Download Effect of metronidazole on spermatogenesis and FSH, LH and

Indi an Journal of Experimental Bio logy
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
J K Grover, V Vats, M Srinivas*, S N Das t , P Jha+, D K Gupta* & D K Mitra'
- ',
Departments of Pharmaco logy, Pediatric Surgery*, Biotechnolog/ and Reprod ucti ve Biologi
All Indi a Institute of Medi cal Sciences, New Delhi 110049. Indi a
E mail: jkgrover@hotmai l.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, testic ul ar and epid idy mal
spermatid counts and causes abnormal sperm morphology with degeneration of sem iniferous tubules with 6 weeks treatment
of metro nid azo le (400 mg/kg, day). In con trast to DNA fl ow cy tometry (FC M), the histological and grav imetric parameters
do not allow a rapid, sensitive, objective and muitiparameteric evaluation of reprod ucti ve toxicants on spermatogenesis.
Moreover, the exact mechanisms for suc h an effect are not en tirely clear. The present study was therefo re undertaken to
assess th e effects of intraperitonea l (i.p.) administration of metronidazo le 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 spermatotox ic effects of
metronidazole were probably mediated by dec rease in the ci rculating hormones responsible fo r spermatogenesis.
Over the past 50 years, there has been an appreciable
decrease in male fertility in human population t and
si nce it is directly related to sperm count and
morphology, the result may reflect an overall
reduction in this process. Among the many factors
that have been cited as possible causes of such an
effect, environmental factors such as exposure to
drugs and chemicals have particularly gained
recognition in the past two decades 2 . In addition,
environmental presence of certain therapeutic drugs
has been shown 3 .
Metronidazole, the first clinically effective 5-nitroimidazole drug has received widespread therapeutic
use in the treatment of various infections especially
anaerobes. Although in clinical use for many years,
except metallic taste, headache, dry mouth, gastritis,
glossitis and urticaria4 serious side effects with the use
of thi s drug has not been reported. Less common side
5
effects such as motor-sensory neuropath/· ,
8
ototoxicitl, esophageal ulcers7, pancreatitis and
hepatotoxici ty 9 have also been described. Few of its
metabolites have been shown to be mutagenic in
certain bacterial test systems such as Ames testlO.l I . In
addition, genotoxic potenti al has also been shown in
human lymphocyte cultures of the patients exposed to
metronidazole 12 .
Studies have also described the metronidazole as a
·
.
13·15
repro d uctIve
toxicant
. H owever, there are no
reports assessing the effects of metronidazole on
maturating testes. Hence, this study was designed to
assess the effects of metronidazole on pre-pubertal
rats. In addition, the previous studies have assessed
the effects of metronidazole on parameters like
testicular histology, testicular sperm head counts,
cauda sperm counts and testicular and epididymal
weights. These parameters though adequate, do not
allow a rapid, sensitive, objective and mu]tiparametcric
evaluation of reproductive toxicants as compared to
flow cytometric studies of spermatogenesis that
allows identification of germ cell SUb-populations
undergoing both proliferative and maturative
processes in normal and perturbed conditions 16. In
addition, FCM is better for assessing germ cell
.
17
Ch anges In testes .
Therefore the present study was undertaken to
assess the effects of administration (ip) of
metronidazole 400 mg/kg daily for 30 day s on
testicular germ cell changes assessed by DNA FCM
and hormone levels of testosterone, FSH and LH .
Experimental design-A total of 40 pre-pubertal
male rats (30 days old) were procured from the
experimental animal facility of the Institute. The rats
were housed in cages in the centrally air-conditioned
animal laboratory. Before the start and during the
experiment, rats were fed standard chow diet and tap
water ad libitum. After acclimatization period of 3
I
...
NOTES
days, they were randomly divided into two groups A
and B. Whil e group A received metronidazole 400
mg/kg.day daily for 30 day s as i.p. injection , rats in
group B received saline I ml daily as i.p. injection . At
the end of study, the animal s were sacrificed under
li ght ether anesthesia; their blood was collected,
centri fuged and th e serum was sto red at -20°C fo r
assess ment of FSH , LH and testosterone levels. The
testicl es were harvested and tran sported in Rosewell
Park Memorial Institute so lution for DNA FCM.
DNA FCM-Freshly harvested testes were
separated from the tuni ca albuginea and minced in
phosphate buffer saline (PBS ). The resultant sin gle
cell suspension was was hed twice in PBS and a 100!AI aliquot of the suspension was fixed in 70% eth ano l
in PBS. After centrifu gati on, the pellet was resuspended in propidium iodide and subj ected to RNA
digestion by R Aase (S igma, USA) . DNA
histograms were then obtained on a tl owcyto meter
(Becton-Dickinson FAC Scan). The data then
obtained were analyzed by Cell Fit Software.
Radio-illllllllilOassay- FSH , LH, and testosterone
leve ls were determined by radi o- immunoassay. FSH
and LH serum levels were determined by RIA
fo ll owi ng the instructions given with the reagents
generousl y provided by the Nati onal Hormone and
Pituitary Program of NroDK (Baltimore, MD). The
LH reference preparation was LH -RP-2 and the
anti serum was anti-rat LH-RIA-Il. The rFSH
reference preparation was rFSH-RP-2 and the
anti serum was anti-rat FSH-S-Il .
Statistical analysis-Student's T test (GraphPAD,
InStat, 1990, Versi on l.l4, INSERM 920666S, India)
was used for comparing the differences between two
groups and a probability of less than 0.05 was
considered stati stically significant.
The mean percentage of haploid, diploid and
tetraploid cells is shown in Table I along with serum
levels of FSH, LH and testosterone. A significant
reduction (P < 0.001) in haploid cell population in
metronidazo le treated groups as compared to saline
treated controls was observed. In proportion , the
percentage of diploid and tetraploid cells was
increased in metronid azo le treated group. Al so
observed a significant reduction in mean serum FSH,
LH and testostero ne (P < 0.00 I) levels in treated
groups.
Dose of metronidazole was selected on the basis a
J
previous stud/ in which metronidazole (400 mg/kg.day
daily for 6 weeks) resulted in adverse effects on the
mating performance and fertility parameters of rats.
11 6 1
Table I -Effect of intra-peritoneal administration of metronidazole
(400 mg/kg.day) on DNA hi stograms of rat testi s and hormonal
levels of LH , FSH and testosterone
[Values are mean ± SD for groups of 20 animals each]
Saline treated
Metronid azo le
Treated (400 mg/kg)
73 .2±2.04
17.54±1.34
9.03± l.l7
42 .73±3.70
44. 12±2.39
13.07±.47
15.36±1.09
0.461 ±0.0547
1.44±0.22 1
11.65±.90
0.254±0.0463
0.735±0.189
DNA Histograms
Haplo id cell s (%)
Diploid cells (% )
Tetraploid cell s (%)
Hormollaiiel'eis
FSH (ng/IllI )
LH (ng/ml )
Testosterone (nlllo llL)
Treated gro up was co mpared wit h saline con tro ls
All va lues are statistically significant at P < 0.000 I
Seminiferous epithelium cycle in the rat te tes has
14 stages lS and identification of variou s stages of
development is a tedi ous process, which is prone to
inter and intra observer variations. The usual go ld
l9
standard , the John sen testicular maturation score
may thu s not be accurate. DNA FCM analysis
correlates strongly with the current standard of quan titative spermatogenic assessment and is a simp lified
and highly objective method of determining spermatogenesis as a statisticall y significant correlati on has
been shown between the percentage of haploid cell s
and tubular concentration of late spermatids an d
tubular spermatid-to-Sertoli cell rati0 2o .
Testicular
suspension
contains
3
distinct
populations of cells each having a different DNA
content, the tetraploid peak mainly represents primary
spermatocytes, spermatogonial G 2 cells and spermatogonial G I cells while seco ndary spermatocytes
constitute the diploid peak; the spermatids and
spermatozoa are haploid. Thus, the percentage of
haploid cells is directly proportional to sperm
maturation. In this study, metronidazo le caused
dramatic fall in the haploid cell percentage in haploid
cell population around 60 days post-natally implyin g
delayed testicular maturation . The present study
supports the previous findings of McClain et 01 13.
Metronidazole significantly (P < 0.000 I) lowered
the levels of FSH, LH and testosterone. Thus, the
toxic effects of metronidazole on spermatogenesis
were probably mediated by decrease in the circulating
hormones responsible for spermatogenesis. However,
oral metronidazole treatment (500 mg/day) fo r 4
weeks in humans failed to alter the levels of total
testosterone, free testosterone and dihydrotestosterone
1162
IND IAN J EXP BIOL, NOVEMBER 200 1
while th e levels of estradio l and dehydroepiandrosterone sulfate were decreased significantly.21 Neither,
did metronidazole inhibited ill vitro testicular 17 alphahydroxylase and 17,20-lyase activit/ 2 or testosterone
biosynthesis in ill vitro mouse Leydig cells cultures23 .
though other imidazole drugs such as ketoconazo le,
bi fonazole, c1otrimazole, miconazo le and isoconazole
have been shown to inhibit biosy nthesis of testi cul ar 17
al pha-hydrox y lase activi ty .
To our knowledge this is the first study whi ch has
evaluated the ill vivo effect of metronidazole on
reproductive hormones in rats. It needs to be seen
whether the lower doses of this drug have the same
effects on reproductive hormones and testes and
whether this process is reversible.
II
12
13
14
15
16
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