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c Laboratory of Cellular Biology, Department of Morphology, Federal University of Minas Gerais, Brazil
d Department of Morphology, Federal University of Sao Paulo, UNIFESP, Sao Paulo, Brazil
e Apoptosis Laboratory, Department of Pathology, Federal University of Minas Gerais, Minas Gerais, Brazil
f Department of Physiology, Southern Illinois University, School of Medicine, Carbondale, Illinois 62901-6512
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, gametogenesis, spermatogenesis, testes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the testing that preceded registration of cimetidine with the Food and Drug Administration, "testicular atrophy" was cited as an adverse effect (information obtained through the Freedom of Information Act of 1966), but no mechanism of cimetidine action was demonstrated. A well-known side effect of cimetidine is its ability to competitively block dihydrotestosterone (DHT) by occupying androgen receptors [1], making it a weak antiandrogen for tissues requiring DHT. Peripheral accessory organ weights are reduced in rodents, probably due to DHT deficiency. The consequences of the loss of DHT activity in the testis are not known since the role of DHT is currently under investigation in regards to its ability to support spermatogenesis [2–6]. In addition, cimetidine is known to block H2-receptors. H2 receptors are found in vascular smooth muscle cells [7], but the presence of H2 receptors in the testis has not been demonstrated.
The present study in rats is directed at further describing testicular lesions produced by cimetidine and sheds light on the mechanism of cimetidine action. The suggested over-the-counter dosage for humans has been between 400 mg/kg day (hyperacidity) and 800 mg/kg day (acute ulcer treatment) and on a surface area basis is equivalent to a rat dose of about 35 mg/kg/day and 70 mg/kg/day, respectively. We have used an intermediate dose 50 mg/kg/day and a higher dose (250 mg/kg/day) to examine cimetidine's effects in a rat model. Cimetidine can be administered i.v. for acute gastrointestinal disorders or can be taken orally for chronic gastrointestinal problems. In the present study we used intraperitoneal administration since it was the most convenient route for a rodent model. Furthermore, our treatment extended approximately two months since the most common use of cimetidine is over a long duration. We tested the potential of cimetidine to produce testicular effects in rodents and did not attempt to mimic human use. In particular, our objective was to determine the cellular site of cimetidine action. The data strongly point to cimetidine as a testicular toxicant and that the peritubular myoid cells are a target for cimetidine action. The data suggest that more studies in the human are necessary to determine if adverse cimetidine-related reproductive consequences may occur.
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MATERIALS AND METHODS |
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Animals were used in accord with the NIH Guide for Care and Use of Laboratory Animals. Adult Wistar rats weighing over 240–300 g were obtained from the Department of Physiology Vivarium (Federal University of Minas Gerais, Belo Horizonte, MG, Brazil) and maintained in 12L:12D conditions. Three groups of eight randomly selected animals were utilized. Animals were fed laboratory rat chow ad libitum and given water ad libitum.
Treatment
Animals received daily i.p. injections containing 50 mg (low dose, n = 8) or 250 mg (high dose n = 8) of cimetidine per kg of body weight for 59 days. The control group (n = 8) received only saline. The low and high doses were selected based on preliminary experiments in rats and were widely diverse since our preliminary experiments showed response to a wide spread in concentrations of cimetidine. Dosages were not selected to mimic human pharmaceutical use.
Preparation of Tissue for Microscopy
Animals were euthanized with pentobarbital during the perfusion procedure used for fixation of the testis. Animals were subsequently perfusion-fixed by whole body perfusion [8]. Briefly, after i.p. administration of heparin, rats were anesthetized and the abdomen and thoracic cavity opened to expose the heart. A needle was inserted into the left ventricle of the heart and 0.9% saline used to clear blood vessels. After clearance of vessels, a 2-way valve apparatus was used to introduce 4% glutaraldehyde into the vessels of animals without removal of the needle. Animals were perfused for 25–30 min, whereupon testes, epididymides, prostate gland, seminal vesicles and coagulating gland and adrenal glands were removed and weighed. Testis tissue was taken and prepared for embedding either in epoxy or glycol methacrylate by using standard techniques. Some testes were embedded in paraffin and sectioned at 5 µm for demonstration of apoptotic cells by using TUNEL labeling. Epoxy-embedded tissues were examined by both light and electron microscopy. Glycol methacrylate-embedded tissues were utilized for morphometric determinations. All cimetidine-treated and control animals were examined by light and electron microscopy. At least five slides were prepared from each animal for light microscopy and two grids of different areas were prepared for each animal and examined by electron microscopy. Each electron microscope thin section contained a minimum of four entire tubular cross sections and four partial tubules.
Hormone Assays
Before perfusion, blood samples were taken to measure testosterone (T) and FSH levels. Testosterone in plasma of rats was measured by a solid-phase radioimmunoassay, by using a commercial kit (Coat-a-Count) purchased from Diagnostic Products Corporation (Los Angeles, CA). One aliquot of 50 µl of plasma was dispensed in each assay tube, and then 1 ml of buffer containing the tracer (40000 cpm/tube) was added. A standard curve was set up with doses of testosterone ranging from 0.2 to 16 ng/ml. The mixture was incubated for 3 h at 37°C, the tubes were then decanted and the radioactivity bound to the tube was measured in a gamma counter with a built-in computer, which calculated the final values of testosterone in ng/ml of plasma. A single assay was performed; the intra-assay coefficient of variation was 4%.
FSH levels were determined with a double-antibody radioimmunoassay provided by the National Hormone and Pituitary Program of the National Institute of Health. FSH-RP-3 was used as a standard preparation. Purified FSH was labeled with 125I and anti-rat FSH-serum was used as the first antibody. The separation between antibody-bound and free tracer was achieved by the use of an anti-rabbit-gamma globulin obtained in goats, facilitating the addition of 10% polyethyleneglycol. A single assay was performed; the intra-assay coefficient of variation was 6.5%.
TUNEL Labeling
TUNEL labeling was performed on glutaraldehyde-fixed (4%) tissue that was subsequently embedded in paraffin and sectioned at 5 µm in thickness. A commercial in situ cell death detection kit (S7100-KIT, Apop Tag, Oncor, Gaithersburg, MD) was used. After labeling, the slides were dehydrated, stained with hematoxylin, counterstained with methyl green, mounted, and analyzed. The testes from three treated animals in each treated group and the testes from two animals in the control group were examined by the TUNEL method and the peroxidase tag was visualized.
Morphometry
An estimate of damaged tubules was performed by examining 100 randomly selected tubules from each animal. If either/or degenerating cells, abnormally low epithelial height, epithelial vacuolization were recorded in cross-sectioned or slightly obliquely sectioned tubules, these tubules were scored as abnormal. The percentage of abnormal tubules was determined. The tubular diameter and the height of the seminiferous tubule epithelium were measured at x100 magnification by using an ocular micrometer calibrated with a stage micrometer. Twenty to forty tubular profiles that were round or nearly round were measured for each animal and a mean was determined. The volume densities of various testicular tissue components were determined by light microscopy by using a 441-intersection grid placed in the ocular of the light microscope. Fifteen fields (6615 points) were scored for each animal at x400 magnification. Points were classified as one of the following: peritubular tissue, seminiferous epithelium, tubular lumen, Leydig cells, blood vessels, lymphatic space, interstitial macrophages and unidentified interstitial components or "other." The volume of each component of the testis was determined as the product of the volume density and testicular volume. To obtain a more precise measure of the testicular volume, 6.5% of the testis weight was considered to be the testicular capsule and was excluded from the testis weight [9]. As the specific gravity of the testis is nearly unity, testicular weight was transformed to units of volume. The total length of the seminiferous tubules, expressed in meters, was obtained by dividing seminiferous tubule volume by the squared radius of the tubule. Correlation analysis was performed with tunica propria volume with the other parameters measured.
Statistics
All data are presented as the mean ± SEM and analyzed via ANOVA (Newman-Keuls test). The significance level in comparisons was considered to be P < 0.05. An analysis of correlation was performed by using the Statistica Software (StatSoft, Inc., Tulsa, OK).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The body and testis weights of cimetidine-treated animals were not different in any of the groups studied (Table 1). There was a significant decrease in the high dose group in epididymal, ventral prostate, combined seminal vesicle and coagulating gland weights (Table 1). There was a significant increase in adrenal gland weight in the high dose animals, an increase possibly due to drug-related stress since the high dose is clearly pharmacologic. A trend toward a decrease in tubular diameter was noted, but this was not statistically significant. The height of the seminiferous epithelium was significantly reduced in high dose animals compared with controls (Table 1).
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FSH levels were significantly increased in both the 50 and 250 mg/kg/day dose levels compared with controls. Serum testosterone levels were not statistically different after cimetidine treatment (Table 1).
Volume and Volume Density Measurements
The morphometric measurements for the volume density and testiticular volume components were similar except for the tunica propria (peritubular tissue), where there was a significant reduction in high dose groups compared with control group (Table 2).
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Microscopy
Light microscopy revealed that most animals (6 out of 8) in the low dose group and 5 out of 8 animals in the high dose group were without visible histologic change in testis structure. Control testes appeared normal (Fig. 1A). The seminiferous tubules of those showing abnormalities in the testes were highly variable in appearance (Fig. 1, B and C). Most tubules appeared normal, but in some areas many tubular profiles were virtually depleted of germ cells and contained only Sertoli cells (Fig. 1D). Commonly, tubules contained Sertoli cells and basal compartment germ cells. Considering all animals in the treated and control groups, a mean of 16.9% of tubules in the low dose treatment groups and 15.2% in the high dose treatment group showed abnormalities as defined in Materials and Methods; whereas 0.1% showed abnormalities in the control group. Those treated animals with marked changes in seminiferous tubules also showed an expanded lymphatic space throughout the testis (Fig. 4, B–D).
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An electron micrograph of the peritubular tissues of control rats is provided (Fig. 2A). Electron microscopy also confirmed the healthy appearance of spermatogenesis from the seminiferous tubules in all control animals and the healthy appearance of spermatogenesis in most seminiferous tubules of treated animals of both cimetidine dose groups. In the high dose group, tubules that were partially depleted of germ cells and fully depleted of germ cells ("Sertoli cell only") were both noted. There was always duplication or multiple layering of the lamina densa of the basal lamina in those tubules that showed extensive depletion of the germ cells. This duplication was seen in the low dose group within the region of the peritubular tissue between the myoid cell and either the Sertoli cells and/or spermatogonia. There was also a thickening and sometimes multiple layering of the lamina densa of the basal lamina lying between the myoid cell and the parietal layer of lymphatic endothelium (Fig. 2B). There was often cellular debris present in the myoid cell layer in the low dose group (Fig. 2C) suggesting previous cell degeneration. The same results obtained for the low dose group of cimetidine-treated animals were obtained for the high dose group. The presence of normal spermatogenesis did not always mean that the region of the basal lamina was also normal. Generally, the basal lamina showed abnormalities in intertubular profiles where the seminiferous epithelium appeared normal. Thus, a close examination revealed focal areas of duplication of the amorphous layer (lamina densa) similar to that seen around germ cell depleted tubules (Fig. 2D).
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A test of correlation showed that FSH levels correlated negatively and significantly (r =-0.86) with the volume of peritubular tissue in animals treated with the high dose.
In the course of examination of many tubules by electron microscopy (4-h examination), one of us (LDR) found two cells of the peritubular tissue early in the process of apoptotic death and six profiles of advanced cell degeneration. The apoptotic myoid cell nuclei showed the typical margination of chromatin classically associated with apoptosis. An examination of the densified cytoplasm indicated that the cell was clearly in the process of degeneration (Fig. 3). Other features including swollen myoid cells and advanced degenerative cells were also seen (Fig. 3). Examination of control tissues for a 4-h period revealed no early apoptotic cells or, in fact, any profiles of advanced cell degeneration. One of us (LDR) has extensive experience in viewing normal and pathological testes and has never observed apoptotic myoid cells or advanced cell degeneration in the peritubular cell layer. Neither are we aware of any literature that has demonstrated apoptotic peritubular cells in normal or treated animals.
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TUNEL Labeling for Apoptotic Cells
Testis cells in control animals usually displayed few labeled cells by using the TUNEL method (Fig. 4A). Labeling of germ cells was increased in both low and high dose animals (Fig. 4B). In treated tissues, cells in the peritubular region (Fig. 4, C and D) were also labeled as were smooth muscle cells of blood vessels (arterioles; Fig. 4, E and F). There was great labeling variability from animal to animal and area to area within the testis. The control showed no labeled peritubular cells. There was considerable animal-to-animal variability in labeling in the treated groups; those animals that showed more tubular degeneration in each group demonstrated myoid cell staining in approximately 10% of tubules when 50 tubular cross sections were examined. No control animals showed TUNEL labeled peritubular cells. TUNEL positive somatic and germ cells were noted almost exclusively in cimetidine-treated tissues, although rare labeling was noted in germ cells of control animals. Since only 2 of 5 animals in the low dose group and 3 of 5 animals in the high dose group showed markedly increased TUNEL labeling, a statistical analysis could not be performed.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Could cimetidine be acting via reduction in gonadotropin secretion? There is conflicting information whether or not cimetidine affects plasma gonadotropins systematically [1, 10]. If effects are present, they are minor in the testis. For example, Leydig, Sertoli, and germ cells show none of the stage-related effects of classical gonadotropin withdrawal [11]. Some studies report decreased testosterone levels in cimetidine-treated rats [12, 13], a finding not supported by the present study or some other previous studies [10, 14–16].
This study has confirmed the well-known effects of cimetidine on accessory sex organ weights [1, 12–17]. These findings may be related to the weak anti-androgenic properties of cimetidine, acting to block DHT from binding to the androgen receptor [1] and thus preventing DHT action. Myoid cells and testicular arteriole cells are known to possess nuclear androgen receptors [18], but neither have not been shown to respond to DHT. A second possible mechanism of cimetidine may be the result of chronic antagonism of H2 receptors. Vascular smooth muscle cells of the body are generally known to be H2 responsive [7], and we believe those of the testis are likewise responsive although we have not found a report describing that peritubular cells possess H2 receptors. That these two cells of the testis undergo apoptosis after cimetidine administration is perhaps not coincidental. Further work is needed to determine if there are H2 receptors in the smooth muscle cells of arterioles and around seminiferous tubules. Finally, peritubular cells, in vitro, show repressed activity when exposed to glucocorticoids [19]. The present study demonstrated adrenal enlargement after cimetidine administration.
Chronic treatment with cimetidine has not been reported to reduce weight in rats [1, 13–17, 20]; the present study provided similar results. However, in most of the aforementioned studies, the mean value for testis weights in cimetidine-treated animals is always numerically below that of controls, although in a statistical comparison with controls the mean level does not reach the level of significance. Likewise, the present study as well as that conducted by the manufacturer (SmithKline Beecham), for the purposes of Federal Drug Administration (FDA) registration of the compound, also detected mean testis weights numerically lower than controls, but the mean values that were recorded were not significantly different. Although the bulk of the data suggest no significant difference in testis weights, we believe that testis weights are slightly lowered by the treatment, based upon rather consistent trends in numerical values and observed testicular "atrophy" (FDA report) or decrease in seminiferous epithelial height in the present and in another study [21]. We also note that testis weight is not a sensitive parameter for demonstration of toxic effects [22]. Taken collectively, we believe that the data support a cimetidine-related decrease in testis weight.
The cause of the patchy germ cell loss is not apparent. No stage-related susceptibility to cimetidine was noted. It is not unusual to see extreme variability in degeneration of spermatogenesis after toxic insult. It is possible that a few of the 20 or so tubules in the rat testis are affected by cimetidine and that what one is viewing is cross sections of tubular profiles from only a few damaged tubules.
To our knowledge there are no studies showing xenobiotic effects on peritubular myoid. There are a few reports of effects on cultured peritubular myoid cell physiology [23, 24]. The present study provides several lines of evidence that the peritubular myoid cell is the primary cell affected by cimetidine. Since the microanatomy of the peritubular tissue in most rodents is not well known, some review is useful [25]. The peritubular tissue is composed of two cell types concentrically arranged around the seminiferous tubules, the outermost having been termed the parietal endothelium of the lymphatic space or parietal lymphatic endothelium [26]. There is an amorphous component of the basal lamina (lamina densa) sandwiched between this so-called endothelial cell and innermost flattened cells, the peritubular myoid cell. Collagen is usually associated within two layers of the lamina densa. The myoid cell is separated from the seminiferous epithelium by another similar appearing, but larger, basal lamina.
A role for the myoid cell in tubular contractions was proposed many years ago [27], and later contractile abilities were demonstrated [28]. More recently, Skinner and colleagues [29] have suggested that the presence of a factor called P-mod S, emanating from the peritubular myoid cell, would be stimulatory to Sertoli cell. Inability to purify such a factor has led to decreasing attention to this factor in recent years.
What is the evidence that the myoid cell is a target of cimetidine toxicity? First, defective basement membranes in damaged and apparently healthy spermatogenic areas were only present in cimetidine-treated animals. The basal lamina that resides between the myoid and the Sertoli cell is jointly produced by these cells [30]. The basal lamina peripheral to the myoid cell is also altered and, given that this basal lamina is not in contact with Sertoli cells, it is most likely not produced in cooperation with the Sertoli cell. Morphometry shows a loss of peritubular tissue, in which the peritubular myoid cell is the major component. Apoptosis of peritubular myoid cells was also demonstrated by electron microscopy, as well as by TUNEL labeling. Thus, the evidence points to the myoid cell as the primary cell affected by cimetidine treatment.
Loss of peritubular tissues or in numbers of myoid cells in seasonally breeding animals does not occur [31], suggesting that cimetidine treatment is not related to lack of gonadotropin or testosterone secretions. Furthermore, our observations of long-term hypophysectomized rats suggest that the peritubular cells are simply enfolded, rather than lost during hormonal withdrawal (personal observations).
Duplication of the basement membrane is not an uncommon finding in some testes that are abnormal [32–34], for example, in genetic cryptorchidism in the rat ([33], unpublished observations). Many pathologies cause enfolding of the basal lamina. We believe that the effect of enfolding of the basal lamina is a phenomenon quite different from a duplication of the basal lamina. Extensive enfolding is usually found after a seminiferous tubule has greatly atrophied [31]; whereas duplication of the amorphous material (lamina lucida) is a phenomenon that is not well understood. Duplication appears to be due to a defective synthetic process, whereas enfolding is more likely a passive result of tubular shrinkage [34]. In the present study, duplication of the amorphous layer of the basal lamina was seen in apparently healthy tubules suggesting that it precedes loss of germ cells.
Cimetidine is considered a week antiandrogen and its antiandrogenic properties are due to inhibition of DHT binding to the androgen receptor [1]. DHT has a greater affinity for the androgen receptor and dissociates more slowly from this receptor. It is often assumed that DHT has its role in peripheral secondary sex organs, while testosterone is more related to support of spermatogenesis. This assumption is not necessarily correct. Relatively current studies suggest that DHT has potential effects on spermatogenesis in experimental situations [2–6], although the relative roles of testosterone and DHT or, specifically, the physiological role of DHT is not completely understood. Could the effects on spermatogenesis, as seen in the current study, be due to a direct effect of the lack of DHT on spermatogenesis? We believe that it is not likely the case, given that there is a well-known stage-related pattern of regression of the testis after hormonal withdrawal [11]. After complete hormonal withdrawal, germ cells up to spermatocytes are the only cells present [35], a situation differing from the tubular atrophy seen in cimetidine-treated rats. In addition, such loss of hormones affects all tubules and does not present the tubule-to-tubule variability described herein.
Apoptosis of smooth muscle cells of the testis has not been reported to our knowledge. Myoid cells and vascular smooth muscle cells share the property of contractility and have the subcellular contractile apparatus in common [36]. It is well know that the testicular vasculature can be lost and regained [37], and that the rat testis has major regenerative properties after certain conditions and presumably the vasculature comes and goes with testis mass [37–39]. Thus, the consequence of apoptotic muscle-like cells, as seen in the present study, is not known. If the vascular changes were the primary defect in cimetidine action, then one would expect all tubules to be similarly affected. The negative correlation of FSH and tunica propria volume suggests that there may be a relationship between the two findings. Although the meaning of this correlation analysis may point to a lack of germ cells causing depression of inhibin [40], the negative correlation of FSH and tunica propria in the highest dose is the strongest of the correlations performed (correlations not shown). Yet, when we examined animals on an individual basis, those with the lower testis weights showed the highest FSH levels (data not shown). There is currently no information in the literature to suggest that the tunica propria regulates FSH, directly or indirectly.
It should be emphasized that cimetidine is still a widely utilized drug in humans under the brand name of Tagamet and that its use is no longer strictly regulated, as when it was medication requiring prescription. The effect on testis weight in experimental animals is minimal, but nevertheless, present. The only known clinical test of cimetidine action on the testis in the literature shows that it reduces sperm counts [41]. Current regulatory requirements would not allow registration of a compound with reproductive side effects unless the risk-to-benefit ratio was sufficiently high to necessitate its use. The present study does not directly relate to cimetidine use in humans but these results indicate that further clinical studies are needed to determine the putative toxicological effects of cimetidine in humans. We are currently investigating a primate model to determine if the results presented herein can be extended to a species more closely akin to humans.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by NIH HD35494. 
2 Correspondence. FAX: 618 453 1517; lrussell{at}siumed.edu
Accepted: June 9, 2000.
Received: February 8, 2000.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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