Keywords: diethylstilbestrol; polyamines; ornithine decarboxylase; spermidine/spermine N1-acetyltransferase

Abstract

Aim: To investigate the effect of diethylstilbestrol (DES), one of the most potent endocrine disruptors, on the metabolism of polyamines in hamster epididymis. Methods: Male golden hamsters of 7-week-old were kept under a light and dark cycle of 14 h and 10 h for 1 week to stimulate maximally the gonadal function. DES was injected subcutaneously at doses of 0.01 mg·kg^-1·day^-1, 0.1 mg·kg^-1·day^-1 and 1 mg·kg^-1·day^-1 for one week. Results: DES treatment caused a significant decrease in the weight of epididymis. The activity of epididymal ornithine decarboxylase (ODC) increased 1 day after DES treatment, kept at a high level for 4 days and then decreased to nearly normal level at day 7. The activity of spermidine/spermine N1-acetyltransferase (SSAT) also increased transiently after DES treatment. The contents of putrescine, spermidine, spermine and N1-acetylspermidine were increased 1 day ~ 4 days after DES treatment and restored to normal at day 7. All these changes showed a marked difference between the caput and the cauda. Conclusion: The polyamine biosynthesis in the hamster epididymis can be affected by DES, a xenoestrogen. DES may probably affect polyamine metabolism in the epididymis by regulating the rate-limiting enzymes involved in the polyamine biosynthesis.

1 Introduction

Polyamines occur in quite high concentrations in hamster epididymis [8]. The appropriate levels of intracellular polyamines are regulated strictly by a combination of crucial enzymes, such as ornithine decarboxylase (ODC) and spermidine and spermine N1-acetyltransferase (SSAT). ODC, the initial and rate-limiting enzyme in polyamine biosynthetic pathway, is readily induced by growth-promoting stimuli in resting cells, making its activity a highly sensitive marker of the action of trophic hormones and growth factors on their target tissues [9]. SSAT is an enzyme that catalyzes acetylation of spermidine and spermine and involved in the conversion of these polyamines into putrescine [10]. The estrogen concentration in the caput epididymidis is approximately 25 times higher than in the plasma [11], suggesting that estrogen plays an important role in epididymal function. However, the role of estrogen on epididymal polyamine metabolism is still poorly understood.

Male hamsters have frequently been used as a model for the research of endocrine disruptors, including DES, on estrogen-sensitive organs, because the hamster is much more responsive to DES than the rat [12]. The hamster epididymis, therefore, can be used as a good model for evaluation of the effect of estrogen on poly-amine metabolism. Previous reports mainly dealt with the effects of DES on the prenatal and neonatal animals and the present investigation was designed to study the effect of DES on the polyamine metabolism in the epididymis of mature hamsters.

2 Materials and methods

DES, dansylchloride, dl-proline, l-ornithine, putrescine, spermine, spermidine and N1-acetylspermi-dine were purchased from Sigma (USA); l-[1-¹⁴C]-ornithine, from NEWTM Life Science Products (Japan); [1-¹⁴C]acetyl-CoA, from Amersham Pharmacia Biotech (Japan); Aquasol-2, from Packard Bioscience (Japan) and other regents, from Wako Pure Chemical Industries (Japan).

Firstly, different doses of DES, 0.01 mg·kg^-1·day^-1, 0.1 mg·kg^-1·day^-1 and 1.0 mg·kg^-1·day^-1, were injected s.c. to three groups of hamsters of 5 animals each, respectively, for one week and 5 animals without treatment served as the control. On day 8 hamsters were sacrificed by decapitation and the reproductive organs were removed, dissected free of surrounding fatty tissues and weighed. A second series of study included 3 additional groups of animals received subcutaneous injection of DES, 1.0 mg·kg^-1·day^-1 for 1 days, 4 days and 7 days, respectively. On the next day, they were sacrificed and the epididymis was dissected and sectioned into the caput and cauda and weighed. Tissues were stored at -20 until use.

Putrescine, spermidine, spermine and N1-acetylsper-midine were assayed by the thin-layer chromatographic method [13]. Briefly, tissues were thoroughly washed with saline to remove the semen and homogenized with a homogenizer (Polytron PT 1200C, Switzerland) in five volumes of ice-cold 5 % trichloroacetic acid (TCA). The whole homogenate was centrifuged at 700g, 4 for 10 min. Dansyl-chloride was added to the supernatant overnight and the sample was applied on a Silica gel 60 (MERCK, Germany) for 2 h. The solvent system used was cyclohexane-ethyl acetate (3:2, v/v). The fluorescence was determined by a spectrofluorometer (JASCO FP-777, Japan) and polyamine contents calculated and expressed as nmol/g wet tissue.

The ODC activity was measured as described previously [14]. Briefly, after being washed the epididymides were homogenized in four volumes of ice-cold 50 mmol/L sodium phosphate buffer (pH 7.2) containing 5 mmol/L dithiothreitol. The post-mitochondrial fractions were obtained after centrifugation at 10,000g, 4 for 20 minutes. One hundred mL of the reaction mixture contained 50 mmol/L sodium phosphate buffer (pH 7.2), 5 mmol/L dithiothreitol, 1 mmol/L pyridoxal 5-phosphate, 1mmol/L ornithine, 0.2 mmol/L EDTA and 7.4 kBq l-[14C]-ornithine. One hundred mL of enzyme solutions were preincubated at 37 for 3 min and mixed with 100 mL reaction mixture at 37 . After 1 h incubation, 100 mL of 2 mol/L H₂SO₄ was added to stop the reaction. Incubation was continued for another 40 min to allow the absorption of the released ¹⁴CO₂ by the 2 mol/L KOH in the glass filter paper disks (GB-100R, TOYO Roshi Kaisha, Japan). The disks were transferred to counting vials with 10 mL Aquasol and the radioactivity was counted in an ALOKA liquid scintillation counter (LSC-6100). The enzyme activity was expressed as nmol ¹⁴CO₂ released·h^-1·mg^-1 protein. Protein was measured using a commercial kit purchased from Bio-Rad Laboratories (USA) with bovine serum albumin (BSA) as standard.

The SSAT activity was assayed by measuring the conversion of [1-¹⁴C]acetyl-CoA into [1-¹⁴C]acetylsper-midine at 30 for 10 min as described by Matsui et al. [15]. The homogenizing solution contained 0.25 mmol/L sucrose, 25 mmol/L KCl, 5 mmol/L MgCl2 and 50 mmol/L Tris-HCl (pH 7.5). After incubation, 50 mL of the reaction mixture containing 3 mmol/L spermidine, 50 mmol/L Tris-HCl (pH 7.8) and 8 mmol/L [1-14C]acetyl-CoA (50 mCi/mL) was spotted on the 2.3 cm disk of P-81 cellulose phosphate paper (Whatman International, Japan). The discs disks were then thoroughly washed and counted for their radioactivity in LSC-6100 in vials containing 10 mL of scintillation solvent. The SSAT activity was expressed as nmol/g wet tissue per 10 min.

Data were expressed as meanSEM. Analysis of variance (ANOVA) and Scheffe's multiple comparison test were used to analyze the significance of differences between groups. P<0.05 was considered significant.

3 Results

Table 2. Effect of 1.0 mg.kg^-1.day^-1 DES on epididymal weight.  ^bP<0.05, ^cP<0.01, compared with controls.

Figure 1. Changes in polyamines levels in hamster epididymis after DES treatment 1 mg.kg^-1.day^-1. (A) Putrescine (B) Spermidine (C) Spermine (D) N1-acetylspermidine (n=5; ^bP<0.05, ^cP<0.01, compared with the corresponding controls).

Figure 3. Epididymal spermidine/spermine N1-acetyltransferase (SSAT) activity in hamster epididymis during 1 mg.kg^-1.day^-1 DES treatment (n=5; ^bP<0.05, ^cP<0.01, compared with the corresponding controls)

All the three polyamines, putrescine, spermidine and spermine, were detected in intact hamster epididymis at the concentration of 200 nmol/g ~ 1000 nmol/g and were shown to be quite sensitive to DES exposure. The poly-amine concentrations are regulated precisely by a combination of enzymes, such as ODC, S-adenosylmethionine decarboxylase and SSAT [18]. Putrescine is formed from ornithine by ODC and putrescine in turn is converted to spermidine. Spermidine is converted to spermine by aminopropyltransferase at the expense of decarboxylated S-adenosylmethionine [10]. Although both ODC activity and putrescine levels were increased at day 1 of DES treatment, the peak of putrescine concentration at day 1 did not coincide with the maximum of ODC activity at day 4, whereas spermidine levels were also increased by day 4 (Figure 1A & 2). Therefore, these data suggest that in the epididymis of DES-exposed hamster, the synthesis of putrescine from ornithine by ODC and N1-acetylspermidine by polyamine oxidase is less prominent than its transformation to spermidine by aminopropyl-transferase during the early stage of DES administration. It is also possible that ornithine available for putrescine synthesis may be rate-limiting in the epididymis.

N1-acetylspermidine and SSAT showed characteristic alterations in the caput epididymidis in the course of DES treatment (Figure 1D & 3). SSAT is a key enzyme in the degradation of polyamines, which is markedly affected by polyamines and their analogues and catalyzes the conversion of sperdimine and spermine to their N1-acetyl derivatives [10]. In SSAT transgenic mice, the alterations of gene transcript were related to uterine and ovarian growth [20], suggesting the overexpression of SSAT gene can induce abnormalities of reproductive organs. In the present study, DES may have regulated the N1-acetylsperimidine levels by acting on SSAT gene expression. However little is known about the relationship between estrogens and SSAT in the epididymis.

The present study has revealed a considerable difference in polyamine contents and SSAT activity in the caput and cauda in both the control and DES-treated groups. It is known that N1-acetylsperimidine levels are higher in the epididymal caput of the hamster than those in the epididymal cauda [8], which may be due to differences in the cell types and/or hormone levels in the two segments of epididymis [21]. As in the hamsters, a higher ODC activity was also seen in the cauda than that in the caput epididymidis of rats[22]. Although more marked changes in polyamine metabolism were observed in DES-treated hamster caput than those in the cauda, the cauda weight decreased more dramatically. It may have been resulted from the fact that the epididymal cauda serves as a repository of mature spermatozoa in a metabolically quiescent state. Because the epididymis is dependent on the presence of the testis to maintain its physiological function, the atrophic testis following DES administration is further helpful to understand the decrease of the lumenal contents and the weight of epididymis [7].

In conclusion, the present study suggests that the polyamine metabolism in the hamster epididymis can be affected by exogenous endocrine disruptors like DES through changing the activities of the rate-limiting enzymes in the polyamine pathway. The changes in epididymal ODC activity in response to DES is so great that it can be used for assessing the responsiveness of different environment estrogens.

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Correspondence to: Dr. Shigeru Matsuzaki, Department of Biochemistry, Dokkyo University School of Medicine, Mibu, 321-0293 Tochigi, Japan.
Tel: +81-282-872 127, Fax: +81-282-867 268
E-mail: matuzaki@dokkyomed.ac.jp
Received 2003-04-15 Accepted 2003-08-04