Received: 15 December 2020 Revised: 18 June 2021 Accepted: 28 June 2021

DOI: 10.1111/bcpt.13633

ORIGINAL ARTICLE

Effects of dipyrone and acetylsalicylic acid on contractions

of distal cauda epididymis duct, serum testosterone and
sperm count in rats

Ana Beatriz Melo Martins1 | Mayara Samala Bezerra1 |

Luana Talinne da Costa Gomes1 | Francisco Mateus Gonçalves Trajano1 |
Pedro Brüch Dantas1 | Maele Oliveira de Sena1 | Elaine Cristina Gavioli2 |
Edilson Dantas da Silva Junior1,2

1
Mode of Drug Action Laboratory,
Federal University of Rio Grande do
Norte, Natal, Brazil
2
Department of Biophysics and
Pharmacology, Federal University of Rio
Grande do Norte, Natal, Brazil
Correspondence
Edilson Dantas da Silva Junior,
Department of Biophysics and
Pharmacology, Federal University of Rio
Grande do Norte, Av. Senador Salgado
Filho, s/n Campus Universit
ario – Lagoa
Nova, Natal, 59072-970, RN. Brazil.
Email: edsjunior9@gmail.com
Funding information
Conselho Nacional de Desenvolvimento
Científico e Tecnol
ogico, Grant/Award
Number: 406996/2018-0; National Council
for Scientific and Technological
Development; Universidade Federal do
Rio Grande do Norte, Grant/Award
Number: Young Investigator Grant
03/2018; Federal University of Rio Grande
do Norte
Abstract
The effects of dipyrone and acetylsalicylic acid (ASA) on male fertility are still
not fully understood, mainly considering the epididymis as a putative target
for their anti-fertility effects. Therefore, this study aimed to investigate the
effects of dipyrone and ASA on the contractions of distal cauda epididymis
duct, serum testosterone levels and sperm parameters in rats. Firstly, we
checked the in vitro effects of dipyrone and ASA (10–1000 μM) on the contrac-
tions of distal cauda epididymis duct by pharmacological experiments. We also
evaluated the effects of in vivo treatment with dipyrone and ASA 100 mg/kg
(p.o.) for 15 days on epididymal duct contractions, serum testosterone levels
and sperm parameters. In vitro dipyrone or ASA decreased the epididymal
duct contractions induced by phenylephrine or carbachol. We observed that
in vivo treatment with both drugs decreased the daily sperm production,
serum testosterone levels and sperm count through epididymis without alter-
ing the epididymal duct contractions and sperm transit time through epididy-
mis. In conclusion, in vitro dipyrone and ASA were able to diminish the
contractions of epididymal duct, whilst in vivo administration decreased the
sperm count throughout epididymis as a consequence of a low sperm produc-
tion caused by reduced testosterone levels.

KEYWORDS
acetylsalicylic acid, dipyrone, distal cauda epididymis duct contraction, sperm parameters,
testosterone levels

1 | INTRODUCTION AND frequently used for pain relief.1–4 The mechanism by

BACKGROUND which these drugs induce their therapeutic or adverse/
toxic effects is through the inhibition of cyclooxygenase
Non-opioid analgesic drugs such as dipyrone (COX), leading to decreased prostaglandin synthesis.5,6
(metamizole) or acetylsalicylic acid (ASA or aspirin) are It is noteworthy that although dipyrone had been

Ana Beatriz Melo Martins and Mayara Samala Bezerra contributed equally.

Basic Clin Pharmacol Toxicol. 2021;1–13. wileyonlinelibrary.com/journal/bcpt © 2021 Nordic Association for the Publication of BCPT 1
(former Nordic Pharmacological Society)
2 MARTINS ET AL.
withdrawn from the US and Canadian markets, it is still
available and one of the most used non-opioid analgesic
drugs in many countries (Brazil, Mexico, Germany,
Russia, Italy, Switzerland and China).1,2,7
Both cyclooxygenase isoforms (COX-1 and COX-2),
prostaglandins and their receptors were reported in the
male reproductive tract,8–10 with robust evidence indicat-
ing an important role of prostaglandins on regulating
steroidogenesis,11–13 spermatogenesis,14 sperm func-
tion12,15,16 and smooth muscle contraction.8,17 As such, it
is expected that prostaglandin synthesis inhibition,
induced by non-opioid analgesic drugs, could have pro-
found effects on male fertility. In fact, previous studies
demonstrate that ASA decreased testosterone production
by human Leydig cells,18 including epididymal sperm
concentration and sperm motility in rats.19 Similar
effects were described for other COX inhibitors, such as
paracetamol and nonsteroidal anti-inflammatory drugs
(naproxen, indomethacin, meloxicam, etc.) in humans
and rodents (for review see Drobnis and Nangia11). Yet,
the adverse effects of dipyrone on male fertility have not
been fully evaluated, thus remaining elusive.
There is growing evidence that the epididymis is an
important target for anti-fertility effects of different
drugs.8,11 This organ exerts a central role on male fertility
by promoting sperm transport, maturation and storage
until the moment of ejaculation.20 The sperm transiting
through the epididymis is a result of smooth muscle
motor activity of the epididymal duct, which is regulated
by androgens, neurotransmitters (acetylcholine via mus-
carinic receptors or noradrenaline via α1A-adrenoceptors)
and autacoids, including prostaglandins.8,21,22 Moreover,
it is during sperm transit in the epididymal duct that
these cells undergo the maturation process, acquiring the
ability to move forward and fertilise oocytes.20 Several
studies demonstrate that alterations in the motor activity
of the epididymal duct, due to exogenous drugs that can
affect the quality and quantity of sperm cells in the ejacu-
late, impair male fertility (as reviewed by Drobnis and
Nangia11 and Elfgen et al8). However, there is a lack of
information of COX inhibition, exerted by dipyrone or
ASA, as a reduction of prostaglandin levels is related to
changes in epididymal duct contractions and alterations
in the quality and quantity of sperm cells.
In this study, two of the most frequently used non-
opioid analgesic drugs (dipyrone and ASA) were evalu-
ated in order to check their effects on epididymal duct
contractions and various sperm parameters. First, we
checked the in vitro effects of dipyrone and ASA on the
contractions evoked by potassium chloride (KCl) or exog-
enous autonomic drugs (phenylephrine-adrenergic drugs,
carbachol-cholinergic drugs) in segments of the distal
cauda epididymis duct that are isolated from adult Wistar
rats. Second, we evaluated in vivo treatment with
dipyrone and ASA for 15 days, regarding contractions
from autonomic drugs in segments of the rat distal cauda
epididymis duct, serum testosterone levels, sperm pro-
duction and count and transit time in different parts of
the epididymis.
2 | MATERIAL A ND METHODS
2.1 | Animals and treatment
Male Wistar rats with 60–90 days old and weighing 200–
300 g were obtained from Animal Facility of Bioscience
Center from Federal University of Rio Grande do Norte
(Natal, Brazil). The animals were kept under controlled
conditions (25 C, 12/12 h light/dark cycle) with food and
water ad libitum. All experimental procedures were pre-
viously approved by the local Ethics Committee for de
Use of Experimental Animals of Federal University of
Rio Grande do Norte (Protocol number 0023/2019). The
study was conducted in accordance with the Basic &
Clinical Pharmacology and Toxicology policy for experi-
mental and clinical studies.23 All the animal experiments
were performed in a blinded fashion. A total number of
40 male rats were used in this study.
In a first series of experiments, animals (n = 19)
were euthanised by decapitation in order to check the
in vitro effects of dipyrone and ASA on the contractions
of distal cauda epididymis duct. Thus, another set of
animals were randomly divided into three experimental
groups: control (treated with drug free-vehicle, p.o.,
daily gavage for 15 days; n = 7), dipyrone (100 mg/kg,
p.o., daily gavage for 15 days; n = 7) and ASA-treated
animals (100 mg/kg, p.o., daily gavage for 15 days;
n = 7). The choice of dose, route of administration and
duration of the treatment were based on previous
reports that assess the effects of both drugs on male
reproductive system of rodents.11,24–28 It is also note-
worthy to mention that the animal dose of 100 mg/kg
for dipyrone or ASA corresponds to an equivalent dose
in human of 16 mg/kg or 967 mg (considering 60 kg
human)29 which is lower than the maximal daily dose
for both drugs (4000 mg).30,31
The animals were weighed daily and used in the
experiments 24 h after the last drug administration. All
animals were euthanised by decapitation between 8:00
and 9:00 AM, and blood samples from control or treated
animals were collected for serum testosterone determina-
tion. Moreover, reproductive organs (testis, epididymis,
ventral lobe prostate, empty seminal vesicle and vas
deferens) were isolated from connective tissues
and weighted (wet weight), segments of distal cauda
MARTINS ET AL.
epididymis duct used for functional experiments as well
as the sperm cells recovered from testis or epididymis
(caput/corpus and cauda) for analysing of daily sperm
production, sperm reserves and sperm transit time trough
epididymis.
2.2 | Isolation of distal cauda epididymis
duct of rats for functional experiments
The rats were euthanised by decapitation and then the
whole epididymis were exposed, cleaned from connective
tissues, and 1.5 cm of distal cauda epididymis duct (site
7 according to Hinton et al)32 were isolated and mounted
under 1 g of resting tension22,33,34 in a 10-ml organ bath
containing nutrient solution with composition (mM):
138.0 NaCl; 5.7 KCl; 1.8 CaCl2
2H2O; 15.0 NaHCO3; 0.36
NaH2PO4
H2O and 5.5 glucose, prepared in glass distilled
deionised water, bubbled with normal air and kept at
32 C, pH 7.4.33,35 Alterations in isometric tension of dis-
tal cauda epididymis duct isolated from rats were
detected by a force displacement transducer (Ugo Basile,
Italy) coupled to a Gemini two-channel physiographic
recorder (Ugo Basile, Italy). After mounting of prepara-
tion, the tissues were allowed to stabilise for 30 min and
then KCl 80 mM were incubated for 5 min in order to
check tissue viability and maximal response stabilisation.
Thereafter, the preparation was carefully washed out and
after further 40 min of stabilisation the experiments were
carried out.
2.2.1 | Evaluation of in vitro effects of
dipyrone and ASA on contractions of distal
cauda epididymis duct
Distal cauda epididymis duct from untreated animals
were isolated and mounted in an organ bath preparation
as described in Section 2.2. After checking tissue viability
with KCl and 40-min stabilisation time, time-course
response for KCl 80 mM or cumulative concentration
response curves for phenylephrine (agonist of α1-
adrenoceptors) or carbachol (agonist of acetylcholine
muscarinic receptors) in the absence (control curves) or
presence of three distinct concentrations of dipyrone
(10, 100 and 1000 μM) or ASA (10, 100 and 1000 μM).
Each concentration of dipyrone or ASA was equilibrated
with tissues for 30–40 min. The dipyrone and ASA con-
centrations used in this study were based on published
IC50 values of both drugs for COX inhibition: dipyrone,
IC50 (COX-1) = 350 μM36 and IC50 (COX-2) > 1000 μM37;
ASA, IC50 (COX-1) = 500 μM38,39 and IC50 (COX-2)
= 1413 μM.39
3
Additionally, pharmacological parameters Emax
(maximal contraction)35,40 and pEC50 (agonist potency -
the negative log of EC50)41 were graphically determined
from concentration response curves in order to allow
comparisons between agonist curves in the absence or
presence of dipyrone or ASA.
2.2.2 | Analysis of in vivo treatment with
dipyrone and ASA on contractions of distal
cauda epididymis duct
A set of animals were treated with drug-free vehicle,
dipyrone or ASA for 15 days followed by the isolation of
distal cauda epididymis duct as described in section 2.1.
Then, segments of distal cauda epididymis duct from all
experimental groups were mounted in a standard organ
bath preparation and after 30 min of stabilisation time,
the tissues were challenged with KCl 80 mM for 5 min to
check viability. Next, the tissues were washed out and
allowed to equilibrate for further 40 min (the presence
of spontaneous contractions was verified for the last
5–10 min of equilibration time). After equilibration time,
cumulative concentration response curves for phenyleph-
rine or carbachol were performed in different tissues
from experimental groups. Pharmacological parameters
described above (Section 2.2.1) were obtained from con-
centration response curves for agonists in order to allow
comparisons between experimental groups.
2.3 | Serum testosterone determination
Blood samples were collected at the end of the treatment
from all experimental groups. Then, serum was obtained
by centrifugation and frozen until the moment of hor-
mone measurements. The analysis of serum testosterone
levels was performed at DNA Center Laboratory (Natal,
Brazil) by means of chemiluminescence immunoassay
(Access Testosterone kit, Beckman Coulter), with a sensi-
tivity of 10 ng/dl.
2.4 | Daily sperm production, sperm
reserves and sperm transit time through
epididymis
The testes from drug-free vehicle, dipyrone or ASA were
isolated, decapsulated, weighed and then homogenised in
5 ml of NaCl 0.9% containing Triton X 100 0.5%. Then,
the homogenates were sonicated for 30 s and after a
10-fold dilution, one sample was transferred to Neubauer
chambers (four samples per animal) for counting the
4 MARTINS ET AL.
homogenisation-resistant testicular spermatids (stage
19 of spermiogenesis). The daily sperm production (DSP)
was calculated by dividing the number of spermatids (per
testis) at stage 19 by 6.1 (which corresponds to the num-
ber of days these spermatids are found in the seminifer-
ous epithelium). In addition, caput/corpus and cauda
epididymis from control, dipyrone or ASA-treated ani-
mals were separated, weighed and cut into small pieces
and then homogenised. Next, sperm cells were counted
as described above for the testis. Sperm transit time
through the caput/corpus and cauda epididymis was
obtained by dividing the number of sperm cells found in
each of these regions by DSP.33,42
2.5 | Data and statistical analysis
The contractile responses were calculated and expressed
as a percentage of the maximum response observed in the
preparation, using KCl 80 mM (% KCl contraction - to
control for unwanted sources of response variation as tis-
sue length, excessive tissue handling during isolation or
organ bath mounting) or whenever appropriated as gra-
mme of tension (g). Curve fitting by non-linear regression
for the calculation of pEC50 or Emax was performed with
GraphPad Prism v.6 software (San Diego, CA, USA).
Values are expressed as means  standard error of mean
(S.E.M.). Unpaired Student’s t-test was used for compari-
sons between two groups. Additionally, one-way ANOVA
followed by the Dunnet’s posttest was also used for com-
parisons between groups three or more groups. A p value
of less than 0.05 was considered to be statistically signifi-
cant. The results were obtained from groups of at least five
experiments with different tissues from distinct animals.
2.6 | Drugs and reagents
The following drugs were used: phenylephrine and carba-
chol from Sigma Chemical Co. (St. Louis, MO, USA),
sodium dipyrone from Purifarma (Brazil) and ASA from
Valdequímica (Brazil). All reagents used for nutrient
solutions were from Dinâmica (Brazil).
3 | R E SUL T S
3.1 | In vitro effects of dipyrone and ASA
on contractions of distal cauda
epididymis duct
The in vitro effects of different concentrations of
dipyrone or ASA (10, 100 and 1000 μM) were checked in
the contractions of rat distal cauda epididymis duct
induced by KCl 80 mM for 5 min (Figure 1A,D, respec-
tively) or by cumulative addition of agonists phenyleph-
rine (Figure 1B,E, respectively) or carbachol (Figure 1C,
F, respectively). Neither dipyrone nor ASA was able to
significant alter the KCl-induced epididymal duct con-
tractions (Figure 1A,D). On the other hand, dipyrone
100 and 1000 μM decrease the Emax for phenylephrine by
about 20% and 40%, respectively (Figure 1B and Table 1).
No significant alteration was found in the potency for
phenylephrine in the presence of dipyrone (Table 1). In
addition, the pre-incubation of dipyrone 1000 μM
decreased the Emax for carbachol by 30% (Figure 1C and
Table 1) and elicited a threefold reduction on the potency
of this agonist (Table 1). ASA 100 and 1000 μM also
reduced the Emax for phenylephrine (by 20% and 30%,
respectively) without changing the potency of this agonist
(Figure 1E and Table 2). The Emax and the potency for
carbachol were significantly reduced (20% and 2.5
fold, respectively) by the presence of ASA 1000 μM pre-
incubated for 40 min (Figure 1F and Table 2).
3.2 | Effects of in vivo treatment with
dipyrone and ASA on animals and
reproductive organ weight
The control animals showed a weight gain of
25.8 g  10.25 (n = 7) after 15 days of the treatment.
Similarly, dipyrone-treated animals also showed a weight
gain of 48.6 g  8.7 (n = 7) which was not different from
control group (P > 0.05). On the other hand, the treat-
ment with ASA for 15 days induced a significant weight
loss of 62.7 g  24.12 (P < 0.05 compared to control).
The in vivo treatment with dipyrone also decreased the
relative weight of the testis and epididymis compared to
control (Table 3). On the other hand, the in vivo treat-
ment with ASA did not modify the relative reproductive
organ weight (Table 3).
3.3 | Effects of in vivo treatment with
dipyrone and ASA on contractions of distal
cauda epididymis duct
The in vivo treatment with dipyrone or ASA for 15 days
was unable to modify the contractions of rat distal cauda
epididymis duct evoked by depolarizing agent KCl 80 mM
for 5 min (Figure 2A,D). Thus, in vivo treatment with
dipyrone or ASA 100 mg/kg for 15 days did not modify
the contractions induced by the adrenergic agonist phen-
ylephrine or cholinergic agonist carbachol (Figure 2B,C,E
and F), as demonstrated by unaltered values of Emax or
MARTINS ET AL. 5

F I G U R E 1 Effects of dipyrone or acetylsalicylic acid (ASA) 10, 100 or 1000 μM pre-incubated for 40 min on rat distal cauda
epididymis duct contractions induced by KCl 80 mM for 5 min (panels A and D, respectively) or cumulative addition of crescent
concentrations of phenylephrine (panels B and E, respectively) or carbachol (panels C and F, respectively). Each bar graph (panels A and D)
or point (panels B, C, E and F) represents means  S.E.M. of 6–7 independent experiments performed with tissues from different animals.
*P < 0.05 in relation to Emax of control. n = number of experiments

pEC50 (potency) for these agonists between control and
treated animals (Table 4). It is worthy to note that the
treatment with ASA showed a non-significant increase
(P = 0.09) of the maximal contraction induced by phenyl-
ephrine in the distal cauda epididymis duct of rats
(Figure 2E and Table 4). In addition, the in vivo treatment
with dipyrone or ASA did not induce any changes in the
spontaneous contractions of distal cauda epididymis duct.
Tissues from control or treated animals showed low spon-
taneous motor activity with frequency inferior to 1 sponta-
neous contraction/min (Figure 3).
testosterone levels by about 60% for both drugs
(Figure 4). Furthermore, the number of spermatids in the
testis and daily sperm production were decreased after
the in vivo treatment with dipyrone or ASA (Table 5).
The sperm reserves in caput/corpus and cauda epididy-
mis were also reduced in the dipyrone or ASA-treated
animals, whilst no significant alteration was found in the
sperm transit time through caput/corpus or cauda epidid-
ymis compared to control (Table 5).
4 | DISCUSSION

3.4 | Effects of in vivo treatment with
dipyrone and ASA on serum testosterone
levels and sperm parameters
The animals treated with dipyrone or ASA 100 mg/kg
for 15 days presented a drastic reduction on serum
This study sought to evaluate anti-fertility effects of
dipyrone and ASA (COX inhibitors and widely used non-
opioid analgesic drugs) by investigating their effects on
contractions of the distal cauda epididymis duct, testos-
terone levels and sperm parameters in rats. We show that
in vitro dipyrone or ASA could alter the contractions of
6 MARTINS ET AL.

T A B L E 1 Maximal contractions (Emax) and pEC50 values for in sperm production and reserves through the epididy-
phenylephrine or carbachol in the absence (control) or presence of mis. We argue that these effects may be related to
dipyrone (10, 100 or 1000 μM; pre-incubated for 40 min) in distal decreased testosterone levels in treated animals, with a
cauda epididymis duct of rats minor role in the epididymal duct’s motor activity.
Pharmacological parameters The pharmacological effects of dipyrone and ASA are
largely associated with the inhibition of cyclooxygenase
Emax pEC50
(COX-1 and COX-2) and consequent decrease in prosta-
Phenylephrine glandin synthesis,4–6 although other mechanisms are also
Control (n = 6) 135.2  9.1 5.7  0.17 reported (modulation of the endocannabinoid system,
+ Dipyrone 10 μM (n = 6) 117.3  6.6 5.6  0.19 nitric oxide [NO] synthesis, and the noradrenergic and
+ Dipyrone 100 μM (n = 6) 108.8  6.3 a
5.4  0.26 cholinergic system, etc.).6 Both COX-1 and COX-2 were
found in different parts of the rodent epididymis. For
+ Dipyrone 1000 μM (n = 6) 79.1  5.7 a
5.5  0.11
instance, COX-1 and COX-2 immunoreactivity were
Carbachol
found in the caput, corpus and cauda of the rat epididy-
Control (n = 6) 116.7  7.2 5.4  0.08 mis and in different cell types (COX-1 on basal cells and
+ Dipyrone 10 μM (n = 6) 113.1  6.1 5.2  0.13 COX-2 on principal cells).10 In the mouse epididymis,
+ Dipyrone 100 μM (n = 6) 98.2  8.8 5.1  0.09 COX-1 was found in all parts of the epididymis, whilst
+ Dipyrone 1000 μM (n = 6) 80.6  8.7 a
4.9  0.09a COX-2 was found only in the distal cauda region.9
It is shown that different prostaglandins (PGD2,
Note: Emax (maximal contraction) expressed as % of KCl contraction and
pEC50 (potency, measured as the negative log of EC50) obtained from the
PGE2 and PGF2alpha) can induce epididymal duct con-
nonlinear regression curves shown in Figure 1B and C. n, number of tractions (for review see Elfgen et al8) and modulate
experiments. spontaneous contractions of the bovine epididymal
a
P < 0.05 in relation to control. duct,43 as well as affect the contractions induced by auto-
nomic neurotransmitters in the distal cauda epididymis
T A B L E 2 Maximal contractions (Emax) (expressed as % KCl duct of rats.44 In the latter case, Hib and Oscar,44 using
contraction) and pEC50 values for phenylephrine or carbachol in in vivo measurements of epididymal duct contractions in
the absence (control) or presence of acetylsalicylic acid (ASA; 10, anaesthetised rats, show that exogenous administration
100 or 1000 μM; pre-incubated for 40 min) in distal cauda of PGE2 and PGF2alpha potentiated contractions of nor-
epididymis duct of rats adrenaline or acetylcholine, whilst indomethacin (COX-
Pharmacological parameters inhibitor) infusion decreased contractile responses, in
turn affected by both agonists.
Emax pEC50
In this study, we found that in vitro incubation of
Phenylephrine both COX inhibitors dipyrone (at 1000 μM) and ASA
Control (n = 7) 143.8  4.7 5.7  0.10 (>100 μM) decreased contractions of the distal cauda epi-
+ ASA 10 μM (n = 7) 130.8  4.5 5.6  0.11 didymis duct, induced by phenylephrine (agonist of α1-
+ ASA 100 μM (n = 7) 112.8  5.8 a
5.5  0.12 adrenoceptors) or carbachol (agonist of acetylcholine
muscarinic receptors), as seen by a significant reduction
+ AAS 1000 μM (n = 7) 99.2  6.7a 5.6  0.12
of the maximal effect of both agonists, without altering
Carbachol
contractile responses of the depolarizing agent KCl.
Control (n = 6) 138.0  8.0 5.5  0.08 These data may imply that cyclooxygenase-mediated
+ ASA 10 μM (n = 6) 131.2  5.5 5.3  0.08 prostaglandin synthesis is part of the signalling pathway
+ ASA 100 μM (n = 6) 121.8  6.1 5.2  0.08 for α1-adrenoceptors and muscarinic receptors in the dis-
+ ASA 1000 μM (n = 6) 109.3  8.1 a
5.1  0.07a tal cauda epididymis duct of rats, contributing to contrac-
tile responses evoked by autonomic agents and
Note: Emax (maximal contraction) expressed as % of KCl contraction and
pEC50 (potency, measured as the negative log of EC50) obtained from the
reinforcing the role of autacoids on general function. We
nonlinear regression curves shown in Figure 1E and F. n, number of noted that stimulation of prostaglandin synthesis, as a
experiments. consequence of α1-adrenoceptors or muscarinic receptor
a
P < 0.05 in relation to control. activation, was found in other isolated tissues as well
(the aorta and trachea) and/or cells (primary cell culture
of the splenic pulpa and smooth muscle cells, etc.).45–48
the distal cauda epididymis duct by autonomic drugs but In vivo treatment with ASA for 15 days reduced body
not by the depolarizing agent KCl. Thus, we observed weight, whilst dipyrone-treated animals showed a
that in vivo treatment with both drugs created alterations body weight gain similar to that of controls. We
MARTINS ET AL. 7

T A B L E 3 Reproductive organs weight (g tissue/100 g animal) for animals treated with drug-free vehicle (control), dipyrone or
acetylsalicylic acid (ASA) 100 mg/kg for 15 days

Experimental groups
Reproductive organ weight
(g tissue/100 g animal) Control (n = 7) Dipyrone 100 mg/kg (n = 7) ASA 100 mg/kg (n = 7)
Testis 0.46  0.01 0.40  0.01 a
0.49  0.01
Epididymis 0.15  0.007 0.13  0.004 a
0.16  0.007
Vas deferens 0.018  0.002 0.018  0.001 0.025  0.001
Empty seminal vesicle 0.11  0.007 0.10  0.007 0.11  0.007
Ventral lobe prostate 0.08  0.005 0.08  0.07 0.09  0.07

Note: Values are means  S.E.M. (number of experiments).

a
P < 0.05 in relation to control.

F I G U R E 2 Effects of in vivo treatment with drug-free vehicle (control), dipyrone or acetylsalicylic acid 100 mg/kg for 15 days on rat
distal cauda epididymis duct contractions induced by KCl 80 mM for 5 min (panels A and D) or cumulative addition of crescent concentrations
of phenylephrine (panels B and E) or carbachol (panels C and F). Each bar graph (panels A and D) or point (panels B, C, E and F) represents
means  S.E.M. of 6–7 independent experiments performed with tissues from different rats. n = number of experiments

speculated that body weight loss in rats in conjunction
with treatment of ASA could be related to a gastrointesti-
nal adverse effect, via the daily administration of this
drug, suggested by other studies.49–52 Dipyrone-treated
animals presented a decrease in the testes and
epididymal relative weight (possibly due to alterations in
sperm count, discussed below), whilst no significant dif-
ferences were found in ASA-treated animals. Absence of
ASA effects on the relative weight of the testes and
epididymis could indicate that another mechanism
8 MARTINS ET AL.

(dependent on COX inhibition or not), although not
explored on this paper, can prevent organ weight loss
with altered sperm counts.
COX inhibitors, including non-opioid analgesic drugs
(paracetamol and dipyrone) or nonsteroidal anti-
inflammatory drugs (aspirin, ibuprofen, naproxen, etc.),
are commonly used drugs, with impact on the reproduc-
tive system. For instance, animal studies indicate
negative effects of COX inhibitors on testosterone levels
(paracetamol and naproxen), spermatogenesis (ASA),
quantity and quality (motility, DNA fragmentation
and morphology alteration) of sperm (indomethacin,
ibuprofen and paracetamol) and pregnancy rates (indo-
methacin).11 In humans, COX inhibitors such as ASA,
paracetamol or ibuprofen were related to poor sperm
quality.11,53,54 Yet, effects of dipyrone on the male repro-
ductive system and the role of the epididymis in anti-
fertility effects on COX inhibitors must be carefully
studied.
In our study, rats submitted to the in vivo treatment
with dipyrone or ASA for 15 days presented marked
reduction in the number of spermatids, daily sperm pro-
duction and testosterone levels. Previous studies demon-
strated that both drugs could reduce testosterone levels in
rodents or isolated cells28,55 as this anti-androgenic effect
was reported for other COX-inhibitors, such as
paracetamol,56 indomethacin,57 or ibuprofen.58 The
reduction of testosterone levels by dipyrone or ASA
could also induce an impairment of spermatogenesis,
with a decrease in daily sperm production; this is
also reported for other agents with similar ability to inter-
fere with androgen synthesis, such as fluoxetine,33
dexamethasone,59 bisphenol-A,60 amongst others.11,61
The exact mechanism underlying the reduction of tes-
tosterone levels by non-opioid analgesics and nonsteroi-
dal anti-inflammatory drugs is still uncertain and under
investigation. It is described that ASA and paracetamol
decreased testosterone synthesis in rat foetal testes, with

T A B L E 4 Maximal contractions (Emax) and pEC50 values for

phenylephrine or carbachol in distal cauda epididymis duct from
rats treated with drug-free vehicle (control), dipyrone or
acetylsalicylic acid (ASA) 100 mg/kg for 15 days

Pharmacological parameters

Emax pEC50
Phenylephrine
Control (n = 6) 0.64  0.10 5.9  0.16
Dipyrone 100 mg/kg (n = 7) 0.60  0.07 5.9  0.11
Control (n = 6) 0.51  0.09 6.0  0.09
ASA 100 mg/kg (n = 7) 0.67  0.08 5.8  0.12
Carbachol
Control (n = 6) 0.59  0.10 5.3  0.08
Dipyrone 100 mg/kg (n = 6) 0.62  0.06 5.3  0.12
Control (n = 6) 0.43  0.05 5.3  0.14
ASA 100 mg/kg (n = 7) 0.58  0.06 5.2  0.11 F I G U R E 4 Serum testosterone levels (ng/dl) from rats
submitted to the in vivo treatment with drug-free vehicle (control;
Note: Emax (maximal contraction) expressed as % of KCl contraction and
pEC50 (potency, measured as the negative log of EC50) obtained from the n = 5), dipyrone (n = 7) or acetylsalicylic acid (n = 5) 100 mg/kg
nonlinear regression curves shown in Figure 1B, C, E and F. n, number of for 15 days. Each bar graph represents means  S.E.M. *P < 0.05 in
experiments. relation to control. n = number of experiments

F I G U R E 3 Original tracings showing the spontaneous contractions of distal cauda epididymis duct from control (left panel), dipyrone
(middle panel) or acetylsalicylic acid (ASA) (right panel) treated animals. Tissues from control or treated animals showed low spontaneous
motor activity with frequency inferior to 1 spontaneous contraction/min
MARTINS ET AL. 9

TABLE 5 Sperm parameters obtained from rats treated with drug-free vehicle (control), dipyrone or acetylsalicylic acid 100 mg/kg for
15 days

Experimental groups

Sperm parameters Control (n = 7) Dipyrone 100 mg/kg (n = 7) ASA 100 mg/kg (n = 7)

Testis
Number of spermatids, x106/testis 320.5  47.5 176.4  10.0a 181.5  8.7a
6
Number of spermatids, x10 /g/testis 189.2  17.7 97.9  5.9 a
106.2  6.4a
Daily sperm production 52.5  7.8 28.9  1.6a 29.7  1.4a
Caput/corpus of epididymis
Sperm number, x106/organ 155.0  24.7 74.4  5.8a 79.9  12.2a
7
Sperm number, x10 /g/organ 40.8  5.0 18.2  1.6 a
21.2  1.7a
Transit time (days) 3.1  0.3 2.6  0.3 2.7  0.4
Cauda of epididymis
Sperm number, x106/organ 158.4  14.3 72.6  5.6a 78.0  5.2a
7
Sperm number, x10 /g/organ 66.7  5.4 28.5  2.0 a
32.5  2.5a
Transit time (days) 3.4  0.5 2.6  0.3 2.6  0.14

Note: Values are Means  S.E.M. n, number of experiments.

a
P < 0.05 in relation to control.

no connection to COX inhibition and prostaglandin syn-
thesis.56 Thus, van den Driesche et al62 showed that para-
cetamol reduced intratesticular levels of testosterone by
decreasing gene expression of enzymes involved in ste-
roidogenesis. Similarly, dipyrone decreased testosterone
synthesis by inhibiting enzymes in the different steps of
steroidogenesis in NCI-H295R cells.24 On the other hand,
indomethacin reduced testosterone levels in the rat foetal
testes, associated with a decrease in testicular prostaglan-
din synthesis through COX inhibition.56 Similarly, ibu-
profen reduced testosterone and prostaglandin levels in
adult male testes, as this drug can suppress gene expres-
sion of enzymes involved in cholesterol transport and ste-
roidogenesis.58 As such, it is plausible to assume that
COX inhibition and the consequent decrease in prosta-
glandin synthesis could be involved in anti-androgenic
effects of non-opioid analgesics, including dipyrone and
ASA, and nonsteroidal anti-inflammatory drugs; particu-
lar off-target effects of each drug could be relevant here.
Dipyrone and ASA-treated animals also showed a sig-
nificant decrease in sperm count in all parts of the epidid-
ymis. Similar results were previously described for ASA
in rodents.19,27 This is the first study, to our knowledge,
yielding evidence about a decrease in sperm count in dif-
ferent regions of the rat epididymis caused by in vivo
treatment with dipyrone. The diminished daily sperm
production caused by low testosterone levels in treated
animals could be an obvious mechanism behind alter-
ations in sperm count through the epididymis. Yet, it has
been shown that alterations in epididymal duct contrac-
tility affected by different drugs are an important mecha-
nism associated with alterations in sperm count and
quality: this occurs by accelerating or retarding the tran-
sit time of sperm cells through this organ.33,35,63,64 We
hypothesise that prostaglandin synthesis disruption in
the epididymis, a function of in vivo treatment with
COX-inhibitors dipyrone and ASA, could affect epididy-
mal smooth muscle contractions, with alterations in
sperm transit time, and contribute to the low sperm cell
count in this organ.
Although our in vitro experiments demonstrated an
inhibitory effect evoked by dipyrone or ASA, the in vivo
treatment with both drugs failed to affect contractions of
the distal cauda epididymis duct from KCl, phenyleph-
rine (adrenergic agonist) or carbachol (cholinergic
agonist). Further, we were unable to find any significant
alteration in sperm transit time through the caput/corpus
and cauda epididymis in treated animals compared to
controls, indicating no significant changes in epididymal
duct motor activity. The long-term effects of COX-
inhibitors, including dipyrone and ASA on epididymal
contractions and consequences in sperm transport, were
poorly investigated and few published data present con-
trasting information. For instance, oral administration of
paracetamol 500 mg/kg or 1000 mg/kg for 30 days did
not alter epididymal contractility65, whilst indomethacin
10 mg/kg (2x daily) for 35 days decreased epididymal
contractions by noradrenaline or acetylcholine.44 For
10
both studies, no information was provided about sperm
transit time through the epididymis in treated animals.
More studies are needed to further evaluate the role of
COX inhibition on epididymal function, mainly after
long-term treatment with different agents.
There are some limitations in this study that should
be addressed in future studies. First, different doses
(50, 100 and 200 mg/kg) and time of drug treatment
(7, 15 and 30 days) could be used to check the dose and
time dependency of anti-fertility effects, considering the
epididymis to be a potential target. Second, evaluation of
LH, FSH, prolactin and inhibin levels in animals submit-
ted to in vivo treatment with dipyrone or ASA should
also be performed. It is possible that dipyrone and ASA
could affect these hormones, leading to a differential out-
come in reproductive organ weight. Finally, we found a
tendency to change in the contractions of distal cauda
epididymis duct induced by KCl 80 mM after in vivo
treatment with ASA 100 mg/Kg. In this case, cumulative
or non-cumulative concentration response curves for this
depolarizing agent could be performed in order to evalu-
ate a putative involvement of potassium channels on
ASA effects.
We must mention that the clinical relevance of male
reproductive effects of COX inhibitors in humans is still a
matter of debate. Hoyer et al66 showed no association
between paracetamol or nonsteroidal anti-inflammatory
drug intake and semen impairment (sperm concentration
in the ejaculate, sperm motility, semen volume, etc.).
There is also evidence about an alteration in semen qual-
ity (sperm morphology or a reduction in sperm motility)
after the the use of paracetamol67 or other nonsteroidal
anti-inflammatory drugs.68 It is argued that reproductive
toxicity of non-opioid analgesic or nonsteroidal anti-
inflammatory drugs in humans seems dependent on the
duration of exposure, doses, or even the type of drug.68
Further studies are necessary to unveil the impact of
these drugs on male fertility, for example, dipyrone,
which is one of the most prescribed or over-the-counter
non-opioid analgesic drugs in different countries,1–4
where the reproductive effects in humans are open to
investigation.
In conclusion, in vitro dipyrone and ASA negatively
affected contractions of the rat distal cauda epididymis
by autonomic drugs; this indicates that prostaglandins
are involved in signal transduction of adrenoceptors
and muscarinic receptors in this tissue. Daily adminis-
tration of both drugs for 15 days decreased sperm
count through the epididymis, which may be associated
with impaired sperm production by reduced testoster-
one levels, with minor involvement of epididymal duct
contractions.
MARTINS ET AL.
A C KN O WL ED G EME N T S
Thanks are due to Fl
avio Maurílio dos Santos Lima,
Cesar Augusto Trevisan Bordignon and Ramon Tadeu
Galv~ao Alves Rodrigues for excellent technical support
and to the Pro-Rectory of Research of Federal University
of Rio Grande do Norte (Portuguese: Pro-Reitoria de Pes-
quisa da Universidade Federal do Rio Grande do Norte)
(Young Investigator Grant 03/2018) and The Brazilian
National Council for Scientific and Technological
Development (Portuguese: Conselho Nacional de
Desenvolvimento Científico e Tecnol
ogico - CNPq)
(Grant number 406996/2018-0) for financial support.
CONFLICT OF INTEREST
The authors declare no conflict of interests.
ORCID
Edilson Dantas da Silva Junior https://orcid.org/0000-
0001-5505-8056
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