MATERIALS AND METHODS

This study was carried out from February to September, 2009 in the Animal Vivarium and the Laboratory of Hormones of the Metropolitan University-Xochimilco of Mexico.

Hormones
Testosterone was supplied by Steraloids (Wilton, NH, U.S.A.). Finasteride was obtained by extraction from Proscar (Merck, Sharp and Dohme). The tablets were crushed, extracted with chloroform and the solvent was eliminated in vacuum; the crude product was purified by silica gel column chromatography. Solvents used were laboratory grade or better.

Animals and Tissues
Prepuberal male Syrian golden hamsters 3 weeks old (108-115 g) and adult male Syrian golden hamsters 2.5 month of age (150-200 g) were obtained from the Metropolitan University in Xochimilco, Mexico. Prepuberal and adult hamster’s gonadectomies were performed under pentobarbital anesthesia (70-80 mg kg^-1) and the castrated hamsters were housed in micro isolator cages (Allentown Caging Equipment); environmental enrichment was provided. Environmental conditions were maintained as recommended in the Guide for the Care and Use of Laboratory animals and the Mexican Low NOM-067-ZOO-1999 (National Research Council, 1996): Controlled temperature (22°C) in at 12/12 h light-dark cycles (lights on 07:00 to 19:00); humidity 45-55%. Food (PMI 5053, irradiated lab diet) and sterile water were provided ad libitum.

After 7 days maintaining these conditions, prepuberal hamsters were separated in three experimental castrated groups of 4 animals/each; one was kept as a castrated control. At the end of the experiment the prepuberal hamsters were euthanized with CO₂.

After 30 days under the above mentioned conditions, the experiment with the adult gonadectomized hamsters was carried out on three groups of 4 animals/experiment and one group was kept as a control. After the treatment, the adult hamsters were euthanized with CO₂. These protocols were approved by the Institutional Care and Use Committee of the Metropolitan University of Mexico (UAM) and the facility was accredited by SAGARPA (Government Organization for Agriculture and Livestock).

Flank Organ Test
Three experimental groups of prepuberal or adult gonadectomized hamsters were used; one was injected daily with 200 μL of sesame oil (control), the second one with 1 mg kg^-1 of testosterone and the third with T plus 1 mg kg^-1 of finasteride for 6 days. One group of prepuberal or intact animals was used to compare the effect of castration. After the treatment, the hamsters were sacrificed. The diameter of the pigmented spot of the flank organs from the animals of each group (4) was measured, as previously reported for adult hamsters (Chen et al., 1995) but not for prepuberal animals. The experiment was repeated under the same conditions twelve different times for each group of prepuberal intact, treated and control animals and nineteen different times for each group of adults intact, treated and control animals. The results were analyzed using one-way analysis of variance and Dunnett’s Method for comparing mean values, with JMP IN 5.1 software (Sokal and Rohlf, 1994).

Determination of the Weight of Prostate and Seminal Vesicles
Three experimental groups of prepuberal or adult gonadectomized hamsters were used; one was injected daily with 200 μL of sesame oil (control), the second with 1 mg kg^-1 of testosterone and the third with T plus 1 mg kg^-1 of finasteride for 6 days. One group of prepuberal and adult intact animals was used to compare the effect of castration. After the treatment, the prepuberal and adult animals were sacrificed. The weight of prostate and seminal vesicles from the animals of each group (4) was determined. The experiment was repeated in the same conditions twelve different times for each group of prepuberal intact, treated and control animals and nineteen different times for each group of adults intact, treated and control animals. The results were analyzed using one-way analysis of variance and Dunnett’s Method for comparing mean values (Sokal and Rohlf, 1994) with JMP IN 5.1 software.

RESULTS

Flank Organ Test
The results from this experiment showed that there are no significant differences in the diameter of the pigmented spot of the flank organs between intact and control castrated prepuberal hamsters. However, the size of flank organs from castrated and treated daily with y s.c. injections of 1 mg kg^-1 of T for 6 days, was significantly increased (p<0.005) as compared with the intact and castrated and vehicle treated control (Table1). These results indicated that flank organs of prepuberal hamsters possess androgen receptors. When testosterone (T) and finasteride (1 mg kg^-1) were injected together, the diameter of the pigmented spot did not significantly decrease in prepuberal animals as compared with that of the flank organs from prepuberal hamsters treated with T (Table 1).

On the other hand, the diameter of the pigmented spot of the male flank organs from intact adults was significantly larger than those of castrated hamsters. Daily s.c. injections of 1 mg kg^-1 of T for 6 days to castrated animals, significantly increased (p<0.005) the size of flank organs as compared with the vehicle treated castrated control (Table 2). These results indicated that flank organs of adult hamsters possess androgen receptors as previously had been reported by Takayasu and Adachi (1975). However, 6 days of treatment with T to the castrated adult males, is not enough time to produce a similar growth of the flank organs than that observed for intact adult hamsters.

When testosterone (T) and finasteride (1 mg kg^-1) were injected together, the diameter of the pigmented spot significantly decreased in the adult hamsters as compared with the flank organs from adult hamsters treated with T (Table 2).

Weight of Prostate and Seminal Vesicles
The results from these experiments showed that there are not significant differences in the weight of prostate and seminal vesicles between intact and control castrated prepuberal hamsters.

Table 1:	Effect of testosterone and finasteride on the diameter of the pigmented spot of the flank organs and on the weight of prostate and seminal vesicles from castrated prepuberal hamsters
The results are shown as mm or mg± of standard deviations. The animals were castrated a week prior to the experiment and treated with daily sc injections of the steroids for 6 days. There are not significant differences in the diameter and the weight of these glands between intact and control castrated prepuberal hamsters. Intact: Non castrated hamsters. Control: Castrated hamsters vehicle-treated. *Significant differences with the animals treated with T. n is indicative of the number of times that each experiment was repeated separately in the same conditions for each group

Table 2:	Effect of testosterone and finasteride on the diameter of the pigmented spot of the flank organs and on the weight of prostate and seminal vesicles from castrated adult hamsters
The results are shown as mm or mg± of standard deviations. The hamsters were castrated a month prior to the experiment and treated with daily sc injections of the steroids for 6 days. There are significant differences in the diameter and the weight of these glands between intact and control castrated adult hamsters. *Significant differences with the hamsters treated with T. n is indicative of the number of times that each experiment was repeated separately in the same conditions for each group

The weight of prostate and seminal vesicles from prepuberal hamsters castrated and treated with daily s.c. injections of 1 mg kg^-1 of T for 6 days, was significantly increased (p<0.005) as compared with the vehicle treated control (Table1). These results indicated that the prostate and seminal vesicles from prepuberal hamsters possess androgen receptors. When testosterone (T) and finasteride (1 mg kg^-1) were injected together, the weight of prostate and seminal vesicles significantly decreased as compared with the weight of the glands from prepuberal hamsters treated with T (Table 1).

On the other hand, the results from this study also showed that the weight of prostate and seminal vesicles from intact adults was significantly larger than those from adult castrated hamsters. When adult castrated animals were treated daily with s.c. injections of 1 mg kg^-1 of T for 6 days, the weight of prostate and seminal vesicles was significantly increased (p<0.005) as compared with the vehicle treated control (Table 2). However, 6 days of treatment with T to castrated adult males, was not enough time to produce a similar growth than that observed for intact adult hamsters. When testosterone (T) and finasteride (1 mg kg^-1) were injected together, the weight of prostate and seminal vesicles was significantly decreased as compared with the weight of the glands from adult hamsters treated with T (Table 2).

DISCUSSION

The aim of this study is to compare the effect of different treatments on the diameter of the pigmented spot of the hamster’s flank organs as well as on the weight of seminal vesicles and prostate gland. This study was carried out in two different animal models: prepuberal and adult hamsters, in order to determine the advantage of each animal model for the pharmacological evaluation of antiandrogens and 5α-reductase inhibitors.

It is important to consider the fact that in prepuberal hamsters after 7 days of castration they did not present androgen dependent characters such as: Increase in the diameter of the pigmented spot of the flank organs as well as growth of prostate and seminal vesicles. This fact was also observed in intact prepuberal hamsters. However, in adult hamsters it was necessary to wait 30 days after castration to observe a decrease of the diameter of the pigmented spot of the flank organs as well as on the growth of prostate and seminal vesicles (Bratoeff et al., 2008, 2009; Cabeza et al., 2001, 2008).

Flank organs of prepuberal hamsters of 4 weeks of age, responded to T stimulus thus, indicating that these glands possess androgen receptor as had previously been reported for adult (Takayasu and Adachi, 1975) but not for prepuberal hamsters. This finding is important since, it indicates that the gene which codifies for the androgen receptor expresses in an independent way the presence of androgens in the blood.

When prepuberal castrated hamsters were treated with T and F together, the flank organs’ diameter size did not decrease. Finasteride is a 4-aza steroid that inhibits 5α-reductase enzyme present in the flank organs of adult hamsters (Chen et al., 1995). These facts taking together indicated that the enzyme 5α-reductase was not present in flank organs from prepuberal hamsters, because a formation of dihydrotestosterone (DHT) was not observed. This fact hasn’t been previously reported in the literature and it is very interesting since, it indicates that the gene that codifies for 5α-reductase enzyme in the flank organs requires the presence of T or DHT for the expression in these glands. On the other hand, the growth of the flank organs in prepuberal hamsters treated with T can be explained by considering the presence of androgen receptors in these organs.

In the model of castrated adult hamsters it was shown that T significantly increased the diameter of the pigmented spot on the flank organs; however, when the hamsters were treated with T plus finasteride a significant decrease of the diameter of the pigmented spot was observed. Therefore, this model (castrated adult hamsters) has advantage over the prepuberal type since, the effect of the 5α-reductase inhibitors in the flank organs can be observed (Bratoeff et al., 2008, 2009; Cabeza et al., 2001, 2008).

On the other hand, the injection of T into the castrated prepuberal hamsters increased the weight of prostate and seminal vesicles. This study indicated that, T stimulated the growth of these glands. These results suggested the presence of androgen receptors in prostate and seminal vesicles a fact which previously has not been reported and indicates that the gene which codifies for the androgen receptor as well as in flank organs expresses in an independent way the presence of androgens in the blood. The model of castrated adult hamsters showed a similar response to T than the prepuberal; therefore, both models are appropriate for use as pharmacological assays for the determination of the antiandrogenic effect (Bratoeff et al., 2008, 2009; Cabeza et al., 2001, 2008).

Treatment with T plus finasteride inhibited the growth of the prostate and seminal vesicles produced by DHT in prepuberal hamsters. This fact indicates very clearly that the 5α-reductase enzyme is present in the glands of these animals (finasteride is an inhibitor of 5α-reductase enzyme); this finding has not previously been reported for prepuberal hamsters. This evidence could be explained on the bases that local steroidogenesis from sodium acetate, as a precursor, takes place in the prostate and seminal vesicles (Luu-The et al., 2008). Then, the presence of low amounts of T produced in this way could activate the codification of 5α-reductase’s gene in these tissues. Furthermore, this study also demonstrates that dihydrotestosterone is responsible for the weight increase of seminal vesicles and prostate gland observed in T-treated animals. The seminal vesicles are more sensitive for the 5α-reductase enzyme inhibition produced by finasteride as compared to the prostate gland, since, a higher decrease of the weight of seminal vesicles than of the weight of prostate was observed in both hamster’s model used (Bratoeff et al., 2008, 2009; Cabeza et al., 2001, 2008).

The treatments administrated to prepuberal hamsters, after 7 days of castration produced similar results in the growth of prostate and seminal vesicles as compared to adult hamsters, after 30 days of castration. However, the response to finasteride from prepuberal hamsters in the flank organs was different than that observed for adult hamsters. This difference in the response of the flank organs to finasteride between prepuberal and adults hamsters is important since, the adult model allows us to determine the effect of androgens and antiandrogens on the prostate, seminal vesicles and flank organs in the same animal. (Bratoeff et al., 2008, 2009; Cabeza et al., 2001, 2008).

In conclusion, the flank organs from prepuberal hamsters are a good model for the assay of the androgenic and antiandrogenic activity of different steroids; unfortunately this model did not give satisfactory results for the 5α-reductase inhibitors in vivo experiments. However, both adults and prepuber hamsters prostate and seminal vesicles are good models for the in vivo determination of these effects in new drugs.

ACKNOWLEDGMENT

This study was supported by a Grant from CONACYT (Project number 54853).