Gonadotoxicity Evaluation of Oral Artemisinin Derivative in Male Rats
I.O. Osonuga ,
O.S. Akinsomisoye ,
Male Wister albino rats were exposed to artemether by gavage at dosages of 25, 50 and 75 mg kg-1 day-1 for 1, 2 and 3 days. The control groups received sterile water (control 1) and 5% ethanol (vehicle for artemether, control 2). The maximum volume injected in all groups was 0.5 mL. Rats administered the highest dose for three days were mated with female rats to determine the fertilizing capacity of their epididymal sperms and fertility status. Artemether significantly reduced (p<0.05) the progressive sperm motility, viability, sperm count and serum testosterone levels in dose and duration dependent manners, factors that may impair fertility. None of the untreated cohabited female rats got pregnant throughout the period of the study. These changes were restored in recovery experiments. The results suggest that artemether could induce reversible infertility in rats.
Artemether is an antimalarial drug used in the treatment of all forms of malaria due to Plasmodium falciparum, Plasmodium ovale and Plasmodium malariae and it is the most active derivative of a new class of antimalarial drugs that are chemically unrelated to existing drugs. Its efficacy against multi-drug resistant falciparum malaria and its potential to delay antimalarial drug resistance have led to its increasing use in recent years.
Many antimalarial drugs have been implicated in male infertility. For instance, chloroquine, quinine and quinacrine have been reported to inhibit Leydig cell steroidogenesis and fertility in male. Chloroquine has also been reported to reduce sperm motility and hence fertility by a reduction in the average number of fetuses of cohabited female rats. Pyrimethamine, an anaphylactic antimalarial drug has been shown to cause spermatogenic arrest and male infertility in mice. Interestingly antimalarial medicinal plant extracts have also been reported in experimental male infertility. For example, Quassia amara which was reported to be highly potent against chloroquine resistant Plasmodium falciparum produced significant reduction in epididymal sperm counts, serum levels of testosterone, luteinizing hormone and follicle stimulating hormone in male rats[7,8]. Joshi et al. also reported a mass atrophy of the spermatogenic elements and Leydig cells when Azadirachta indica extract was administered to male rats. More recently, Raji et al.[10,11] reported a dose dependent decrease in serum testosterone and luteinizing hormone when Azadirachta indica and Morinda lucida extracts were individually administered to male rats. These medicinal plants are commonly used in folkloric medicine to treat malaria and have been reported to possess antiplasmodium activities in mice.
The strong efficacy of artemether to various forms of malarial parasites made its introduction and use in malarial chemotherapy globally acceptable. It is therefore imperative to evaluate the reproductive activities of the drug in experimental model, since none is currently available. This study was therefore designed to evaluate the effects of artemether on male reproductive functions in albino rats.
MATERIALS AND METHODS
Animals: Wistar strain albino rats (190-230 g) obtained from the Central Animal house, College of Medicine, University of Ibadan were used for the study. The animals were fed with standard rat cubes (Ladokun feeds Nig. Ltd.) and water ad libitum. They were housed at room temperature under photoperiod-controlled environment (12 h light: 12 h dark light cycles).
Drug and dose regimens: Artemether (Rhone-Poulene Rorer International, France) was obtained from the University of Ibadan Health center and administered in 5% ethanol (vehicle for artemether) orally at doses of 25, 50 and 75 mg kg-1 day -1 to male rats for 1, 2 and 3 days. Each group has its corresponding positive and negative controls whose rats received sterile water and 5% ethanol, respectively.
Study protocol: The study was divided into three experimental sections as follows:
Experiment 1: Five male rats each were treated with 25, 50 and 75 mg kg-1 artemether. Each group was run for 1, 2 and 3 days.
Experiment 2- Recovery studies: Twenty-five male rats were divided into
five equal groups and each group was administered sterile water only, 5% ethanol
only, 25, 50 and 75 mg kg-1day-1 artemether, for three
days and allowed to recover from the drug for another three days.
Experiment 3- Fertility studies: Twenty-five male rats divided into
three equal groups were treated, respectively with sterile water only, 5% ethanol
only, 25, 50 and 75 mg kg-1day-1 artemether, for three
days. They were then introduced to proestrous female rats (200 g) of proven
fertility at a ratio of 2 males to 3 females. A single time point fertility
test for each rat was carried out using the following formula:% Fertility success
= number of pregnant female rats divided by number of mated female rats multiplied
by 100 as earlier described.
Rats were weighed and sacrificed by exsanguinations under 25% urethane anesthesia (0.6 mL/100 g body weight) about 24 h after the last artemether administration. Blood was collected from each rat via cardiac puncture from which the serum was separated. Testosterone concentration in the serum was measured using the enzyme immunoassay (EIA) technique as previously described.
Sperm characteristic analysis: Semen collected from the cauda epididymis was used in the determination of the progressive sperm motility, viability, count and morphology[11,13].
Statistical analysis: Data were presented as Mean±SEM and statistical analyses were carried out using the Students t-test and ANOVA. Significant difference was accepted at p<0.05.
Effect of artemether on body weight: There was no significant change
in body and reproductive organ weights of artemether treated rats when compared
with the controls (Data not shown).
||Effects of artemether on sperm parameters after 1, 2 and 3
days of treatments and recovery
|| Effects of artemether on sperm morphological parameters
|| Effects of artemether on serum testosterone levels
This trend was also observed in the recovery group.
Effect of artemether on sperm parameters (sperm counts, viability and motility
and morphology): Administration of artemether at 25, 50 and 75 mg kg-1
day-1 significantly reduced (p<0.05) the progressive sperm motility,
sperm count and sperm viability in dose and duration dependent manners when
compared with the controls (Table 1). Drug withdrawal resulted
in gradual restoration of sperm parameters (Table 1). In almost
all the test groups, the most common abnormality encountered was the simple
bent tail. Administration of artemether caused dose and duration dependent
increase in the number of abnormal sperms when compared with the controls. However
there was a significant decrease (p<0.05) in the number of abnormal sperms
encountered in the recovery group (Table 2).
Effect of artemether on serum testosterone level: Serum testosterone levels of artemether treated rats significantly reduced in dose and duration dependent manners when compared with the controls. However there was an appreciable increase in serum testosterone levels of rats in the recovery group (Table 3).
Fertility studies: All rats that received the various doses of the drug did not produce any litter. The control groups sired 8.00±2.00 and 7.10±1.80 physically normal litters, respectively.
The results suggest that artemether could cause reversible impairment to reproductive
activity in male albino rats. Gonadotoxic effects of many antimalarial agents
have been demonstrated in many animal species including human. Antimalarial
drugs such as chloroquine and quinines[3,4,14] have been reported
to be toxic to germ cells and cause spermatogenic arrest. Chloroquine has also
been demonstrated having varied effects on male reproductive functions including
fertility reduction in the male rats and complete obliteration
of Leydig cell response to leutropin and hormones having leutropin like activity
in vitro. Chloroquine was reported to have an activating
influence on bovine sperm respiration and motility in vitro as well as
stimulatory to aged porcine spermatozoa motility. Adeeko and
Dada also reported that chloroquine caused a dose dependent reduction
in fertility of male rats as evidenced by a reduction in average number of fetuses
of cohabited females. Okanlawon and Ashiru showed that in chloroquine
treated rats; there was a disruption of spermatogenesis, which was accompanied
by a decline in serum concentrations of testosterone in these rats. These effects
of chloroquine were reported in all cases to be reversible.
In the present study artemether caused significant reduction in sperm motility
in dose and duration dependent manners while there was an increase in the number
of dead caudal epididymal sperm. Sperm motility is usually acquired partly in
the epididymis raising the suspicion that the deleterious effect of artemether
is on the storage of sperms in the epididymis. These observed reductions in
sperm functions might be related to the altered micro-environment in the epididymis
and/or the drug might be toxic to maturing or mature spermatozoa in the epididymis.
The decrease observed in sperm count could then be the result of the increase
in number of dead spermatozoa. Artemether appears to be different from other
antimalarial drugs in its having an endoperoxide bridge thereby generating single
oxygen and free radicals. Free radicals have been proposed as
a major cause of defective sperm function in cases of male infertility.
The reduced serum testosterone concentration during artemether administration
confirmed that the drug could suppress Leydig cell steroidogenesis leading to
reduction in testosterone production and hence reduced sperm motility and fertility
potentials. This probably led to the inability of the untreated female rats
to conceive upon cohabitation with artemether treated male rats. The actions
of artemether appear reversible within the therapeutic doses and durations employed
in this study. It is obvious that spermatogenic cycle was not covered and whether
the reproductive organs could concentrate or store artemether is also not known
yet. Ongoing studies in our laboratory will address these issues and the detail
probable mechanism of action of artemether on reproductive organs.
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