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Protective effects of ursodeoxycholic acid against 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced testicular damage in mice
Toxicology and Applied Pharmacology 194 (2004) 239 – 247 www.elsevier.com/locate/ytaap
Protective effects of ursodeoxycholic acid against 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced testicular damage in mice Young-Il Kwon, a Je-Deuk Yeon, b Seung-Min Oh, b and Kyu-Hyuck Chung b,* a
Department of Clinical Laboratory Science, Shinheung College, Uijeongbu, Kyunggido 480-701, South Korea b College of Pharmacy, Sungkyunkwan University, Suwo˘n, Kyunggido 440-746, South Korea Received 22 July 2003; accepted 17 September 2003
Abstract The protective effect of ursodeoxycholic acid (UDCA), a biliary component found in bears, on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced testicular damage in mice was investigated. Fifty C57BL/6J mice were equally divided into five groups. The mice in the control group received the vehicle and standard chow. The single TCDD treatment group received 27.5 Ag/kg of TCDD subcutaneously. The UDCA-included treatment group received pulverized chow containing 0.125%, 0.25% and 0.5% UDCA, respectively, for 70 days starting 10 days before TCDD injections. The body and testicular weights were shown to be decreased in the single TCDD treatment group, while the decrease was prevented by UDCA added to the chow. In addition, the decrease in the serum-luteinizing hormone (LH) or the follicle stimulating hormone (FSH) secondary to a TCDD injection was not observed in the UDCA-included treatment group. Contrary to the single TCDD treatment group, the germinal epithelium and intercellular space were relatively well preserved in the UDCA-included treatment group. Adding UDCA also normalized TCDD-induced irregular ultrastructural changes such as development of phagolysosomes, inflated smooth endoplasmic reticulum (SER), dilated and altered mitochondria, necrosis and completely damaged seminiferous tubules. Moreover, in the experiment for Arnt expression, UDCA added to the chow suppressed the TCDD-induced relocation of Arnt from the cytoplasm to the nuclei. In conclusion, TCDD-induced testicular toxicity was effectively protected by UDCA. There was almost complete recovery of the testes in the UDCA-included treatment group. Thus, UDCA may be useful for the prevention and treatment of TCDD-induced testicular damage. D 2003 Published by Elsevier Inc. Keywords: TCDD; Testicular toxicity; Ursodeoxycholic acid; Preventive effect
Introduction Halogenated aromatic hydrocarbons including polychlorinated dibenzodioxin and polychlorinated dibenzofuran are the two most typical environmental pollutants exhausted from industrial waste incineration or paper manufacturing processes. 2,3,7,8-Tetrachlorldibenzo-p-dioxin (TCDD) is the most toxic compound among polychlorinated aromatic hydrocarbons. Humans are generally exposed to such compounds, which are incorporated into food, drinking water, soil, dust, smoke and air. Dioxins with very slow biodegradability have long persisted in the environment. These compounds can be concentrated in the adipose tissues of animals in the food chain, and * Corresponding author. College of Pharmacy, SungKyunKwan University, #300 Chonchon-dong, Changan-ku, Suwo˘n, Kyunggido 440-746, South Korea. Fax: +82-31-292-8800. E-mail address: [email protected] (K.-H. Chung). 0041-008X/$ - see front matter D 2003 Published by Elsevier Inc. doi:10.1016/j.taap.2003.09.024
are often found in human breast milk (Sharara et al., 1998). Among various TCDD toxicities, reproductive toxicity may be seen in both males and females. TCDD is classified as an environmental hormone because it has been found to cause endocrine disruption in various biotas (Astroff and Safe, 1988; Mebus et al., 1987; Umbreit et al., 1988). The toxic effects of TCDD in male reproductive systems include a reduction in the size of the testes, prostate gland and seminal vesicle, as well as a decrease in the number of sperm count (Johnson et al., 1992; Khera and Ruddick, 1973; Moore et al., 1985; Rune et al., 1991). It has also been reported that TCDD causes separation and rupture of spermatogonia and the Sertoli cells, which in turn impedes spermatogenesis (Kim et al., 1999; Rune et al., 1991). In recent years, several natural formulations relating to reducing or eliminating TCDD toxicity have been in focus. Resveratrol (Casper et al., 1999), flavonoids (Ashida et al., 2000), green tea (Kang et al., 2000) and panax ginseng (Kim et al., 1999) may provide protection against TCDD toxic-
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ities. Resveratrol extracted from red wine has a preventive effect against dioxin toxicity by binding to aromatic hydrocarbon receptors, to which dioxin compounds bind. Although this compound has competitive inhibitory activities for the dioxins previously absorbed in the body, it cannot suppress continuous absorption of dioxin per se, nor can it enhance the excretion thereof. Additionally, activated carbon may be used as an absorbent of dioxin. Nevertheless, it is not recommended for common use because of a low selectivity in adsorption. Thus, it has a drawback by interfering in the absorption of essential lipophilic vitamins. Ursodeoxycholic acid (UDCA), a component found in the bear’s bile, has been used in the treatment of liver disease. Some of the UDCA pills are used for liver protection and as a cholagogue. UDCA has also been known to enhance immunity and have anticancer effects (Suh et al., 1997), anti-inflammation effects (Seraj et al., 1997), antiprimary sclerosing cholangitis effects (Beuers et al., 1992) and antistress effects (Kawamura et al., 1989). In addition, UDCA as a cholic acid induced from cholesterol may have an effect in the sperm’s development and motility. However, no research has been done on its effect on the male productive organs, particularly in steroidogenesis and spermatogenesis. The objective of this study is to identify whether UDCA can suppress testicular toxicities of TCDD in C57BL/6J male mice. Therefore, we investigated the preventive effect of UDCA, added to chow, against the TCDD-induce testicular damage such as body and testicular weight losses, reduced hormonal concentrations, histopathological and immunohistochemical changes.
Materials and methods Animals and treatment. Male C57BL/6J mice (4 –5 weeks old, 16 –17 g, Charles River Laboratories in Japan) were used throughout the experiments. This experiment was performed in a controlled environment (maintained at 23 F 3 jC, a relative humidity of 60 F 10 jC, in 12-h light – dark cycle). The mice were provided with feed and tap water ad libitum. Water was sterilized with a UV-sterilizer and feed (Hae Eun Trade Co., Korea) was sterilized under 2.0 M rad of radioactive rays. Fifty C57BL/6J mice were equally divided into five groups. The normal control group received the vehicle and standard chow. The TCDD treatment group received 27.5 Ag/ kg TCDD (2,3,7,8-tetrachloro-dibenzo-p-dioxin, 95% purity, ChemServices, Inc., PA, USA), which had been dissolved in acetone and corn oil (1:6, v/v) in volumes of 5 ml/kg body weight subcutaneously. The UDCA-included treatment group received pulverized chow containing 0.125%, 0.25% and 0.5% UDCA (98% purity; Daewoong Pharm. Co., Ltd., Korea), respectively, for 70 days starting 10 days before the TCDD injection. Appropriate doses of TCDD and UDCA were determined in the preliminary experiment. Acetonecorn oil (1:6, v/v) alone was administered subcutaneously as a vehicle-control group.
Change in body weight, testicular weight and hormonal concentration. Body weight was checked twice a week during the treatment period. Feed and water were removed 1 day before the animals were sacrificed. Blood samples drawn directly from a heart puncture were taken. The testes were removed under general anesthesia with ethyl ether and the testicles were weighed. Serum was separated by centrifugation and stored at 80 jC until an assay was performed. The serum concentrations of the testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) of six mice in each group were determined using ACS: 180 (automated chemiluminescence system, Bayer, NY, USA) (n = 6). Observation of histopathological and ultrastructural change. To assess histological and ultrastructural changes, the fixed testicular tissues harvested from 10 surviving mice in each group (except seven mice of 27.5 Ag/kg single TCDD treatment group) were paraffin embedded. Then, they were prepared as 2 – 3-Am-thick sections using a microtome (microm HM 440E). They were double stained with hematoxylin and eosin, and were observed by light microscope. About 50 horizontally sectioned seminiferous tubules from five mice in each group (total of 250 seminiferous tubules) were measured to determine the mean diameter of the seminiferous tubules. All seminiferous tubules of one histological section from a testicular specimen were evaluated and scored from 1 to 10 using the Johnsen scoring system. In using electron microscopy, the testicular tissues were resected at 1 mm3 thickness and fixed in a 2.5% glutaldehyde solution for 4 h at 4 jC. Then, the specimens were washed with a phosphate buffer solution at pH 7.3, and placed in 1% osmium tetroxide (pH 7.3) for 2 h. They were then dehydrated in ethanol and propylene oxide, and were embedded in an epon-mixed solution. The ultrathin sections (60 – 70 nm) severed with an ultra-microtome (Reichert-Jung, Ultracut E) were stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (H-600, Hitachi). Immunohistochemistry. To assess immunohistochemical changes, the testicles removed from 10 surviving mice in each group (except seven mice of 27.5 Ag/kg single TCDD treatment group) were fixed in fresh Bouin’s solution (for cytochrome P450scc expression) and formaldehyde solution (AhR, Arnt, Cyp1a1 and Cyp1a2 expression) for 12 and 24 h, respectively. These tissues were subsequently dehydrated, embedded in paraffin and sectioned at 5 Am (n = 6). Briefly, these sections were deparaffinized and rehydrated through graded alcohol to Tris-buffered saline (TBS), and nonspecific binding was blocked by inactivating them for 10 min using a heat-inactivated normal goat serum (Rockland, Gilbertsville, CA, USA) after being diluted at the proportion of 1:5. The primary antibody was diluted in the blocking solution as described (AhR, (Santa Cruz Biotechnology, Inc., CA, USA), diluted at 1:50; Arnt (Santa Cruz Biotechnolo-
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gy), diluted at 1:50; Cyp1a1 (CHEMICON International Inc., CA, USA), diluted at 1:100; Cyp1a2 (CHEMICON International Inc.), diluted at 1:100; cytochrome P450scc (CHEMICON International Inc.), diluted at 1:100 and incubated over the sections in humidified chamber overnight (AhR and Arnt) at 4 jC or 40 min (Cyp1a1, Cyp1a2 and cytochrome P450scc) at room temperature). These sections were subsequently washed with TBS containing 0.1% Tween 20, followed by TBS for 5 min, incubated with appropriate secondary antibodies (biotin conjugated affinity purified anti-goat IgG [Rockland] for AhR and Arnt; biotinylated anti-rabbit IgG [Vector laboratories, CA, USA] for Cyp1a1 and cytochrome P450scc), then washed again with TBS and incubated with a streptavidin-peroxidase complex (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA). After washing them three times for 5 min using TBS, immunoreaction was localized using hydrogen peroxide-activated 3.3V-diaminobenzidine-tetrahydrochloride (Sigma) as a peroxidase substrate. These sections were counterstained with Mayer’s hematoxylin and were dehydrated with graded series of alcohol, rinsed with xylene and mounted with Permount. In the negative controls for which primary antibodies were substituted with normal goat IgG, normal rabbit IgG or normal sheep IgG showed little to no background staining. Johnsen’s score counts. To measure the degree of spermatogenesis, the Johnsen’s testicular biopsy score was used. The degree of spermatogenesis was scored from 1 to 10 depending on the presence or absence of cell forms arranged according to the extent of maturity of their spermatogenesis. For these observations, an objective lens (magnification: 40) was used. A total of 250 seminiferous tubules (50 seminiferous tubules for five subjects in each group) were used.
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Statistical analysis. One-way analysis of variance (ANOVA) test was carried out for the analyses of body weight, weights of the testes and hormone levels of the acute toxicity test. If the ANOVA test results showed a significant difference, an unpaired Student’s t test was performed for cases with P < 0.05 and P < 0.01.
Results Change in body weight and testicular weight As shown in Fig. 1, body weight was decreased in the single TCDD treatment group than that of the vehicle control group after 12 days of TCDD administration. However, there was no significant difference in body weight change between the UDCA (0.125%, 0.25% and 0.5%) included treatment group and vehicle control group. It was revealed that the TCDD-induced body weight loss was prevented by UDCA. As shown in Fig. 2, a significant decrease in testicular weight was induced by a single TCDD treatment. However, TCDDinduced testicular weight loss was significantly inhibited in UDCA-included treatment group. The gain in the body and testicular weight was enhanced by UDCA-included treatment group at the similar rate as that of the vehicle control group, which suggested that UDCA had both protective and therapeutic effects against the TCDD-induced testicular atrophy. Changes of serum hormones (Testosterone, LH and FSH) As shown in Table 1, the concentration of testosterone and FSH was decreased in the single TCDD treatment group in contrast to the vehicle control group. However, there was no loss of serum testosterone concentration in 0.125% and
Fig. 1. The effects of adding UDCA to chow on body weights of C57BL/6J mice during 60 days after a single dose of TCDD. The graph shows the mean and SD of the data obtained from survival mice of 7 – 10 animals (the number of single TCDD treatment group = 7, the number of others = 10).
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Fig. 2. The effects of adding UDCA to chow on testicular weights of C57BL/6J mice 60 days after a single dose of TCDD. The graph shows the mean and SD of the data obtained from survival mice of 7 – 10 animals (the number of 27.5 Ag/kg TCDD single treated group = 7, the number of others = 10). *Significantly different from vehicle control group (V-C) at P < 0.05. **Significantly different from vehicle control group (V-C) at P < 0.01. # Significantly different from TCDD single treated group at P < 0.05. ## Significantly different from TCDD single treated group at P < 0.01.
0.5% UDCA-included treatment groups and FSH concentration in 0.25% UDCA-included treatment group compared with vehicle control group. Therefore, it was suggested that serum hormonal changes caused by TCDD could be prevented by the administration of UDCA. Histopathological change Cell differentiation showed a reduction in most cells, including spermatogonia in the single TCDD treatment group (Fig. 3b). Separation of spermatogonia near the basement membrane, secondary to dissolution of the germinal epithelium, and expansion of intercellular space among neighboring germ cells, were observed. Enlargement of the Table 1 The effects of adding UDCA to chow on serum hormonal concentrations 60 days after a single dose of TCDD in C57BL/6J mice TCDD dose (Ag/kg)
% UDCA in chow
Testosterone (ng/dl)
LH (mIU/ml)
FSH (mIU/ml)
None 27.5
None None 0.125 0.250 0.500
86.42 53.86 73.92 69.02 86.76
0.49 0.47 0.53 0.58 0.48
1.52 0.75 1.19 1.44 1.00
F F F F F
15.49 11.94** 6.62# 15.57 6.72##
F F F F F
0.10 0.12 0.05 0.06 0.12
F F F F F
0.60 0.30* 0.41 0.15## 0.50
Data are mean F SD, n = 6. LH, luteinizing hormone; FSH, follicle stimulating hormone. * Significantly different from vehicle control group at P < 0.05. ** Significantly different from vehicle control group at P < 0.01. # Significantly different from single TCDD treatment group, given pulverized-chow without UDCA, at P < 0.05. ## Significantly different from single TCDD treatment group, given pulverized-chow without UDCA, at P < 0.01.
Fig. 3. Tubule cross-sections of testis. Histopathological changes; (a) vehicle control (acetone: corn oil = 1:6, v/v). Seminiferous tubules, tubule cross-sections of normal testis exhibited the usual arrangement of cells at different stages; (b) 27.5 Ag/kg single TCDD treatment. The basic compartments of the seminiferous epithelium are seen as wide gaps between neighboring cells and enlargement of the intercellular spaces (arrows). No germ cells but Sertoli cells are presented in few sections (arrowhead); (c) 27.5 Ag/kg TCDD and 0.125% UDCA-included treatment. The germ cells of the seminiferous tubules are normally seen at different stages, but Leydig cell hyperplasia is observed in the interstitium (arrow); original magnification: 200.
spaces between the basic and abdominal compartments was also observed, together with a maturation arrest in the spermatocytes and spermatids. Thus, normal sperm development stages in the tubules were barely observed. In most individual subjects, the tubules in which only the Sertoli cells had been in existence with few germ cells along with completely ruptured or necrotized tubules (Fig. 3b). In the UDCA-included treatment group (Fig. 3c), all sperm devel-
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opmental stages were almost completely preserved in contrast with damages found in the single TCDD treatment group (Fig. 3b). Sperm development was normal in all developmental stages similar to that of the vehicle control group (Fig. 3a). Ultrastructural changes As a result of ultrastructural change, in the single TCDD treatment group (Fig. 4b), phagolysosomes were observed in the Leydig cell, smooth endoplasmic reticulum (SER) was inflated and mitochondria were dilated and altered. Also, necrosis was observed near the basement membrane. The basement membrane was separated from germ cells, and vacuolation was characteristically observed both in the
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cytoplasm of the degenerating Sertoli cell and in the sperm head, showing some dissolution near the sperm head. Largesized lipid droplets and phagolysosome were also observed in the Sertoli cells. Abnormal deficiency in development of inner mitochondria was also found (Fig. 4b). In the UDCAincluded treatment group (Fig. 4c), abnormalities were reduced and their recovery was closer to what was found in the vehicle control group (Fig. 4a). Immunohistochemical changes Arnt expression was observed in the spermatids, but it was not seen in most cells including Leydig cells in the vehicle-control group. In contrast with the vehicle control group, Arnts were relocated from the cytoplasm to the
Fig. 4. Tubule cross-sections of testis. Ultrastructural changes; (a) vehicle control (acetone: corn oil = 1:6, v/v). Cells have close contact with each other. The existence of a tight junction is seen in common under physiological conditions (arrows); (b) 27.5 Ag/kg single TCDD treatment. Sertoli cell cytoplasm showed large vacuoles with large phagolysosomes (arrows); (c) 27.5 Ag/kg TCDD and 0.25% UDCA-included treatment. Intact Sertoli cells (s) with smooth endoplasmic reticulum (arrowhead) and pachytene spermatocyte (arrow).
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margin of the nuclei in most of the spermatids in the single TCDD treatment group (Fig. 5a). This indicates that Arnts were concentrated in the edge of nuclei secondary to TCDD. In the UDCA-included treatment group, Arnts of the spermatids were not relocated from the cytoplasm to the margin of the nuclei (Fig. 5b), in a similar pattern to that of the vehicle control group. No CYP1A1 expression was found in germ cells of the seminiferous tubules in the single TCDDtreatment group. However, CYP1A1 was found in all the Leydig cells and spermatids. CYP1A1 in the endothelial cells of the blood vessels was strongly induced in the single TCDD treatment group (Fig. 6b). Although induction of CYP1A1 was seen in Leydig cells and spermatids in the UDCA-included treatment group (Fig. 6c) compared to the vehicle-control group (Fig. 6a), stain intensity was somewhat reduced compared to that of the single TCDD treatment group (Fig. 6a). Moreover, induction of CYP1A1 in the
Fig. 6. Immunohistochemical analysis of cyp1a1 expression in C57BL/6J male mouse testes; (a) vehicle control (acetone: corn oil = 1:6, v/v); (b) 27.5 Ag/kg single TCDD treatment. Leydig cells were negative but showed particularly strong staining endothelial cell cytoplasm in blood vessels (arrow). Spermatids were slightly positive; (c) 27.5 Ag/kg TCDD and 0.25% UDCA-included treatment. Spermatids and Leydig cells were slightly positive and endothelial cell cytoplasm was negative; original magnification: 400. Fig. 5. Immunohistochemical change of ARNT in seminiferous tubules of C57BL/6J male mouse testis; (a) 27.5 Ag/kg single TCDD. Arnt staining showed a trend to converge in a corner of nuclei in the spermatids (arrow); (b) 27.5 Ag/kg TCDD and 0.25% UDCA-included treatment. Arnt exhibited spread around the nucleus of spermatids (arrow); original magnification: 1000.
endothelial cells in blood vessels was not observed (Figs. 6b and 6c). Induction of P450scc was regularly observed in the cytoplasm of Leydig cells in the vehicle control group. Nevertheless, owing to rupture and decrease of the Leydig
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cells, stain intensity was reduced in the single TCDD treatment group (Fig. 7b) compared to that of the vehiclecontrol group (Fig. 7a). However, stain intensity was increased in the UDCA-included treatment group (Fig. 7c)
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Table 2 The effects of adding UDCA to chow on mean Johnsen’s scores 60 days after a single dose of TCDD in C57BL/6J mice TCDD dose (Ag/kg)
% UDCA in chow
Score
None 27.5
None None 0.125 0.250 0.500
9.22 7.42 8.81 8.42 8.40
F F F F F
1.17 1.13* 1.04# 1.29# 1.58#
Data are mean F SD, n = 250. * Significantly different from vehicle control at P < 0.01. # Significantly different from single TCDD treatment group, given pulverized-chow without UDCA, at P < 0.01.
compared to that of the single TCDD treatment group, and was similar to that of the vehicle control group. Johnsen’s testicular biopsy score count The scores were significantly decreased in the single TCDD treatment group ( P < 0.01) in contrast to that of the vehicle control group, indicating that spermatogenesis had been suppressed by TCDD. However, the count was significantly increased in the UDCA-included treatment group compared to the single TCDD treatment group, suggesting that the TCDD-suppressed spermatogenesis was protected by UDCA (Table 2).
Discussions TCDD-induced testicular damage
Fig. 7. Immunohistochemical changes of cytoplasm P450scc in C57BL/6J male mouse testis; (a) vehicle control (acetone: corn oil = 1:6, v/v); (b) 27.5 Ag/kg single TCDD treatment. Disruption and reduction of Leydig cells were observed in the interstitium, but showed no decrease in staining intensity compared to vehicle control; (c) 27.5 Ag/kg TCDD and 0.25% UDCA-included treatment. In the interstitium, most Leydig cells were positive and observed no difference compared to control; original magnification: 400.
Although TCDD has been studied in a variety of animal models, toxicities observed upon TCDD injection varied depending on the dose, duration of exposure and species of the animal. It has been reported in past publications that male rats exposed to TCDD have also been implicated in causing similar adverse effects in the testes of several other animals, such as weight loss of testicles, seminal vesicle and prostate gland, and changes in the form of testes. In addition, it also implicates the fact that epididymis increases upon occurrence of sperm glanuloma, reduction in fertility and occurrence of reproductive toxicity. It is clear that TCDD reaches the testes (Johnson et al., 1992; Khera and Ruddick, 1973; Moore et al., 1985; Rune et al., 1991). In this study, body or testicular weight in the single TCDD treatment group was significantly lower than that of the vehicle control group. In addition, it was observed that serum FSH and LH had been significantly decreased by TCDD administered for 60 days in contrast to that of the vehicle control group. However, FSH and LH of the blood plasma were not affected in rats exposed to TCDD (Moore et al., 1989, Li et al., 1997). This difference in the toxic effect was presumed to be due to the dissimilarity of the dose, duration of exposure and the species of animal.
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Leydig cells are in the interstitium of the testis and function as the primary site of testosterone biosynthesis. Leydig cell development and steroid production depend on the pituitary-derived Luteinizing Hormone. It requires activation of four steroid enzymes, that is, cytochrome P450scc, 3h-hydroxysteroid dehydrogenase/45-44-isomerase (3hHSD), 17a-hydroxylase/c17-20 lyase (P45017a) and 17-ketosteroid reductase (Dufau, 1988; Payne and Youngblood, 1995). When LH reaches the high affinity receptors on the surface of the Leydig cells, adenylate cyclase becomes activated and the production of cAMP in the cell increases, carrying cholesterols to the inner mitochondrial membrane. Here, cytochrome P450scc is activated, and cholesterols are converted into pregnenolones, the C21 steroids, in the first stage of testosterone synthesis. TCDD has been known to primarily suppress cytochrome P450scc in Leydig cells (Dufau, 1988; Moore et al., 1991). In the immunohistochemical observations made in this study, cytochrome P450scc was suppressed in Leydig cells, indicating that TCDD had interrupted the transformation of cholesterol by cytochrome P450scc. Arnt was relocated to the margin of the nuclei of the spermatids in the single TCDD treatment group, indicating that Arnt was concentrated in the edge of the nuclei by TCDD. It had been reported that Arnt was relocated to the nuclei in the cytoplasm by TCDD exposure (Wormke et al., 2000). In the single TCDD treatment group, CYP1A1 expression was found in Leydig cell, spermatids and endothelial cells of the blood vessels. It is reported that TCDD may cause morphological lesions and a decrease in steroidogenesis (Kim et al., 1999; Rune et al., 1991). Similarly, in this study, a single dose treatment of 27.5 Ag/kg TCDD caused morphological and immunohistochemical changes. In most of the TCDD treatment group, seminiferous tubules, where only the Sertoli cells exist with few germ cells found, were shown to be necrosed and completely damaged. Thus, it suggests that the toxicity of TCDD in the testicles caused abnormal spermatogenesis. The Johnsen’s score (Johnsen, 1970) is a devised method in which the seminiferous epithelia of human testes are observed to ascertain the degree of spermatogenesis in a convenient and prompt way. It scores morphological changes in the seminiferous tubules from 1 to 10 by the presence or absence of the main cell types arranged in the order of maturity. As a result of Johnsen’s score, it was evident that TCDD had caused damages in spermatogenesis. Prevention of the TCDD-induced testicular toxicity by UDCA supplementation Activated carbons have a strong absorptive activity for dioxin. Accordingly, dioxin may be excreted effectively by these activated carbons (Manara et al., 1984). However, they have a drawback of interfering with the absorption of essential lipophilic vitamins. Thus, it is not recommended to use them commonly because of a low selectivity in adsorption. It has been reported that natural formulations
such as resveratrol, flavonoids, green tea and panax ginseng have protective and therapeutic effects against the TCDDinduced testicular toxicity, with respect to its potential benefits to humans chronically exposed to TCDD by binding to aromatic hydrocarbon receptors to which dioxin compounds are bound (Ashida et al., 2000; Kang et al., 2000; Kim et al., 1999; Casper et al., 1999). UDCA has various pharmacological effects in vivo. It is currently used for the clinical treatment of cholelithiasis, biliary tract diseases, chronic hepatic diseases, hepatic insufficiency, post enterectomy dyspepsia, fatty liver and the like. The chemoprotective mechanism of UDCA has been reported mainly on inhibition of the hydrophobic bile acidinduced toxicity (Setchell et al., 1997), anti-apoptotic effects (Benz et al., 1998) and immune-modulating effects (Calmus et al., 1992). In addition, there are reports that UDCA forms micelles together with phospholipids to create the ‘‘cholesterol-like’’ membrane-stabilizing effects and to reduce toxic effects by promoting excretion of toxic substances (Guldutuna et al., 1993; Heuman et al., 1996) as part of its function of stabilizing and protecting cell membranes. In this study, the preventive effects of UDCA against the toxicity of TCDD in C57BL/6J mice were investigated. The results confirm the preventive effect of UDCA on inadequate development caused by TCDD. The protective effect of UDCA was not dose-dependent, suggesting that the range of UDCA doses administered in this study was established to achieve maximum recovery. However, the increase in the body and testicular weights of 0.5% UDCA-included treatment group was less than that of 0.125 and 0.25% UDCAincluded treated groups. Accordingly, the assumption is that weight loss occurred due to a reduction in fat absorption secondary to the biliary excretion effect of a large dose of UDCA administration. UDCA has both protective and therapeutic effects against TCDD-induced testicular atrophy. Morphologically, the germinal epithelia and intercellular space were relatively well-preserved in the UDCA-included treatment group when compared with the vehicle control group. Addition of UDCA also normalized the ultrastructural alterations caused by TCDD such as occurrence of phagolysosomes, inflated smooth endoplasmic reticulum, dilated and altered mitochondria, necrosis and completely damaged seminiferous tubules. There were no differences between the UDCA-included treatment group and that of the vehicle control group in the Johnsen’s scores indicating that UDCA protected the TCDD-damaged spermatogenesis. Moreover, in the experiment for Arnt expression, UDCA added to chow suppressed the TCDD-caused Arnt relocation from the cytoplasm to the nuclei. In conclusion, the TCDD-induced testicular toxicity was effectively protected by UDCA. There was almost complete recovery of the testes in the UDCAincluded treatment group. Therefore, UDCA may be useful in the prevention and treatment of TCDD-induced testicular damage. The ability of UDCA in protecting a wide range of endogenous endpoints presumably occurred through increased excretions or cyto-
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protective membrane stabilization. Further study on the toxicokinetics of TCDD after feeding UDCA chow is needed.
Acknowledgments This study was supported by the Brain Korea 21 grants from Korea Research Foundation in Korea.
References Ashida, H., Fukuda, I., Yamashita, T., Kanazawa, K., 2000. Flavones and flavonols at chowary levels inhibit a transformation of aryl hydrocarbon receptor induced by dioxin. FEBS Lett. 476, 213 – 217. Astroff, B., Safe, S., 1988. Comparative antiestrogenic activities of 2,3,7,8tetrachlorodibenzo-p-dioxin and 6-methyl-1, 3, 8-thichlorobenzofuran in the female rat. Toxicol. Appl. Pharmacol. 95, 435 – 443. Benz, C., Angermu¨ller, S., To¨x, U., Klo¨ters-Plachky, P., Riedel, H., Sauer, P., Stremmel, W., Stiehl, A., 1998. Effect of tauroursodeoxycholic acid on bile acid-induced apoptosis and cytolysis in rat hepatocytes. J. Hepatol. 28, 99 – 106. Beuers, U., Spengler, U., Kruis, W., Aydemir, U., Wiebecke, B., Heldwein, W., Weinzierl, M., Pape, G.R., Sauerbruch, T., Paumgartner, G., 1992. Ursodeoxycholic acid for treatment of primary sclerosing cholangitis: a placebo-controlled trial. Hepatology 16, 707 – 714. Calmus, Y., Weill, B., Ozier, Y., Chereau, C., Houssin, D., Poupon, R., 1992. Immunosuppressive properties of chenodeoxycholic and ursodeoxycholic acids in the mouse. Gastroenterology 103, 617 – 621. Casper, R.F., Quesne, M., Rogers, I.M., Shirota, T., Jolivet, A., Milgrom, E., Savouret, J.F., 1999. Resveratrol has antagonist activity on the aryl hydrocarbon receptor: implications for prevention of dioxin toxicity. Mol. Pharmacol. 56, 784 – 790. Dufau, M.L., 1988. Endocrine regulation and communicating functions of Leydig cell. Annu. Rev. Physiol. 50, 483 – 508. Guldutuna, S., Zimmer, G., Imhof, M., Bhatti, S., You, T., Leuschner, U., 1993. Molecular aspects of membrane stabilization by ursodeoxycholate. Gastroenterology 104, 1736 – 1744. Heuman, D.M., Bajaj, R., Lin, Q., 1996. Adsorption of mixtures of bile salt taurine conjugates to lecithin-cholesterol membranes: implications for bile salt toxicity and cytoprotection. J. Lipid Res. 37, 562 – 573. Johnsen, S.G., 1970. Testicular biopsy score count—A method for registration of spermatogenesis in human testes: normal values and results in 335 hypogonadal males. Hormones 1, 2 – 25. Johnson, L., Dickerson, R., Safe, S.H., Nyberg, C.L., Lewis, R.P., Welsh, T.H., 1992. Reduced Leydig cell volume and function in adult rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin without a significant effect on spermatogenesis. Toxicology 76, 103 – 118. Kang, K.S., Li, G.X., Park, J.S., Lee, B.J., Che, J.H., Lee, D.S., Lee, Y.S., 2000. Effect of green tea on the male reproduction system of rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Microbiol. Biotechnol. 10, 281 – 286. Kawamura, T., Koizumi, F., Ishimori, A., 1989. Effect of ursodeoxycholic
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acid on water immersion restraint stress ulcer of rats. Nippon Shokakibyo Gakkai Zasshi 86, 2373 – 2378. Khera, K.S., Ruddick, J.A., 1973. Polychlorodibenzo-p-dioxins. Perinatal effects and the dominant lethal test in Wistar rats. In: Blair, E.H. (Ed.), Chlorodioxins: Origin and Fate. American Chemical Society, Washington, DC, pp. 70 – 84. Kim, W., Hwang, S., Lee, H., Song, H., Kim, S., 1999. Panax ginseng protects the testis against 2,3,7,8-tetrachlorodibenzo-p-dioxin induced testicular damage in guinea pigs. BJU Int. 83, 842 – 849. Kleeman, J.M., Moore, R.W., Peterson, R.E., 1990. Inhibition of testicular steroidogenesis in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats: evidence that the key lesion occurs prior to or during pregnenolone formation. Toxicol. Appl. Pharmacol. 106, 112 – 125. Li, X., Johnson, D.C., Rozman, K.K., 1997. 2,3,7,8-Tetrachlorodibenzo-pdioxin (TCDD) increases release of luteinizing hormone and folliclestimulating hormone from the pituitary of immature female rats in vivo and in vitro. Toxicol. Appl. Pharmacol. 142, 264 – 269. Manara, L., Coccia, P., Croci, T., 1984. Prevention of TCDD toxicity in laboratory rodents by addition of charcoal or cholic acids to chow. Food Chem. Toxicol. 22, 815 – 818. Mebus, C.A., Reddy, V.R., Piper, W.N., 1987. Depression of rat testicular 17-hydroxylase and 17, 20-lyase after administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Biochem. Pharmacol. 36, 727 – 731. Moore, R.W., Potter, C.L., Theobald, H.M., Robinson, J.A., Peterson, R.E., 1985. Androgenic deficiency in male rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 79, 99 – 111. Moore, R.W., Parsons, J.A., Bookstaff, R.C., Peterson, R.E., 1989. Plasma concentrations of pituitary hormones in 2,3,7,8-tetrachlorodibenzo-pdioxin treated male rats. J. Biochem. Toxicol. 4, 165 – 172. Moore, R.W., Jefcoate, C.R., Peterson, R.E., 1991. 2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits steroidogenesis in the rat testis by inhibiting the mobilization of cholesterol to cytochrome P450scc. Toxicol. Appl. Pharmacol. 109, 85 – 97. Payne, A.H., Youngblood, G.L., 1995. Regulation of expression of steroidogenic enzymes in Leydig cells. Biol. Reprod. 52, 217 – 225. Rune, G.M., de Souza, Ph., Krowkw, R., Merker, H.J., Neubert, D., 1991. Morphological and histochemical pattern of response in rat testes after administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Histol. Histopathol. 6, 459 – 467. Seraj, M.J., Umemoto, A., Kajikawa, A., Mimura, S., Kinouchi, T., Ohnishi, Y., Monden, Y., 1997. Effects of chowary bile acids on formation of azoxymethane-induced aberrant crypt foci in F344 rats. Cancer Lett. 115, 97 – 103. Setchell, K.D., Rodrigues, C.M., Clerici, C., Solinas, A., Morelli, A., Gartung, C., Boyer, J., 1997. Bile acid concentrations in human and rat liver tissue and in hepatocyte nuclei. Gastroenterology 112, 226 – 235. Sharara, F.I., Seifer, D.B., Flaws, J.A., 1998. Environmental toxicants and female reproduction. Fertil. Steril. 70, 613 – 622. Suh, H., Jung, E.J., Kim, T.H., Lee, H.Y., Park, Y.H., Kim, K.W., 1997. Antiangiogenic activity of ursodeoxycholic acid and its derivatives. Cancer Lett. 113, 117 – 122. Umbreit, T.H., Hesse, E.J., Macdonald, G.J., Gallo, M.A., 1988. Effects of TCDD – estradiol interactions in three strains of mice. Toxicol. Lett. 40, 1 – 9. Wormke, M., Castro-Rivera, E., Chen, I., Safe, S., 2000. Estrogen and arylhydrocarbon receptor expression and crosstalk in human Ishikawa endometrial cancer cells. J. Steroid Biochem. Mol. Biol. 72, 197 – 207.
Protective effects of ursodeoxycholic acid against 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced testicular damage in mice Young-Il Kwon, a Je-Deuk Yeon, b Seung-Min Oh, b and Kyu-Hyuck Chung b,* a
Department of Clinical Laboratory Science, Shinheung College, Uijeongbu, Kyunggido 480-701, South Korea b College of Pharmacy, Sungkyunkwan University, Suwo˘n, Kyunggido 440-746, South Korea Received 22 July 2003; accepted 17 September 2003
Abstract The protective effect of ursodeoxycholic acid (UDCA), a biliary component found in bears, on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced testicular damage in mice was investigated. Fifty C57BL/6J mice were equally divided into five groups. The mice in the control group received the vehicle and standard chow. The single TCDD treatment group received 27.5 Ag/kg of TCDD subcutaneously. The UDCA-included treatment group received pulverized chow containing 0.125%, 0.25% and 0.5% UDCA, respectively, for 70 days starting 10 days before TCDD injections. The body and testicular weights were shown to be decreased in the single TCDD treatment group, while the decrease was prevented by UDCA added to the chow. In addition, the decrease in the serum-luteinizing hormone (LH) or the follicle stimulating hormone (FSH) secondary to a TCDD injection was not observed in the UDCA-included treatment group. Contrary to the single TCDD treatment group, the germinal epithelium and intercellular space were relatively well preserved in the UDCA-included treatment group. Adding UDCA also normalized TCDD-induced irregular ultrastructural changes such as development of phagolysosomes, inflated smooth endoplasmic reticulum (SER), dilated and altered mitochondria, necrosis and completely damaged seminiferous tubules. Moreover, in the experiment for Arnt expression, UDCA added to the chow suppressed the TCDD-induced relocation of Arnt from the cytoplasm to the nuclei. In conclusion, TCDD-induced testicular toxicity was effectively protected by UDCA. There was almost complete recovery of the testes in the UDCA-included treatment group. Thus, UDCA may be useful for the prevention and treatment of TCDD-induced testicular damage. D 2003 Published by Elsevier Inc. Keywords: TCDD; Testicular toxicity; Ursodeoxycholic acid; Preventive effect
Introduction Halogenated aromatic hydrocarbons including polychlorinated dibenzodioxin and polychlorinated dibenzofuran are the two most typical environmental pollutants exhausted from industrial waste incineration or paper manufacturing processes. 2,3,7,8-Tetrachlorldibenzo-p-dioxin (TCDD) is the most toxic compound among polychlorinated aromatic hydrocarbons. Humans are generally exposed to such compounds, which are incorporated into food, drinking water, soil, dust, smoke and air. Dioxins with very slow biodegradability have long persisted in the environment. These compounds can be concentrated in the adipose tissues of animals in the food chain, and * Corresponding author. College of Pharmacy, SungKyunKwan University, #300 Chonchon-dong, Changan-ku, Suwo˘n, Kyunggido 440-746, South Korea. Fax: +82-31-292-8800. E-mail address: [email protected] (K.-H. Chung). 0041-008X/$ - see front matter D 2003 Published by Elsevier Inc. doi:10.1016/j.taap.2003.09.024
are often found in human breast milk (Sharara et al., 1998). Among various TCDD toxicities, reproductive toxicity may be seen in both males and females. TCDD is classified as an environmental hormone because it has been found to cause endocrine disruption in various biotas (Astroff and Safe, 1988; Mebus et al., 1987; Umbreit et al., 1988). The toxic effects of TCDD in male reproductive systems include a reduction in the size of the testes, prostate gland and seminal vesicle, as well as a decrease in the number of sperm count (Johnson et al., 1992; Khera and Ruddick, 1973; Moore et al., 1985; Rune et al., 1991). It has also been reported that TCDD causes separation and rupture of spermatogonia and the Sertoli cells, which in turn impedes spermatogenesis (Kim et al., 1999; Rune et al., 1991). In recent years, several natural formulations relating to reducing or eliminating TCDD toxicity have been in focus. Resveratrol (Casper et al., 1999), flavonoids (Ashida et al., 2000), green tea (Kang et al., 2000) and panax ginseng (Kim et al., 1999) may provide protection against TCDD toxic-
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ities. Resveratrol extracted from red wine has a preventive effect against dioxin toxicity by binding to aromatic hydrocarbon receptors, to which dioxin compounds bind. Although this compound has competitive inhibitory activities for the dioxins previously absorbed in the body, it cannot suppress continuous absorption of dioxin per se, nor can it enhance the excretion thereof. Additionally, activated carbon may be used as an absorbent of dioxin. Nevertheless, it is not recommended for common use because of a low selectivity in adsorption. Thus, it has a drawback by interfering in the absorption of essential lipophilic vitamins. Ursodeoxycholic acid (UDCA), a component found in the bear’s bile, has been used in the treatment of liver disease. Some of the UDCA pills are used for liver protection and as a cholagogue. UDCA has also been known to enhance immunity and have anticancer effects (Suh et al., 1997), anti-inflammation effects (Seraj et al., 1997), antiprimary sclerosing cholangitis effects (Beuers et al., 1992) and antistress effects (Kawamura et al., 1989). In addition, UDCA as a cholic acid induced from cholesterol may have an effect in the sperm’s development and motility. However, no research has been done on its effect on the male productive organs, particularly in steroidogenesis and spermatogenesis. The objective of this study is to identify whether UDCA can suppress testicular toxicities of TCDD in C57BL/6J male mice. Therefore, we investigated the preventive effect of UDCA, added to chow, against the TCDD-induce testicular damage such as body and testicular weight losses, reduced hormonal concentrations, histopathological and immunohistochemical changes.
Materials and methods Animals and treatment. Male C57BL/6J mice (4 –5 weeks old, 16 –17 g, Charles River Laboratories in Japan) were used throughout the experiments. This experiment was performed in a controlled environment (maintained at 23 F 3 jC, a relative humidity of 60 F 10 jC, in 12-h light – dark cycle). The mice were provided with feed and tap water ad libitum. Water was sterilized with a UV-sterilizer and feed (Hae Eun Trade Co., Korea) was sterilized under 2.0 M rad of radioactive rays. Fifty C57BL/6J mice were equally divided into five groups. The normal control group received the vehicle and standard chow. The TCDD treatment group received 27.5 Ag/ kg TCDD (2,3,7,8-tetrachloro-dibenzo-p-dioxin, 95% purity, ChemServices, Inc., PA, USA), which had been dissolved in acetone and corn oil (1:6, v/v) in volumes of 5 ml/kg body weight subcutaneously. The UDCA-included treatment group received pulverized chow containing 0.125%, 0.25% and 0.5% UDCA (98% purity; Daewoong Pharm. Co., Ltd., Korea), respectively, for 70 days starting 10 days before the TCDD injection. Appropriate doses of TCDD and UDCA were determined in the preliminary experiment. Acetonecorn oil (1:6, v/v) alone was administered subcutaneously as a vehicle-control group.
Change in body weight, testicular weight and hormonal concentration. Body weight was checked twice a week during the treatment period. Feed and water were removed 1 day before the animals were sacrificed. Blood samples drawn directly from a heart puncture were taken. The testes were removed under general anesthesia with ethyl ether and the testicles were weighed. Serum was separated by centrifugation and stored at 80 jC until an assay was performed. The serum concentrations of the testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) of six mice in each group were determined using ACS: 180 (automated chemiluminescence system, Bayer, NY, USA) (n = 6). Observation of histopathological and ultrastructural change. To assess histological and ultrastructural changes, the fixed testicular tissues harvested from 10 surviving mice in each group (except seven mice of 27.5 Ag/kg single TCDD treatment group) were paraffin embedded. Then, they were prepared as 2 – 3-Am-thick sections using a microtome (microm HM 440E). They were double stained with hematoxylin and eosin, and were observed by light microscope. About 50 horizontally sectioned seminiferous tubules from five mice in each group (total of 250 seminiferous tubules) were measured to determine the mean diameter of the seminiferous tubules. All seminiferous tubules of one histological section from a testicular specimen were evaluated and scored from 1 to 10 using the Johnsen scoring system. In using electron microscopy, the testicular tissues were resected at 1 mm3 thickness and fixed in a 2.5% glutaldehyde solution for 4 h at 4 jC. Then, the specimens were washed with a phosphate buffer solution at pH 7.3, and placed in 1% osmium tetroxide (pH 7.3) for 2 h. They were then dehydrated in ethanol and propylene oxide, and were embedded in an epon-mixed solution. The ultrathin sections (60 – 70 nm) severed with an ultra-microtome (Reichert-Jung, Ultracut E) were stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (H-600, Hitachi). Immunohistochemistry. To assess immunohistochemical changes, the testicles removed from 10 surviving mice in each group (except seven mice of 27.5 Ag/kg single TCDD treatment group) were fixed in fresh Bouin’s solution (for cytochrome P450scc expression) and formaldehyde solution (AhR, Arnt, Cyp1a1 and Cyp1a2 expression) for 12 and 24 h, respectively. These tissues were subsequently dehydrated, embedded in paraffin and sectioned at 5 Am (n = 6). Briefly, these sections were deparaffinized and rehydrated through graded alcohol to Tris-buffered saline (TBS), and nonspecific binding was blocked by inactivating them for 10 min using a heat-inactivated normal goat serum (Rockland, Gilbertsville, CA, USA) after being diluted at the proportion of 1:5. The primary antibody was diluted in the blocking solution as described (AhR, (Santa Cruz Biotechnology, Inc., CA, USA), diluted at 1:50; Arnt (Santa Cruz Biotechnolo-
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gy), diluted at 1:50; Cyp1a1 (CHEMICON International Inc., CA, USA), diluted at 1:100; Cyp1a2 (CHEMICON International Inc.), diluted at 1:100; cytochrome P450scc (CHEMICON International Inc.), diluted at 1:100 and incubated over the sections in humidified chamber overnight (AhR and Arnt) at 4 jC or 40 min (Cyp1a1, Cyp1a2 and cytochrome P450scc) at room temperature). These sections were subsequently washed with TBS containing 0.1% Tween 20, followed by TBS for 5 min, incubated with appropriate secondary antibodies (biotin conjugated affinity purified anti-goat IgG [Rockland] for AhR and Arnt; biotinylated anti-rabbit IgG [Vector laboratories, CA, USA] for Cyp1a1 and cytochrome P450scc), then washed again with TBS and incubated with a streptavidin-peroxidase complex (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA). After washing them three times for 5 min using TBS, immunoreaction was localized using hydrogen peroxide-activated 3.3V-diaminobenzidine-tetrahydrochloride (Sigma) as a peroxidase substrate. These sections were counterstained with Mayer’s hematoxylin and were dehydrated with graded series of alcohol, rinsed with xylene and mounted with Permount. In the negative controls for which primary antibodies were substituted with normal goat IgG, normal rabbit IgG or normal sheep IgG showed little to no background staining. Johnsen’s score counts. To measure the degree of spermatogenesis, the Johnsen’s testicular biopsy score was used. The degree of spermatogenesis was scored from 1 to 10 depending on the presence or absence of cell forms arranged according to the extent of maturity of their spermatogenesis. For these observations, an objective lens (magnification: 40) was used. A total of 250 seminiferous tubules (50 seminiferous tubules for five subjects in each group) were used.
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Statistical analysis. One-way analysis of variance (ANOVA) test was carried out for the analyses of body weight, weights of the testes and hormone levels of the acute toxicity test. If the ANOVA test results showed a significant difference, an unpaired Student’s t test was performed for cases with P < 0.05 and P < 0.01.
Results Change in body weight and testicular weight As shown in Fig. 1, body weight was decreased in the single TCDD treatment group than that of the vehicle control group after 12 days of TCDD administration. However, there was no significant difference in body weight change between the UDCA (0.125%, 0.25% and 0.5%) included treatment group and vehicle control group. It was revealed that the TCDD-induced body weight loss was prevented by UDCA. As shown in Fig. 2, a significant decrease in testicular weight was induced by a single TCDD treatment. However, TCDDinduced testicular weight loss was significantly inhibited in UDCA-included treatment group. The gain in the body and testicular weight was enhanced by UDCA-included treatment group at the similar rate as that of the vehicle control group, which suggested that UDCA had both protective and therapeutic effects against the TCDD-induced testicular atrophy. Changes of serum hormones (Testosterone, LH and FSH) As shown in Table 1, the concentration of testosterone and FSH was decreased in the single TCDD treatment group in contrast to the vehicle control group. However, there was no loss of serum testosterone concentration in 0.125% and
Fig. 1. The effects of adding UDCA to chow on body weights of C57BL/6J mice during 60 days after a single dose of TCDD. The graph shows the mean and SD of the data obtained from survival mice of 7 – 10 animals (the number of single TCDD treatment group = 7, the number of others = 10).
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Fig. 2. The effects of adding UDCA to chow on testicular weights of C57BL/6J mice 60 days after a single dose of TCDD. The graph shows the mean and SD of the data obtained from survival mice of 7 – 10 animals (the number of 27.5 Ag/kg TCDD single treated group = 7, the number of others = 10). *Significantly different from vehicle control group (V-C) at P < 0.05. **Significantly different from vehicle control group (V-C) at P < 0.01. # Significantly different from TCDD single treated group at P < 0.05. ## Significantly different from TCDD single treated group at P < 0.01.
0.5% UDCA-included treatment groups and FSH concentration in 0.25% UDCA-included treatment group compared with vehicle control group. Therefore, it was suggested that serum hormonal changes caused by TCDD could be prevented by the administration of UDCA. Histopathological change Cell differentiation showed a reduction in most cells, including spermatogonia in the single TCDD treatment group (Fig. 3b). Separation of spermatogonia near the basement membrane, secondary to dissolution of the germinal epithelium, and expansion of intercellular space among neighboring germ cells, were observed. Enlargement of the Table 1 The effects of adding UDCA to chow on serum hormonal concentrations 60 days after a single dose of TCDD in C57BL/6J mice TCDD dose (Ag/kg)
% UDCA in chow
Testosterone (ng/dl)
LH (mIU/ml)
FSH (mIU/ml)
None 27.5
None None 0.125 0.250 0.500
86.42 53.86 73.92 69.02 86.76
0.49 0.47 0.53 0.58 0.48
1.52 0.75 1.19 1.44 1.00
F F F F F
15.49 11.94** 6.62# 15.57 6.72##
F F F F F
0.10 0.12 0.05 0.06 0.12
F F F F F
0.60 0.30* 0.41 0.15## 0.50
Data are mean F SD, n = 6. LH, luteinizing hormone; FSH, follicle stimulating hormone. * Significantly different from vehicle control group at P < 0.05. ** Significantly different from vehicle control group at P < 0.01. # Significantly different from single TCDD treatment group, given pulverized-chow without UDCA, at P < 0.05. ## Significantly different from single TCDD treatment group, given pulverized-chow without UDCA, at P < 0.01.
Fig. 3. Tubule cross-sections of testis. Histopathological changes; (a) vehicle control (acetone: corn oil = 1:6, v/v). Seminiferous tubules, tubule cross-sections of normal testis exhibited the usual arrangement of cells at different stages; (b) 27.5 Ag/kg single TCDD treatment. The basic compartments of the seminiferous epithelium are seen as wide gaps between neighboring cells and enlargement of the intercellular spaces (arrows). No germ cells but Sertoli cells are presented in few sections (arrowhead); (c) 27.5 Ag/kg TCDD and 0.125% UDCA-included treatment. The germ cells of the seminiferous tubules are normally seen at different stages, but Leydig cell hyperplasia is observed in the interstitium (arrow); original magnification: 200.
spaces between the basic and abdominal compartments was also observed, together with a maturation arrest in the spermatocytes and spermatids. Thus, normal sperm development stages in the tubules were barely observed. In most individual subjects, the tubules in which only the Sertoli cells had been in existence with few germ cells along with completely ruptured or necrotized tubules (Fig. 3b). In the UDCA-included treatment group (Fig. 3c), all sperm devel-
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opmental stages were almost completely preserved in contrast with damages found in the single TCDD treatment group (Fig. 3b). Sperm development was normal in all developmental stages similar to that of the vehicle control group (Fig. 3a). Ultrastructural changes As a result of ultrastructural change, in the single TCDD treatment group (Fig. 4b), phagolysosomes were observed in the Leydig cell, smooth endoplasmic reticulum (SER) was inflated and mitochondria were dilated and altered. Also, necrosis was observed near the basement membrane. The basement membrane was separated from germ cells, and vacuolation was characteristically observed both in the
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cytoplasm of the degenerating Sertoli cell and in the sperm head, showing some dissolution near the sperm head. Largesized lipid droplets and phagolysosome were also observed in the Sertoli cells. Abnormal deficiency in development of inner mitochondria was also found (Fig. 4b). In the UDCAincluded treatment group (Fig. 4c), abnormalities were reduced and their recovery was closer to what was found in the vehicle control group (Fig. 4a). Immunohistochemical changes Arnt expression was observed in the spermatids, but it was not seen in most cells including Leydig cells in the vehicle-control group. In contrast with the vehicle control group, Arnts were relocated from the cytoplasm to the
Fig. 4. Tubule cross-sections of testis. Ultrastructural changes; (a) vehicle control (acetone: corn oil = 1:6, v/v). Cells have close contact with each other. The existence of a tight junction is seen in common under physiological conditions (arrows); (b) 27.5 Ag/kg single TCDD treatment. Sertoli cell cytoplasm showed large vacuoles with large phagolysosomes (arrows); (c) 27.5 Ag/kg TCDD and 0.25% UDCA-included treatment. Intact Sertoli cells (s) with smooth endoplasmic reticulum (arrowhead) and pachytene spermatocyte (arrow).
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margin of the nuclei in most of the spermatids in the single TCDD treatment group (Fig. 5a). This indicates that Arnts were concentrated in the edge of nuclei secondary to TCDD. In the UDCA-included treatment group, Arnts of the spermatids were not relocated from the cytoplasm to the margin of the nuclei (Fig. 5b), in a similar pattern to that of the vehicle control group. No CYP1A1 expression was found in germ cells of the seminiferous tubules in the single TCDDtreatment group. However, CYP1A1 was found in all the Leydig cells and spermatids. CYP1A1 in the endothelial cells of the blood vessels was strongly induced in the single TCDD treatment group (Fig. 6b). Although induction of CYP1A1 was seen in Leydig cells and spermatids in the UDCA-included treatment group (Fig. 6c) compared to the vehicle-control group (Fig. 6a), stain intensity was somewhat reduced compared to that of the single TCDD treatment group (Fig. 6a). Moreover, induction of CYP1A1 in the
Fig. 6. Immunohistochemical analysis of cyp1a1 expression in C57BL/6J male mouse testes; (a) vehicle control (acetone: corn oil = 1:6, v/v); (b) 27.5 Ag/kg single TCDD treatment. Leydig cells were negative but showed particularly strong staining endothelial cell cytoplasm in blood vessels (arrow). Spermatids were slightly positive; (c) 27.5 Ag/kg TCDD and 0.25% UDCA-included treatment. Spermatids and Leydig cells were slightly positive and endothelial cell cytoplasm was negative; original magnification: 400. Fig. 5. Immunohistochemical change of ARNT in seminiferous tubules of C57BL/6J male mouse testis; (a) 27.5 Ag/kg single TCDD. Arnt staining showed a trend to converge in a corner of nuclei in the spermatids (arrow); (b) 27.5 Ag/kg TCDD and 0.25% UDCA-included treatment. Arnt exhibited spread around the nucleus of spermatids (arrow); original magnification: 1000.
endothelial cells in blood vessels was not observed (Figs. 6b and 6c). Induction of P450scc was regularly observed in the cytoplasm of Leydig cells in the vehicle control group. Nevertheless, owing to rupture and decrease of the Leydig
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cells, stain intensity was reduced in the single TCDD treatment group (Fig. 7b) compared to that of the vehiclecontrol group (Fig. 7a). However, stain intensity was increased in the UDCA-included treatment group (Fig. 7c)
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Table 2 The effects of adding UDCA to chow on mean Johnsen’s scores 60 days after a single dose of TCDD in C57BL/6J mice TCDD dose (Ag/kg)
% UDCA in chow
Score
None 27.5
None None 0.125 0.250 0.500
9.22 7.42 8.81 8.42 8.40
F F F F F
1.17 1.13* 1.04# 1.29# 1.58#
Data are mean F SD, n = 250. * Significantly different from vehicle control at P < 0.01. # Significantly different from single TCDD treatment group, given pulverized-chow without UDCA, at P < 0.01.
compared to that of the single TCDD treatment group, and was similar to that of the vehicle control group. Johnsen’s testicular biopsy score count The scores were significantly decreased in the single TCDD treatment group ( P < 0.01) in contrast to that of the vehicle control group, indicating that spermatogenesis had been suppressed by TCDD. However, the count was significantly increased in the UDCA-included treatment group compared to the single TCDD treatment group, suggesting that the TCDD-suppressed spermatogenesis was protected by UDCA (Table 2).
Discussions TCDD-induced testicular damage
Fig. 7. Immunohistochemical changes of cytoplasm P450scc in C57BL/6J male mouse testis; (a) vehicle control (acetone: corn oil = 1:6, v/v); (b) 27.5 Ag/kg single TCDD treatment. Disruption and reduction of Leydig cells were observed in the interstitium, but showed no decrease in staining intensity compared to vehicle control; (c) 27.5 Ag/kg TCDD and 0.25% UDCA-included treatment. In the interstitium, most Leydig cells were positive and observed no difference compared to control; original magnification: 400.
Although TCDD has been studied in a variety of animal models, toxicities observed upon TCDD injection varied depending on the dose, duration of exposure and species of the animal. It has been reported in past publications that male rats exposed to TCDD have also been implicated in causing similar adverse effects in the testes of several other animals, such as weight loss of testicles, seminal vesicle and prostate gland, and changes in the form of testes. In addition, it also implicates the fact that epididymis increases upon occurrence of sperm glanuloma, reduction in fertility and occurrence of reproductive toxicity. It is clear that TCDD reaches the testes (Johnson et al., 1992; Khera and Ruddick, 1973; Moore et al., 1985; Rune et al., 1991). In this study, body or testicular weight in the single TCDD treatment group was significantly lower than that of the vehicle control group. In addition, it was observed that serum FSH and LH had been significantly decreased by TCDD administered for 60 days in contrast to that of the vehicle control group. However, FSH and LH of the blood plasma were not affected in rats exposed to TCDD (Moore et al., 1989, Li et al., 1997). This difference in the toxic effect was presumed to be due to the dissimilarity of the dose, duration of exposure and the species of animal.
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Leydig cells are in the interstitium of the testis and function as the primary site of testosterone biosynthesis. Leydig cell development and steroid production depend on the pituitary-derived Luteinizing Hormone. It requires activation of four steroid enzymes, that is, cytochrome P450scc, 3h-hydroxysteroid dehydrogenase/45-44-isomerase (3hHSD), 17a-hydroxylase/c17-20 lyase (P45017a) and 17-ketosteroid reductase (Dufau, 1988; Payne and Youngblood, 1995). When LH reaches the high affinity receptors on the surface of the Leydig cells, adenylate cyclase becomes activated and the production of cAMP in the cell increases, carrying cholesterols to the inner mitochondrial membrane. Here, cytochrome P450scc is activated, and cholesterols are converted into pregnenolones, the C21 steroids, in the first stage of testosterone synthesis. TCDD has been known to primarily suppress cytochrome P450scc in Leydig cells (Dufau, 1988; Moore et al., 1991). In the immunohistochemical observations made in this study, cytochrome P450scc was suppressed in Leydig cells, indicating that TCDD had interrupted the transformation of cholesterol by cytochrome P450scc. Arnt was relocated to the margin of the nuclei of the spermatids in the single TCDD treatment group, indicating that Arnt was concentrated in the edge of the nuclei by TCDD. It had been reported that Arnt was relocated to the nuclei in the cytoplasm by TCDD exposure (Wormke et al., 2000). In the single TCDD treatment group, CYP1A1 expression was found in Leydig cell, spermatids and endothelial cells of the blood vessels. It is reported that TCDD may cause morphological lesions and a decrease in steroidogenesis (Kim et al., 1999; Rune et al., 1991). Similarly, in this study, a single dose treatment of 27.5 Ag/kg TCDD caused morphological and immunohistochemical changes. In most of the TCDD treatment group, seminiferous tubules, where only the Sertoli cells exist with few germ cells found, were shown to be necrosed and completely damaged. Thus, it suggests that the toxicity of TCDD in the testicles caused abnormal spermatogenesis. The Johnsen’s score (Johnsen, 1970) is a devised method in which the seminiferous epithelia of human testes are observed to ascertain the degree of spermatogenesis in a convenient and prompt way. It scores morphological changes in the seminiferous tubules from 1 to 10 by the presence or absence of the main cell types arranged in the order of maturity. As a result of Johnsen’s score, it was evident that TCDD had caused damages in spermatogenesis. Prevention of the TCDD-induced testicular toxicity by UDCA supplementation Activated carbons have a strong absorptive activity for dioxin. Accordingly, dioxin may be excreted effectively by these activated carbons (Manara et al., 1984). However, they have a drawback of interfering with the absorption of essential lipophilic vitamins. Thus, it is not recommended to use them commonly because of a low selectivity in adsorption. It has been reported that natural formulations
such as resveratrol, flavonoids, green tea and panax ginseng have protective and therapeutic effects against the TCDDinduced testicular toxicity, with respect to its potential benefits to humans chronically exposed to TCDD by binding to aromatic hydrocarbon receptors to which dioxin compounds are bound (Ashida et al., 2000; Kang et al., 2000; Kim et al., 1999; Casper et al., 1999). UDCA has various pharmacological effects in vivo. It is currently used for the clinical treatment of cholelithiasis, biliary tract diseases, chronic hepatic diseases, hepatic insufficiency, post enterectomy dyspepsia, fatty liver and the like. The chemoprotective mechanism of UDCA has been reported mainly on inhibition of the hydrophobic bile acidinduced toxicity (Setchell et al., 1997), anti-apoptotic effects (Benz et al., 1998) and immune-modulating effects (Calmus et al., 1992). In addition, there are reports that UDCA forms micelles together with phospholipids to create the ‘‘cholesterol-like’’ membrane-stabilizing effects and to reduce toxic effects by promoting excretion of toxic substances (Guldutuna et al., 1993; Heuman et al., 1996) as part of its function of stabilizing and protecting cell membranes. In this study, the preventive effects of UDCA against the toxicity of TCDD in C57BL/6J mice were investigated. The results confirm the preventive effect of UDCA on inadequate development caused by TCDD. The protective effect of UDCA was not dose-dependent, suggesting that the range of UDCA doses administered in this study was established to achieve maximum recovery. However, the increase in the body and testicular weights of 0.5% UDCA-included treatment group was less than that of 0.125 and 0.25% UDCAincluded treated groups. Accordingly, the assumption is that weight loss occurred due to a reduction in fat absorption secondary to the biliary excretion effect of a large dose of UDCA administration. UDCA has both protective and therapeutic effects against TCDD-induced testicular atrophy. Morphologically, the germinal epithelia and intercellular space were relatively well-preserved in the UDCA-included treatment group when compared with the vehicle control group. Addition of UDCA also normalized the ultrastructural alterations caused by TCDD such as occurrence of phagolysosomes, inflated smooth endoplasmic reticulum, dilated and altered mitochondria, necrosis and completely damaged seminiferous tubules. There were no differences between the UDCA-included treatment group and that of the vehicle control group in the Johnsen’s scores indicating that UDCA protected the TCDD-damaged spermatogenesis. Moreover, in the experiment for Arnt expression, UDCA added to chow suppressed the TCDD-caused Arnt relocation from the cytoplasm to the nuclei. In conclusion, the TCDD-induced testicular toxicity was effectively protected by UDCA. There was almost complete recovery of the testes in the UDCAincluded treatment group. Therefore, UDCA may be useful in the prevention and treatment of TCDD-induced testicular damage. The ability of UDCA in protecting a wide range of endogenous endpoints presumably occurred through increased excretions or cyto-
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protective membrane stabilization. Further study on the toxicokinetics of TCDD after feeding UDCA chow is needed.
Acknowledgments This study was supported by the Brain Korea 21 grants from Korea Research Foundation in Korea.
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