In vivo evidence for an increase in 5alpha-reductase activity in the rat central nervous system following morphine exposure

In vivo evidence for an increase in 5alpha-reductase activity in the rat central nervous system following morphine exposure

Int. J. Devl Neuroscience 23 (2005) 621–626 www.elsevier.com/locate/ijdevneu In vivo evidence for an increase in 5alpha-reductase activity in the rat...

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Int. J. Devl Neuroscience 23 (2005) 621–626 www.elsevier.com/locate/ijdevneu

In vivo evidence for an increase in 5alpha-reductase activity in the rat central nervous system following morphine exposure Hossein Amini, Abolhassan Ahmadiani * Department of Pharmacology, Neuroscience Research Center, Shaheed Beheshti University of Medical Sciences, P.O. Box 19835-355, Tehran, Iran Received 27 April 2005; received in revised form 5 July 2005; accepted 6 July 2005

Abstract In the present study, the effects of acute and chronic morphine exposure on testosterone concentrations in the central nervous system (CNS) and serum were investigated in rats. Acute morphine administration (5 mg/kg, sc) reduced significantly testosterone levels in serum and spinal cord but not in the brain. Following chronic morphine administration (orally for 21 days), the brain testosterone was also significantly reduced as well as serum and spinal cord. Since, the decrease in testosterone levels following morphine exposure was more obvious in the CNS than serum, we suggested that it cannot be caused by only a direct decline in testosterone levels in periphery, and an increased local metabolism of testosterone in the CNS might be attributed in these effects. This hypothesis was supported with the findings that pretreatment with finasteride, a 5alpha-reductase inhibitor (5 mg/kg, sc) blocked testosterone elimination from the CNS following morphine exposure. Moreover, the serum concentration of 5alpha-reduced metabolites of testosterone, dihydrotestosterone and 3alpha-diol glucuronide was increased significantly following chronic morphine exposure, but not after co-treatment with finasteride. These results suggest that morphine exposure increase the CNS activity of 5alpha-reductase, which is an important metabolizing enzyme for testosterone. # 2005 ISDN. Published by Elsevier Ltd. All rights reserved. Keywords: Morphine; Testosterone; 5Alpha-reductase; Finasteride; Dihydrotestosterone; 3Alpha-diol glucuronide

1. Introduction It is well known that chronic morphine exposure decrease serum testosterone (Barraclough and Sawyer, 1955; Cicero et al., 1976; Morley, 1981; Millan and Herz, 1985; Yilmaz et al., 1999; Abs et al., 2000; Rajagopal et al., 2003), but the exact underlying mechanisms have not fully elucidated and need further investigations. Morphine may affect testicular testosterone formation by inhibiting LH secretion, which is centrally mediated through inhibition of hypothalamic GnRH release (Blank and Roberts, 1982; Drouva et al., 1981; Mehmanesh et al., 1988), although additional effects at the hypophyseal level may also contribute (Blank et al., 1986; Kalra et al., 1988). Castration of male rats affects morphine ability to suppress serum LH. While, it has been shown that morphine was * Corresponding author. Fax: +98 21 22403154. E-mail address: [email protected] (A. Ahmadiani).

apparently more effective than testosterone in lowering serum LH in the initial stages of castration, but it was completely ineffective in long-term castrated rats (Cicero et al., 1980, 1982; Bhanot and Wilkinson, 1983). Both the delayed loss of the response to naloxone after castration (Masotto and Negro-Vilar, 1988) and lack of effect of castration (Cicero et al., 1982) on naloxone-induced increase in serum LH levels have been reported. Morphine induces supersensitivity to the effects of naloxone on LH (Cicero et al., 1983). Moreover, morphine and naloxone exert age-dependent effects on secretion of LH. It has been shown that morphine suppresses LH secretion at very early stages of development, well before puberty, whereas naloxone not only does not increase LH, but also does not reverse the inhibitory effect of morphine on LH in prepubescent male rats (Cicero et al., 1993). Naloxone treatment was found to elevate plasma FSH levels but not plasma LH levels in immature pigs (Trudeau et al., 1988).

0736-5748/$30.00 # 2005 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2005.07.001

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There is also another possibility that morphine-induced decrease in serum testosterone may be due to its direct effect on leydig cell function (Gerendai et al., 1984). It has been reported that intratesticular injections of naloxone increases serum testosterone levels without increasing LH (Cicero et al., 1989), and morphine suppresses serum testosterone after pretreatment with human chorionic gonadotropin, which reverse morphine’s suppression of LH (Adams et al., 1993). Similarly, it has been shown that in hypophysectomized rats, b-endorphin decreases testosterone levels (Chandrashekar and Bartke, 1992). It has been also reported that opioid peptides synthesized in the testis are components of the intratesticular regulatory system and that local opioid actions are modulated by testicular nerves (Gerendai, 1991). Recently, we have reported that serum testosterone is not a predictor of testosterone concentration in the central nervous system (CNS). Moreover, the brain enzyme 5alphareductase which metabolizes testosterone, is activated by formalin-induced tonic pain (Amini and Ahmadiani, 2002). The CNS is a target for morphine to induce analgesia and interest in this study was based on the notion that the effect of morphine on testosterone concentration in the CNS had not been studied. The objective of the present study was to evaluate acute and chronic morphine administration on testosterone levels in the rat CNS and periphery and to investigate possible involved mechanisms.

2. Experimental procedures 2.1. Animals and materials All experiments were performed using adult male Sprague–Dawley rats (250–300 g), kept on a 12-h dark/12h light cycle (lights on at 05:00 h) with ad libitum access to food (pellet from Pars Co., Tehran, Iran) and water. Experiments were executed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, revised 1985). Finasteride was from sigma (St. Louis, MO, USA). Morphine sulfate was kindly donated by Temad Co. (Tehran, Iran). All used solvents and salts were of analytical grade and obtained from Merck (Darmstadt, Germany). 2.2. Drug treatment For acute morphine exposure, morphine sulfate (5 mg/ kg) was dissolved in saline and injected sc in volume of 1 ml/kg, 2 h before decapitation. For chronic morphine exposure, morphine was added to feed water. The first 3 days were started with a concentration of 0.1 mg/ml of morphine followed by 0.2 mg/ml in the second 3 days

period and 0.3 mg/ml in the third 3 days period. From the days 9 to 21, a concentration of 0.4 mg/ml morphine was used. From the days 1 to 6, glucose in a concentration of 0.5% was added to feed water to compensate bitter taste of morphine. After 21 days oral morphine treatment, the rats were decapitated while morphine addiction was apparent in all. Finasteride (5 mg/kg) and its vehicle ethanol–caster oil (20:80, v/v) were given in two sc injections at 2 h apart, i.e. 2 and 4 h before decapitation. 2.3. Assay method for steroids All animals were killed by rapid decapitation between 10:30 and 11:30 h. The brain and spinal cords were rapidly removed and kept frozen at 20 8C. Trunk blood was collected and centrifuged at 900  g for 20 min, after 1 h room temperature. Following centrifugation, serum was separated and kept frozen. Testosterone assays were performed using commercially available RIA kit (Immunotech, Marseilles, France), directly in serum but following extraction from the brain and spinal cords using previously described method (Amini and Ahmadiani, 2002). Serum determination of dihydrotestosterone and 3alpha-diol glucuronide was done using a direct ELISA kit (Diagnostic Biochem Canada, Canada). 2.4. Statistical analysis Data are presented as mean  S.E.M. The statistical significance of differences was assessed by analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparisons tests. Statistical significance was accepted at level of P < 0.05.

3. Results 3.1. Effects of morphine exposure and finasteride treatment on testosterone levels in serum and CNS Testosterone levels in the brain (F (6, 43) = 4.38, P < 0.0015), spinal cord (F (6, 42) = 15.77, P < 0.0001) and serum (F (6, 40) = 3.07, P < 0.014) were significantly different between treatment groups (Fig. 1). A significant reduction of the brain testosterone (97%, P < 0.01) was observed after chronic morphine exposure compared to intact group. To determine whether decrease in testosterone levels might be attributed to increased testosterone metabolism via 5alpha-reductase pathway, finasteride (an inhibitor of 5alpha-reductase) was used. Finasteride completely reversed decrease in brain testosterone induced by chronic morphine. In spinal cord, both acute and chronic morphine exposure significantly (90% in comparison to intact group, P < 0.01) reduced testosterone levels, which were reversed by finasteride treatment. It was also observed that the injection of finasteride and its

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Fig. 1. Effects of morphine and finasteride treatment on the serum (upper panel), brain (middle panel) and spinal cord (lower panel) testosterone concentrations. For acute morphine exposure, morphine sulfate (5 mg/kg) was dissolved in saline and injected sc, 2 h before decapitation. For chronic morphine exposure, morphine was added to feed water with increasing concentration from 0.1 to 0.4 mg/ml for 21 days. Finasteride (5 mg/kg) and its vehicle ethanol–caster oil (20:80, v/v) were given in two sc injections at 2 h apart, i.e. 2 and 4 h before decapitation. Results are expressed as mean  S.E.M. and are from six to eight rats. One-way ANOVA followed by Tukey–Kramer multiple comparison tests. *P < 0.05; **P < 0.01 vs. the intact group.

vehicle significantly (P < 0.05) increased and decreased testosterone in spinal cord, respectively. Serum testosterone concentrations were significantly decreased following both acute and chronic morphine administration (P < 0.05) compared to intact rats, but not after co-treatment with finasteride.

3.2. Effects of chronic morphine and finasteride on serum dihydrotestosterone and 3alpha-diol glucuronide concentrations To determine whether the testosterone metabolites in serum can be influenced by morphine exposure, the serum

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concentration of dihydrotestosterone and 3alpha-diol glucuronide was measured following morphine and finasteride administration. Significant difference in serum dihydrotestosterone (F(2, 18) = 4.27, P < 0.03) and serum 3alpha-diol glucuronide (F(2, 18) = 5.46, P < 0.014) was observed between intact, chronic morphine exposed and chronic morphine exposed rats treated with finasteride. Chronic exposure to morphine significantly (P < 0.05) increased serum concentrations of dihydrotestosterone and 3alphadiol glucuronide. In finasteride treated rats, chronic morphine administration was not able to significantly increase dihydrotestosterone and 3alpha-diol glucuronide concentrations in serum (P > 0.05) compared to intact rats.

4. Discussion As expected, morphine exposure decreased serum testosterone concentrations to significant values. However, for the first time, it was shown that the concentration of testosterone in the CNS is also affected by morphine. A significant reduction in the brain testosterone levels was observed after chronic but not acute morphine exposure. In spinal cord, both acute and chronic morphine exposure significantly reduced testosterone concentrations. Although it might be argued that the CNS data could simply reflect direct decline in testosterone levels in periphery, a number of evidences argue against this conclusion: first, the decrease in testosterone levels following morphine exposure was more obvious in the CNS than serum; second, acute morphine exposure reduced testosterone levels in spinal cord more than brain, which may be explained by greater activation of testosterone metabolizing enzymes in the spinal cord following acute administration of morphine. An important metabolic pathway for testosterone in the CNS is the 5alphareductase pathway (Celotti et al., 1997). The enzyme 5alpha-reductase is widely distributed in the various CNS areas, especially spinal cord (MacLusky et al., 1987; Thigpen et al., 1993; Celotti et al., 1997). These evidences raised the question of whether morphine administration can increase testosterone metabolism via 5alpha-reduction pathway. We pretreated rats with finasteride in doses that could inhibit the CNS 5alpha-reductase (Celotti et al., 1997). Finasteride completely inhibited the decrease of the CNS testosterone following morphine exposure and this observation indicated that morphine exposure increases the activity of the 5alpha-reductase of the CNS. The finasteride vehicle administration decreased testosterone concentration which requires further investigation of its mechanism. The results also show that finasteride and its vehicle, respectively, increase and decrease testosterone levels in spinal cord. Like the obtained results following acute morphine, this indicates that the concentration of testosterone in the brain and spinal cord could be changed independently. Increase in the serum concentration of 5alpha-reduced metabolites of testosterone dihydrotestosterone and 3alpha-diol glucuronide following

chronic morphine exposure and partial inhibition of these effects by finasteride (Fig. 2) is an evidence of possible increase in 5alpha-reductase activity in periphery by morphine which is in turn in agreement with increasing in CNS 5alpha-reductase activity. Although there are numerous reports concerning decreased serum testosterone levels following morphine exposure, in our knowledge, there was no report of possible increase in testosterone metabolism by morphine. Possible decreased synthesis of testosterone as a result of morphine exposure has been proposed mostly because of its known effect on LH (Barraclough and Sawyer, 1955; Morley, 1981; Millan and Herz, 1985). However, we suggested that morphine affect LH secretion indirectly, although the resulting decreased serum LH by morphine could decrease testicular testosterone synthesis. It has been shown that morphine exerts effects on testicular function that are independent of its effects on LH (Cicero et al., 1989; Adams

Fig. 2. Effect of chronic morphine exposure on serum concentrations of dihydrotestosterone (upper panel) and 3alpha-diol glucuronide (lower panel) and effect of finasteride pretreatment. Results are expressed as mean  S.E.M. and are from six to eight rats. One-way ANOVA followed by Tukey–Kramer multiple comparison tests. *P < 0.05 vs. the control group.

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et al., 1993; Chandrashekar and Bartke, 1992). In addition, the hypothesis that morphine has a direct effect on LH cannot explain some observations such as induction of supersensitivity to the effects of naloxone on LH by morphine (Cicero et al., 1983), age-dependent effects of morphine and naloxone on secretion of LH (Cicero et al., 1993) and effects of castration on morphine ability to suppress serum LH (Cicero et al., 1980, 1982; Bhanot and Wilkinson, 1983). Increase in 5alpha-reductase activity, as suggested by the results of the present study, should be considered for future studies to investigate its importance in endocrine effects of morphine. It is desirable that the activity of the enzyme should be measured ‘‘in vitro’’ in the brain and spinal cord in the future studies. The mechanism that control the enzymatic pattern and activity of 5alpha-reductase in the brain have not been fully elucidated (Gao et al., 2002). It has been reported that the diencephalons 5alpha-reductase activity shows a highly significant increase after a single administration of carbamazepine, reserpine, diazepam, phenytoin, Phenobarbital and disulfiram (Kaneyuki et al., 1979). However, there is another report that neither hypothalamic deafferentations, nor the treatment with reserpine, p-chlorophenylalanine, atropine, morphine or naloxone produce any significant modification in the metabolism of testosterone in the hypothalamus of long-term castrated male rats (Celotti et al., 1983). This controversy may be solved by considering some additional factors such as effect of short- and long-term castration and selected neuronal tissue. In conclusion, our data provide the biochemical evidence for a decreasing effect of morphine exposure on the CNS concentrations of testosterone. It is suggested that morphine exposure increase the CNS activity of 5alpha-reductase, which is an important metabolizing enzyme for testosterone. Acknowledgements We wish to thank the Vice Chancellorship Office for Research Affairs of Shaheed Beheshti University of Medical Sciences for the grant supporting this work. We also thank Dr. Rahimi (Razak Pharmaceutical, Tehran, Iran) for donating finasteride to us.

References Abs, R., Verhelst, J., Maeyaert, J., Buyten, J.P.V., Opsomer, F., Adriaensen, H., Verlooy, J., Havenbergh, T.V., Smet, M., Acker, K.V., 2000. Endocrine consequences of long-term intrathecal administration of opioids. J. Clin. Endocrinol. Metab. 85, 2215–2222. Adams, M.L., Sewing, B., Forman, J.B., Meyer, E.R., Cicero, T.J., 1993. Opioid-induced suppression of rat testicular function. J. Pharmacol. Exp. Ther. 266, 323–328. Amini, H., Ahmadiani, A., 2002. Increase in testosterone metabolism in the rat central nervous system by formalin-induced tonic pain. Pharmacol. Biochem. Behav. 74, 199–204.

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Barraclough, C.A., Sawyer, C.H., 1955. Inhibition of the release of pituitary ovulating hormone in the rat by morphine. Endocrinology 57, 329–337. Bhanot, R., Wilkinson, M., 1983. Opiatergic control of LH secretion is eliminated by gonadectomy. Endocrinology 112, 399–401. Blank, M.S., Fabbri, A., Catt, K.J., Dufau, M.L., 1986. Inhibition of luteinizing hormone release by morphine and endogenous opiates in cultured pituitary cells. Endocrinology 118, 2091–2101. Blank, M.S., Roberts, D.L., 1982. Antagonist of gonadotropin-releasing hormone blocks naloxone-induced elevations in serum luteinizing hormone. Neuroendocrinology 35, 309–312. Celotti, F., Negri-Cesi, P., Limonata, P., Melcangi, C., 1983. Is the 5alphareductase of the hypothalamus and of the anterior pituitary neurally regulated? Effects of hypothalamic deafferentations and of centrally acting drugs. J. Steroid Biochem. 19, 229–234. Celotti, F., Negri-Cesi, P., Poletti, A., 1997. Steroid metabolism in the mammalian brain: 5alpha-reduction and aromatization. Brain Res. Bull. 44, 365–375. Chandrashekar, V., Bartke, A., 1992. The influence of B-endorphine on testicular endocrine function in adult rats. Biol. Reprod. 47, 1–5. Cicero, T.J, Adams, M.L., OConnor, L.H., Nock, B., 1989. In vivo evidence for a direct effect of naloxone on testicular steroidogenesis in the male rat. Endocrinology 125, 957–963. Cicero, T.J., Meyer, E.R., Gabriel, S.M., Bell, R.D., Wilcox, C.E., 1980. Morphine exerts testosterone-like effects in the hypothalamus of the castrated male rat. Brain Res. 202, 151–164. Cicero, T.J., Meyer, E.R., Schmoeker, P.F., 1982. Development of tolerance to the effects of morphine on luteinizing hormone secretion as a function of castration in the male rat. J. Pharmacol. Exp. Ther. 223, 784–789. Cicero, T.J., Nock, B., OConnor, L., 1993. Naloxone does not reverse the inhibitory effect of morphine on luteinizing hormone secretion in prepubescent male rats. J. Pharmacol. Exp. Ther. 264, 47–53. Cicero, T.J., Owens, D.P., Schmoeker, P.F., Meyer, E.R., 1983. Morphineinduced supersensitivity to the effects of naloxone on luteinizing hormone secretion in the male rat. J. Pharmacol. Exp. Ther. 225, 35–41. Cicero, T.J., Wilcox, C.E., Bell, R.D., Meyer, E.R., 1976. Acute reduction in serum testosterone levels by narcotics in the male rat: stereospecificity, blockade by naloxone and tolerance. J. Pharmacol. Exp. Ther. 198, 340– 346. Drouva, S.V., Epelbaum, J., Tapia-Arancibia, L., Laplante, E., Kordon, C., 1981. Opiate receptors modulate LHRH and SRIF release from mediobasal hypothalamic neurons. Neuroendocrinology 32, 163–167. Gao, C.Q., Dhooge, W.S., Kaufman, J.M., Weyne, J.J., Eechaute, W.P., 2002. Hypothalamic 5alpha-reductase and 3alpha-oxidoreductase activity in the male rat. J. Steroid Biochem. Mol. Biol. 80, 91–98. Gerendai, I., 1991. Modulation of testicular functions by testicular opioid peptides. J. Physiol. Pharmacol. 42, 427–437. Gerendai, I., Shaha, C., Thau, R., Bardin, C.W., 1984. Do testicular opiates regulate leydig cell function? Endocrinology 115, 1645–1647. Kaneyuki, T., Kohsaka, M., Shohmori, T., 1979. Sex hormone metabolism in the brain: influence of central acting drugs on 5alpha-reduction in rat diencephalons. Endocrinol. Jpn. 26, 345–351. Kalra, P.S., Sahu, A., Kalra, S.P., 1988. Opiate-induced hypersensitivity to testosterone feedback: pituitary involvement. Endocrinology 122, 997– 1003. MacLusky, N.J., Clark, C.R., Shanabroug, M., Naftolin, F., 1987. Metabolism of androgens in the spinal cord of the rat. Brain Res. 422, 83–89. Masotto, C., Negro-Vilar, A., 1988. Gonadectomy influences the inhibitory effect of the endogenous opiate system on pulsatile gonadotropin secretion. Endocrinology 123, 747–752. Mehmanesh, H., Almedia, O.F.X., Nikolarakis, K.E., Herz, A., 1988. Hypothalamic LH-RH release after acute and chronic treatment with morphine studied in a combined in vivo/in vitro model. Brain Res. 451, 69–76. Millan, M.J., Herz, A., 1985. The endocrinology of Opioids. Int. Rev. Neurobiol. 26, 1–83. Morley, J.E., 1981. The endocrinology of the opiates and opioid peptides. Metabolism 30, 95–209.

626

H. Amini, A. Ahmadiani / Int. J. Devl Neuroscience 23 (2005) 621–626

Rajagopal, A., Vassilopoulou-Sellin, R., Palmer, J.L., Kaur, G., Bruera, E., 2003. Hypogonadism and sexual dysfunction in male cancer survivors receiving chronic opioid therapy. J. Pain Symptom Manage. 26, 1055– 1061. Thigpen, A.E., Silver, R.I., Guileyardo, J.M., Casey, M.L., McConnel, J.D., Russell, D.W., 1993. Tissue distribution and ontogenity of steroid 5alpha-reductase isozyme expression. J. Clin. Invest. 92, 903–910.

Trudeau, V.L., Meijer, J.C., van de Wiel, D.F., Bevers, M.M., Erkens, J.H., 1988. Effects of morphine and naloxone on plasma levels of LH, FSH, prolactin and growth hormone in the immature male pig. J. Endocrinol. 119, 501–508. Yilmaz, B., Konar, V., Kutlu, S., Sandal, S., Canpolat, S., Gezen, M.R., Kelestimur, H., 1999. Influence of chronic morphine exposure on serum LH, FSH, testosterone levels, and body and testicular weights in the developing male rat. Arch. Androl. 43, 189–196.