EFFECTS OF TAMOXIFEN, MIFEPRISTONE AND CYPROTERONE ON THE ELECTROCONVULSIVE THRESHOLD AND PENTETRAZOLE-INDUCED CONVULSIONS IN MICE

Copyright 2002 by Institute of Pharmacology Polish Academy of Sciences Polish Journal of Pharmacology Pol. J. Pharmacol., 2002, 54, 103 109 ISSN 1230-6002 EFFECTS OF TAMOXIFEN, MIFEPRISTONE AND CYPROTERONE ON THE ELECTROCONVULSIVE THRESHOLD AND PENTETRAZOLE-INDUCED CONVULSIONS IN MICE Kinga K. Borowicz 1,#, Jarogniew uszczki 1, Mariusz Matuszek 2, Zdzis³aw Kleinrok 3, Stanis³aw J. Czuczwar 1,4 Department of Pathophysiology, Ist Clinic of General Surgery and Transplantology,! Department of Pharmacology and Toxicology, Medical University School, Jaczewskiego 8, PL 20-090 Lublin, Poland, " Isotope Laboratory, Institute of Agricultural Medicine, Jaczewskiego 2, PL 20-090 Lublin, Poland Effects of tamoxifen, mifepristone and cyproterone on the electroconvulsive threshold and pentetrazole-induced convulsions in mice. K.K. BORO- WICZ, J. USZCZKI, M. MATUSZEK, Z. KLEINROK, S.J. CZUCZWAR. Pol. J. Pharmacol., 2002, 54, 103 109. The aim of this study was to evaluate the efficacy of three antihormones, tamoxifen (TXF, an antiestrogen), mifepristone (MIF, an antiprogesterone) and cyproterone (CYP, an antiandrogen) in two major models of experimental epilepsy, electrically and pentetrazole (PTZ)-evoked seizures in mice. TXF (20 50 mg/kg) significantly raised the threshold for electroconvulsions in female mice, whereas CYP was active in male mice. Similar effects were observed in castrated mice. Different data were obtained in sexually immature animals since both TXF and CYP exerted anticonvulsive effects in animals of both genders. MIF (5 50 mg/kg) remained without effect on electrically evoked seizures in mice. The anticonvulsive action of TXF was reversed by aminophylline, bicuculline, kainic acid and N-methyl-D-aspartic acid, but not by estradiol or strychnine. The protective action of CYP was reversed by aminophylline and bicuculline, but not by testosterone, kainic acid, N-methyl-D-aspartic acid or strychnine. All three antihormones were ineffective against PTZ-induced convulsions in mice. Our results suggest that the action of TXF and CYP might be indirectly associated with the respective hormonal receptor-mediated events, but the nature of this dependence is unclear and further investigations are needed to elucidate this phenomenon. Key words: tamoxifen, mifepristone, cyproterone, electroconvulsive threshold, pentetrazole-evoked seizures correspondence; e-mail: kornel@asklepios.am.lublin.pl

K.K. Borowicz, J. uszczki, M. Matuszek, Z. Kleinrok, S.J. Czuczwar Abbreviations: AMI aminophylline, BIC bicuculline, CD # the dose of a convulsant necessary to produce seizures in 50% of animals, CS # the current strength necessary to produce seizures in 50% of animals, CYP cyproterone, E estradiol, KA kainic acid, MIF mifepristone, NMDA N-methyl-D-aspartate, PTZ pentetrazole, STR strychnine, T testosterone, TXF tamoxifen INTRODUCTION Findings from animal and human studies indicate that gonadal steroids influence seizure processes. Increases of circulating estradiol (E) exacerbate catamenial epilepsy, particularly complex partial and tonic-clonic seizures [2]. Physiological levels of estrogens decrease the threshold and increase the magnitude of seizure activity in experimental animals [5, 27]. Intraperitoneal (ip) [21] and intravenous (iv) [26] E can increase the firing of a seizure focus [24], lower the electroconvulsive threshold [35], and shorten the stimulation to kindling threshold seizures evoked by amygdala stimulation and pentetrazole (PTZ) [5, 19]. Increases in endogenous progesterone level or exogenous progesterone treatment elevate the threshold and decrease the magnitude of seizure activity [39]. Cyclic increases in progesterone during the luteal phase are associated with decreases in tonic-clonic seizures in women with catamenial epilepsy [12]. Progesterone increases kindling [18] and electroconvulsive threshold [40]. This hormone is also effective against PTZ-induced seizures [22]. Moreover, cyclic withdrawal from endogenous and exogenous progesterone potentiates kainic acid (KA)- and perforant pathway stimulation-induced seizures [11, 29]. Thus, in general, E has proconvulsant, and progesterone anticonvulsant effects. However, progesterone was also reported to exacerbate absence seizures in women [15]. On the other hand, E reduced frequency of vestibular seizures in C57/EL mice [20]. Finally, testosterone exerted antiseizure effects against KA-evoked convulsions in rats [13]. However, this androgen enhanced the development of amygdala-kindled seizures [7] and attenuated the anticonvulsant effect of flurazepam against electrically produced seizures in mice [32]. There is no data available on the influence of the steroid hormone antagonists upon seizure phenomena. This is why we decided to examine the activity of tamoxifen (TXF, an estrogen antagonist), mifepristone (MIF, a progesterone antagonist) and cyproterone (CYP, an androgen antagonist) in two major models of experimental epilepsy, electrically and PTZ-evoked seizures in mice. The first model is recommended as a model of generalized tonic- -clonic seizures, the second one reflects myoclonic convulsions in humans [8]. Animals MATERIALS and METHODS The experiments were carried out on male and female Swiss mice weighing 20 25 g. The animals were housed in colony cages under standard laboratory conditions with free access to chow pellets and tap water. All experiments were done at the same time of a day (between 9.00 a.m. and 12.00 a.m.) to minimize circadian influences on seizure susceptibility. The animals were chosen randomly and assigned to experimental groups of 8 mice. Surgery For experiments on castrated animals, mice were respectively orchidectomized or ovariectomized under ketamine (100 mg/kg) anesthesia. All animals were allowed a 2 week recovery period prior to inclusion in the experiment. Electroconvulsive threshold Electroconvulsions were produced according to Swinyard et al. (36) by using ear-clip electrodes and alternating current delivered by a Hugo Sachs (Type 221, Freiburg, Germany) stimulator, with a stimulus duration of 0.2 s. Tonic hindlimb extension was taken as the end point. The convulsive threshold was evaluated as CS 50, which is a current strength (in ma) necessary to produce tonic hindlimb extension in 50% of animals tested. To estimate the convulsive threshold, at least four groups of mice (8 animals per group) were challenged with electroshocks of various intensities. Subsequently, an intensity-response curve was calculated on the basis of the percentage of mice convulsing. In order to evaluate the influence of endogenous gonadal hormones on the action of antihormones, the experiments were also done on castrated or sexually immature animals (14-day old mice pups). 104 Pol. J. Pharmacol., 2002, 54, 103 109

SEXUAL ANTIHORMONES AND SEIZURES Pentetrazole-induced convulsions At least 32 mice (8 animals per group) were injected ip with TXF, MIF or CYP (all three drugs at the same dose of 50 mg/kg), and then subcutaneously (sc) with various doses of PTZ. Following the injection of the convulsant, mice were placed separately in transparent cages (25 15 10 cm) and observed for 30 min for the occurrence of clonic seizures. Clonic seizure activity was defined as clonus of the whole body lasting over 3 s with an accompanying loss of righting reflex. To estimate the CD 50 value for PTZ alone or in combination with an antihormone, an intensity-response curve was calculated on the basis of the percentage of mice with clonic convulsions. Drugs Tamoxifen (TXF; Polfa, Kutno, Poland), cyproterone (CYP; Schering, Berlin, Germany), mifepristone (MIF), pentetrazole (PTZ), kainic acid (KA), N-methyl-D-aspartic acid (NMDA), bicuculline (BIC), aminophylline (AMI), strychnine (STR), estradiol (E), testosterone (T) (all drugs from Sigma, St. Louis, MO, USA) were used in this study. TXF, MIF and CYP were suspended in a 1% solution of Tween 80 (Sigma, St. Louis, MO, USA). All remaining drugs were brought into solution with sterile saline. BIC was dissolved with a drop of glacial acetic acid and adjusted to ph of 5.5 with 0.1 M NaOH and made up to the desired volume with sterile saline. The ph of solutions of excitatory amino acids was adjusted to 7.2 with 0.1 M NaOH. Except for PTZ which was administered sc in a volume of 5 ml/kg, all the remaining agents were given ip in a volume of 10 ml/kg. Hormones were administered on 2 consecutive days (5 injections twice daily, the last one 1 h before convulsive tests). Except for BIC which was injected 15 min before electroconvulsions, all remaining chemoconvulsants were given 30 min prior to this test. TXF, MIF and CYP were administered 120 min before convulsive tests. The doses refer to the free drug forms. Statistics CS 50 and ED 50 values with respective confidence limits and statistical analysis of the results were calculated by fitting the data with the use of computerized probit analysis based on the method of Litchfield and Wilcoxon [23]. RESULTS Effects of TXF, MIF and CYP on the electroconvulsive threshold in naive, castrated and sexually immature mice TXF administered at doses of 20 and 50 mg/kg, 120 min before the test, significantly raised the electroconvulsive threshold in naive female, but not male mice. At a dose of 10 mg/kg, it did not affect the threshold for electroconvulsions in female mice. Conversely, CYP applied at 40 and 50 mg/kg, 120 min before the test, raised the threshold for electroconvulsions in naive male but not female mice (Tab. 1). Table 1. Effects of TXF, MIF and CYP on the electroconvulsive threshold in female (F) and male (M) mice Treatment (mg/kg) F M Control 6.4 (6.1 6.7) 6.1 (5.8 6.4) TXF (50) 9.1 (8.7 9.4)*** 6.4 (6.1 6.8) TXF (20) 7.4 (6.8 7.9)** 6.5 (6.1 6.9) TXF (10) 6.3 (6.0 6.6) ND Control 5.6 (5.3 5.9) 5.6 (5.3 5.8) CYP (50) 5.8 (5.4 6.3) 6.9 (6.7 7.2)*** CYP (40) 5.5 (5.3 5.8) 6.6 (6.3 6.8)*** CYP (20) ND 5.2 (4.8 5.7) MIF (5) 5.7 (5.3 6.2) 5.4 (5.1 5.7) MIF (50) 5.9 (5.6 6.2) 5.9 (5.6 6.2) TXF, MIF and CYP were given ip 120 min before testing. The data are CS 50 values, i.e. current strength (in ma, 95% confidence limits in parentheses) necessary to produce tonic hindlimb extension in 50% of animals tested. CS 50 values and statistical comparisons were calculated according to the method of Litchfield and Wilcoxon [23]. TXF tamoxifen; MIF mifepristone; CYP cyproterone; ND not determined. ** p < 0.01, *** p < 0.001 vs. respective control group TXF (50 mg/kg) and CYP (50 mg/kg) also significantly raised the electroconvulsive threshold in ovariectomized female and orchidectomized male mice, respectively (Tab. 2). Finally, TXF (50 mg/kg) and CYP (50 mg/kg) produced a significant rise in the electroconvulsive threshold in sexually immature animals of both genders, although CYP exerted more pronounced effect upon male than female animals (Tab. 3). ISSN 1230-6002 105

K.K. Borowicz, J. uszczki, M. Matuszek, Z. Kleinrok, S.J. Czuczwar Table 2. Effects of TXF, MIF and CYP on the electroconvulsive threshold in ovariectomized (o) female and orchidectomized (o) male mice Treatment (mg/kg) o FEMALES o MALES Control 7.6 (7.4 7.9) 7.8 (7.6 8.0) TXF (50) 10.3 (9.5 11.1)*** 7.6 (7.4 7.9) CYP (50) 7.7 (7.4 8.0) 9.3 (9.0 9.6)*** MIF (50) 7.8 (7.4 8.2) 7.4 (7.1 7.7) *** p < 0.001 vs. control group. See Table 1 for details and abbreviations Table 3. Effects of TXF, MIF and CYP on the electroconvulsive threshold in sexually immature (i) female and male mice Treatment (mg/kg) i FEMALES i MALES Control 6.8 (6.3 7.4) 7.4 (7.2 7.9) TXF (50) 8.1 (7.6 8.7)** 8.2 (7.8 8.8)** CYP (50) 7.8 (7.2 8.3)* 8.8 (8.5 9.2)*** MIF (50) 6.6 (6.0 7.1) 7.3 (6.9 7.8) * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group. See Table 1 for details and abbreviations Table 4. Effects of estradiol and some chemoconvulsants on the protection provided by TXF against electroconvulsions in female mice Treatment (mg/kg) CS 50 (ma) Control 1 6.4 (5.7 7.1) TXF (50) 8.7 (8.1 9.2)*** E (5) 6.2 (5.4 7.0) TXF (50) + E (5) 8.5 (8.0 8.9)*** Control 1 7.1 (6.8 7.5) TXF (50) control 2 9.7 (9.3 10.2)*** TXF (50) + BIC (2) 7.0 (6.6 7.4) ns,a TXF (50) + AMI (5) 7.9 (7.4 8.4)*,b TXF (50) + AMI (50) 7.0 (6.6 7.4) ns,a TXF (50) + KA (5) 7.4 (6.6 8.4) ns,a TXF (50) + NMDA (30) 7.2 (6.8 7.7) ns,a TXF (50) + STR (0.3) 9.2 (8.8 9.8)***,ns TXF tamoxifen; E estradiol; BIC bicuculline; AMI aminophylline; KA kainic acid; NMDA N-methyl-D-aspartic acid; STR strychnine. * p < 0.05, *** p < 0.001 vs. respective control 1 group, b p < 0.01, a p < 0.001 vs. control 2, ns not significant MIF did not affect the threshold for electroconvulsions in naive, castrated or immature animals (Tab. 1 3). Effects of estradiol on the TXF-induced protection against electrically evoked seizures in female mice E applied at the dose of 5 mg/kg failed to influence the threshold for electroconvulsions and to reverse the protection offered by TXF against electrically induced seizures in female mice (Tab. 4). Effects of testosterone on the CYPinduced protection against electrically evoked seizures in male mice T (25 mg/kg) affected neither the electroconvulsive threshold nor the protective activity of CYP against electrically evoked convulsions in male mice (Tab. 5). Table 5. Effects of testosterone and some chemoconvulsants on the CYP-produced protection against electrically evoked convulsions in male mice Treatment (mg/kg) CS 50 (ma) Control 1 6.0 (5.6 6.4) CYP (50) 7.1 (6.7 7.5)*** T (25) 6.0 (5.5 6.6) CYP (50) + T (25) 6.9 (6.4 7.2)*** Control 1 6.6 (6.2 7.1) CYP (50) control 2 7.8 (7.3 8.1)*** CYP (50) + BIC (2) 6.9 (6.5 7.3) ns,a CYP (50) + AMI (5) 7.2 (6.8 7.8)*,c CYP (50) + AMI (50) 6.5 (6.1 7.0) ns,a CYP (50) + KA (5) 7.8 (7.4 8.2)***,ns CYP (50) + NMDA (30) 8.0 (7.6 8.6)***,ns CYP (50) + STR (0.3) 7.7 (7.3 8.0)***,ns * p < 0.05, *** p < 0.001 vs. respective control 1 group, c p < 0.05, a p < 0.001 vs control 2, ns not significant. CYP cyproterone; T testosterone. For more details see the legend to Table 3 Effects of some chemoconvulsants on the protective action of TXF and CYP against electrically evoked seizures in mice The protective action of TXF in female mice was partially reversed by AMI at 5 mg/kg and at- 106 Pol. J. Pharmacol., 2002, 54, 103 109

SEXUAL ANTIHORMONES AND SEIZURES tenuated by AMI at 50 mg/kg, BIC (2 mg/kg), KA (5 mg/kg) and NMDA (30 mg/kg). However, STR (0.4 mg/kg) did not affect the protection offered by TXF (Tab. 4). The anticonvulsive activity of CYP in male mice was partially reversed by AMI (5 mg/kg) and abolished by AMI (50 mg/kg) and BIC (2 mg/kg). KA (5 mg/kg), NMDA (30 mg/kg) and STR (0.4 mg/kg) were ineffective in this respect (Tab. 5). All chemoconvulsants were given at subconvulsive doses also unable to affect the electroconvulsive threshold in mice of both genders (data not shown). Effects of TXF, MIF and CYP against PTZ-induced convulsions in mice None of the three antihormones used at the dose of 50 mg/kg influenced the CD 50 value of PTZ for the induction of clonic convulsions in female and male mice (Tab. 6). Table 6. Effect of TXF, MIF and CYP on PTZ-evoked seizures in female and male mice Treatment (mg/kg) FEMALES MALES PTZ + saline 73.9 (65.3 83.6) 86.0 (77.1 96.0) PTZ + TXF (50) 77.0 (67.6 87.9) 80.7 (72.0 90.5) PTZ + CYP (50) 81.2 (73.9 89.4) 78.9 (69.6 89.5) PTZ + MIF (50) 79.5 (71.6 88.3) 74.0 (64.3 85.2) Table data are 50% convulsive doses of PTZ (CD 50 values; in milligrams per kilogram; 95% confidence limits) for the production of clonic convulsions in mice. PTZ pentetrazole; TXF tamoxifen; CYP cyproterone; MIF mifepristone DISCUSSION In this study, we show for the first time that sexual antihormones may influence seizure phenomena. Among all gonadal receptor antagonists used in these experiments, only MIF remained without effect on seizure activity evaluated in two basic models of experimental epilepsy. TXF (an antiestrogen, 20 mg/kg) significantly raised the electroconvulsive threshold in female mice, and CYP (an antiandrogen, 40 mg/kg) exerted comparable effects in male mice. Both drugs remained ineffective against convulsions produced by PTZ. The protective efficacy of TXF against electrically induced seizures was reversed by AMI, BIC and excitatory amino acids, but not by E or STR. Furthermore, the anticonvulsive efficacy of CYP was reduced by AMI, BIC, but not by T, excitatory amino acids or STR. It must be underlined that all chemoconvulsants and gonadal hormones were used at their highest doses ineffective against electrically evoked seizures in mice. Above results suggest that antiseizure activity of antihormones may depend on adenosine- and GABA A receptor-evoked events, but not glycinergic ones. Additionally, in case of TXF, NMDA and KA receptor-mediated events seem to be associated with the anticonvulsive properties of this drug. It is noteworthy that according to Saberi et al. [33], TXF applied at the dose of 3 mg/kg reversed, whereas, when administered at 10 mg/kg, it significantly enhanced the proconvulsive effect of E (30 g/kg) in amygdala-kindled rats. This dual effect may result from the partial agonistic property of TXF at estrogen receptors. However, in the present study TXF (1 10 mg/kg) and E (20 g/kg 5 mg/kg) remained without effect in electroconvulsive threshold seizures in mice. This discrepancy may be, at least partially, explained by species differences (rats and mice) and the distinct nature of amygdala-kindling and electroconvulsive threshold models of experimental seizures. Our finding that the action of antihormones may not be dependent only on the respective hormonal receptors, does not seem to be astonishing. In fact, anti- or proconvulsive properties of some steroid hormones are probably not due to their interaction with intracellular hormonal receptors [10]. It has been proposed that progesterone ability to rapidly alter neuronal firing [34] occurs through interactions with GABA A -benzodiazepine receptor complexes [14, 16, 25]. This may explain the rapid analgesic [12], anxiolytic [4], anticonvulsant [3] and anesthetic actions of progesterone. Progesterone may also potentiate the effects of endogenous anticonvulsants such as adenosine [31]. Pretreatment with the progesterone antagonist, MIF (3 mg/kg), did not inhibit the protective activity of progesterone against amygdala-kindled seizures in rats, although the GABA A receptor antagonist, BIC (2 mg/kg), blocked the antiseizure action of progesterone in this model of epilepsy [28]. Furthermore, estrogens are thought to produce the positive modulation of NMDA-type glutamate receptors [17]. E selectively regulates agonist binding sites on the NMDA receptor complex in the CA1 region of the hippocampus [38]. Estrogens ISSN 1230-6002 107

K.K. Borowicz, J. uszczki, M. Matuszek, Z. Kleinrok, S.J. Czuczwar may also be responsible for the negative modulation of the GABA A receptor complex in the central nervous system [30]. On the other hand, T has two major metabolites: E, which can exacerbate seizures, and dihydrotestosterone, which blocks NMDA- -type glutamate transmission [17]. It is noteworthy that, according to Czuczwar et al. [6], STR administered at doses ineffective in the electroconvulsive threshold test, significantly diminished the protective action of valproate, phenobarbital, and diphenylhydantoin, remaining without effect on the efficacy of carbamazepine against maximal electroshock in mice. Thus, there is a possibility that the mechanism of action of the three antiepileptics may be dependent upon glycine-mediated inhibition. On the other hand, the nature of the protective activity of TXF and CYP does not seem to be related to the enhancement of glycinergic neurotransmission, sharing this property with carbamazepine. However, it is intriguing that the antiestrogen exhibited the protective efficacy only in female (naive or castrated) mice, and the antiandrogen only in male (naive or castrated) ones. In immature animals both antihormones possessed anticonvulsive efficacy in males as well as in females. It might be possible that these antihormones interact with their own steroid receptors, whose stimulation triggers subsequently other inhibitory mechanisms in the central nervous system, including adenosine or GABA A -mediated events. Before sexual maturity the process of hormonal receptor differentiation is not yet completed, and this may be the reason for the unspecific action of the two antihormones in 14-day-old mice pups. TXF, similarly to estrogens, may also negatively modulate excitatory amino acid receptors. In fact, TXF behaves as an ago-antagonist of estrogen receptors, and might share some pharmacological properties with female gonadal hormones. Moreover, TXF was also reported to inhibit protein kinase C (PKC) [9]. Since PKC inhibitors exert anticonvulsive action, this effect may be, at least partially, responsible for antiseizure properties of the antiestrogen. At present, it is difficult to discuss (because of the lack of available data), why the antiprogesterone remained ineffective in seizure phenomena. However, MIF exerts also antiglucocorticoid effects [37]. Bearing in mind that glucocorticoids may exacerbate seizures induced by intrahippocampal injection of KA, there is a reason to consider that the antiprogesterone (potentially proconvulsive) and the antiglucocorticoid (potentially anticonvulsive) effects of MIF abolish each other. 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