, caspase activation . Therefore, additionally to a possible antiandrogenic effect of cimetidine (as discussed below), it is possible that smooth muscle cell death of vas deferens is caused by increased intracellular calcium influx due to cimetidine antagonist action on H2 receptors. Although cimetidine also exerts antiandrogenic and antiangiogenic actions, further studies focusing on the comparison of cimetidine effects with other histamine H2 receptors antagonist effects would be advisable.
Besides H2 receptors, ARs have also been detected in the smooth muscle cells of vas deferens  and testicular arterioles . Type L calcium channels of smooth muscle cells are inhibited by testosterone [3, 4]. In the rat vas deferens, 5α-DHT inhibits Ca2+ influx through the voltage-dependent calcium channels and, then, inhibits smooth muscle contraction . As caspase-stimulated smooth muscle cell apoptosis occurs by increased calcium influx [43, 44], the muscular layer atrophy of vas deferens following cimetidine treatment could also be related to the antiandrogenic effect of this drug on the smooth muscle cells.
Another point to be emphasized is that the high incidence of TUNEL-positive cells was parallel to the overexpression of NF-kB in the muscular layer of rat vas deferens from CMTG. NF-kB transcription and activation play a crucial role in regulating the process of cell death by apoptosis in several cell types including smooth muscle cells [25, 31]. In most cell types, NF-kB remains bound to IkBα protein and thereby is inactive in the cytoplasm [47, 48]. After stimulation by various reagents, IkBα is rapidly phosphorylated by the IkB kinase (IKK) complex and degraded by the proteasome, allowing NF-kB to translocate to the nucleus and activate its target gene [27, 49, 50]. Thus, NF-kB activates the transcription of many genes capable of suppressing cell death . Studies have demonstrated that vascular smooth muscle cells apoptosis has been commonly observed in response to inhibition of NF-kB by IkBα , by Propionyl L-carnitine  and also cimetidine. This drug (cimetidine) inhibits the translocation of NF-kB to the nucleus, decreasing the transcription of antiapoptotic genes and inducing apoptosis in salivary gland tumor cells . Pretreatment with roxatidine (anti-H2 receptor and AR antagonist) has also demonstrated to inhibit the translocation of the activated NF-kB subunits, p65 and p50, to the nucleus . These findings are consonant to our results since an enhanced NF-kB immunoexpression was found in the cytoplasm, but not nuclei, of smooth muscle cells following cimetidine treatment. A similar increased immunolabeling has also been demonstrated in a study, in which NF-kB p50 was used, and has been related to a possible epitope unmasking . The antibody used in the present study bounds around to phosphorylation site of Serine 276 that belongs to the amino acids sequence 1–313 of p65. This sequence containing the nuclear localization signal is essential for IkBα binding . Therefore, the enhanced NF-kB immunostaining in the muscle cells of cimetidine treated rats could be resulted from the unmasking epitope and indicates that this factor is in its active form in the cytoplasm.
The decreased AR immunolabeling in the cimetidine-treated rats also reinforces the idea that cimetidine may have interfered in the translocation of the activated NF-kB to the nucleus. AR gene is a NF-kB target gene; thus, overexpression of NF-kB factor p65 causes increased AR mRNA and protein levels . Therefore, our results indicate that muscle cell apoptosis can be related to a possible interference of cimetidine on the translocation of the activated NF-kB to the nucleus. A plausible explanation for this interference can be related to the antiandrogenic action (AR antagonist) of cimetidine since studies focusing on the effects of AR on NF-kB activity in the prostate cancer cell lines have demonstrated that NF-kB activity depends on the availability of androgens. Thus, in the presence of androgens, NF-kB activity is decreased by AR while in the absence of ligand, AR increases NF-kB activity .
Regarding the collagen content, smooth muscle tissue substitution by collagen fibers (fibrous tissue) has been detected in the smooth muscular wall of vas deferens of dogs and rats subjected to different injuries [57–59]. In the present study, it was supposed that an increase of fibrous tissue (collagen fibers) would occur among the muscle cells during smooth muscular layer involution. However, no collagen content difference was detected between the groups after the quantitative analysis of the birefringent collagen under polarized light. This finding can explain the significant reduction of the muscular area. The absence of fibrosis during the involution of muscular layer, observed in the present study, could be related to a possible histamine antagonist effect of cimetidine on the fibroblasts. This is reinforced by the fact that histamine stimulates fibroblasts proliferation via H2 receptors . Moreover, type I collagen synthesis by fibroblasts, stimulated by histamine via H2 receptors, is inhibited by cimetidine . However, further studies are necessary to confirm this possibility.