Effect of Metronidazole on Spermatogenesis and FSH, LH and Testosterone Levels of Pre-Pubertal Rats

Indi an Journal of Experimental Biology Vo l. 39. November 2001 , pp. 11 60- 11 62

Effect of metronidazole on spermatogenesis and FSH, LH and testosterone levels of pre-pubertal rats

t J K Grover, V Vats, M Srinivas*, S N Das , P Jha+, D K Gupta* & D K Mitra' Departments of Pharmacology, Pediatric Surgery*, Biotechnolog/ and Reproducti ve Biologi - ', All Indi a Institute of Medi cal Sciences, New Delhi 110049. Indi a E mail: jkgrover@hotmail. com. Fax: 9 1- 11-6862663 Received 14 February 200/; revised 16 May 200J

Metronidazole, a 5-nitroimidazole drug has been reported to decrease testi cular weight, testicul ar and epididy mal spermatid counts and causes abnormal sperm morphology with degeneration of seminiferous tubules with 6 weeks treatment of metro nidazole (400 mg/kg, day). In contrast to DNA fl ow cytometry (FCM), the histological and gravimetric parameters do not all ow a rapid, sensitive, objective and muitiparameteric evaluation of reproducti ve toxicants on spermatogenesis. Moreover, the exact mechanisms for such an effect are not entirely clear. The present study was therefore undertaken to assess th e effects of intraperitoneal (i.p.) administration of metronidazole 400 mg/kg dail y for 30 days on testi cul ar germ cell changes assessed by DNA (FCM) and hormone levels of testosterone, FSH and LH in pre-pubertal rats. A significant reduction in the haploid cell population in metronidazole treated groups as compared to saline treated controls was observed. The mean serum FSH, LH and testosterone value were also lowered in treated animals. Thus, the spermatotoxic effects of metronidazole were probably mediated by decrease in the ci rculating hormones responsible fo r spermatogenesis.

Over the past 50 years, there has been an appreciable reports assessing the effects of metronidazole on decrease in male fertility in human population t and maturating testes. Hence, this study was designed to si nce it is directly related to sperm count and assess the effects of metronidazole on pre-pubertal morphology, the result may reflect an overall rats. In addition, the previous studies have assessed reduction in this process. Among the many factors the effects of metronidazole on parameters like that have been cited as possible causes of such an testicular histology, testicular sperm head counts, effect, environmental factors such as exposure to cauda sperm counts and testicular and epididymal drugs and chemicals have particularly gained weights. These parameters though adequate, do not 2 recognition in the past two decades . In addition, allow a rapid, sensitive, objective and mu]tiparametcric environmental presence of certain therapeutic drugs evaluation of reproductive toxicants as compared to 3 has been shown . flow cytometric studies of spermatogenesis that ... Metronidazole, the first clinically effective 5-nitro­ allows identification of germ cell SUb-populations I imidazole drug has received widespread therapeutic undergoing both proliferative and maturative use in the treatment of various infections especially processes in normal and perturbed conditions 16. In anaerobes. Although in clinical use for many years, addition, FCM is better for assessing germ cell . 17 except metallic taste, headache, dry mouth, gastritis, Ch anges In testes . glossitis and urticaria4 serious side effects with the use Therefore the present study was undertaken to of thi s drug has not been reported. Less common side assess the effects of administration (ip) of 5 effects such as motor-sensory neuropath/· , metronidazole 400 mg/kg daily for 30 days on ototoxicitl, esophageal ulcers7, pancreatitis8 and testicular germ cell changes assessed by DNA FCM hepatotoxicity 9 have al so been described. Few of its and hormone levels of testosterone, FSH and LH. metabolites have been shown to be mutagenic in Experimental design-A total of 40 pre-pubertal certain bacterial test systems such as Ames testlO.l I . In male rats (30 days old) were procured from the addition, genotoxic potential has also been shown in experimental animal facility of the Institute. The rats human lymphocyte cultures of the patients exposed to were housed in cages in the centrally air-conditioned 12 metronidazole . animal laboratory. Before the start and during the Studies have also described the metronidazole as a experiment, rats were fed standard chow diet and tap repro ductIve· toxicant. 13·15 . H owever, t h ere are no water ad libitum. After acclimatization period of 3 NOTES 11 6 1 days, they were randomly divided into two groups A Table I -Effect of intra-peritoneal administration of metronidazole and B. While group A received metronidazole 400 (400 mg/kg. day) on DNA hi stograms of rat testi s and hormonal mg/kg.day daily for 30 days as i.p. injection, rats in levels of LH , FSH and testosterone [Values are mean ± SD for groups of 20 animals each] group B received saline I ml daily as i.p. injection. At the end of study, the animals were sacrificed under Saline treated Metronidazole li ght ether anesthesia; their blood was collected, Treated (400 mg/kg) centri fuged and th e serum was stored at -20°C fo r DNA Histograms assessment of FSH, LH and testosterone levels. The Hapl oid cell s (%) 73.2±2.04 42.73±3.70 testicles were harvested and transported in Rosewell Diploid cells (%) 17.54±1.34 44. 12±2.39 Park Memorial Institute solution for DNA FCM. Tetraploid cell s (%) 9.03± l.l7 13.07±.47 DNA FCM-Freshly harvested testes were Hormoll aiiel'eis separated from the tunica albuginea and minced in FSH (ng/IllI ) 15. 36±1.09 11.65±.90 phosphate buffer saline (PBS). The resultant sin gle LH (ng/ml ) 0.461 ±0.0547 0.254±0.0463 cell suspension was washed twice in PBS and a 100- Testosterone (nlllollL) 1.44±0.221 0.735±0.189 !AI aliquot of the suspension was fixed in 70% eth anol Treated group was compared with saline contro ls in PBS. After centrifugati on, the pellet was re­ All valu es are statistically significant at P < 0.000 I suspended in propidium iodide and subjected to RNA digestion by R Aase (S igma, USA). DNA Seminiferous epithelium cycle in the rat te tes has lS histograms were then obtained on a tl owcytometer 14 stages and identification of various stages of (Becton-Dickinson FAC Scan). The data then development is a tedious process, which is prone to obtained were analyzed by Cell Fit Software. inter and intra observer variations. The usual gold l 9 Radio-illllllllilOassay- FSH , LH, and testosterone standard, the Johnsen testicular maturation score levels were determined by radi o-immunoassay. FSH may thus not be accurate. DNA FCM analysis and LH serum levels were determined by RIA correlates strongly with the current standard of quanti­ foll owing the instructions given with the reagents tative spermatogenic assessment and is a simplified generousl y provided by the National Hormone and and highly objective method of determining spermato­ Pituitary Program of NroDK (Baltimore, MD). The genesis as a statistically significant correlati on has LH reference preparation was LH-RP-2 and the been shown between the percentage of haploid cell s anti serum was anti-rat LH-RIA-Il. The rFSH and tubular concentration of late spermatids and 2o reference preparation was rFSH-RP-2 and the tubular spermatid-to-Sertoli cell rati0 . anti serum was anti-rat FSH-S-Il . Testicular suspension contains 3 distinct Statistical analysis-Student's T test (GraphPAD, populations of cells each having a different DNA InStat, 1990, Version l.l4, INSERM 920666S, India) content, the tetraploid peak mainly represents primary

was used for comparing the differences between two spermatocytes, spermatogonial G2 cells and spermato­ groups and a probability of less than 0.05 was gonial G I cells while secondary spermatocytes considered stati stically significant. constitute the diploid peak; the spermatids and The mean percentage of haploid, diploid and spermatozoa are haploid. Thus, the percentage of tetraploid cells is shown in Table I along with serum haploid cells is directly proportional to sperm levels of FSH, LH and testosterone. A significant maturation. In this study, metronidazole caused reduction (P < 0.001) in haploid cell population in dramatic fall in the haploid cell percentage in haploid metronidazole treated groups as compared to saline cell population around 60 days post-natally implyin g treated controls was observed. In proportion, the delayed testicular maturation. The present study percentage of diploid and tetraploid cells was supports the previous findings of McClain et 01 13. increased in metronidazole treated group. Al so Metronidazole significantly (P < 0.000 I) lowered observed a significant reduction in mean serum FSH, the levels of FSH, LH and testosterone. Thus, the LH and testosterone (P < 0.00 I) levels in treated toxic effects of metronidazole on spermatogenesis groups. were probably mediated by decrease in the circulating Dose of metronidazole was selected on the basis a hormones responsible for spermatogenesis. However, previous stud/J in which metronidazole (400 mg/kg.day oral metronidazole treatment (500 mg/day) fo r 4 daily for 6 weeks) resulted in adverse effects on the weeks in humans failed to alter the levels of total mating performance and fertility parameters of rats. testosterone, free testosterone and dihydrotestosterone 1162 INDIAN J EXP BIOL, NOVEMBER 200 1

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