Short communication
Effects of androsterone on convulsions in various seizure models in mice
Katarzyna Mróz, Tomasz Mróz, Marian Wielosz, Piotr Tutka
Department of Experimental and Clinical Pharmacology, Medical University of Lublin, Jaczewskiego 8, PL 20-090 Lublin, Poland
Correspondence:Piotr Tutka, e-mail: tutka@am.lublin.pl
Abstract:
It is believed that a deficiency of androgens, including free testosterone, may promote the development of convulsions.The present study revealed differences in the action of androsterone (AND), amajor excreted metabolite of testosterone and a neurosteroid,in three commonly used seizure models in mice.AND administered intraperitoneally exhibited dose-dependent protection against tonic-clonic convulsions caused by maximal electroshock (MES) with ED# (effective dose# ) of 227 mg/kg. The compound also in- hibited the convulsive action of pentylenetetrazole (PTZ), increasing its CD# (convulsive dose# ) for clonic convulsions from 77.2 (PTZ + saline) to 93.9 (p < 0.05) for PTZ + AND 40 mg/kg and 113.9 mg/kg (p < 0.001) for PTZ + AND 60 mg/kg. In mice pretreated with 60 mg/kg AND, the CD# for PTZ-induced tonic convulsions increased from 102 to 127.6 mg/kg (p < 0.01). Surprisingly, doses of 50 and 100 mg/kg AND lowered the CD# for kainate (KA)-induced convulsions from 40.8 to 28.7 (p < 0.05) and 25.4 mg/kg (p < 0.001), respectively. In summary, for two of the mouse seizure models, our findings confirmed previous studies that demon- strated protective activity of AND. However, the potentiation of KA-induced convulsions by AND was somewhat unexpected and suggested that AND may also possess proconvulsant activity.
Key words:
androgens, androsterone, epilepsy, kainate, maximal electroshock, pentylenetetrazole, seizures
Introduction
Reproductive endocrine disorders that can be attrib- uted to reduced androgen levels (such as subnormal free testosterone), are common in epileptic men [4].
In addition, a deficiency of androgens may promote the development of epileptic discharges and increase seizure susceptibility [3].
Subnormal levels of plasma free testosterone result in reduced production of its major excreted metabolite – androsterone (5a-androstan-3a-ol-17-one, AND).
AND has been found in the adult brain, and its action as a neurosteroid withg-aminobutyric acid (GABA)A- receptor-modulating activity has been proposed [20].
As the role of GABAergic transmission in the patho- genesis of convulsions is crucial [1], alterations in AND levels are relevant to seizure control [18]. In- deed, AND has been found to exert protective activity in bothin vitro and in vivo experiments [5, 7, 10].
In this study, we investigated the effects of AND on convulsions elicited in mice using three commonly accepted seizure models: maximal electroshock (MES), pentylenetetrazole (PTZ) and kainate (KA).
Materials and Methods
Animals
The experiments were performed on adult male Swiss mice weighing 20–25 g. The animals were kept under standard laboratory conditions on a natural light-dark cycle, with ambient temperature of 18–22°C, relative humidity of 52–58%, and unlimited access to chow pellets and water. All animals were acclimatized to their home cages for 1 week before testing. The ex- perimental groups, consisting of 8 mice, were chosen by means of a randomized schedule. Each mouse was used only once. The tests were performed between 8:00 and 14:00. The control groups were always tested on the same day as the corresponding experi- mental groups. The experimental protocol and proce- dures were followed according to “Principles of Labo- ratory Animal Care” (NIH publication No. 86–23, re- vised 1985), approved by the Medical University of Lublin Ethics Committee for the use of experimental animals and confirmed with the European Communi- ties Council Directive (86/609/EEC).
Drugs
AND was suspended in a 1% aqueous solution of Tween 80, whereas KA and PTZ (all from Sigma, St.
Louis, MO, USA) were dissolved in sterile saline for immediate administration, either intraperitoneally (ip) at a volume of 10 ml/kg of body weight, or subcutane- ously (sc) at a volume of 5 ml/kg of body weight.
Fresh drug solutions or suspensions were preparedex tempore on each experimental day. Control animals were injected with equivalent amounts of sterile sa- line or 1% solution of Tween 80 in water using the same route.
Electroconvulsions
Electroconvulsions were produced with a current de- livered via ear-clip electrodes by a Rodent Shocker generator (constant-current stimulator Type 221, Hugo Sachs Elektronik, Freiburg, Germany). The criterion for convulsant activity was tonic hindlimb extension (i.e., the hind limbs of animals became extended at 180° to the plane of the body axis). The protective ac- tivity of AND against MES-induced convulsions
(elicited with 0.2 s stimulus duration and fixed current intensity of 25 mA) was determined.
Pentylenetetrazole-induced convulsions
Clonic convulsions were induced bysc administration of PTZ at doses ranging from 60 to 140 mg/kg. Tonic convulsions were induced by injection of higher doses (90–160 mg/kg) of PTZ. Following injection of the convulsant, mice were placed separately into transpar- ent Plexiglas cages (25 × 15 × 10 cm) and observed for 30 min for the occurrence of seizures. The clonic seizure activity was defined as clonus of all four limbs for at least 5 s, with an accompanying loss of the righting reflex. The tonic seizure was definied as the tonic extension of all four limbs. The number of ani- mals convulsing out of the total number of mice tested was recorded for each treatment condition. An inten- sity-response curve was constructed from the percent- age of convulsing mice. The convulsive action of PTZ was quantified as the CD50 (convulsive dose50; the dose producing convulsions in 50% of mice). To de- termine the CD50 value, the effects of five different doses of PTZ were tested.
Kainate-induced convulsions
In the KA model, seizures were induced following ad- ministration of the convulsant at doses of 30–60 mg/kg.
As described above, mice were placed in cages and observed for 2 h. The limbic convulsions were char- acterized by repetitive rearing and falling, whole body jerks, occasional opisthotonus, Straub tail, and some- times explosive running, that progressed into severe clonic movements involving all four limbs, and, in most cases, status epilepticus. The number of con- vulsing mice out of the total number of mice tested was recorded for each treatment condition. Four groups of mice were injected with various doses of KA and the CD50dose (indicating the convulsive ac- tion of KA) was calculated from a dose-response curve with four data points.
Experimental design
At first, the same dose of AND was injected ip into five groups of mice. The duration of AND pretreat- ment was based on information concerning its bio- logical activity obtained from the literature and con- firmed in our pilot experiments. The doses ranged as
Effect of androsterone on seizures in mice
Katarzyna Mróz et al.
follows: 150–350 mg/kg for the MES, 20–60 mg/kg for PTZ, and 25–100 mg/kg for the KA model. Fifteen minutes after administration of AND, mice were chal- lenged with MES or injected with varying doses of KA or PTZ. The protective effect of AND against MES- induced convulsions was evaluated and expressed as its ED50(effective dose50; the dose protecting 50% of mice against convulsions).The CD50s of PTZ or KA for mice pretreated with AND were calculated and compared with the CD50s of the convulsants adminis- tered separately.
Statistics
The CD50and ED50values with their respective 95%
confidence limits were calculated and statistically analyzed by the computer-assisted log-probit method, according to Litchfield and Wilcoxon [11]. The index of probability of less than 0.05 (p < 0.05) was consid- ered significant in comparative analysis.
Results
Effects of androsterone on maximal electroshock-induced convulsions
As shown in Figure 1, AND administeredip at doses of 150–350 mg/kg exhibited dose-dependent protec- tion against convulsions in the MES test. There was a linear trend between increasing doses of AND and a resultant increase in protection, thus enabling deter- mination of the ED50value (227 mg/kg).
Effects of androsterone on pentylenetetrazole- induced clonic and tonic convulsions
AND injectedip in a dose-dependent manner consid- erably decreased the convulsive action of PTZ (Fig.
2). The CD50of PTZ for clonic convulsions increased from 77.2 mg/kg (PTZ + saline) to 93.9 (p < 0.05) and 113.9 mg/kg (p < 0.001) in mice pretreated with 40 and 60 mg/kg of AND, respectively. Likewise, ad-
age of mice protected against tonic convul- sions. Five groups consisting of 8 mice were tested
Fig. 2.Effects of androsterone (AND) on pentylenetetrazole (PTZ)-induced clonic (¦) and tonic (¾) convulsions.
AND was administered ip in doses of 20, 40, and 60 mg/kg for 15 min before sc injection of PTZ. Each bar reflects the CD# value of PTZ. Four or five groups consisting of 8 mice were tested to calculate each CD# value
ministration of 60 mg/kg AND markedly reduced the potency with which PTZ could induce tonic convul- sions, raising the CD50 of PTZ from 102 (PTZ + sa- line) to 127.6 mg/kg (p < 0.01).
Effect of androsterone on kainate-induced convulsions
AND dose-dependently potentiated KA-induced con- vulsions (Fig. 3). AND administeredip at doses of 50 and 100 mg/kg lowered the CD50values of KA from 40.8 (KA + saline) to 28.7 (p < 0.05) and 25.4 mg/kg (p < 0.001), respectively. Administration of a lower dose of AND (25 mg/kg) had no statistically signifi- cant effect on KA-induced convulsions.
Discussion
The present results clearly demonstrate that AND, a metabolite of testosterone, affects the convulsant ac- tivity in several seizure models in mice. First, AND, significantly and dose-dependently, protected against convulsions evoked by MES, which may serve as a model for human generalized tonic-clonic seizures.
Second, the dose-dependent, anticonvulsant effect of AND was also demonstrated in PTZ-induced clonic convulsions, a model commonly used as a screen to test potential antiepileptic drugs for human myoclonic seizures. Furthermore, AND also inhibited tonic con- vulsions induced by PTZ. Third, AND actedin a dose- dependent manner to significantly increase the inci- dence of convulsions induced by ip KA (a model for complex partial seizures and/or status epilepticus) [17].
Our results confirm and extend the findings of a previous report which demonstrated the ability of AND to prevent convulsions evoked by MES and PTZ in mice [5].In the study by Kaminski et al. [5], the anticonvulsant effect of AND was also docu- mented in 4-aminopyridine- and pilocarpine-induced convulsions. The authors of the study concluded that AND, due to its anticonvulsant activity in experimental models, is worthy of further investigation as a promis- ing candidate for adjunctive treatment in epilepsy.
It is commonly accepted that the mechanism of PTZ-induced convulsant action involves inhibition of GABAA receptor mediated responses [15]. Antiepi- leptic drugs, such as clonazepam, phenobarbital, val- proate, and vigabatrin, that act, at least partially, via potentiation of GABAergic mechanism are highly ef- fective against PTZ-induced clonic convulsions [8, 14]. It has also been suggested that centrally-acting neurosteroids, such as allo-tetrahydrodeoxycorticos- terone, allo-tetrahydrocorticosterone, 5a-androstan- 17b-ol-3-one, 5a,3a-pregnanolone or 5b,3a-pregna- nolone, exert their anticonvulsant actionsvia modula- tion of GABAA-receptor function [2, 8, 20]. Thus, the anticonvulsant effect of AND in the PTZ clonic seizure model may be related to its positive GABAA-receptor modulating activity. Moreover, the hypothesis attrib- uting the anticonvulsant action of AND to the modu- lation of GABAA-receptor function is supported by the findings that AND is effective against convulsions caused by 3-mercaptopropionic acid (3-MPA) in ham- sters [16]. 3-MPA is believed to inhibit glutamic acid decarboxylase (GAD), the enzyme responsible for GABA synthesis, and, to a lesser extent to increase GABA degradation [12]. Agents that increase GAD activity, such as valproate, protect against seizures in- duced by this convulsant [13].
Effect of androsterone on seizures in mice
Katarzyna Mróz et al.
Fig. 3.Effect of androsterone (AND) on kainate (KA)-induced convulsions. AND was administered ip in doses of 25, 50, and 100 mg/kg. KA was injected ip 15 min after saline or AND administration.
Each bar reflects the CD# value of KA.
Four groups consisting of 8 mice were tested to calculate each CD# value
have proconvulsant activity. In view of the aforemen- tioned studies, which clearly outlined the anticonvul- sive properties of AND, the finding of a proconvul- sant effect of this compound is somewhat unexpected.
Following ip injection into mice, neurosteroids with 5a,3a-configurations have been reported to produce a dose-dependent delay in the onset of KA-evoked limbic convulsions, but complete protection from these convulsions was not attained [7]. In another re- port, Leœkiewicz et al. [10] found AND to be ineffec- tive against KA-induced convulsions in mice when it was administered at doses of 10 and 20 mg/kg. This is in agreement with the present study, as the lowest dose of AND tested (25 mg/kg) did not affect the con- vulsive activity of KA. However, administration of higher doses of AND (50 and 100 mg/kg), markedly decreased the CD50 of KA (by 30 and 38%, respec- tively), thus clearly exerting a proconvulsant effect.
On the basis of our results, it is impossible to ex- plain a mechanism for the proconvulsant effect of AND in KA-induced convulsions. Agents blocking N-methyl-D-aspartate and/or a-amino-3-hydroxy-5- methyl-4-isoxazole propionate/kainate (AMPA/KA) receptors are the most effective protectants against KA-induced convulsions [9]. However, drugs that po- tentiate GABAAreceptor responses, such as benzodi- azepines or barbiturates, have been also reported to be effective against these convulsions [6]. In turn, the GABAA receptor antagonist picrotoxin lowered the threshold for KA convulsions in mice [19]. Thus, it seems rather unlikely that the proconvulsant effect of AND is related to an increase of GABAergic system activity; however, modulation of GABAA receptor function cannot be excluded.To clarify this issue, fur- ther studies are needed.
Acknowledgments:
This study was supported by grant PW 525/2007 from Medical University of Lublin, Lublin, Poland.
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Received:
September 26, 2008; in revised form: May 13, 2009.
Effect of androsterone on seizures in mice
Katarzyna Mróz et al.
