* Department of Hygiene, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan;
"Research Area" CREST, Japan Science and Technology Corporation, Kawaguchi Center Building, 1-8, Honcho 4-Chome, Kawaguchi City, Saitama 332-0012, Japan;
Department of Integrative Physiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan; and
Laboratory of Marine Environmental Science, Institute of Marine Biological Chemistry, Department of Bioscience and Biotechnology, Division of Bioresource and Bioenvironmental Sciences, Kyushu University Graduate School, Fukuoka 812-8581, Japan
Received May 30, 2001; accepted August 21, 2001
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ABSTRACT |
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Key Words: tributyltin chloride; 2-generation reproductive toxicity study; male reproductive toxicity; ventral prostate; 17ß-estradiol; aromatase inhibition.
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INTRODUCTION |
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TBT is well known to masculinize the sex organs of the female causing the development of a penis and a vas deferens along with the female sex organs in some mollusks (IPCS, 1990; Matthiessen and Gibbs, 1998
; Oberdörster and Cheek, 2000
). Hypothetically, this phenomenon (imposex) is attributed to the inhibition of aromatization and conjugation of testosterone by TBT (Matthiessen and Gibbs, 1998
). Both the aromatization of testosterone for the synthesis of 17ß-estradiol and the conjugation of testosterone for the excretion of testosterone are common metabolic pathways in mammals. Estrogen has various roles in the reproductive function of male mammals besides the sex differentiation of the brain and the regulation of sexual behavior (Hess et al., 1997
; Sharpe, 1998
). Disturbance of aromatization of testosterone was reported to damage spermatogenesis in the studies using aromatase inhibitor (Shetty et al., 1998
) and aromatase-deficient mice (Robertson et al., 1999
). Therefore, it is possible that TBT also affects male reproduction in mammals. There are a few studies concerning the male reproductive toxicity of organotin compounds. Gaines and Kimbrough (1968) reported that fertility was reduced in the male rats given the feed containing either 100 or 200 ppm of triphenyltin hydroxide for 64 days and Snow and Hays (1983) reported that the 20-day administration of 20 mg/kg body weight of triphenyltin acetate or triphenyltin chloride via feed severely affected spermatogenesis in rats. However, the male reproductive function was not evaluated in detail in these studies. We therefore carried out a 2-generation reproductive toxicity study of tributyltin chloride (TBTCl) in male rats and examined its adverse effects on sexual development and reproductive function in detail (results with female rats are reported by Ogata et al., 2001
). With respect to the effects of prenatal TBT exposure on the development of offspring after birth, Crofton et al. (1989) reported that po administration of bis(tri-n-butyltin)oxide to pregnant rats retarded the growth of offspring at a dosage of 10.0 mg/kg body weight, but did not affect growth at a dosage of 2.5 mg/kg body weight. We therefore used dietary concentrations of 5, 25, and 125 ppm TBTCl in this study because, based on the assumption that adult male rats eat 80 g diet/kg body weight/day, the daily intake of TBTCl in the 5, 25, and 125 ppm TBTCl groups was estimated to be 0.4, 2.0, and 10.0 mg/kg body weight, respectively.
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MATERIALS AND METHODS |
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Animals and treatments.
Animals were bred in an air-conditioned room. In this room, the light cycle was 12-h light:12-h dark, the temperature was 2426°C, and the air humidity was 4080%. This experiment was reviewed by the Committee on the Ethics of Animal Experiments in the Faculty of Medicine, Kyushu University and was carried out under the Guidelines for Animal Experiments in the Faculty of Medicine, Kyushu University and the Law (No. 105) and Notification (No. 6) of the Government of Japan.
F1 Generation
Parent rats (Kud:Wistar) of F1 generation were purchased at 9 weeks of age from Kyudo Co., Ltd., Tosu, Japan. During the acclimation period, the parent rats were provided with CE-2 feed and tap water ad libitum. After a 2-week acclimation period, these rats were housed as breeding pairs (1 male and 1 female per cage) in polypropylene resin cages with wood shavings as bedding. Copulation was examined every morning and was confirmed by the presence of a vaginal plug and/or sperm in a vaginal smear. The cohabitation period was 4 days. On the day when copulation was confirmed, pregnant female rats were moved into aluminum cages and were housed individually. These rats were randomly assigned to the control group and the 3 TBTCl groups and the rats in each TBTCl group were provided with the prescribed TBTCl diet ad libitum until the day of weaning of the F1 generation rats. On postnatal day 1 (PND 1), body weight and anogenital distance (AGD) of the F1 generation rats were recorded and litters were randomly reduced to 4 males and 4 females, where possible. The body weight of the rats was recorded on PNDs 1, 4, 14, and 21, AGD was recorded on PNDs 1 and 4, eye opening was examined from PND 14, and the testes descent was examined from PND 20.
The F1 generation rats were weaned on PND 22 and the male rats were housed on litter bases in polypropylene resin cages with wood shavings as bedding. The number of rats in each cage was 4 until PND 35, and 2 thereafter. The rats were provided with the same TBTCl diet as their mother until the termination of the experiment. Body weight and mean food consumption were recorded weekly. On PND 92, rats in the same treatment groups and in the different litters were housed as breeding pairs to be parents of the F2 generation. The cohabitation period was 14 days and the exposure continued during this period. When copulation was confirmed or the 14-day cohabitation period was over, the male rats were housed by sex again and the exposure continued. On PND 119, the F1 generation male rats were killed by inhalation of carbon dioxide. Blood was collected from the posterior vena cava and serum was separated and stored at 80°C. The testes, epididymis, ventral prostate, and seminal vesicle were removed and weighed. The seminal vesicle was weighed without fluid.
F2 Generation
The exposure to F1 dams continued during gestation and lactation. The treatment and examination of the F2 generation male rats were the same as those of the F1 generation rats except that the rats in this generation were not mated. One F2 generation male rat randomly selected from each litter was killed on PND 91 and was examined as the F1 generation male rat.
Homogenization-resistant spermatid and sperm count.
The decapsulated testis was homogenized in 150 ml saline containing 0.05% (v/v) Triton X-100 with Waring blender (Polytron, Kinematica, Littau/Luzern, Switzerland) for 1 min and homogenization-resistant spermatids were counted using a hemocytometer. Sperm collected from the cauda epididymidis was also counted in the same manner. Spermatids and sperm were counted blind to the treatment.
Sperm motility.
The epididymis was clamped at the corpus-cauda junction. The vas deferens was clamped as well. After that, the distal cauda epididymidis was cut with a razor, and sperm was allowed to diffuse into 10 ml M199 with Hanks' salts and L-glutamine (GIBCO, Grand Island, NY) containing 0.5% BSA (Katayama Chemical, Osaka, Japan) and was incubated in a glass chamber for 15 min at 37°C. The sperm sample was diluted with the M199 solution mentioned above (1:3) and the motility of about 100150 sperm was observed with an optical microscope equipped with a stage warmer at 37°C. Sperm with progressive motility, sperm with nonprogressive motility, and immotile sperm were counted and percentages of motile sperm and progressively motile sperm were calculated.
Tissue preparation and histopathology of the testis.
The testis was fixed in Bouin's solution, embedded in paraffin wax, thinly sectioned, and stained with periodic acid Schiff reagent (PAS) and hematoxylin. In the histopathologic examination, seminiferous tubules were classified into 14 stages. All cross-sections of seminiferous tubule in 1 transverse section of the testis were examined and the histopathologic changes were evaluated. Degeneration of 1 or 2 germ cells in 1 seminiferous tubule was not regarded as a histopathologic change. Histopathologic examination was carried out blind to the treatment.
Hormone determinations.
Serum concentrations of testosterone, 17ß-estradiol, and luteinizing hormone (LH) were measured by radioimmunoassay. Test kits used were DPC 17ß-estradiol kit and DPC total testosterone kit (Diagnostic Products Corporation, Los Angeles, CA), and Biotrak rat luteinizing hormone (rLH) [125I] assay system (Amersham Life Science Ltd., Buckinghamshire, England). Double antibody assay was applied in the DPC 17ß-estradiol kit. For each assay, all samples were randomized prior to analysis.
Statistical analysis.
We divided this study into 3 blocks, although no discernible block effects were observed. Statistical analysis of the offspring data during the lactational period was carried out using the litter as a unit. Cumulative chi-squares test was used for the analysis of the copulation index, fertility index, and the ratio of the rats with abnormal histopathologic finding of the testis. Regarding the other data, statistical differences were analyzed with Fisher's least significant difference procedure after 1-way ANOVA. The results were interpreted as significant below a level of 0.05.
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RESULTS |
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Body Weight Gain and Food Consumption after Weaning
Body weights of the F1 and F2 generations after weaning are shown in Figure 1 and food consumptions of the 2 generations are shown in Figure 2
. In both the F1 and F2 generations, body weight gain in the 125 ppm TBTCl group was consistently suppressed. The body weight of the 125 ppm TBTCl group was approximately 7080% of the control value in the F1 generation and was approximately 6575% of the control value in the F2 generation. However, food consumption (g/kg body weight/day) was not decreased in this group (Fig. 2
). In the F1 generation, body weight gain was also slightly suppressed in the 25 ppm TBTCl group.
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DISCUSSION |
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TBT causes imposex in some mollusks and this effect was attributed to the inhibition of aromatization and conjugation of testosterone (IPCS, 1990; Maatthiessen and Gibbs, 1998). Disturbance of aromatization of testosterone was reported to severely damage spermatogenesis especially at spermiogenic stages in monkeys and mice (Robertson et al., 1999
; Shetty et al., 1998
). Serum testosterone concentration showed a 10-fold increase in the study of Shetty et al. (1998) and Leydig cell hyperplasia/hypertrophy was evident in the study of Robertson et al. (1999). These studies indicated that aromatase inhibition induced "hyper-androgenic" status in male mammals. In this study, histopathologic changes were evident in the TBTCl-treated rats, although the changes were minimal. Serum concentrations of LH and testosterone increased in the TBTCl-treated rats. However, these increases were less than twofold even in the highest dose group and these values were within historical control ranges. In addition, atrophy of the ventral prostate was evident in the rats fed the TBTCl diet and this is seemingly contrary to "hyper-androgenic" status. However, the prostatic atrophy caused by aromatase inhibition has been frequently reported (Habenicht et al., 1986
; Kawashita et al., 2000
; O'Connor et al., 1998
; Suzuki et al., 1996
). Along with the androgen receptor, the estrogen receptor also exists in the prostate (Mawhinney and Neubauer, 1979
; Sciarra and Toscano, 2000
). Estrogen is thought to be important for the normal functioning of this organ probably by stimulating androgen receptor expression (Bouton et al., 1981
; Collins et al., 1994
). In fact, antagonization of the estrogen receptor also causes the prostatic atrophy (Dhar et al., 1998
; Hoyt et al., 1998
; Neubauer et al., 1992
). It is thought that aromatase inhibition decreases the production of 17ß-estradiol and then disturbs the functioning of the prostate. We measured serum 17ß-estradiol concentration in this study and found that serum 17ß-estradiol concentration was decreased in the TBTCl-treated rats. Body weight was decreased to approximately 70% of the control value in the 125 ppm TBTCl group in this study. However, O'Connor et al. (2000) reported that feed-restricted CD Sprague-Dawley rats, the body weight of which was 74% of the control value, did not show a decrease in serum 17ß-estradiol concentration. Therefore, a decrease in serum 17ß-estradiol concentration in TBTCl-treated rats was caused by TBTCl itself in this study. Decreased circulating 17ß-estradiol without decreases in serum concentrations of LH and testosterone strongly indicated that the production of 17ß-estradiol was suppressed by TBTCl treatment. This might indicate that TBTCl is an aromatase inhibitor in male rats. In the validation study of tier 1 screening battery for detecting endocrine disruptors, O'Connor et al. (1998) found that aromatase inhibitor anastazole decreased accessory sex organ weights and serum 17ß-estradiol concentration without the decreases in serum concentrations of LH and testosterone in rats. The effects of aromatase inhibitors observed in their study were similar to those of TBTCl in this study. In this study, the TBTCl-treated female rats showed masculinization (Ogata et al., 2001
) as did TBT-treated female mollusks (IPCS, 1990
; Matthiessen and Gibbs, 1998
; Oberdörster and Cheek, 2000
) and this is consistent with our speculation. If TBTCl is an aromatase inhibitor, it seems to be a weak inhibitor because TBTCl did not cause severe damage on spermatogenesis and did not induce remarkable increase in serum testosterone concentration as shown in aromatase-knockout mice (Fisher et al., 1998
; Robertson et al., 1999
). There are some studies that did not show the prostatic atrophy by aromatase inhibition (Fisher et al., 1998
;Habenicht and el Etreby, 1989
; Oesterling et al., 1988
). In these studies, 4- to 10-fold increases in serum testosterone concentration were observed. We think that a remarkable increase in circulating androgen counterbalanced the effect of the estrogen reduction (Sciarra and Toscano, 2000
) and suppressed the prostatic atrophy in these studies. In spite of the decrease in the ventral prostate weight, the weight of another accessory sex organ, the seminal vesicle, was not decreased in the TBTCl-treated rats in this study. We weighed the seminal vesicle without fluid. We thought that this related to the absence of the seminal vesicle weight reduction in the TBTCl-treated rats in this study. We speculated that TBTCl is a weak aromatase inhibitor in rats but could not conclude so because we did not obtain direct evidence that TBTCl inhibited aromatase gene expression and/or aromatase enzyme activity in TBTCl-treated rats in this study. In fact, we could not deny the possibility that TBTCl did not inhibit aromatase but accelerated the metabolism and/or excretion of estrogen in this study. In addition, Yamabe et al. recently reported that TBT activated androgen-dependent response of human prostate cancer cells (Yamabe et al., 2000
). TBTCl may have multiple effects on the sex endocrine system in mammals as in mollusks. Further studies are needed to clarify the mechanism of the effects of TBTCl on the male reproductive system observed in this study.
In this study, the effects of TBTCl on the male reproductive system were more apparent in the F2 generation than in the F1 generation. In the F2 generation, the decreases in the reproductive organ weights and the spermatid/sperm count in the 125 ppm TBTCl group were more potent, the ventral prostate weight and the spermatid count also decreased in the 25 ppm TBTCl group, and the histopathologic change in the testis of TBTCl-treated rat was more apparent. The mothers of the TBTCl-treated F1 rats were exposed to TBTCl just from day 0 of pregnancy but the mothers of the TBTCl-treated F2 rats were exposed to TBTCl from their prenatal period. TBTCl is a lipophilic substance that is reported to bioaccumulate in rodents (IPCS, 1990) and TBTCl is reported to be transferred via placenta from dam to fetuses (Iwai et al., 1982
). The F2 rats might be exposed to higher concentration of TBTCl than the F1 rats in utero and this may explain the difference in the potency of the effects on the male reproductive system in the TBTCl groups between the 2 generations. We are now examining the concentrations of TBT and its metabolites in the organs of the TBTCl-treated neonates and adult rats in this study. Other possible cause of the intergenerational difference is the male-mediated developmental toxicity of TBTCl in the F2 generation. The fathers of the TBTCl-treated F1 rats were not exposed to TBTCl but the fathers of the TBTCl-treated F2 rats were exposed to TBTCl from their prenatal period. The frequency of morphologically abnormal sperm was not increased in the TBTCl-treated rats in the F1 generation. However, sperm of the F1 rats might be affected by TBTCl treatment and this might potentiate the male reproductive effect of TBTCl in the F2 generation.
In conclusion, TBTCl was proved to have male reproductive effects and might be an aromatase inhibitor in rats. Sex differentiation of the brain is reported to be more vulnerable to disruption of sex endocrine system than the reproductive system (Kubo et al., 2001). Therefore, we think it important to examine the effects of TBT on brain development for understanding its endocrine-disrupting effect in mammals.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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