The effect of gabapentin and phenytoin on sperm morphology in Wistar rats

Transcription

1 Vol. 7, No short communication The effect of gabapentin and phenytoin on sperm morphology in Wistar rats Akhila J. Shetty 1 Department of Pharmacology, Kasturba Medical College, Manipal University, Light House Hill Road, Mangalore, Karnataka, India Received 28 August 2007; accepted: 31 October 2007 SUMMARY The objective of the present study was to investigate the effects of the antiepileptic drugs, gabapentin and phenytoin, on sperm morphology in Wistar rats. Groups (n=5) of rats were treated with cyclophosphamide (20 mg/day), gabapentin (16, 25, 32 mg/day) and phenytoin (3.5, 5.5, 7 mg/ day) for five consecutive days. 14 and 35 days after the last exposure, sperm morphology was evaluated by standard procedure. Gabapentin and phenytoin did not induce significant changes in sperm morphology. The results suggest that phenytoin and gabapentin are not germ cell mutagens in males, and do not appear to adversely affect male fertility. Reproductive Biology : Key words: gabapentin, phenytoin, spermatozoa, Wistar rats 1 Address for correspondence: Kasturba Medical College, Light House Hill Road, Mangalore 1, Karnataka, India; shettyakhila@yahoo.com Copyright 2007 by the Society for Biology of Reproduction

2 248 Antiepileptic drugs and sperm morphology INTRODUCTION Information concerning the mechanisms by which antiepileptic drugs affect spermatogenesis or sperm function are scarce. Men with epilepsy have reduced fertility, and antiepileptic drugs may affect semen quality as well [6]. Gabapentin (2-[1-(aminomethyl)cyclohexyl]acetic acid), C 9 H 12 NO 2 [2] and phenytoin (5,5-diphenyl-2,4-imidazolidinedione), C 15 H 12 N 2 O 2 [2] are effective for partial and secondarily generalized seizures [1]. Gabapentin releases inhibitory neurotransmitter GABA from neuronal nerve endings, and phenytoin decreases neuronal excitability by delaying the recovery from inactivation of sodium channels [9]. Gabapentin is an antiepileptic with anxiolytic property [4]. Phenytoin is also used in treating digitalis-induced cardiac arrhythmias [10]. This study was undertaken to evaluate the effect of these two drugs on sperm morphology using the rodent model. MATERIALS AND METHODS The study was undertaken after receiving approval from local Ethical Committee For Animal Research. Male Wistar albino rats (9-13 weeks old, g; [11, 12]) were kept in the facilities of Kasturba Medical College (Mangalore, Karnataka, India) under standard laboratory conditions. The rodents had ad libitum access to food and water. Rats were segregated into control and experimental groups (n=5 per group). Control groups were treated with sterile water (0.5 ml; a negative control) or cyclophosphamide (20 mg/day; a positive control). The following drug doses were tested: gabapentin - 16, 25 and 32 mg/day which corresponds to human dose of 900, 1400 and 1800 mg per day, and phenytoin - 3.5, 5.5 and 7.0 mg/day which corresponds to human dose of 200, 300 and 400 mg per day [8]. Both drugs were administered intraperitoneally (ip) every day for five consecutive days [11, 12]. On days 14 and 35 after the last treatment, the animals were anaesthetized with 30 mg/kg pentobarbital sodium (ip) and then sacrificed by cervical dislocation. Laprotomy was conducted, and the right epididymis was

3 Shetty 249 removed and minced in 1 ml phosphate buffered saline (PBS, ph 7.2). The suspension was filtered through 80 µm nylon mesh. One drop of 1% aqueous eosin Y was added to the filtrate and incubated for 30 minutes [11, 12]. For sperm morphology evaluation, one drop of the stained sperm suspension was used to prepare a slide smear. The slides were air-dried, coded and analyzed. 500 spermatozoa per animal were observed under high power objective (magnification 400 ) and classified into normal and abnormal types. The abnormal spermatozoa were classified into two categories: 1/ spermatozoa with defective heads (amorphous, hook less, banana shaped, double headed, microcephaly and cephalocaudal junction defects; and 2/ spermatozoa with defective tails (two-tailed, coiled/bent tails). Data were presented as means+sem and analyzed by Mann-Whitney test. RESULTS AND DISCUSSION There was no statistically significant difference in the number of normal and defective spermatozoa between the control rats and the rats treated with gabapentin or phenytoin (tab. 1). However, the administration of cyclophosphamide (a positive control) caused on day 35 a significant increase (p<0.01) in the number of spermatozoa with tail defects and a decrease (p<0.001) in the number of normal spermatozoa. Antiepileptic drugs affect reproductive and endocrine functions, but their impact on fertility is not well known. Limited information is available concerning the mechanisms by which antiepileptic drugs affect spermatogenesis or sperm functioning [5]. Spermatozoa collected on days 14 and 35 post-treatment, at the time of drug exposure were app. spermatids and spermatogonia, respectively, [11, 12]. Several agents that induce transmissible sperm damage interfere with sperm head morphology. The compounds that induce an increase in sperm head damage are highly correlated with known germ cell mutational activity. This suggests that chemicals yielding positive results in sperm morphology tests should be regarded as potential germ cell mutagens in mammals. The increase in the number of spermatozoa with head defects may be an indication of point mutations. The results of most studies on laboratory

4 250 Antiepileptic drugs and sperm morphology Table 1. Rat sperm morphology parameters (mean±sem) after exposure to gabapentin or phenytoin Drug Dose (mg/ day) Sampling time (day) Normal spermatozoa Spermatozoa with head defects Spermatozoa with tail defects Control group ± ± ±2.7 Phenytoin ± ± ±2.5 Phenytoin ± ± ±3.4 Phenytoin ± ± ±7.0 Gabapentin ± ±2.4 Gabapentin ± ± ±7.0 Gabapentin ± ± ±2.5 Cyclophosphamide ι ± ± ±12.3 Phenytoin ± ± ±3.8 Phenytoin ± ± ±4.4 Phenytoin ± ± ±2.36 Gabapentin ± ± ±6.3 Gabapentin ± ± ±2.2 Gabapentin ± ± ± 6.0 Cyclophosphamide ι ±25.1** 5.0± ±25.1* ι a positive control; each time point and dose represents data from 5 animals (500 spermatozoa per animal) *p<0.01, **p<0.001, significantly different from control group animals and humans suggest that abnormally shaped spermatozoa may not reach the oviduct and/or participate in fertilization. Moreover, an increased percentage of tail abnormalities positively correlates with infertility [11]. Daud et al. [3] reported that gabapentin significantly reduced rat sperm motility and density as well as serum testosterone and FSH levels. These effects were attributed to the higher dosage of the drug used (100 mg/kg bw for 60 days; [3]). The results of several other studies targeting the level of sex hormones in both human females and males showed no significant changes in sex hormones after exposure to gabapentin [3, 7]. The study suggests that gabapentin and phenytoin are not germ cell mutagens in males and that they do not adversely affect male fertility.

5 Shetty 251 REFERENCES 1. Aminoff M 2007 Nervous System. In: Current Medical Diagnosis and Treatment, Eds LM Tierney Jr, SJ McPhee and MA Papadakis, Mc Graw Hill, New York. 2. Budavari S, O Neill MJ, Smith A and Heckelman P 1996 The Merck Index (An Encyclopedia of Drugs Chemicals, and Biologicals). Merck Research Laboratories, New Jersey. 3. Daoud AS, Bataineh H, Otoom S, and Abdul Zahara E 2004 The effect of vigabatrin, lamotrigine and gabapentin on the fertility, weights, sex hormones and biochemical profiles of male rats. Neuro Endocrinology Letters Eisendrath SJ and Lichtmacher J 2007 Psychiatric Disorders. In: Current Medical Diagnosis and Treatment, LM Tierney Jr, SJ McPhee, MA Papadakis, Mc Graw Hill, New York. 5. Hayashi T, Yoshinaga A, Ohno R, Ishii N, Kamata S, Watanabe T and Yamada T 2005 Asthenozoospermia: possible association with long term exposure to an antiepileptic drug carbamazepine. International Journal of Urology Hong CY, Chaput de Saintonge DM and Turner P 1982 Effects of chlorpromazine and other drugs acting on the central nervous system on human sperm motility. European Journal of Clinical Pharmacology Morrell MJ, Sarto GE, Shafer PO, Borda EA, Herzog A and Callanan N 2000 Health issues for women with epilepsy: a descriptive survey to assess knowledge and awareness among healthcare providers. Journal of Women s Health and Gender-Based Medicine Paget GE and Barnes JM 1964 Toxicity tests. In: Evaluation of Drug Activities: Pharmacometrics, Eds DR Laurence, AL Bacharach, Academic press, London. 9. Satoskar RS, Bhandarkar SD, and Nirmala NR 2005 Pharmacology and Pharmacotherapeutics, Popular Prakashan, Mumbai, India. 10. Seth SD 1999 Text Book of Pharmacology. B. I. Churchill Livingstone Pvt. Ltd, New Delhi, India. 11. Shetty AJ and Narayana K 2007 The effects of carbamazepine on sperm morphology in Wistar rats. Indian Journal of Physiology and Pharmacology Shetty AJ, Narayana K, Bairy KL, Bhat P, and D souza U 2007 The effect of carbamazepine on sperm counts in Wistar rats - reflecting upon its mitogenic potential. Reproductive Biology ACKNOWLEDGEMENTS Author wishes to thank Drs C.V. Raghuveer and Khadilkar U., Kasturba Medical College, Mangalore, Karnataka, India for their support during the conduct of the study.