Accepted: 9 June 2016 DOI: 10.1111/and.12669
ORIGINAL ARTICLE
Testicular toxicity following separate and combined administration of PDE5 inhibitors and opioid: assessment of recovery following their withdrawal V. U. Nna | E. E. Osim Department of Physiology, Faculty of Basic Medical Sciences, College of Medical Sciences, University of Calabar, Calabar, Cross River State, Nigeria Correspondence Victor U. Nna, Department of Physiology, Faculty of Basic Medical Sciences, College of Medical Sciences, University of Calabar, Cross River State, Nigeria. Email:
[email protected]
Summary We previously observed that PDE5 inhibitors and opioids were widely abused in Nigeria. Here, we examined the effect of high doses of sildenafil, tadalafil, tramadol and sildenafil + tramadol on reproductive toxicity in male rats. Rats were either administered normal saline (0.2 ml), sildenafil (10 mg/kg), tadalafil (10 mg/kg), tramadol (20 mg/kg) or sildenafil + tramadol (10 and 20 mg/kg respectively) p. o. for 8 weeks. The recovery groups were allowed 8-week recovery period before sacrifice. Results showed that body weight change, testicular and epididymal weights, epididymal sperm count and sperm viability were significantly reduced in all treated groups compared with the control. Spermatozoa with abnormal morphology were significantly increased in all treated groups compared with the control. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) were significantly reduced, while malondialdehyde (MDA) was significantly increased in all treated groups compared with the control. The severity of toxicity was highest in sildenafil + tramadol group. There was no complete recovery from reproductive toxicity following withdrawal of the various treatments. High doses of sildenafil, tadalafil, tramadol or sildenafil + tramadol result in testicular oxidative stress-induced reproductive toxicity with poor reversal following withdrawal. KEYWORDS
erectile dysfunction, oxidative stress, PDE5 inhibitors, spermatogenesis, tramadol
1 | INTRODUCTION
action in delaying ejaculation. The recreational use of these medications has been reported to be on the rise (Nna, Ofem, & Osim, 2016).
The maintenance of sexual potency is a matter of great concern for
So far, the desire to achieve a harder and longer lasting erection, the
the male population. As a result, several sex stimulants have been
need to delay ejaculation and the quest for an increased genital size
employed to manage the two widely known male sexual act-related
are all reported reasons why young men resort to taking sex stimulants
problems: erectile dysfunction (ED) and premature ejaculation (PE).
recreationally (Bechara, Casabé, De Bonis, Helien, & Bertolino, 2010;
Several drugs have been produced to treat ED by inhibiting phospho-
Nna et al., 2014, 2016). Furthermore, our previous studies report-
diesterase-5 (PDE5) which is responsible for downregulating cGMP (a
ed high incidence of abuse of ED and PE medications, with respon-
key component of the pathway for penile erection). Tramadol hydro-
dents confessing to taking far above recommended doses (probably
chloride, developed in the late 1970s, has been undergoing various
due to psychological dependence) (Bechara et al., 2010; Nna et al.,
clinical trials in a bid to assess its suitability in treating PE since it inhib-
2016). Recently, a new combination therapy (sildenafil + tramadol) has
its serotonin and norepinephrine re-uptake—a possible mechanism of
evolved in Nigeria. This combination therapy was formulated following
Andrologia. 2017;49:e12669. https://doi.org/10.1111/and.12669
wileyonlinelibrary.com/journal/and
© 2016 Blackwell Verlag GmbH
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previous speculations that erectile and ejaculatory dysfunctions often
administration (recovery test), but were given ad libitum access to rat
occurred together (Blanker, Bohnen, et al., 2001; Blanker, Bosch, et al.,
chow and water. After the recovery period, the animals were sacri-
2001).
ficed and their left testis and epididymis also harvested for analysis as
Users of sex stimulants have reported their side effects to include
in treatment groups.
stomach pain and headache (Nna et al., 2016). Several authors have reported detrimental effects not easily discernible like headache and stomach pain, but serious enough to impair reproductive function
2.3 | Assessment of sperm motility
parameters (Ahmed & Kurkar, 2014; Bliesener et al., 2005). However,
Sperm motility was assessed by placing 10 μl of sperm suspension
it is unclear whether reproductive deficits associated with prolonged
collected from the left epididymis on a clean pre-warmed slide, cov-
use of these drugs at far above recommended doses are reversible
ered with a coverslip and examined using a light microscope (Leica
following their withdrawal. Furthermore, there are no reports on the
DM 750, Switzerland) equipped with a heated stage (37°C), at 100×
effects of the combination therapy (sildenafil + tramadol) in vogue in
magnification (Atashfaraz, Farokhi, & Najafi, 2013).
Nigeria today on reproductive function parameters. Therefore, this present study was aimed at assessing the effect of long-term use of these medications (including the combination therapy) on reproduc-
2.4 | Determination of epididymal sperm count
tive function parameters, as well as the effect of withdrawal of treat-
Assessment of epididymal sperm count was done using the method
ment on same.
described by Freud and Carol (1964). The left cauda epididymis from each rat was placed in 2 ml of normal saline, pre-warmed to 37°C.
2 | MATERIALS AND METHODS 2.1 | Laboratory animals Seventy (70) male albino Wistar rats aged 8 weeks and weighing
Small incisions were made in the cauda epididymis and spermatozoa were obtained and suspended in saline solution. Two hundred microlitres of the suspension was transferred to both chambers of a Neubauer haemocytometer using a Pasteur pipette by touching the edge of the coverslip and allowing each chamber to be filled by cap-
180–200 g were used for this study. The animals were purchased
illary action. The epididymal sperm count for each animal was then
from the Department of Agriculture, Faculty of Science, and housed
obtained and recorded.
in the Department of Physiology Animal house, University of Calabar, Nigeria. Standard animal cages with wood dust as bedding were used in keeping the animals. They were allowed ad libitum access to rat chow and clean water, and exposed to 12/12-hr light/dark cycle.
2.5 | Assessment of sperm viability and morphology Sperm viability was evaluated using the method described by Wyrobek
The animals were acclimatised for 7 days. Indeed, the animals were
et al. (1983). Twenty microlitres of 0.05% eosin Y–nigrosin was added
kept in line with laid down principles for animal care as prescribed
to an equal volume of sperm suspension and incubated at room tem-
in Helsinki’s 1964 declaration. The animal ethics committee of the
perature for 2 min. After incubation, all slides were viewed under a
University of Calabar graciously approved our study protocol.
light microscope (Leica DM 750) at magnifications of ×100 and ×400. Live spermatozoa were not stained, while dead spermatozoa were
2.2 | Experimental design and drug administration
stained pink. For each assay, 400 spermatozoa were counted and viability percentages were calculated (Wyrobek et al., 1983).
The rats were assigned into 2 major groups: (A) treatment and (B) recovery groups (n = 35). Each group was further divided into 5 subgroups (n = 7): control (0.2 ml normal saline), sildenafil-treated (10
2.6 | Antioxidant and lipid peroxidation assessment
mg/kg), tadalafil-treated (10 mg/kg), tramadol-treated (20 mg/kg) and
The left testes of the animals were homogenised using a Potter-
sildenafil + tramadol-treated group (10 and 20 mg/kg respectively).
Elvehjem homogenizer. A 20% (1/5 w/v) homogenate of the tissue
Sildenafil citrate (Maxheal Laboratories Pvt Ltd, India), tadalafil (Pfizer,
was prepared in 50 mm Tris–HCl buffer (pH 7.4) containing 1.15%
India) and tramadol hydrochloride (Glow Pharma Pvt Ltd, India) were
potassium chloride and centrifuged at 10,000 g at 4°C for 10 min. The
purchased from Unipervit Pharmacy, Ikot Omin, Calabar, Nigeria. The
supernatant was collected for the assessment of catalase (CAT) activ-
different drugs were orally administered, once, every two days as
ity using hydrogen peroxide as the substrate. The H2O2-mediated
used in our previous reports (Nna, Akpan, Okon, & Atangwho, 2015;
oxidation of Fe+2 to Fe+3 was measured spectrophotometrically at
Nna, Akpan, & Osim, 2015), while the control group received normal
560 nm using xylenol orange dye (FOX-1) (Clairborne, 1995). Lipid
saline as vehicle. The drugs were administered to both treatment and
peroxidation was quantified as malondialdehyde (MDA) accord-
recovery groups for 8 weeks, after which animals in the treatment
ing to the method described by Ohkawa, Ohishi, and Yagi (1979).
groups (A) were sacrificed under chloroform anaesthesia and the left
Briefly, 300 μl of 10% trichloroacetic acid was mixed with 150 μl of
testis and epididymis carefully harvested for semen analysis, oxida-
the sample and centrifuged at 1,000 g at 4°C for 10 min. Thereafter,
tive stress assessment and histopathological examination. Animals in
300 μl of the supernatant was transferred to a test tube with 300 μl
the recovery groups (B) were allowed another 8 weeks without drug
of 67% thiobarbituric acid and incubated for 25 min at 100°C. Five
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minutes after cooling the solution, a pink colour appeared because
weight, significantly (p
