Middle East Fertility Society Journal 22 (2017) 323–328

Contents lists available at ScienceDirect

Middle East Fertility Society Journal

journal homepage: www.sciencedirect.com

Original Article

Short and long term effects of different doses of paracetamol on sperm

parameters and DNA integrity in mice
Nahid Abedi a, Ali Nabi b, Esmat Mangoli b, Ali Reza Talebi b,⇑
a
Department of Biology, Faculty of Basic Sciences, University of Mazandaran, Iran
b
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

a r t i c l e i n f o a b s t r a c t

Article history: The aim was to survey the impact of normal and high doses of paracetamol consumption on sperm
Received 8 December 2016 parameters and DNA integrity in mice. A total of 36 adult male mice were divided into three groups: mice
Revised 25 April 2017 of group A served as control fed on basal diet, group B received normal dosage of Paracetamol (66 mg kg/
Accepted 1 June 2017
day) and basal diet, group C received high dosage of Paracetamol (100 mg kg/day) and basal diet for 35,
Available online 22 June 2017
70 and 105 days. The cauda epididymitis of each mouse was dissected and placed in 1 ml of pre-warm
Ham’s F10 culture medium for 20 min. The swim-out spermatozoa were analyzed for count, motility,
Keywords:
morphology and viability. Sperm chromatin quality was evaluated by chromomycin A3 staining
Paracetamol
Acetaminophen
(CMA3), aniline blue staining and sperm chromatin dispersion test (SCD). The results showed that almost
Sperm parameters all of the sperm parameters significantly decrease following consumption of normal and high dosage of
Mice Paracetamol in three periods of experiments in mice (p < 0.05). Regarding to SCD test, we found a highly
DNA integrity significant difference only in dose effect, but in CMA3 test and aniline blue staining there was a signifi-
cant difference (p < 0.05) in both dose and time effects. According to our results, paracetamol as an anal-
gesic and antipyretic may have detrimental effects on sperm parameters and DNA integrity in mice.
! 2017 Middle East Fertility Society. Production and hosting by Elsevier B.V. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction NSAIDs or COX-2 selective inhibitors but is often chosen because

of its better tolerance [4].
Infertility is a major problem in up to 15% of the sexually active It is shown that overdosing with acetaminophen can cause hep-
population and male factor is responsible in 50% of these cases [1]. atic necrosis in both humans and laboratory animals [5], and pro-
As far as, lifestyle has some directly impresses on male infertility, longed human use has been implicated in chronic renal disease [6]
sperm DNA quality and sperm parameters [2], we study paraceta- necrotic changes in lung [7], testis, lymphoid tissue of mice [8] and
mol as a common pain killer. Acetaminophen (paracetamol) is asthma in children [9]. Moreover, genotoxic effects of acetamino-
widely used as an analgesic and antipyretic without prescription. phen have been observed [10]. In vivo, acetaminophen has been
The most commonly used over-the-counter (OTC) pain medication shown to cause chromosome aberrations in bone marrow cells
is acetaminophen along with aspirin and non-steroidal anti- from exposed mice [3]. High doses of acetaminophen have also
inflammatory drugs (NSAIDs) [3]. Paracetamol has a range of been reported to lead to testicular atrophy and decrease of testos-
action like that of NSAIDs and be similar to particularly the COX- terone hormone in vitro in rat and human [11–13].
2 selective inhibitors. It is usually accepted that it inhibits COX-1 There is a clear negative relationship between sperm chro-
and COX-2 through metabolism by the peroxidase function of matin/DNA damage and reproductive outcomes. Furthermore, it
these isoenzymes. This results in inhibition of phenoxyl radical for- is generally accepted that the sperm chromatin condensation has
mation from a critical tyrosine remains needed for the cyclooxyge- a key role in male fertility, early embryonic growth and pregnancy
nase activity of COX-1 and COX-2 and prostaglandin (PG) outcomes [14]. The inter and intra molecular disulphide bonds of
synthesis. Paracetamol is, on average, a weaker analgesic than protamine molecules are essential for sperm nuclear compaction
and stabilization. It is believed that this kind of nuclear compaction
protects sperm genome from external damages including oxidative
Peer review under responsibility of Middle East Fertility Society.
stress; temperature height and acid-induced DNA denaturation
⇑ Corresponding author at: Research and Clinical Center for Infertility, Bouali [15]. There are some kinds of tests for sperm chromatin/DNA eval-
Ave., Safaeyeh, Yazd, Iran. uation which show different forms of damages. Chromatin struc-
E-mail address: [email protected] (A.R. Talebi).

http://dx.doi.org/10.1016/j.mefs.2017.06.001
1110-5690/! 2017 Middle East Fertility Society. Production and hosting by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
324 N. Abedi et al. / Middle East Fertility Society Journal 22 (2017) 323–328

tural probes by nuclear dyes with cytochemical bases are sensitive, 2.4.1. Chromomycin A3 staining
easy and economical which do not need exclusive device like flow CMA3 is a fluorochrome antibiotic which competes with the
cytometry [16]. There are some studies that showed effects of protamines for binding to the minor groove of DNA and show pro-
paracetamol on sperm parameters in mice and Rat [3,12,17]. tamine deficiency. Briefly, the smears were dried and fixed in Car-
Hence, we designed this study for the first time to evaluate Short noy’s solution at 4 "C for 10 min. The slides were treated with
and long term effects of different doses of paracetamol on sperm 150 ll of CMA3 (0.25 mg/ml) in McIlvain buffer for 20 min. After
parameters and chromatin condensation in three period of sper- staining in darkroom, they were washed in buffer and mounted
matogenesis in mice by cytocemical tests. with buffered glycerol. In each sample, at least 200 spermatozoa
were counted under fluorescent microscope with a 460-nm filter
2. Materials and methods and 100X eyepiece magnification and the percentage of CMA3+
spermatozoa was reported. Bright yellow-stained chromomycin-
2.1. Animal reacted spermatozoa (CMA3+) were considered as abnormal and
yellowish green-stained or no reacted spermatozoa (CMA3$) were
In this experimental study, totally 36 adult male NMRI mice considered as mature sperm with normal protamination [23].
with average weight 37 g and 11 ± 0 weeks old which were
obtained from the animal house of Research & Clinical Center for 2.4.2. Sperm chromatin dispersion (SCD) test
Infertility of Shahid Sadoghi University of Medical Sciences. These We used the sperm chromatin dispersion test for the assess-
mouse were divided into 3 groups; control (group I, n = 12), normal ment of sperm DNA fragmentation. The SCD test was performed
dosage (group II, n = 12) and high dosage (group III, n = 12) and via the Halosperm# Kit (INDAS laboratories, Madrid, Spain). Briefly,
each group was treated during three different periods with consid- after adding 50 ll of semen to 100 ll of low melting agarose, 8 ll
ering of mice spermatogenesis duration. Hence each group was of this suspension was decanted on coated slide of the kit. A small
divided into three subgroups (each n = 4) by 35, 70 and 105 days lamella was put on it and kept on 4 "C for 5 min. The slide was
with or without treatments. Group II received normal dosage of immersed in Denaturant Agent (for 7 min) and Lysis Solution (for
acetaminophen which was calculated by the formula 20 min). After dehydrating, the slides were stained with Staining
Solution A & B for 7 min [24].
D ¼ MTR ð1-1Þ

where M is maximum recommended daily human dosage in tablets, 2.4.3. Aniline blue
R is ratio of weight of rat to average adult human weight of 60 kg Aniline blue staining was indicated the surplus histone in chro-
and Tis weight of each tablet [18]. So, 66 mg/kg body weight acet- matin structure. After preparing sperm and spread onto glass side,
aminophen was dissolved in daily water. The last group was given wait to air-dry. The smears slides were fixed in 3% buffered glu-
100 mg/kg acetaminophen [19], but, in control group, we did not taraldehyde in 0.2 M phosphate buffer (pH 7.2) for 30 min at room
receive any medication. As we know acetaminophen is very bitter temperature. Afterwards, the smears slides were stained by aniline
is the reason why we dissolved 0.002% and 0.003% saccharine in blue solution for 15 min.
the daily water of group II and III. During experiments, animals
were kept in standard condition with a temperature range of 3. Calculation
25 ± 3 "C and mean relative humidity of 50 ± 5% in the animal
house. This experimental study was approved by ethical committee Statistical analysis was performed by SPSS version 20 for Win-
of clinical center for infertility Shahid Sadoghi University. dows (SPSS Inc., Chicago, IL, USA). Repeated measures analysis of
variance (ANOVA) was applied to evaluate the significant differ-
2.2. Epididymal sperm preparation ences between dose and time effects. The term ‘statistically signif-
icant’ was used to signify a two- sided P-value <0.05 for sperm
We studied the spermatozoa after 1, 2 and 3 durations of sper- parameters and cytochemical tests. All data were expressed in
matogenesis following drug treatments [21]. So, the mice were mean ± SD.
killed after 35, 70 and 105 days by cervical dislocated and the
cauda epididymis of each animal was cut and placed in 1 ml 4. Results
Ham’sF10 medium. The dishes were incubated at 37 "C and 5%
CO2 for 10 min to make spermatozoa swim out [21]. It is significant to be mentioned that our study was performed
in three different periods of spermatogenesis and the p-value of
2.3. Sperm analysis Mauchly’s Test of Sphericity were more than 0.05 in all obtained
results. Hence, we could use repeated measures of variance
The sperm count, motility, normal morphology and viability (%) (ANOVA) with three levels (three different periods) as the most
were evaluated for at least 200 spermatozoa from each animal. suitable test. As far as we were willing to figure out the effect of
Sperm count and motility were evaluated by Meckler Chamber two different subjects effect (time and dose) on sperm quality,
(Sefi Medical Co., Haifa) and light microscopy (Olympus Co., Tokyo, two-way repeated measures of variance analysis was performed.
Japan). Motility indices were expressed as the percentages of pro- Summary of this analysis results was expressed separately in two
gressive motility (rapid and slow), non-progressive and immotile different tables.
spermatozoa. The morphologically normal spermatozoa and the Table 4.1 demonstrates means, standard deviation and pairwise
percentage of viable sperm cells were assessed by Papanicolau comparisons of sperm parameters. The part of pairwise compar-
staining and Eosin test respectively [22]. isons in Table 4.1 has two subparts. One of them illustrates the
comparison among three levels of time (one, two, and three peri-
2.4. Sperm chromatin and DNA study ods of spermatogenesis). The results of this part will be able to
answer a question that acetaminophen consumption for three peri-
Sperm DNA integrity and chromatin condensation were ods of spermatogenesis can affect male fertility more than using it
assessed using Sperm Chromatin Dispersion (SCD) test and chro- for two or less periods of spermatogenesis. A significant change
momycin A3 (CMA3) staining respectively. between using paracetamol for one and three period of spermato-
 N. Abedi et al. / Middle East Fertility Society Journal 22 (2017) 323–328 325

Table 4.1
Semen analyses data showing means, standard deviations & pairwise comparisons.

Parameters Control (group A) 66 mg kg/day normal dosage 100 mg kg/day high dosage Pairwise comparisons
Mean ± SD (group B) acetaminophen (group C) Acetaminophen
Time p. Value Treatment p.Value
Mean ± SD Mean ± SD
(1, 2, 3) (group A, B, C)
Count (%106) 1 11.25 ± 0.96 7.5 ± 1.29 6.0 ± 1.41 2 0.173 B 0.004*
2 10.25 ± 0.96 8.5 ± 0.58 6.75 ± 1.26 1 A
3 10.75 ± 0.5 6.5 ± 2.4 5.0 ± 1.41 3 0.423 C 0.022*
Viability 1 70.75 ± 3.4 67.5 ± 2.08 65.25 ± 3.30 2 1.000 B 0.000*
2 79.75 ± 4.11 61.75 ± 3.95 60.25 ± 4.5 1 A
3 70.25 ± 0.96 58.25 ± 3.40 55.75 ± 7.04 3 0.042* C 0.007*
Rapid motility (%) 1 20.25 ± 1.5 6.0 ± 1.82 4.75 ± 0.5 2 0.723 B 0.001*
(grade a)
2 20.25 ± 1.25 8.75 ± 1.71 4.75 ± 2.5 1 A
3 34.0 ± 3.37 5.0 ± 0.82 3.5 ± 0.58 3 0.044* C 0.000*
Slow motility (%) 1 16.5 ± 2.38 15.25 ± 0.96 8.0 ± 1.15 2 0.216 B 0.003*
(grade b)
2 16.0 ± 2.16 8.0 ± 1.41 4.25 ± 2.63 1 A
3 13.0 ± 2.45 5.0 ± 0.82 4.25 ± 1.71 3 0.032* C 0.006*
Non progressive 1 28.5 ± 2.64 13.75 ± 2.06 26.5 ± 3.70 2 1.000 B 0.602
motility (%)
(grade c)
2 25.5 ± 1.29 21.25 ± 2.5 22.5 ± 1.29 1 A
3 22.0 ± 3.56 36.5 ± 5.74 36.0 ± 2.71 3 0.010* C 0.026
Immotile sperm (%) 1 34.75 ± 2.75 64.25 ± 0.96 60.75 ± 4.79 2 0.134 B 0.001*
(grade d)
2 38.25 ± 2.21 62.75 ± 1.71 68.5 ± 2.08 1 A
3 31.0 ± 2.16 53.5 ± 4.79 56.25 ± 3.86 3 0.025* C 0.001*
Normal morphology 1 58.0 ± 2.16 44.75 ± 2.5 40.5 ± 2.08 2 1.000 B 0.003*
2 58.0 ± 2.83 45.0 ± 2.94 36.25 ± 4.272 1 A
3 58.0 ± 3.46 33.25 ± 4.42 48.0 ± 3.83 3 1.000 C 0.004*
2 30.25 ± 4.79 47.50 ± 7.85 52.75 ± 5.38
3 30.50 ± 5.45 52.25 ± 7.54 59.75 ± 5.74

1 After one period of spermatogenesis.

2 After two period of spermatogenesis.
3 After three period of spermatogenesis.

Table 4.2
DNA and chromatin assays with means, standard deviations & pairwise comparisons.

Parameters Control (group A) 66 mg kg/day normal 100 mg kg/day high Pairwise comparisons
Mean ± SD dosage (group B) dosage (group C)
Time p. Value Treatment p.Value
acetaminophen Mean ± SD Acetaminophen Mean ± SD
(1,2,3) (group A, B, C)
SCD test 1 53.75 ± 2.99 71.5 ± 3.70 67.5 ± 6.24 2 0.636 B 0.012*
2 52.25 ± 5.12 72 ± 5.94 77.75 ± 3.77 1 A
3 50.25 ± 2.06 66.25 ± 4.19 72.00 ± 5.29 3 1.000 C 0.001*
AB staining 1 41.25 ± 2.99 49.75 ± 5.38 57.5 ± 4.79 2 0.111 B 0.378
2 42.75 ± 4.99 63.5 ± 4.65 70.75 ± 5.74 1 A
*
3 48.25 ± 0.5 57.25 ± 2.22 61.5 ± 2.64 3 0.023 C 0.004*
CMA3 staining 1 24.25 ± 3.30 36.75 ± 6.13 48.75 ± 7.85 2 0.457 B 0.009*
2 30.25 ± 4.79 47.50 ± 7.85 52.75 ± 5.38 1 A
3 30.50 ± 5.45 52.25 ± 7.54 59.75 ± 5.74 3 0.022* C 0.012*

1 After one period of spermatogenesis.

2 After two period of spermatogenesis.
3 After three period of spermatogenesis.

genesis is observed in viability, and all parameters which related to CMA3 in three various periods of spermatogenesis show statisti-
motility of sperm. On the contrary, no significant differences are cally significant changes. While, there is no meaningful different
reported in count and normal morphology. The other part of pair- among SCD test results in all times. As we mentioned above, the
wise comparisons gives up genuine comparison among three doses second part of pairwise comparisons reveals critical information
of treatments (group A, B, and C). It is clear that almost all of the about three dosage of drug treatments. The eye-catching related
sperm parameters except non-progressive blue staining had signif- point to be heeded is that approximately all of the DNA assays
icant differences (P < 0.05) between three groups. The data showed show significant differences between three groups.
that acetaminophen could impair sperm parameters after one or Tables 4.3 and 4.4 indicates the summery of the tests within
more spermatogenesis periods even in normal dosage. subject’s effects among sperm parameters and DNA tests respec-
The results of DNA tests by pairwise comparisons are displayed tively. The effect of time, dose, and both elements were analyzed
in Table 4.2. The comparisons of the results of aniline blue and for each parameter of male fertility. The results of analysis indicate
326 N. Abedi et al. / Middle East Fertility Society Journal 22 (2017) 323–328

Table 4.3
Semen analyses with summery of the tests of within subjects effects.

Source Sum of squares Degree of freedom Mean square F ratio p. Value Partial eta squared
6 *
Count (%10 ) Treatment 145.72 2 72.86 28.00 0.001 0.903
Error (treatment) 15.61 6 2.60
Time 7.72 2 3.86 4.68 0.059 0.610
Error (time) 4.94 6 0.824
Treatment * Time 8.44 4 2.11 1.14 0.384 0.275
Error (treatment * time) 22.22 12 1.85
Viability Treatment 1202.17 2 601.08 59.83 0.000* 0.952
Error (treatment) 60.27 6 10.05
Time 302.17 2 151.08 13.41 0.006* 0.817
Error (time) 67.61 6 11.27
*
Treatment * Time 281.66 4 70.42 4.95 0.014 0.623
Error (treatment * Time) 170.56 12 14.21
Rapid motility (%) (grade a) Treatment 3033.5 2 1516.75 355.33 0.000* 0.992
Error (treatment) 25.61 6 4.27
Time 96.17 2 48.08 21.20 0.002* 0.876
Error (time) 13.61 6 2.27
Treatment * Time 442.33 4 110.58 32.19 0.000* 0.915
Error (treatment * Time) 41.22 12 3.43
Slow motility (%) (grade b) Treatment 567.39 2 283.69 81.70 0.000* 0.965
Error (treatment) 20.83 6 3.47
*
Time 210.89 2 105.44 15.30 0.004 0.836
Error (time) 41.33 6 6.89
Treatment * Time 77.44 4 19.36 14.83 0.000* 0.832
Error (treatment * Time) 15.67 12 1.31
Non progressive motility (%) (grade c) Treatment 126.00 2 63.00 21.00 0.002* 0.875
Error (treatment) 18.00 4 3.00
*
Time 578.17 2 289.08 46.67 0.000 0.940
Error (time) 37.167 6 6.19
Treatment * Time 966.33 4 241.38 20.27 0.000* 0.871
Error (treatment * Time) 143.00 12 11.91
Immotile sperm (%) (grade d) Treatment 5564.22 2 2782.11 397.44 0.000* 0.993
Error (treatment) 42.0 6 7.00
Time 570.06 2 285.03 65.36 0.000* 0.956
Error (time) 26.17 6 4.36
Treatment * Time 113.44 4 28.36 2.77 0.077 0.480
Error (treatment * Time) 123.00 12 10.25
Normal morphology Treatment 2235.39 2 1117.69 90.96 0.000* 0.968
Error (treatment) 73.72 6 12.29
Time 14.22 2 7.11 0.532 0.613 0.151
Error (time) 80.22 6 13.370
*
Treatment * Time 629.44 4 157.36 15.05 0.000 0.834
Error (treatment * Time) 125.44 12 10.45

Table 4.4
DNA analysis with summary of the tests of within subjects effects.

Source Sum of squares Degree of freedom Mean square F ratio p. Value Partial eta squared
SCD Treatment 2950.89 2 1475.44 88.53 0.000* 0.967
Error (treatment) 100.00 6 16.67
Time 127.05 2 63.53 3.67 0.091 0.550
Error (time) 103.83 6 17.30
Treatment * Time 189.94 4 47.48 2.13 0.139 0.416
Error (treatment * Time) 267.17 12 22.26
AB staining Treatment 819.39 2 409.69 18.88 0.003* 0.863
Error (treatment) 130.17 6 21.69
*
Time 1177.56 2 588.78 18.79 0.003 0.862
Error (time) 188.00 6 31.33
Treatment * Time 1779.79 4 444.94 28.91 0.000* 0.906
Error (treatment * Time) 184.67 12 15.39
CMA3 staining Treatment 4035.05 2 2017.52 60.31 0.000* 0.953
Error (treatment) 200.72 6 33.45
*
Time 732.05 2 366.03 8.50 0.018 0.739
Error (time) 258.39 6 43.06
Treatment * Time 120.61 4 30.153 0.860 0.515 0.223
Error (treatment * Time) 420.94 12 35.08

that some parameters have a highly significant differences in all staining, and mobility parameters. This table reveals that time does
effects but the dose is more prominent (has larger partial eta num- not affect on some others parameters like count and SCD test.
ber) than time and treatment ⁄ time such as viability, aniline blue Although, time does not influence in normal morphology, it can
 N. Abedi et al. / Middle East Fertility Society Journal 22 (2017) 323–328 327

enhance the role of treatment. The data show that long-term use of the other hand, hepatotoxic dose of acetaminophen in mice was
acetaminophen in normal and high dosages causes damage to accompanied by a dramatic decrease in mRNA for histone [3,6].
sperm quality and DNA integrity and increases sperm DNA frag- So, it is possible that the altered chromatin structure observed in
mentation and protamine deficiency in mice. the sperm could be related to errors in histone synthesis and fal-
lowed by disruption in histone–protamines replacement during
the testicular phase of sperm chromatin packaging. Further studies
5. Discussion are required to illuminate the mechanism of action and the effects
of different dosage of acetaminophen on human spermatozoa.
paracetamol as an analgesic and antipyretic without prescrip-
tion commonly used during pregnancy and no significant relations 6. Conclusion
were seen between acetaminophen use and low birth weight or
preterm delivery and it was safe [25,26]. In some cases Paraceta- Our study showed that paracetamol or acetaminophen as an
mol caused hematological effects [17], increased childhood wheeze analgesic and antipyretic drug may impact sperm parameters
and asthma risk [9], hepatotoxicity [20], multiple endocrine distur- and chromatin/DNA integrity in mice. It should be noted that these
bances in the human adult testis [11]. effects are dose dependent and are seen both in short and long-
The findings of present study showed that almost all of the terms of drug consumption. We advise that men especially in fer-
sperm parameters were decreased following consumption of nor- tility period be careful in acetaminophen usage without prescrip-
mal and high dosage of paracetamol in three period of spermatoge- tion of their physician.
nesis in mice. A similar study has shown that the treatment
paracetamol causes a significant decrease in sperm motility and Acknowledgment
sperm count in rat that is in accordance with our results. It is also
shown that paracetamol may cause a significant decrease in mor- The authors were grateful Dr. Aflatoonian and his colleagues in
phologically normal spermatozoa. These effects could be due to clinical center for infertility Shahid Sadoghi University for all their
impacts of paracetamol on testis and epididymis [17]. Beside, Aksu supporting during this project.
et al. claim that their study demonstrates decrease in sperm motil-
ity and live sperm rate in male rat by consumption of paracetamol References
[27].
Ratnasooriya et al. declared that long-term administration of [1] A.R. Talebi, A.A. Sarcheshmeh, M.A. Khalili, N. Tabibnejad, Effects of ethanol
consumption on chromatin condensation and DNA integrity of epididymal
high doses of paracetamol damages the reproductive competence
spermatozoa in rat. Alcohol. [Research Support, Non-U.S. Gov’t] 45(4) (2011)
of male rats. They presented that this effects are reversible and Jun pp. 403–419.
was not due to a general toxicity but due to an increase in oligo- [2] F.B. Choucair, E.G. Rachkidi, G.C. Raad, E.M. Saliba, N.S. Zeidan, R.A. Jounblat, I.F.
zoospermia, deficiencies of normal and hyper-activated sperm Jaoude, M.M. Hazzouri, High level of DNA fragmentation in sperm of Lebanese
infertile men using Sperm Chromatin Dispersion test, Middle East Fertility Soc.
motility, and reduction in the fertilizing potential of spermatozoa J. 21 (4) (2016) 269–276, December 31.
[28]. In our study we also saw that long term consumption of [3] R. Wiger, J.K. Hongslo, D.P. Evenson, P. De Angelis, P.E. Schwarze, J.A. Holme,
paracetamol may disrupt sperm fertility potential. Effects of acetaminophen and hydroxyurea on spermatogenesis and sperm
chromatin structure in laboratory mice. Reprod Toxicol. [Comparative Study].
To the best of our knowledge, this is the first investigation on 9(1) (1995) Jan-Feb pp. 21–33.
the relationship between sperm chromatin condensation and short [4] G.G. Graham, M.J. Davies, R.O. Day, A. Mohamudally, K.F. Scott, The modern
and long-term paracetamol administration in normal and high pharmacology of paracetamol: therapeutic actions, mechanism of action,
metabolism, toxicity and recent pharmacological findings.
doses. Regarding to the DNA integrity tests, firstly in SCD test, Inflammopharmacology [Comparative Study Review] 21(3) (2013 Jun) pp.
we found a significant difference among groups in three period 201–232.
of spermatogenesis. This showed that paracetamol consumption [5] F. Bessone, Non-steroidal anti-inflammatory drugs: What is the actual risk of
liver damage? World J Gastroenterol. [EditorialReview] 16(45) (2010 Dec 7)
in normal and high doses may cause sperm DNA fragmentation. pp. 5651–5661.
Secondly, In CMA3 staining, in the first 35 days we found signifi- [6] A.D. Vliegenthart, J.M. Shaffer, J.I. Clarke, L.E. Peeters, A. Caporali, D.N. Bateman,
cant differences between groups only when high dosage of parac- et al., Comprehensive microRNA profiling in acetaminophen toxicity identifies
novel circulating biomarkers for human liver and kidney injury, Sci. Rep. 5
etamol was treated. On the other hand, there were significantly
(2015) 15501.
differences between groups in the rates of sperm protamine defi- [7] G.J. Smith, J.A. Cichocki, B.J. Doughty, J.E. Manautou, S.E. Jordt, J.B. Morris, Effect
ciency in both normal and high dosage of paracetamol after 70 of acetaminophen on oxidant and irritant respiratory tract responses to
and 105 days. Thus, it seems that long-term use of paracetamol environmental tobacco smoke in female mice, Environ. Health Perspect. (2015
October 9).
has detrimental effects on histone–protamines replacement during [8] J.R. Reel, A.D. Lawton, JCt Lamb, Reproductive toxicity evaluation of
the testicular phase of sperm chromatin packaging and cause acetaminophen in Swiss CD-1 mice using a continuous breeding protocol,
sperm protamine deficiency in mice. To compare our data with Fundam Appl. Toxicol. 18 (2) (1992 February) 233–239 (Comparative
StudyResearch Support, U.S. Gov’t, P.H.S.).
others, although we did not find any similar study in the literature, [9] J.E. Sordillo, C.V. Scirica, S.L. Rifas-Shiman, M.W. Gillman, S. Bunyavanich, C.A.
but, there was a study on testicular cells populations with tetra- Camargo Jr., et al., Prenatal and infant exposure to acetaminophen and
ploid, diploid, and haploid DNA content using acridine orange ibuprofen and the risk for wheeze and asthma in children, J. Allergy Clin.
Immunol. 135 (2) (2015 February) 441–448 (Research Support, N.I.H.,
staining with flow cytometry in mice. The results of mentioned Extramural).
study showed that daily treatments with either hydroxyurea or [10] R.M. Rodrigues, A. Heymans, V. De Boe, A. Sachinidis, U. Chaudhari, O. Govaere,
acetaminophen caused an increased frequency of cells with altered et al., Toxicogenomics-based prediction of acetaminophen-induced liver injury
using human hepatic cell systems, Toxicol. Lett. (2015 October 20).
sperm chromatin structure and increased sperm DNA denaturation [11] O. Albert, C. Desdoits-Lethimonier, L. Lesne, A. Legrand, F. Guille, K. Bensalah,
in mice [3] which confirmed our results. et al., Paracetamol, aspirin and indomethacin display endocrine disrupting
There are some possible reasons for sperm DNA damage follow- properties in the adult human testis in vitro, Hum. Reprod. 28 (7) (2013 July)
1890–1898 (Research Support, Non-U.S. Gov’t).
ing acetaminophen treatments. It is shown that acetaminophen
[12] A. Jaqueson, H. Semont, M. Thevenin, J.M. Warnet, R. Prost, J.R. Claude, Effect of
causes an inhibition of both DNA replication and DNA repair by a daily high doses of paracetamol given orally during spermatogenesis in the rat
specific inhibition of enzyme ribonucleotide reductase [10]. In fact, testes, Arch Toxicol. Suppl. 7 (1984) 164–166.
these effects provide a reason for acetaminophen’s ability to make [13] S. van den Driesche, J. Macdonald, R.A. Anderson, Z.C. Johnston, T. Chetty, L.B.
Smith, et al., Prolonged exposure to acetaminophen reduces testosterone
sister chromatid exchanges, micronuclei, chromosomal aberrations production by the human fetal testis in a xenograft model, Sci. Transl. Med. 7
and apoptosis in cells with low drug metabolism activity [3]. On (288) (2015 May 20) 288ra80 (Research Support, Non-U.S. Gov’t).
328 N. Abedi et al. / Middle East Fertility Society Journal 22 (2017) 323–328

[14] A.R. Talebi, M.A. Khalili, S. Vahidi, J. Ghasemzadeh, N. Tabibnejad, Sperm [22] A.R. Talebi, E. Mangoli, H. Nahangi, M. Anvari, M. Pourentezari, I. Halvaei,
chromatin condensation, DNA integrity, and apoptosis in men with spinal cord Vitamin C attenuates detrimental effects of diabetes mellitus on sperm
injury, J. Spinal Cord Med. 36 (2) (2013 March) 140–146. parameters chromatin quality and rate of apoptosis in mice, Eur. J. Obstet.
[15] J.R. Gannon, B.R. Emery, T.G. Jenkins, D.T. Carrell, The sperm epigenome: Gynecol. Reprod. Biol. 181 (2014 October) 32–36 (Research Support, Non-U.S.
implications for the embryo, Adv. Exp. Med. Biol. 791 (2014) 53–66 (Review). Gov’t).
[16] M. Rahimipour, A.R. Talebi, M. Anvari, A.A. Sarcheshmeh, M. Omidi, Effects of [23] M. Pourentezari, A.R. Talebi, E. Mangoli, M. Anvari, M. Rahimipour, Additional
different doses of ethanol on sperm parameters, chromatin structure and deleterious effects of alcohol consumption on sperm parameters and DNA
apoptosis in adult mice, Eur J Obstet Gynecol Reprod Biol. 170 (2) (2013 integrity in diabetic mice, Andrologia (2015 September 10).
October) 423–428 (Research Support, Non-U.S. Gov’t). [24] E.I. Cortes-Gutierrez, M.I. Davila-Rodriguez, J.L. Fernandez, C. Lopez-
[17] K.O. Oyedeji, A.F. Bolarinwa, S.S. Ojeniran, Effect of paracetamol Fernandez, A.R. Aragon-Tovar, L.C. Urbina-Bernal, et al., DNA damage in
(acetaminophen) on haematological and reproductive parameters in male spermatozoa from infertile men with varicocele evaluated by sperm
albino rats, Res. J. Pharmacol. 7 (2) (2013) 21–25. chromatin dispersion and DBD-FISH, Arch. Gynecol. Obstet. (2015 July 30).
[18] U.B. Ekaluo, E.V. Ikpeme, A.E. Udokpoh, Sperm head abnormality and [25] A.R. Scialli, R. Ang, J. Breitmeyer, M.A. Royal, A review of the literature on the
mutagenic effects of aspirin, paracetamol and caffeine containing analgesics effects of acetaminophen on pregnancy outcome, Reprod. Toxicol. 30 (4) (2010
in rats, Internet J. Toxicol. 7 (1) (2009) 1–9. December) 495–507 (Review).
[19] S. Hunskaar, O.B. Fasmer, K. Hole, Formalin test in mice, a useful technique for [26] L. de Fays, K. Van Malderen, K. De Smet, J. Sawchik, V. Verlinden, J. Hamdani,
evaluating mild analgesics, J. Neurosci. Methods 14 (1) (1985) 69–76. et al., Use of paracetamol during pregnancy and child neurological
[20] R. Agarwal, L.A. MacMillan-Crow, T.M. Rafferty, H. Saba, D.W. Roberts, E.K. development, Dev. Med. Child Neurol. 57 (8) (2015 August) 718–724 (Review).
Fifer, et al., Acetaminophen-induced hepatotoxicity in mice occurs with [27] E.H. Aksu, M. Özkaraca, F.M. Kandemir, A.D. Ömür, E. Eldutar, S. Küçükler, S. Ç
inhibition of activity and nitration of mitochondrial manganese superoxide omaklı, Mitigation of paracetamol-induced reproductive damage by chrysin in
dismutase, J. Pharmacol. Exp. Ther. 337 (1) (2011 April) 110–116 (Comparative male rats via reducing oxidative stress, Andrologia (2016 February 1).
Study Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov’t). [28] W.D. Ratnasooriya, J.R. Jayakody, Long-term administration of large doses of
[21] E. Mangoli, A.R. Talebi, M. Anvari, M. Pourentezari, Effects of experimentally- paracetamol impairs the reproductive competence of male rats, Asian J.
induced diabetes on sperm parameters and chromatin quality in mice, Iran J. Androl. 2 (4) (2000 December) 247–255.
Reprod. Med. 11 (1) (2013 January) 53–60.