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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Abstract: Tributyltin and 1, 1-dichloro-2, 2 bis (p-chlorophenyl) ethylene (p,p′-DDE) have been ubiquitously distributed over the world. In Japan, p,p′-DDE and tributyltin are ingested through marine products, in which these substances are accumulated through bio-concentration and the food chain. However, the consequence of potential combined hazards of these substances remains unknown. Therefore, the effects of concurrent exposure to 125 ppm p,p′-DDE and 25 ppm tributyltin were investigated in immature male Wistar rats by oral administration during puberty. In this study, tributyltin promoted the growth of pubertal male rats, while p,p′-DDE itself did not affect the growth but inhibited the growth enhancement by tributyltin. Furthermore, tributyltin reduced thymus weight but p,p′-DDE also prevented this weight reduction. Neither development of male sexual accessory organs nor sexual maturation was affected even by concurrent exposure to p,p′-DDE and tributyltin. No significant changes of serum testosterone, luteinizing hormone, follicle-stimulating hormone concentrations, and epididymal sperm numbers were observed with the administration of p,p′-DDE and/or tributyltin. These results indicate that sexual maturation, male reproductive organ development and sperm production is scarcely affected in immature male Wistar rats even by concurrent exposure to p,p′-DDE and tributyltin at a daily dose of ca. 2 mg/kg tributyltin and 10 mg/kg p,p′-DDE. Moreover, the simultaneous administration of p,p′-DDE with tributyltin counterbalanced the effects that were attributed to tributyltin alone.

Some synthetic chemicals may affect growth and development in wildlife and human beings by imitoting the actions of endogenous hormones (Gray & Kelce 1996; Gray & Ostby 1998; Vos et al. 2000). Such phenomena as genital abnormalities or reproductive impairments have been reported in wildlife or human beings over the past 30 years. These chemicals are called endocrine disrupters or endocrine disrupting chemicals (Colborn & Clement 1992).

Organotin compounds are used primarily as antifouling agents. Tributyltin oxide is added as an antifouling agent in numerous formulations to marine paints, which are applied to the hulls of ships to retard the growth of barnacles (Boyer 1989). However, tributyltin is slowly released from the painted surface and thus pollutes the seawater. Prolonged release of tributyltin from ship-bottom coatings has been documented in the Japanese sea, which has been polluted up to the level such that sexual mutations, so-called imposex changes, were reported in sea snails (Horiguchi et al. 1994 & 1995; Bettin et al. 1996; Oehlmann et al. 1996; Takahashi et al. 2000). Thus, tributyltin is at present suspected to be one of the endocrine disrupting chemicals (Vos et al. 2000).

1,1-Dichloro-2,2bis(p-chlorophenyl) ethylene (p,p′-DDE) is a persistent metabolite of DDT, which was used as a pesticide for over 30 years and is still being used for malarial control in tropical developing countries (Lopes-Carrillo et al. 1996; Rivero-Rodriguez et al. 1997). Once exposure occurs, p,p′-DDE can persist and accumulate for decades in the human body (Morgan & Roan 1974). It has been reported that some abnormalities in sexual development in rats and wildlife might be associated with exposure to p,p′-DDE (Kelce et al. 1995; Gray & Kelce 1996).

p,p′-DDE and tributyltin are ubiquitous as environmental contaminants. In Japan, these substances are generally taken up through marine products, especially fish or shellfish where these substances are abundant due to the bio-concentration and accumulation (Horiguchi et al. 1994; Oshima et al. 1997; Takahashi et al. 2000; Ema & Harazono 2001). As a result, concurrent exposure to p,p′-DDE and tributyltin will inevitably develop in mammals, especially human beings. If these substances may mimic endogenous hormone activities, immature male rats would be more vulnerable during the pubertal period where sexual maturation and reproductive organs are still under development. This study is undertaken to elucidate any effects resulting from concurrent exposure to these two substances on immature male Wistar rats during the pubertal period.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals. Twenty-four male Wistar rats, 4 weeks of age were purchased from Kyudo Breeding Co., Ltd. (Tosu, Japan). The rats were acclimatized to animal facilities for two weeks. The animals were kept in a room with a temperature of 20–24° and humidity of 30–60%. The light cycle was a 12 hr light: dark cycle. The rats were fed rat chow; CE-2 (Clea Japan Inc., Tokyo, Japan) and tap water ad libitum. Three rats were housed in a plastic cage with laboratory grade pine shavings as bedding. Normal sexual development is completed by 8 weeks of age in Wistar rat. Therefore, the pubertal period was considered to be around this time. This experiment was approved by the Committee of Ethics on Animal Experiments in the Faculty of Medicine, Kyushu University and conducted under the Guidelines for Animal Experiments in the Faculty of Medicine, Kyushu University and the Law (No. 105) and Notification (No.6) of the Japanese Government.

Preparation of special chow. p,p′-DDE (1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene) with a purity of 99% was purchased from Aldrich Chemical Co. (Milwaukee, WI, USA) and tributyltin chloride (>95% purity) from Tokyo Kasei Co. (Tokyo, Japan).

Special rat chow containing tributyltin chloride and/or p,p′-DDE was prepared as follows; tributyltin chloride or p,p′-DDE was dissolved in ethanol and mixed with CE-2 to form food pellets. During this process, ethanol was removed by evaporation. The concentration of tributyltin chloride in CE-2 was measured using a gas chromatograph equipped with a flame photometric detector (GC-FPD). The concentration of p,p′-DDE was determined by a gas chromatograph with an electron capture-detector and an autoinjector using the internal standard method. The relative peak areas of individual peaks and the internal standard peak were determined using a Shimadzu Chromatopac C-R7A automatic peak area determinator. Approximately 0.04 ng/g (0.04 ppb) tributyltin and 0.21 ppb p,p′-DDE were present in normal rat chow, CE-2. Tributyltin and p,p′-DDE concentrations in tap water were below the detection limit. Tributyltin and p,p′-DDE concentrations in the special chow were verified by random samplings during the experiment and at designated concentrations.

Treatment. Immature male Wistar rats were randomly divided into four groups: control, DDE, tributyltin, and DDE+tributyltin, with 6 rats per group. The control group was fed only CE-2; the DDE group was given CE-2 containing 125 μg p,p′-DDE/g (125 ppm); the tributyltin group was administered CE-2 containing 25 μg tributyltin chloride/g (25 ppm); and the DDE+tributyltin group was given CE-2 containing 125 ppm p,p′-DDE and 25 ppm tributyltin chloride. The rats were fed prepared chow for 42 days from 6 weeks to 12 weeks of age. Body weights of rats were measured every 7 days.

Tissue sampling and histological examination. The rats were sacrificed at 12 weeks of age by CO2 inhalation and autopsied. Blood samples were collected from the posterior vena cava. Serum was separated by centrifugation and stored −80° until measurement of parameters. Organ weights measured at autopsy included body, liver, kidneys, spleen, thymus, testes, epididymides, seminal vesicles and prostate. These organs were fixed in 10% neutral buffered formalin solution and then embedded in paraffin, cut into 6-micrometer section and stained with haematoxylin and eosin for histopathological study. In the case of testis, it was fixed in Bouin's solution, embedded in paraffin, sectioned at 3-micrometer and stained with periodic acid Schiff (PAS) and haematoxylin. The left epididymis was used for sperm count. Decapsulated epididymis was homogenized in saline containing 0.05% (v/v) Triton X-100 in a blender and homogenization-resistant sperm was counted using a haemocytometer.

Hormone assay. Testosterone was measured using a No-Extraction Coat-a-Count RIA kit obtained from Diagnostic Products Company (Los Angeles, CA, USA). Measurement of luteinizing hormone and follicle-stimulating hormone was performed using rat luteinizing hormone (rLH) and rat follicle-stimulating hormone (rFSH) radioimmunoassay kit purchased from Amersham International plc (Amersham, UK).

Statistical analysis. Statistical significance was determined by Fisher's least significant difference procedure after a one-way analysis of variance (one-way ANOVA). The significant level was set at P<0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The administration of p,p′-DDE and/or tributyltin did not give rise to any overt signs of toxicity. The body weight changes in all groups during the observation period are shown in fig. 1. At 6 weeks of age, the body weight was 170.5±12.9 g in the control group, 171.1±11.9 g in the DDE group, 169.3±13.0 g in the tributyltin group and 169.6±11.1 g in the DDE+tributyltin group. It reached 430.1±24.3 g in the control group, 443.8±34.1 g in the DDE group, and 462.5±33.3 g in the tributyltin group and 441.6±24.2 g in the DDE+tributyltin group at 12 weeks of age, respectively. The body weight increase in the tributyltin group was always much larger than those in other groups. However, this growth enhancement was not seen in the DDE+tributyltin group (fig. 1). Average food consumption during the observation period was not significantly different in any groups.

image

Figure 1. Body weight changes of male Wistar rats exposed to p,p′-DDE and/or tributyltin (TBT) during observation period. *Significantly different from control (P<0.05).

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Weights of organs examined at autopsy were shown in table 1. The liver and spleen weights slightly increased in the p,p′-DDE- and/or tributyltin-administered groups. The kidney weight also increased in the DDE group, while the thymus weight decreased in the tributyltin group. However, a significant change was only observed in the thymus weight in the tributyltin group, whereas its weight reduction was not recognized in the DDE+tributyltin group (table 1). With regard to male reproductive organs, there were no changes in the weight of testis, epididymis, prostate and seminal vesicles as shown in table 1. In order to examine systemic toxicity, serum biochemical parameters of renal and hepatic function were evaluated. These biochemical markers were unaffected by any treatment, while AST and ALT relatively increased in the DDE and/or tributyltin-treated groups. The renal function was not impaired in any group (table 2). Serum testosterone concentration is shown in fig. 2. Its concentration relatively increased in the DDE group, albeit statistically insignificant. No differences were observed in serum luteinizing hormone and follicle-stimulating hormone concentrations in any group and there were no treatment-related effects in epididymal sperm numbers (table 3). In microscopic studies, no pathological changes were recognized in the examined organs in any group.

Table 1.  Organ weights (g) of male Wistar rats exposed to p,p′-DDE and/or tributyltin (TBT) at 12 weeks of age.
 ControlDDETBTDDE+TBT
  1. Values (g) are means±S.D. (n=6). *Significantly different from control (P<0.05).

Liver17.0±2.219.5±2.819.9±2.020.1±2.7
Spleen0.87±0.151.07±0.281.02±0.160.97±0.10
Kidney3.41±0.423.79±0.733.36±0.273.34±0.33
Thymus0.60±0.110.55±0.100.45±0.04*0.52±0.06
Testis1.76±0.111.71±0.391.80±0.061.81±0.04
Epididymis0.53±0.050.47±0.200.57±0.040.54±0.01
Prostate0.43±0.050.45±0.110.44±0.060.46±0.11
Seminal vesicles0.54±0.070.56±0.080.59±0.070.54±0.06
Table 2.  Serum biochemistry of hepatic and renal function.
 ControlDDETBTDDE+TBT
  1. Values are means±S.D. (n=6). BUN: blood urea nitrogen.

AST (IU/l)80.0±8.084.5±13.087.5±10.690.2±11.6
ALT (IU/l)58.8±9.364.5±8.861.7±13.961.8±5.5
Cr (mg/dl)0.73±0.050.65±0.060.72±0.040.72±0.08
BUN (mg/dl)25.8±2.325.5±4.127.5±1.728.6±2.3
image

Figure 2. Serum testosterone concentrations of male Wistar rats exposed to p,p′-DDE and/or tributyltin (TBT) at 12 weeks of age.

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Table 3.  Serum luteinizing hormone, follicle-stimulating hormone concentrations, and epididymal sperm count.
 ControlDDETBTDDE+TBT
  1. Values are means±S.D. (n=6). LH: luteinizing hormone, FSH: follicle-stimulating hormone.

LH (ng/ml)4.93±0.944.76±2.336.22±1.965.76±2.55
FSH (ng/ml)12.38±2.2012.85±2.7715.76±4.8511.30±5.32
Epididymal sperm (millions)194.4±15.3178.7±16.5184.6±8.6189.5±12.9

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

p,p′-DDE is still detected in mammals or human beings due to its persistence and accumulation (Mes 1992; Takahashi et al. 2000) and although tributyltin concentration in the environment has been declining in recent years but are still over the safety level (Oshima et al. 1997; Takahashi et al. 2000; Ema et al. 2001). Tributyltin and p,p′-DDE are, to a considerable extent, taken up by marine products and thus, the concomitant exposure to these substances is insidiously proceeding through the ingestion of marine products in Japan. If these substances mimic endogenous hormone actions, immature animals would be vulnerable, as sexual or reproductive organ maturation is still under development. Therefore, complex effects of concurrent exposure to tributyltin and p,p′-DDE on pubertal male Wistar rats were investigated in this study. The concentrations of tributyltin and p,p′-DDE present in the chow in this study were 25 ppm tributyltin and 125 ppm p,p′-DDE and thus total daily intake resulted in approximately 2 mg/kg tributyltin and 10 mg/kg p,p′-DDE, based on calculations from daily mean chow consumption. Ingested doses in this study are certainly very high compared to daily environmental exposure levels but it is noteworthy that p,p′-DDE or tributyltin concentrations present in the rat are comparable to those detected in human serum or tissues. p,p′-DDE concentrations in human blood and fat were found to be 0.87 ng/g (ppb) in whole blood and 429.2 ppb in fatty tissue in residents of British Columbia (Mes 1992). On the other hand, fat and plasma concentrations in Sprague-Dawley dam rats were reported to be 141 ppm on gestational day (Day) 19 and 29 ppb on Day 21, following gavage p,p′-DDE dosing for 5 days (Day 14–18) at a dose of 10 mg/kg (You et al. 1999). In the case of tributyltin, the detected level in the human or monkey liver was about 2 ppb (Takahashi et al. 1998) and 380 ppb was detected in the rat fat tissue after oral administration of tributyltin chloride at a dose of 2 mg/kg for 28 days (Bressa et al. 1991). These levels are similar to the concentrations achieved in this experiment, although actual tissue concentrations of p,p′-DDE or tributyltin were not determined.

The present study revealed that concurrent exposure to p,p′-DDE and tributyltin during the pubertal period scarcely affected male reproductive organ development, sexual maturation and sperm number in male Wistar rats at the dose administered in this experiment. In the previous studies, tributyltin evidently affected the male reproductive system at the dose of about 10 mg/kg but its effects were minimal at the dose of 2 mg/kg (Omura et al. 2001). On the other hand, p,p′-DDE had definite antiandrogenic actions on the male sexual organs at the dose of 100 mg/kg, while little detectable effect was observed at the dose of 10 mg/kg (You et al. 1998; Makita et al. 2003). Our results were consistent with previous studies and no additive or synergistic effects were observed with concurrent exposure to p,p′-DDE and tributyltin in this experiment.

Tributyltin promoted the growth of pubertal male rats, but p,p′-DDE inhibited the growth enhancement attributed to tributyltin. Usually, tributyltin retards the growth with or without decrease in food consumption (Boyer 1989). In our experiment, growth enhancement occurred without changes in food consumption. However, the increase of the body weight is small and there is some uncertainty due to the small number of animals. In contrast, administered tributyltin did reduce the weight of thymus, which is known to be a target organ of tributyltin (Boyer 1989). Tributyltin is reported to have a direct and selective toxic effect on lymphocytes in the thymus. Therefore, its weight reduction seemed to be attributed to this effect of tributyltin. But p,p′-DDE also prevented the thymus weight reduction by tributyltin. These experimental results indicate that tributyltin and p,p′-DDE had opposite actions. Such a counterbalanced phenomenon was also observed in simultaneous administration of 1,4-dichlorobenzene and p,p′-DDE in our previous study (Makita 2004).

With regard to the interaction of tributyltin and p,p′-DDE, the following explanations would be possible. Both substances have different effects on metabolism. Tributyltin reduces hepatic cytochrome P450 contents (Shim et al. 2003), while p,p′-DDE induces its contents (Yoshioka et al. 1984). Thus, p,p′-DDE antagonized tributyltin actions. Furthermore, tributyltin can exert different actions under the presence of other substances, thus it was shown by Burton et al. (2002) that tributyltin enhances the ability of polychlorinated biphenyl 126 (PCB126) to induce P450 1A (CYP1A), while tributyltin itself inhibited it. This seems to be the case in this study too. A slight elevation of ALT and AST and a increase of the liver weight may reflect the activated metabolism of p,p′-DDE and/or tributyltin due to overload. On the other hand, tributyltin and p,p′-DDE are one of the most prevalent endocrine-disrupting chemicals (Vos et al. 2000). p,p′-DDE was shown to inhibit the binding of androgens to the androgen receptor (Kelce et al. 1995; You et al. 1998) and tributyltin is supposed to be an inhibitor of aromatase enzyme metabolizing testosterone to 17β-oestradiol, which increases testosterone levels (Bettin et al. 1996; Vos et al. 2000; Cooke 2002). However, total testosterone levels were not markedly increased by tributyltin, which did not produce typical androgenic effects on male reproductive organs such as prostate or epididymis in this experiment. Therefore, it is less possible that tributyltin exerts some androgenic action and p,p′-DDE as an androgen receptor antagonist prevented this action.

In conclusion, immature male Wistar rats were resistant to concurrent exposure to tributyltin and p,p′-DDE in this study, which shows no deteriorations of male reproductive system, even at the quite high level of exposure. Furthermore, p,p′-DDE counterbalanced some of the observed effects attributed to tributyltin. Our results indicated that some environmental contaminants have opposite or antagonistic actions like tributyltin and p,p′-DDE on each other. Further investigation should be conducted regarding the complex effects of several environmental contaminants.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We are indebted to Dr. T. Matsuura for supporting this study. We are also grateful to Ms. Y. Hirose and T. Inoue for technical assistances. This study was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (Grant No. 10470096).

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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