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Prevention by 17β-estradiol and progesterone of calcitonin gene-related peptide-induced elevation of skin temperature in castrated male rats
BASIC SCIENCE
PREVENTION BY 17-ESTRADIOL AND PROGESTERONE OF CALCITONIN GENE-RELATED PEPTIDE-INDUCED ELEVATION OF SKIN TEMPERATURE IN CASTRATED MALE RATS MITSUTOSHI YUZURIHARA, YASUSHI IKARASHI, MASAMICHI NOGUCHI, YOSHIO KASE, SHUICHI TAKEDA, AND MASAKI ABURADA
ABSTRACT Objectives. To clarify the relationship between calcitonin gene-related peptide (CGRP) and ovarian hormones (17-estradiol and progesterone) in hot flashes in men who undergo androgen deprivation therapy for prostate cancer, we studied the effects of ovarian hormones on CGRP-induced elevation of skin temperature in castrated male rats. The results were compared with those from rats treated with testosterone replacement. Methods. Adult male rats were castrated by either a single injection of gonadotropin-releasing hormone analogue (Leuplin, 1.0 mg/kg, subcutaneously) or bilateral orchiectomy. The castrated animals were subcutaneously injected daily for 14 days with ovarian hormones, testosterone, or olive oil as the vehicle. On the day after the final administration of the drug, the changes in skin temperature induced by exogenous CGRP (10 g/kg intravenously), serum testosterone concentration, and prostate weight were measured. Results. The CGRP-induced elevation of skin temperature was significantly greater in the castrated rats than in the sham-treated rats. This potentiation was significantly inhibited by treatment with ovarian hormones, as well as by testosterone replacement. The testosterone replacement restored decreases in both the serum testosterone level and the prostate weight due to castration; the treatment with ovarian hormones did not affect them. Conclusions. 17-Estradiol and progesterone, which do not confer testosterone activity on serum, may be useful for the treatment of hot flashes in patients for whom testosterone replacement therapy is contraindicated, such as those with prostate carcinoma. In addition, we suggest that CGRP is closely involved in the amelioration of hot flashes by ovarian hormones in men who undergo androgen deprivation therapy. UROLOGY 64: 1042–1047, 2004. © 2004 Elsevier Inc.
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urgical or medical castration is highly effective as a treatment for prostate cancer. However, up to 80% of castrated men experience hot flashes.1,2 Hot flashes generally begin with a sudden outpouring of sweat and an increase in heart rate and peripheral blood flow, causing a drastic increase in skin temperature.3 This adverse effect often impairs the quality of life for these patients.4,5 It has been demonstrated that ovarian hormones such as estrogen6 and progesterone7 are effective for the amelioration of hot From the Medicinal Evaluation Laboratory and Research Division, Tsumura & Company, Ibaraki, Japan Reprint requests: Mitsutoshi Yuzurihara, Ph.D., Medicinal Evaluation Laboratory, Tsumura & Company, 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-1192, Japan Submitted: March 22, 2004, accepted (with revisions) June 2, 2004 © 2004 ELSEVIER INC. 1042
ALL RIGHTS RESERVED
flashes in men who undergo androgen deprivation therapy (ADT) for prostate cancer, as well as in menopausal women.8 However, the etiology of hot flashes in men who receive ADT for prostate cancer is not understood. Recently, Spetz et al.9 and Wyon et al.10 reported an increase in plasma levels of calcitonin gene-related peptide (CGRP) during hot flashes after castration for carcinoma of the prostate. More recently, we also demonstrated that exogenous injection of CGRP elevated the skin temperature in medically or surgically castrated male rats, and the increases were statistically significantly greater than those in sham-treated rats.11 These studies suggest that CGRP plays an important role in the occurrence of hot flashes or skin temperature increases in androgen-deficient men or male rats treated by castration. 0090-4295/04/$30.00 doi:10.1016/j.urology.2004.06.013
The purpose of the present study was to clarify whether CGRP is involved in the amelioration of 17-estradiol and progesterone to hot flashes in men treated with castration for carcinoma of the prostate. For that purpose, we investigated the effects of 17-estradiol and progesterone on CGRPinduced elevation of skin temperature in surgically and medically castrated rats and compared the results with those due to testosterone replacement. MATERIAL AND METHODS EFFECTS OF TESTOSTERONE, 17-ESTRADIOL, OR PROGESTERONE ON CGRP-INDUCED ELEVATION OF SKIN TEMPERATURE IN ORX RATS OR GnRH ANALOGUE-TREATED RATS Nine-week-old male Sprague-Dawley rats weighing 300 to 350 g (SLC, Hamamatsu, Japan) were anesthetized with 50 mg/kg sodium pentobarbital intraperitoneally and bilaterally orchiectomized (ORX) or underwent a sham operation as controls. The ORX rats (n ⫽ 32) were subcutaneously injected daily for 14 days from the seventh day after surgery with testosterone (0.1, 1.0, and 10.0 mg/kg, n ⫽ 8; Sigma-Aldrich, St. Louis, Mo), 17-estradiol (0.1, 1.0, and 10.0 g/kg, n ⫽ 8; Wako Pure Chemical Industries, Osaka, Japan), progesterone (0.1, 1.0, and 10.0 mg/kg, n ⫽ 8; Wako Pure Chemical Industries), or olive oil (1.0 mL/kg, n ⫽ 8) as the vehicle control. The sham-operated rats (n ⫽ 8) were injected with olive oil (1.0 mL/kg) using the same schedule. In another set of experiments, 1.0 mg/kg of a gonadotropinreleasing hormone (GnRH) analogue (Leuplin, a sustainedrelease formulation of leuprorelin acetate, Takeda Chemical Industries, Osaka, Japan) or saline (2 mL/kg) as the vehicle control was subcutaneously injected into male rats. From the day after the injection of the GnRH analogue or saline, testosterone (10.0 mg/kg, n ⫽ 8), 17-estradiol (10.0 g/kg, n ⫽ 8), progesterone (10.0 mg/kg, n ⫽ 8), or olive oil (1.0 mL/kg, n ⫽ 8) as the vehicle control was subcutaneously administered daily for 14 days to the GnRH analogue-treated rats and olive oil (1.0 mL/kg, n ⫽ 8) to the sham-treated rats (1.0 mL/kg saline, n ⫽ 8). On the day after the final injection of the drug, the changes in skin temperature induced by CGRP were evaluated in all animals. In brief, all rats were anesthetized with intraperitoneal co-injection of urethane (0.75 g/kg, Sigma-Aldrich) and alpha-chloralose (0.06 g/kg, Sigma-Aldrich). A thermistor probe (SXN-54, Technol Seven, Yokohama, Japan) was taped to the plantar face of each hind foot of each animal. Forty minutes later, when the basal skin temperature was stable, 10 g/kg CGRP (Peptide Institute, Osaka, Japan) dissolved in saline was injected into the tail vein. The mean temperature of the two thermistor probes was automatically measured at 5-minute intervals throughout the experiment. The data were recorded by a K932 recording device (Technol Seven). The changes in skin temperature after injection of CGRP were plotted for each animal, and the area under the temperature curve was calculated using Pharmacokinetic Analysis and Graphics for Clinical Pharmacology analysis (Medical Research AS Medica, Osaka, Japan).
MEASUREMENT OF SERUM TESTOSTERONE, 17ESTRADIOL, AND PROGESTERONE CONCENTRATIONS, AND PROSTATE WEIGHT
Testosterone (0.1, 1.0, and 10.0 mg/kg, n ⫽ 8), 17-estradiol (0.1, 1.0, and 10.0 g/kg, n ⫽ 8), progesterone (0.1, 1.0, and 10.0 mg/kg, n ⫽ 8), or olive oil (1.0 mL/kg, n ⫽ 8) was subcutaneously UROLOGY 64 (5), 2004
administered to ORX rats and olive oil (1.0 mL/kg, n ⫽ 8) to sham-operated rats, according to the same schedule described in the experiments of the skin temperature analysis. On the day after the final administration of the drug, the blood of each animal was collected in a polypropylene tube from the abdominal aorta under anesthesia with intraperitoneal co-injection of urethane and alpha-chloralose. The serum was obtained by centrifuging at 1500g and 4°C for 15 minutes. The serum concentrations of 17-estradiol (Diagnostic Products, Los Angeles, Calif), progesterone (Diagnostic Products), and testosterone (Immunoteck, Marseilles, France) were measured using a radioimmunoassay kit. The prostate in each animal was removed and weighed after the blood collection.
ETHICS All experimental procedures were performed according to the “Guidelines for the Care and Use of Laboratory Animals” approved by the Laboratory Animal Committee of Tsumura.
STATISTICAL ANALYSIS
All values are reported as the mean ⫾ the standard error of the mean. The statistical significance of the data was evaluated by one-way analysis of variance followed by Dunnett’s test. For all tests, the statistical significance level was accepted at P ⬍0.05.
RESULTS EFFECTS OF TESTOSTERONE, 17-ESTRADIOL, AND PROGESTERONE ON CGRP-INDUCED ELEVATION OF SKIN TEMPERATURE IN ORX RATS In all groups, the basal skin temperature was stable at a range of 24.01° ⫾ 0.10°C. No statistically significant differences were observed in the basal skin temperature among all groups. The skin temperature in each group was maximally elevated 50 to 60 minutes after injection of CGRP and had recovered to basal levels by 120 minutes. The effects of testosterone, 17-estradiol, and progesterone on CGRP-induced elevation of skin temperature are shown in Figure 1 as the area under the temperature curve data. The elevation of skin temperature was significantly greater in ORX rats than in the sham-operated rats. The enhancement of CGRP-induced skin temperature elevation in ORX rats was inhibited by pretreatment with testosterone, 17-estradiol, and progesterone in a dose-dependent manner. EFFECTS OF TESTOSTERONE, 17-ESTRADIOL, AND PROGESTERONE ON CGRP-INDUCED ELEVATION OF SKIN TEMPERATURE IN GnRH ANALOGUE-TREATED RATS Treatment with the GnRH analogue significantly enhanced the CGRP (10 g/kg)-induced elevation of skin temperature (P ⬍0.01), as well as the skin temperature elevation in ORX rats. Pretreatment with testosterone (10 mg/kg), 17-estradiol (10 g/kg), or progesterone (10 mg/kg) significantly 1043
(A) 1500
p<0.05
AUC
1200 900 600
*
*
300 0
10 (mg/kg)
0.1 1.0
Testosterone Sham
Orchiectomy
(B) 1500
p<0.01
AUC
1200 900 600
**
300 0
0.1 1.0 10 (µg/kg) 17β-Estradiol Sham
Orchiectomy
(C)
AUC
1500
p<0.01
1200 900
*
600
**
300 0 0.1 1.0
10
Progesterone (mg/kg) Sham
Orchiectomy
FIGURE 1. Effects of (A) testosterone, (B) 17-estradiol, and (C) progesterone on CGRP-induced elevation of skin temperature in ORX rats. Each hormone was injected for 14 days before day of temperature measurement. Each value expressed as mean ⫾ SEM (n ⫽ 8). Significance with Dunnett’s test after one-way analysis of variance indicated as *P ⬍0.05 and **P ⬍0.01 versus vehicle-treated group of ORX rats.
inhibited the CGRP-induced elevation of skin temperature (data not shown), as well as the skin temperature in ORX rats. 1044
EFFECTS OF EXOGENOUS TESTOSTERONE, 17ESTRADIOL, AND PROGESTERONE ON SERUM TESTOSTERONE, 17-ESTRADIOL, AND PROGESTERONE CONCENTRATIONS AND PROSTATE WEIGHT IN ORX RATS The effects of each hormonal treatment on the serum concentrations of testosterone, 17-estradiol, and progesterone in the ORX and sham-operated rats are shown in Table I. Orchiectomy significantly decreased the serum concentrations of testosterone and progesterone compared with those in the sham-operated rats. The decrease in the testosterone level (92% to 94%) was greater than the decrease in progesterone (40% to 60%). However, orchiectomy did not affect the serum concentration of 17-estradiol. Supplying testosterone to ORX rats increased the testosterone levels in a dose-dependent manner, but it did not affect the lower progesterone levels. Supplying testosterone also increased the serum concentration of 17-estradiol, which was not affected by orchiectomy, in a dose-dependent manner. The treatment of ORX rats with exogenous 17estradiol increased the serum concentration of estrogen in a dose-dependent manner. However, it did not affect the orchiectomy-induced reduction in serum testosterone and progesterone. The treatment of ORX rats with exogenous progesterone increased the lower concentration of serum progesterone induced by orchiectomy in a dose-dependent manner. However, it did not affect the orchiectomy-induced reduction of serum testosterone or the normal concentration of serum 17-estradiol. The effects of each hormonal treatment on the prostate weight in the ORX rats are also shown in Table I. The prostate weight in the ORX rats was significantly lower statistically (P ⬍0.001) than those in the sham-operated rats. The lower tissue weights were recovered by treatment with exogenous testosterone in a dose-dependent manner, but were not affected by treatment with 17-estradiol or progesterone. COMMENT CGRP is a potent vasodilator and causes a regional blood flow increase in rats12,13 and humans.14 These suggest that the elevation of skin temperature induced by CGRP is mediated by the blood flow increase resulting from the vasodilator effect of this peptide in the skin. We demonstrated in a previous study11 that surgical or medical castration in male rats induced not only a marked decrease in the serum concentration of testosterone but also an enhancement of the CGRP-induced elevation of skin temperature. Because testosterone inhibits the elevation of skin temperature, we UROLOGY 64 (5), 2004
TABLE I. Effects of testosterone, 17-estradiol, and progesterone on prostate weight and concentrations of serum testosterone, 17-estradiol, and progesterone in ORX rats Group Sham-treated control ORX ORX ⫹ testosterone ORX ⫹ testosterone ORX ⫹ testosterone Sham-treated control ORX ORX ⫹ 17-estradiol ORX ⫹ 17-estradiol ORX ⫹ 17-estradiol Sham-treated control ORX ORX ⫹ progesterone ORX ⫹ progesterone ORX ⫹ progesterone
Dose (mg/kg)
0.1 1.0 10.0
0.0001 0.001 0.01
0.1 1.0 10.0
Prostate Weight (g)
Testosterone (ng/mL)
17-Estradiol (pg/mL)
Progesterone (ng/mL)
0.683 ⫾ 0.07 0.132 ⫾ 0.02* 0.419 ⫾ 0.05‡ 0.952 ⫾ 0.08‡ 1.329 ⫾ 0.11‡ 0.733 ⫾ 0.05 0.086 ⫾ 0.01* 0.085 ⫾ 0.01 0.091 ⫾ 0.01 0.087 ⫾ 0.01 0.787 ⫾ 0.03 0.080 ⫾ 0.01* 0.079 ⫾ 0.01 0.079 ⫾ 0.01 0.094 ⫾ 0.01
1.61 ⫾ 0.13 0.10 ⫾ 0.02* 1.00 ⫾ 0.53 10.21 ⫾ 1.28‡ 52.09 ⫾ 2.33‡ 1.29 ⫾ 0.14 0.10 ⫾ 0.04* 0.04 ⫾ 0.01 0.11 ⫾ 0.02 0.19 ⫾ 0.04 1.35 ⫾ 0.12 0.07 ⫾ 0.02* 0.10 ⫾ 0.05 0.10 ⫾ 0.02 0.34 ⫾ 0.05
11.73 ⫾ 1.47 10.29 ⫾ 0.97 11.29 ⫾ 0.92 17.01 ⫾ 3.79‡ 32.98 ⫾ 2.89‡ 14.79 ⫾ 1.10 9.99 ⫾ 0.89 14.16 ⫾ 1.55 26.63 ⫾ 3.12 36.45 ⫾ 3.79‡ 12.97 ⫾ 1.69 10.17 ⫾ 0.91 11.51 ⫾ 2.02 10.99 ⫾ 1.74 18.02 ⫾ 3.02
7.13 ⫾ 0.92 4.48 ⫾ 1.00* 4.87 ⫾ 1.44 6.92 ⫾ 1.31 5.37 ⫾ 0.92 7.76 ⫾ 1.99 3.12 ⫾ 0.73* 4.87 ⫾ 1.11 6.49 ⫾ 1.38 5.65 ⫾ 2.25 5.23 ⫾ 1.35 1.86 ⫾ 0.55* 2.58 ⫾ 0.54 3.25 ⫾ 0.81 12.52 ⫾ 1.71‡
KEY: ORX ⫽ orchiectomy. Controls in surgical castration group underwent sham operation instead of orchiectomy; testosterone, 17-estradiol, or progesterone was administered to each castrated group for 14 days before day of temperature analysis. Data expressed as mean ⫾ SEM (n ⫽ 8). Statistical significance determined by post hoc analyses (Dunnett’s test) after one-way analysis of variance. * P ⬍0.001 compared with corresponding sham-treated control. † P ⬍0.05 compared with corresponding sham-treated control. ‡ P ⬍0.001 compared with ORX group in castrated groups.
have suggested that the elevation is due to the testosterone deficiency resulting from castration. In the present study, we newly examined the effect of orchiectomy on the serum concentrations of ovarian hormones (17-estradiol and progesterone), in addition to testosterone, in male rats. Orchiectomy significantly decreased not only the serum testosterone level but also the progesterone level. Because part of the progesterone in male rats is known to be synthesized in the testes,15 the decreased progesterone level is considered to be due to orchiectomy. These results suggest a possibility that the decrease in the progesterone concentration is also involved in the CGRP-induced elevation of skin temperature in ORX rats. This suggestion was supported by the results of progesterone replacement in both castrated rats; the hormonal replacement clearly restored the castration-induced elevation of skin temperature. An exogenous testosterone supply to ORX rats elevated not only the serum concentration of testosterone but also the serum concentration of 17estradiol. The increase in the estrogen level may have been due to synthesis from exogenous testosterone in a reaction catalyzed by aromatase in extratesticular tissues, such as adipose and adrenal tissue.16,17 Therefore, the inhibition of the elevation of skin temperature by exogenous testosterone in castrated rats may involve the synthesis of estrogen in addition to testosterone. In fact, the serum concentration of 17-estradiol in ORX rats was elevated by supplying exogenous estrogen, UROLOGY 64 (5), 2004
without altering the serum concentrations of testosterone or progesterone, and the CGRP-induced skin temperature rise in castrated rats was clearly inhibited by supplying exogenous estrogen. With regard to the mechanism of CGRP-induced skin temperature elevation, a decline in estrogens18,19 or androgens20 has been reported to decrease CGRP synthesis and release. These findings suggest that both sex hormones similarly regulate CGRP synthesis and release. We demonstrated in previous studies21,22 that ovariectomy (estrogen deficiency) induced a decrease in the plasma concentration of CGRP, supporting these results. In addition, estrogen deficiency increased the number of arterial CGRP receptors. These results suggest that the vascular response by endogenous CGRP is maintained at normal by an increase in the number of arterial CGRP receptors in the sex hormone-deficient rats. In the present study, no statistically significant differences were observed in the basal skin temperature among all groups before CGRP was injected. One possibility is that this result was due to a homeostatic response by upregulation of arterial CGRP receptors in ORX rats, as well as in ovariectomized rats. In fact, the skin temperature elevation induced by exogenous injection of CGRP was significantly greater in ORX rats (present study) or ovariectomized rats21,22 than in sham-operated rats. Thus, hot flashes may be observed when the CGRP level in plasma is increased by certain stimulation under the condition of sex hormone deficiency. Although the stimulant 1045
to release CGRP into the circulation is not yet clear, some stresses may be presumed as the trigger because CGRP secretion is reported to be enhanced in menopausal women with hot flashes by cold load stress.23 To clarify our hypothesis, we will investigate in detail the mechanism in the ORX rat in future studies. Hormone replacement therapy with testosterone for the amelioration of hot flashes is contraindicated in men who undergo ADT for testosteronedependent prostate cancer.1 In the present study, supplemental estrogen or progesterone inhibited the CGRP-induced elevation of skin temperature without increasing the serum concentration of testosterone, suggesting that neither hormone confers any testosterone activity. The exposure of rodents24 or humans25 to estrogens has been reported to induce a proliferative lesion, squamous metaplasia, in their prostates. In contrast to these findings, estrogens have been used as part of treatment regimens for advanced prostate cancer. Estrogen is believed to exert a direct growth-inhibitory effect on prostate cancer cells by induction of apoptosis or cell cycle arrest26 or by estrogen -receptors.27 In the present study, the orchiectomy-induced decrease in prostate weight was recovered by supplying exogenous testosterone, but it was not affected by treatment with 17-estradiol or progesterone. These results suggest that neither hormone results in proliferation of the prostate. CONCLUSIONS The results of the present study support those of some clinical studies,5,7,28 suggesting that estrogen and progesterone are useful for men with specific contraindications, such as a history of testosterone-dependent prostate cancer, who are experiencing hot flashes. Finally, we suggest that CGRP is closely involved with 17-estradiol and progesterone in the amelioration of hot flashes of men who undergo ADT. REFERENCES 1. Miller JI, and Ahmann FR: Treatment of castrationinduced menopausal symptoms with low dose diethylstilbestrol in men with advanced prostate cancer. Urology 40: 499 – 502, 1992. 2. Karling P, Hammer M, and Varenhorst E: Prevalence and duration of hot flushes after surgical or medical castration in men with prostatic carcinoma. J Urol 152: 1170 –1173, 1994. 3. Freedman RR: Physiology of hot flashes. Am J Human Biol 13: 453– 464, 2001. 4. Charig CR, and Rumdle JS: Flushing: long-term side effect of orchiectomy in treatment of prostatic carcinoma. Urology 33: 175–178, 1989. 5. Quella SK, Loprinzi CL, Sloan JA, et al: Long term use of megestrol acetate by cancer survivors for the treatment of hot flashes. Cancer 82: 1784 –1788, 1998. 1046
6. Atala A, Amin M, and Harty JI: Diethylstilbestrol in treatment of postorchiectomy vasomotor symptoms and its relationship with serum follicle-stimulating hormone, luteinizing hormone, and testosterone. Urology 39: 108 –110, 1992. 7. Loprinzi Cl, Michalak JC, Quella SK, et al: Megestrol acetate for the prevention of hot flashes. N Engl J Med 331: 347–352, 1994. 8. Notelovitz M, and Mattox JH: Suppression of vasomotor and vulvovaginal symptoms with continuous oral 17-estradiol. Menopause 7: 310 –317, 2000. 9. Spetz AC, Pettersson B, Varenhorst E, et al: Momentary increase in plasma calcitonin gene-related peptide is involved in hot flashes in men treated with castration for carcinoma of the prostate. J Urol 166: 1720 –1723, 2001. 10. Wyon Y, Spetz AC, Hammar M, et al: Urinary excretion of calcitonin gene-related peptide in males with hot flushes after castration for carcinoma of the prostate. Scand J Urol Nephrol 35: 92–96, 2001. 11. Yuzurihara M, Ikarashi Y, Noguchi M, et al: Involvement of calcitonin gene-related peptide in elevation of skin temperature in castrated male rats. Urology 62: 947–951, 2003. 12. Mears E, Vessey MP, Andolsek L, et al: Preliminary evaluation of four oral contraceptives containing only progestogens. BMJ 21: 730 –734, 1969. 13. Wells M, Sturdee DW, Barlow DH, et al: Effect on endometrium of long term treatment with continuous combined oestrogen-progestogen replacement therapy: follow up study. BMJ 325: 231–232, 2002. 14. Keast JR, and Gleeson RJ: Androgen receptor immunoreactivity is present in primary sensory neurons of male rats. Neuroreport 9: 4137– 4140, 1998. 15. Ganong WF: The gonads: development and function of the reproductive system, in Ganong WF (Ed): Review of Medical Physiology, 10th ed. Singapore, Maruzen Asia, 1981, pp 331–363. 16. Simpson ER, Mahendroo MS, Means GD, et al: Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocr Rev 15: 342–355, 1994. 17. Bulun SE, Zeitoun KM, Takayama K, et al: Molecular basis for treating endometriosis with aromatase inhibitors. Hum Reprod Update 6: 413– 418, 2000. 18. Gangula PR, Lanlua P, Wimalawansa S, et al: Regulation of calcitonin gene-related peptide expression in dorsal root ganglia of rats by female sex steroid hormones. Biol Reprod 62: 1033–1039, 2000. 19. Gangula PR, Wimalawansa S, and Yallampalli C: Pregnancy and sex steroid hormones enhance circulating calcitonin gene-related peptide concentration in rats. Hum Reprod 15: 949 –953, 2000. 20. Sun C, Chen M, Mao J, et al: Biphasic effects of orchiectomy on calcitonin gene-related peptide synthesis and release. Neuroreport 12: 3497–3502, 2001. 21. Noguchi M, Ikarashi Y, Yuzurihara M, et al: Up-regulation of calcitonin gene-related peptide receptors underlying elevation of skin temperature in ovariectomized rats. J Endocrinol 175: 177–183, 2002. 22. Noguchi M, Ikarashi Y, Yuzurihara M, et al: Effects of the Japanese herbal medicine Keishi-bukuryo-gan and 17estradiol on calcitonin gene-related peptide-induced elevation of skin temperature in ovariectomized rats. J Endocrinol 176: 359 –366, 2002. 23. Chen JT, and Shiraki M: Menopausal hot flash and calcitonin gene-related peptide: effect of Keishi-bukuryo-gan, a kampo medicine, related to plasma calcitonin gene-related peptide level. Maturitas 45: 199 –204, 2003. 24. Triche TJ, and Harkin JC: An ultrastructural study of hormonally induced squamous metaplasia in the coagulating gland of the mouse prostate. Lab Invest 25: 596 – 606, 1971. UROLOGY 64 (5), 2004
25. Sugimura Y, Cunha GR, Yonemura CU, et al: Temporal and spatial factors in diethylstilbestrol-induced squamous metaplasia of the developing human prostate. Hum Pathol 19: 133–139, 1988. 26. Robertson CN, Robertson KM, Padilla GM, et al: Induction of apoptosis by diethylstilbestrol in hormone-insensitive prostate cancer cells. J Natl Cancer Inst 88: 908 –917, 1996.
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27. Lau KM, LaSpina M, Long J, et al: Expression of estrogen receptor (ER)-␣ and ER- in normal and malignant prostatic epithelial cells: regulation by methylation and involvement in growth regulation. Cancer Res 60: 3175–3182, 2000. 28. Gerber GS, Zagaja GP, Ray PS, et al: Transdermal estrogen in the treatment of hot flushes in men with prostate cancer. Urology 55: 97–101, 2000.
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PREVENTION BY 17-ESTRADIOL AND PROGESTERONE OF CALCITONIN GENE-RELATED PEPTIDE-INDUCED ELEVATION OF SKIN TEMPERATURE IN CASTRATED MALE RATS MITSUTOSHI YUZURIHARA, YASUSHI IKARASHI, MASAMICHI NOGUCHI, YOSHIO KASE, SHUICHI TAKEDA, AND MASAKI ABURADA
ABSTRACT Objectives. To clarify the relationship between calcitonin gene-related peptide (CGRP) and ovarian hormones (17-estradiol and progesterone) in hot flashes in men who undergo androgen deprivation therapy for prostate cancer, we studied the effects of ovarian hormones on CGRP-induced elevation of skin temperature in castrated male rats. The results were compared with those from rats treated with testosterone replacement. Methods. Adult male rats were castrated by either a single injection of gonadotropin-releasing hormone analogue (Leuplin, 1.0 mg/kg, subcutaneously) or bilateral orchiectomy. The castrated animals were subcutaneously injected daily for 14 days with ovarian hormones, testosterone, or olive oil as the vehicle. On the day after the final administration of the drug, the changes in skin temperature induced by exogenous CGRP (10 g/kg intravenously), serum testosterone concentration, and prostate weight were measured. Results. The CGRP-induced elevation of skin temperature was significantly greater in the castrated rats than in the sham-treated rats. This potentiation was significantly inhibited by treatment with ovarian hormones, as well as by testosterone replacement. The testosterone replacement restored decreases in both the serum testosterone level and the prostate weight due to castration; the treatment with ovarian hormones did not affect them. Conclusions. 17-Estradiol and progesterone, which do not confer testosterone activity on serum, may be useful for the treatment of hot flashes in patients for whom testosterone replacement therapy is contraindicated, such as those with prostate carcinoma. In addition, we suggest that CGRP is closely involved in the amelioration of hot flashes by ovarian hormones in men who undergo androgen deprivation therapy. UROLOGY 64: 1042–1047, 2004. © 2004 Elsevier Inc.
S
urgical or medical castration is highly effective as a treatment for prostate cancer. However, up to 80% of castrated men experience hot flashes.1,2 Hot flashes generally begin with a sudden outpouring of sweat and an increase in heart rate and peripheral blood flow, causing a drastic increase in skin temperature.3 This adverse effect often impairs the quality of life for these patients.4,5 It has been demonstrated that ovarian hormones such as estrogen6 and progesterone7 are effective for the amelioration of hot From the Medicinal Evaluation Laboratory and Research Division, Tsumura & Company, Ibaraki, Japan Reprint requests: Mitsutoshi Yuzurihara, Ph.D., Medicinal Evaluation Laboratory, Tsumura & Company, 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-1192, Japan Submitted: March 22, 2004, accepted (with revisions) June 2, 2004 © 2004 ELSEVIER INC. 1042
ALL RIGHTS RESERVED
flashes in men who undergo androgen deprivation therapy (ADT) for prostate cancer, as well as in menopausal women.8 However, the etiology of hot flashes in men who receive ADT for prostate cancer is not understood. Recently, Spetz et al.9 and Wyon et al.10 reported an increase in plasma levels of calcitonin gene-related peptide (CGRP) during hot flashes after castration for carcinoma of the prostate. More recently, we also demonstrated that exogenous injection of CGRP elevated the skin temperature in medically or surgically castrated male rats, and the increases were statistically significantly greater than those in sham-treated rats.11 These studies suggest that CGRP plays an important role in the occurrence of hot flashes or skin temperature increases in androgen-deficient men or male rats treated by castration. 0090-4295/04/$30.00 doi:10.1016/j.urology.2004.06.013
The purpose of the present study was to clarify whether CGRP is involved in the amelioration of 17-estradiol and progesterone to hot flashes in men treated with castration for carcinoma of the prostate. For that purpose, we investigated the effects of 17-estradiol and progesterone on CGRPinduced elevation of skin temperature in surgically and medically castrated rats and compared the results with those due to testosterone replacement. MATERIAL AND METHODS EFFECTS OF TESTOSTERONE, 17-ESTRADIOL, OR PROGESTERONE ON CGRP-INDUCED ELEVATION OF SKIN TEMPERATURE IN ORX RATS OR GnRH ANALOGUE-TREATED RATS Nine-week-old male Sprague-Dawley rats weighing 300 to 350 g (SLC, Hamamatsu, Japan) were anesthetized with 50 mg/kg sodium pentobarbital intraperitoneally and bilaterally orchiectomized (ORX) or underwent a sham operation as controls. The ORX rats (n ⫽ 32) were subcutaneously injected daily for 14 days from the seventh day after surgery with testosterone (0.1, 1.0, and 10.0 mg/kg, n ⫽ 8; Sigma-Aldrich, St. Louis, Mo), 17-estradiol (0.1, 1.0, and 10.0 g/kg, n ⫽ 8; Wako Pure Chemical Industries, Osaka, Japan), progesterone (0.1, 1.0, and 10.0 mg/kg, n ⫽ 8; Wako Pure Chemical Industries), or olive oil (1.0 mL/kg, n ⫽ 8) as the vehicle control. The sham-operated rats (n ⫽ 8) were injected with olive oil (1.0 mL/kg) using the same schedule. In another set of experiments, 1.0 mg/kg of a gonadotropinreleasing hormone (GnRH) analogue (Leuplin, a sustainedrelease formulation of leuprorelin acetate, Takeda Chemical Industries, Osaka, Japan) or saline (2 mL/kg) as the vehicle control was subcutaneously injected into male rats. From the day after the injection of the GnRH analogue or saline, testosterone (10.0 mg/kg, n ⫽ 8), 17-estradiol (10.0 g/kg, n ⫽ 8), progesterone (10.0 mg/kg, n ⫽ 8), or olive oil (1.0 mL/kg, n ⫽ 8) as the vehicle control was subcutaneously administered daily for 14 days to the GnRH analogue-treated rats and olive oil (1.0 mL/kg, n ⫽ 8) to the sham-treated rats (1.0 mL/kg saline, n ⫽ 8). On the day after the final injection of the drug, the changes in skin temperature induced by CGRP were evaluated in all animals. In brief, all rats were anesthetized with intraperitoneal co-injection of urethane (0.75 g/kg, Sigma-Aldrich) and alpha-chloralose (0.06 g/kg, Sigma-Aldrich). A thermistor probe (SXN-54, Technol Seven, Yokohama, Japan) was taped to the plantar face of each hind foot of each animal. Forty minutes later, when the basal skin temperature was stable, 10 g/kg CGRP (Peptide Institute, Osaka, Japan) dissolved in saline was injected into the tail vein. The mean temperature of the two thermistor probes was automatically measured at 5-minute intervals throughout the experiment. The data were recorded by a K932 recording device (Technol Seven). The changes in skin temperature after injection of CGRP were plotted for each animal, and the area under the temperature curve was calculated using Pharmacokinetic Analysis and Graphics for Clinical Pharmacology analysis (Medical Research AS Medica, Osaka, Japan).
MEASUREMENT OF SERUM TESTOSTERONE, 17ESTRADIOL, AND PROGESTERONE CONCENTRATIONS, AND PROSTATE WEIGHT
Testosterone (0.1, 1.0, and 10.0 mg/kg, n ⫽ 8), 17-estradiol (0.1, 1.0, and 10.0 g/kg, n ⫽ 8), progesterone (0.1, 1.0, and 10.0 mg/kg, n ⫽ 8), or olive oil (1.0 mL/kg, n ⫽ 8) was subcutaneously UROLOGY 64 (5), 2004
administered to ORX rats and olive oil (1.0 mL/kg, n ⫽ 8) to sham-operated rats, according to the same schedule described in the experiments of the skin temperature analysis. On the day after the final administration of the drug, the blood of each animal was collected in a polypropylene tube from the abdominal aorta under anesthesia with intraperitoneal co-injection of urethane and alpha-chloralose. The serum was obtained by centrifuging at 1500g and 4°C for 15 minutes. The serum concentrations of 17-estradiol (Diagnostic Products, Los Angeles, Calif), progesterone (Diagnostic Products), and testosterone (Immunoteck, Marseilles, France) were measured using a radioimmunoassay kit. The prostate in each animal was removed and weighed after the blood collection.
ETHICS All experimental procedures were performed according to the “Guidelines for the Care and Use of Laboratory Animals” approved by the Laboratory Animal Committee of Tsumura.
STATISTICAL ANALYSIS
All values are reported as the mean ⫾ the standard error of the mean. The statistical significance of the data was evaluated by one-way analysis of variance followed by Dunnett’s test. For all tests, the statistical significance level was accepted at P ⬍0.05.
RESULTS EFFECTS OF TESTOSTERONE, 17-ESTRADIOL, AND PROGESTERONE ON CGRP-INDUCED ELEVATION OF SKIN TEMPERATURE IN ORX RATS In all groups, the basal skin temperature was stable at a range of 24.01° ⫾ 0.10°C. No statistically significant differences were observed in the basal skin temperature among all groups. The skin temperature in each group was maximally elevated 50 to 60 minutes after injection of CGRP and had recovered to basal levels by 120 minutes. The effects of testosterone, 17-estradiol, and progesterone on CGRP-induced elevation of skin temperature are shown in Figure 1 as the area under the temperature curve data. The elevation of skin temperature was significantly greater in ORX rats than in the sham-operated rats. The enhancement of CGRP-induced skin temperature elevation in ORX rats was inhibited by pretreatment with testosterone, 17-estradiol, and progesterone in a dose-dependent manner. EFFECTS OF TESTOSTERONE, 17-ESTRADIOL, AND PROGESTERONE ON CGRP-INDUCED ELEVATION OF SKIN TEMPERATURE IN GnRH ANALOGUE-TREATED RATS Treatment with the GnRH analogue significantly enhanced the CGRP (10 g/kg)-induced elevation of skin temperature (P ⬍0.01), as well as the skin temperature elevation in ORX rats. Pretreatment with testosterone (10 mg/kg), 17-estradiol (10 g/kg), or progesterone (10 mg/kg) significantly 1043
(A) 1500
p<0.05
AUC
1200 900 600
*
*
300 0
10 (mg/kg)
0.1 1.0
Testosterone Sham
Orchiectomy
(B) 1500
p<0.01
AUC
1200 900 600
**
300 0
0.1 1.0 10 (µg/kg) 17β-Estradiol Sham
Orchiectomy
(C)
AUC
1500
p<0.01
1200 900
*
600
**
300 0 0.1 1.0
10
Progesterone (mg/kg) Sham
Orchiectomy
FIGURE 1. Effects of (A) testosterone, (B) 17-estradiol, and (C) progesterone on CGRP-induced elevation of skin temperature in ORX rats. Each hormone was injected for 14 days before day of temperature measurement. Each value expressed as mean ⫾ SEM (n ⫽ 8). Significance with Dunnett’s test after one-way analysis of variance indicated as *P ⬍0.05 and **P ⬍0.01 versus vehicle-treated group of ORX rats.
inhibited the CGRP-induced elevation of skin temperature (data not shown), as well as the skin temperature in ORX rats. 1044
EFFECTS OF EXOGENOUS TESTOSTERONE, 17ESTRADIOL, AND PROGESTERONE ON SERUM TESTOSTERONE, 17-ESTRADIOL, AND PROGESTERONE CONCENTRATIONS AND PROSTATE WEIGHT IN ORX RATS The effects of each hormonal treatment on the serum concentrations of testosterone, 17-estradiol, and progesterone in the ORX and sham-operated rats are shown in Table I. Orchiectomy significantly decreased the serum concentrations of testosterone and progesterone compared with those in the sham-operated rats. The decrease in the testosterone level (92% to 94%) was greater than the decrease in progesterone (40% to 60%). However, orchiectomy did not affect the serum concentration of 17-estradiol. Supplying testosterone to ORX rats increased the testosterone levels in a dose-dependent manner, but it did not affect the lower progesterone levels. Supplying testosterone also increased the serum concentration of 17-estradiol, which was not affected by orchiectomy, in a dose-dependent manner. The treatment of ORX rats with exogenous 17estradiol increased the serum concentration of estrogen in a dose-dependent manner. However, it did not affect the orchiectomy-induced reduction in serum testosterone and progesterone. The treatment of ORX rats with exogenous progesterone increased the lower concentration of serum progesterone induced by orchiectomy in a dose-dependent manner. However, it did not affect the orchiectomy-induced reduction of serum testosterone or the normal concentration of serum 17-estradiol. The effects of each hormonal treatment on the prostate weight in the ORX rats are also shown in Table I. The prostate weight in the ORX rats was significantly lower statistically (P ⬍0.001) than those in the sham-operated rats. The lower tissue weights were recovered by treatment with exogenous testosterone in a dose-dependent manner, but were not affected by treatment with 17-estradiol or progesterone. COMMENT CGRP is a potent vasodilator and causes a regional blood flow increase in rats12,13 and humans.14 These suggest that the elevation of skin temperature induced by CGRP is mediated by the blood flow increase resulting from the vasodilator effect of this peptide in the skin. We demonstrated in a previous study11 that surgical or medical castration in male rats induced not only a marked decrease in the serum concentration of testosterone but also an enhancement of the CGRP-induced elevation of skin temperature. Because testosterone inhibits the elevation of skin temperature, we UROLOGY 64 (5), 2004
TABLE I. Effects of testosterone, 17-estradiol, and progesterone on prostate weight and concentrations of serum testosterone, 17-estradiol, and progesterone in ORX rats Group Sham-treated control ORX ORX ⫹ testosterone ORX ⫹ testosterone ORX ⫹ testosterone Sham-treated control ORX ORX ⫹ 17-estradiol ORX ⫹ 17-estradiol ORX ⫹ 17-estradiol Sham-treated control ORX ORX ⫹ progesterone ORX ⫹ progesterone ORX ⫹ progesterone
Dose (mg/kg)
0.1 1.0 10.0
0.0001 0.001 0.01
0.1 1.0 10.0
Prostate Weight (g)
Testosterone (ng/mL)
17-Estradiol (pg/mL)
Progesterone (ng/mL)
0.683 ⫾ 0.07 0.132 ⫾ 0.02* 0.419 ⫾ 0.05‡ 0.952 ⫾ 0.08‡ 1.329 ⫾ 0.11‡ 0.733 ⫾ 0.05 0.086 ⫾ 0.01* 0.085 ⫾ 0.01 0.091 ⫾ 0.01 0.087 ⫾ 0.01 0.787 ⫾ 0.03 0.080 ⫾ 0.01* 0.079 ⫾ 0.01 0.079 ⫾ 0.01 0.094 ⫾ 0.01
1.61 ⫾ 0.13 0.10 ⫾ 0.02* 1.00 ⫾ 0.53 10.21 ⫾ 1.28‡ 52.09 ⫾ 2.33‡ 1.29 ⫾ 0.14 0.10 ⫾ 0.04* 0.04 ⫾ 0.01 0.11 ⫾ 0.02 0.19 ⫾ 0.04 1.35 ⫾ 0.12 0.07 ⫾ 0.02* 0.10 ⫾ 0.05 0.10 ⫾ 0.02 0.34 ⫾ 0.05
11.73 ⫾ 1.47 10.29 ⫾ 0.97 11.29 ⫾ 0.92 17.01 ⫾ 3.79‡ 32.98 ⫾ 2.89‡ 14.79 ⫾ 1.10 9.99 ⫾ 0.89 14.16 ⫾ 1.55 26.63 ⫾ 3.12 36.45 ⫾ 3.79‡ 12.97 ⫾ 1.69 10.17 ⫾ 0.91 11.51 ⫾ 2.02 10.99 ⫾ 1.74 18.02 ⫾ 3.02
7.13 ⫾ 0.92 4.48 ⫾ 1.00* 4.87 ⫾ 1.44 6.92 ⫾ 1.31 5.37 ⫾ 0.92 7.76 ⫾ 1.99 3.12 ⫾ 0.73* 4.87 ⫾ 1.11 6.49 ⫾ 1.38 5.65 ⫾ 2.25 5.23 ⫾ 1.35 1.86 ⫾ 0.55* 2.58 ⫾ 0.54 3.25 ⫾ 0.81 12.52 ⫾ 1.71‡
KEY: ORX ⫽ orchiectomy. Controls in surgical castration group underwent sham operation instead of orchiectomy; testosterone, 17-estradiol, or progesterone was administered to each castrated group for 14 days before day of temperature analysis. Data expressed as mean ⫾ SEM (n ⫽ 8). Statistical significance determined by post hoc analyses (Dunnett’s test) after one-way analysis of variance. * P ⬍0.001 compared with corresponding sham-treated control. † P ⬍0.05 compared with corresponding sham-treated control. ‡ P ⬍0.001 compared with ORX group in castrated groups.
have suggested that the elevation is due to the testosterone deficiency resulting from castration. In the present study, we newly examined the effect of orchiectomy on the serum concentrations of ovarian hormones (17-estradiol and progesterone), in addition to testosterone, in male rats. Orchiectomy significantly decreased not only the serum testosterone level but also the progesterone level. Because part of the progesterone in male rats is known to be synthesized in the testes,15 the decreased progesterone level is considered to be due to orchiectomy. These results suggest a possibility that the decrease in the progesterone concentration is also involved in the CGRP-induced elevation of skin temperature in ORX rats. This suggestion was supported by the results of progesterone replacement in both castrated rats; the hormonal replacement clearly restored the castration-induced elevation of skin temperature. An exogenous testosterone supply to ORX rats elevated not only the serum concentration of testosterone but also the serum concentration of 17estradiol. The increase in the estrogen level may have been due to synthesis from exogenous testosterone in a reaction catalyzed by aromatase in extratesticular tissues, such as adipose and adrenal tissue.16,17 Therefore, the inhibition of the elevation of skin temperature by exogenous testosterone in castrated rats may involve the synthesis of estrogen in addition to testosterone. In fact, the serum concentration of 17-estradiol in ORX rats was elevated by supplying exogenous estrogen, UROLOGY 64 (5), 2004
without altering the serum concentrations of testosterone or progesterone, and the CGRP-induced skin temperature rise in castrated rats was clearly inhibited by supplying exogenous estrogen. With regard to the mechanism of CGRP-induced skin temperature elevation, a decline in estrogens18,19 or androgens20 has been reported to decrease CGRP synthesis and release. These findings suggest that both sex hormones similarly regulate CGRP synthesis and release. We demonstrated in previous studies21,22 that ovariectomy (estrogen deficiency) induced a decrease in the plasma concentration of CGRP, supporting these results. In addition, estrogen deficiency increased the number of arterial CGRP receptors. These results suggest that the vascular response by endogenous CGRP is maintained at normal by an increase in the number of arterial CGRP receptors in the sex hormone-deficient rats. In the present study, no statistically significant differences were observed in the basal skin temperature among all groups before CGRP was injected. One possibility is that this result was due to a homeostatic response by upregulation of arterial CGRP receptors in ORX rats, as well as in ovariectomized rats. In fact, the skin temperature elevation induced by exogenous injection of CGRP was significantly greater in ORX rats (present study) or ovariectomized rats21,22 than in sham-operated rats. Thus, hot flashes may be observed when the CGRP level in plasma is increased by certain stimulation under the condition of sex hormone deficiency. Although the stimulant 1045
to release CGRP into the circulation is not yet clear, some stresses may be presumed as the trigger because CGRP secretion is reported to be enhanced in menopausal women with hot flashes by cold load stress.23 To clarify our hypothesis, we will investigate in detail the mechanism in the ORX rat in future studies. Hormone replacement therapy with testosterone for the amelioration of hot flashes is contraindicated in men who undergo ADT for testosteronedependent prostate cancer.1 In the present study, supplemental estrogen or progesterone inhibited the CGRP-induced elevation of skin temperature without increasing the serum concentration of testosterone, suggesting that neither hormone confers any testosterone activity. The exposure of rodents24 or humans25 to estrogens has been reported to induce a proliferative lesion, squamous metaplasia, in their prostates. In contrast to these findings, estrogens have been used as part of treatment regimens for advanced prostate cancer. Estrogen is believed to exert a direct growth-inhibitory effect on prostate cancer cells by induction of apoptosis or cell cycle arrest26 or by estrogen -receptors.27 In the present study, the orchiectomy-induced decrease in prostate weight was recovered by supplying exogenous testosterone, but it was not affected by treatment with 17-estradiol or progesterone. These results suggest that neither hormone results in proliferation of the prostate. CONCLUSIONS The results of the present study support those of some clinical studies,5,7,28 suggesting that estrogen and progesterone are useful for men with specific contraindications, such as a history of testosterone-dependent prostate cancer, who are experiencing hot flashes. Finally, we suggest that CGRP is closely involved with 17-estradiol and progesterone in the amelioration of hot flashes of men who undergo ADT. REFERENCES 1. Miller JI, and Ahmann FR: Treatment of castrationinduced menopausal symptoms with low dose diethylstilbestrol in men with advanced prostate cancer. Urology 40: 499 – 502, 1992. 2. Karling P, Hammer M, and Varenhorst E: Prevalence and duration of hot flushes after surgical or medical castration in men with prostatic carcinoma. J Urol 152: 1170 –1173, 1994. 3. Freedman RR: Physiology of hot flashes. Am J Human Biol 13: 453– 464, 2001. 4. Charig CR, and Rumdle JS: Flushing: long-term side effect of orchiectomy in treatment of prostatic carcinoma. Urology 33: 175–178, 1989. 5. Quella SK, Loprinzi CL, Sloan JA, et al: Long term use of megestrol acetate by cancer survivors for the treatment of hot flashes. Cancer 82: 1784 –1788, 1998. 1046
6. Atala A, Amin M, and Harty JI: Diethylstilbestrol in treatment of postorchiectomy vasomotor symptoms and its relationship with serum follicle-stimulating hormone, luteinizing hormone, and testosterone. Urology 39: 108 –110, 1992. 7. Loprinzi Cl, Michalak JC, Quella SK, et al: Megestrol acetate for the prevention of hot flashes. N Engl J Med 331: 347–352, 1994. 8. Notelovitz M, and Mattox JH: Suppression of vasomotor and vulvovaginal symptoms with continuous oral 17-estradiol. Menopause 7: 310 –317, 2000. 9. Spetz AC, Pettersson B, Varenhorst E, et al: Momentary increase in plasma calcitonin gene-related peptide is involved in hot flashes in men treated with castration for carcinoma of the prostate. J Urol 166: 1720 –1723, 2001. 10. Wyon Y, Spetz AC, Hammar M, et al: Urinary excretion of calcitonin gene-related peptide in males with hot flushes after castration for carcinoma of the prostate. Scand J Urol Nephrol 35: 92–96, 2001. 11. Yuzurihara M, Ikarashi Y, Noguchi M, et al: Involvement of calcitonin gene-related peptide in elevation of skin temperature in castrated male rats. Urology 62: 947–951, 2003. 12. Mears E, Vessey MP, Andolsek L, et al: Preliminary evaluation of four oral contraceptives containing only progestogens. BMJ 21: 730 –734, 1969. 13. Wells M, Sturdee DW, Barlow DH, et al: Effect on endometrium of long term treatment with continuous combined oestrogen-progestogen replacement therapy: follow up study. BMJ 325: 231–232, 2002. 14. Keast JR, and Gleeson RJ: Androgen receptor immunoreactivity is present in primary sensory neurons of male rats. Neuroreport 9: 4137– 4140, 1998. 15. Ganong WF: The gonads: development and function of the reproductive system, in Ganong WF (Ed): Review of Medical Physiology, 10th ed. Singapore, Maruzen Asia, 1981, pp 331–363. 16. Simpson ER, Mahendroo MS, Means GD, et al: Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocr Rev 15: 342–355, 1994. 17. Bulun SE, Zeitoun KM, Takayama K, et al: Molecular basis for treating endometriosis with aromatase inhibitors. Hum Reprod Update 6: 413– 418, 2000. 18. Gangula PR, Lanlua P, Wimalawansa S, et al: Regulation of calcitonin gene-related peptide expression in dorsal root ganglia of rats by female sex steroid hormones. Biol Reprod 62: 1033–1039, 2000. 19. Gangula PR, Wimalawansa S, and Yallampalli C: Pregnancy and sex steroid hormones enhance circulating calcitonin gene-related peptide concentration in rats. Hum Reprod 15: 949 –953, 2000. 20. Sun C, Chen M, Mao J, et al: Biphasic effects of orchiectomy on calcitonin gene-related peptide synthesis and release. Neuroreport 12: 3497–3502, 2001. 21. Noguchi M, Ikarashi Y, Yuzurihara M, et al: Up-regulation of calcitonin gene-related peptide receptors underlying elevation of skin temperature in ovariectomized rats. J Endocrinol 175: 177–183, 2002. 22. Noguchi M, Ikarashi Y, Yuzurihara M, et al: Effects of the Japanese herbal medicine Keishi-bukuryo-gan and 17estradiol on calcitonin gene-related peptide-induced elevation of skin temperature in ovariectomized rats. J Endocrinol 176: 359 –366, 2002. 23. Chen JT, and Shiraki M: Menopausal hot flash and calcitonin gene-related peptide: effect of Keishi-bukuryo-gan, a kampo medicine, related to plasma calcitonin gene-related peptide level. Maturitas 45: 199 –204, 2003. 24. Triche TJ, and Harkin JC: An ultrastructural study of hormonally induced squamous metaplasia in the coagulating gland of the mouse prostate. Lab Invest 25: 596 – 606, 1971. UROLOGY 64 (5), 2004
25. Sugimura Y, Cunha GR, Yonemura CU, et al: Temporal and spatial factors in diethylstilbestrol-induced squamous metaplasia of the developing human prostate. Hum Pathol 19: 133–139, 1988. 26. Robertson CN, Robertson KM, Padilla GM, et al: Induction of apoptosis by diethylstilbestrol in hormone-insensitive prostate cancer cells. J Natl Cancer Inst 88: 908 –917, 1996.
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27. Lau KM, LaSpina M, Long J, et al: Expression of estrogen receptor (ER)-␣ and ER- in normal and malignant prostatic epithelial cells: regulation by methylation and involvement in growth regulation. Cancer Res 60: 3175–3182, 2000. 28. Gerber GS, Zagaja GP, Ray PS, et al: Transdermal estrogen in the treatment of hot flushes in men with prostate cancer. Urology 55: 97–101, 2000.
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