NF-kB overexpression and decreased immunoexpression of AR in the muscular layer is related to structural damages and apoptosis in cimetidine-treated rat vas deferens

Abstract

Background: Cimetidine, histamine H2 receptors antagonist, has caused adverse effects on the male hormones and reproductive tract due to its antiandrogenic effect. In the testes, peritubular myoid cells and muscle vascular cells death has been associated to seminiferous tubules and testicular microvascularization damages, respectively. Either androgen or histamine H2 receptors have been detected in the mucosa and smooth muscular layer of vas deferens. Thus, the effect of cimetidine on this androgen and histamine-dependent muscular duct was morphologically evaluated.

Methods: The animals from cimetidine group (CMTG; n=5) received intraperitoneal injections of 100 mg/kg b.w. of cimetidine for 50 days; the control group (CG) received saline solution. The distal portions of vas deferens were fixed in formaldehyde and embedded in paraffin. Masson´s trichrome-stained sections were subjected to morphological and the following morphometrical analyzes: epithelial perimeter and area of the smooth muscular layer. TUNEL (Terminal deoxynucleotidyl-transferase mediated dUTP Nick End Labeling) method, NF-kB (nuclear factor kappa B) and AR (androgen receptors) immunohistochemical detection were also carried out. The birefringent collagen of the muscular layer was quantified in picrosirius red-stained sections under polarized light. The muscular layer was also evaluated under Transmission Electron Microscopy (TEM).

Results: In CMTG, the mucosa of vas deferens was intensely folded; the epithelial cells showed numerous pyknotic nuclei and the epithelial perimeter and the area of the muscular layer decreased significantly. Numerous TUNEL-labeled nuclei were found either in the epithelial cells, mainly basal cells, or in the smooth muscle cells which also showed typical features of apoptosis under TEM. While an enhanced NF-kB immunoexpression was found in the cytoplasm of muscle cells, a weak AR immunolabeling was detected in these cells. In CMTG, no significant difference was observed in the birefringent collagen content of the muscular layer in comparison to CG.

Conclusions: Cimetidine induces significant damages in the epithelium; a possible antiandrogenic effect on the basal cells turnover should be considered. The cimetidine-induced muscle cells apoptosis confirms the susceptibility of these cells to this drug. The parallelism between enhanced cytoplasmic NF-kB immunolabeling in the damaged muscular tissue and muscle cell apoptosis suggests that this drug may avoid the translocation of NF-kB to the nucleus and interfere in the control of NF-kB-mediated smooth muscle cell apoptosis. The decreased immunoexpression of ARs verified in the damaged muscular tissue reinforces this possibility.

Figure 1

Photomicrographs of vas deferens of animals from CG (A and C) and CMTG (B and D) stained by Masson's trichrome. In B (CMTG), note that the epithelium (EP) is intensely folded and surrounds a decreased lumen (asterisk) in comparison to CG (A); a reduction in the muscular layer (M) is also evident. Lamina propria (LP). In C, a high magnification of epithelium (EP) shows epithelial columnar cells with normal aspect (arrows). In D, pyknotic nuclei (arrows) and vacuolization (v) are observed in the epithelial cells. Bars: 340 μm (A, B); 33μm (C, D).

Figure 2

Photomicrographs of vas deferens of animals from CG (A, E, F, I) and CMTG (B-D, G-H, J) submitted to TUNEL method (A-D), NF-kB (E-H) and AR (I, J) immunohistochemistry and counterstained by hematoxylin. In A, no TUNEL-labeled cells are observed in the vas deferens. However, in C, TUNEL-positive nuclei are observed in the columnar (thick arrow) and mainly in the basal (thin arrows) cells of epithelium (EP). Lamina propria (LP). In B and D, numerous TUNEL-positive muscle cells are also observed in the muscular layer (arrows). In E and F (CG), portions of the muscular layer show weak (E, arrows) or no (F; asterisks) NF-kB immunolabeling while in G and H (CMTG), a strong immunostaining is noted in the cytoplasm of muscle cells (arrows). In I and J, note the strong (I) and weak (J) AR immunolabeling in the muscle cells of vas deferens of rats from CG and CMTG, respectively. Bars: 50 μm (A); 20 μm (B); 27 μm (C); 12 μm (D); 156 μm (E, G); 38 μm (F, H); 58 μm (I,J).

Figure 3

Electron micrographs of muscular layer of vas deferens of animals from CG(A and B)and CMTG(C and D). In A and B, the smooth muscle cells (M) show nucleus (Nu) with regularly distributed chromatin and cytoplasm with numerous mitochondria (mi), miofilaments (Mf) and dense bodies (arrows). In high magnification, miofilaments (Mf) associated to dense bodies (arrows) are observed. In C, a cross-sectioned smooth muscle cell with numerous miofilaments (Mf) and dense bodies (arrows) is observed. Note that the cytoplasm is apparently more electron dense than in CG; the nucleus shows portions of strongly condensed chromatin (asterisks). In D, the shrunken cytoplasm of a smooth muscle cell (M1) containing dense bodies (white arrows) is more electron dense than CG. The nucleus is irregularly outlined and shows strongly electron dense chromatin in the nuclear periphery (asterisks). Next to this cell, two smooth muscle cells (M2 and M3) with irregular nuclei and portions of chromatin irregularly distributed in the nuclear periphery (arrowheads) are observed. A portion of strongly condensed chromatin is observed close to these nuclei (thick arrow). Bars: 4 μm (A); 1 μm (A, inset); 2.5 μm (B); 2 μm (C and D).

Figure 4

Photomicrographs of vas deferens of animals from CG (A) and CMTG (B) subjected to Picrosirius-polarization method. In A and B, the evident birefringent lamina propria is observed (LP). Note that in both groups (A and B), scarce birefringent collagen fibers (arrows) are observed in the muscular layer of vas deferens (M). Bars: 154 μm (A, B).

Figure 5

Percentage of birefringent collagen fibers in the muscular layer of vas deferens of rats from CG and CMTG groups. No difference is observed between the groups.