Toxicological & Environmental Chemistry

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Histological changes in the reproductive system of

male rats exposed to cigarette smoke or electronic
cigarette vapor

Ewelina Wawryk-Gawda, Michał K. Zarobkiewicz, Katarzyna Chłapek,

Patrycja Chylińska-Wrzos & Barbara Jodłowska-Jędrych

To cite this article: Ewelina Wawryk-Gawda, Michał K. Zarobkiewicz, Katarzyna Chłapek, Patrycja
Chylińska-Wrzos & Barbara Jodłowska-Jędrych (2019): Histological changes in the reproductive
system of male rats exposed to cigarette smoke or electronic cigarette vapor, Toxicological &
Environmental Chemistry, DOI: 10.1080/02772248.2019.1703989

To link to this article: https://doi.org/10.1080/02772248.2019.1703989

Published online: 25 Dec 2019.

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TOXICOLOGICAL & ENVIRONMENTAL CHEMISTRY
https://doi.org/10.1080/02772248.2019.1703989

Histological changes in the reproductive system of

male rats exposed to cigarette smoke or electronic
cigarette vapor
Ewelina Wawryk-Gawdaa , Michał K. Zarobkiewiczb, Katarzyna Chłapekc,

ska-Wrzosd and Barbara Jodłowska-JeRdrychd
Patrycja Chylin
a
Department of Paediatric Pulmonology and Rheumatology, University Children Hospital of
Lublin, Medical University of Lublin, Lublin, Poland; bDepartment of Clinical Immunology,
Medical University of Lublin, Lublin, Poland; cDepartment of Financial Accounting, Cracow
University of Economics, Krakow, Poland; dChair and Department of Histology and
Embryology with Experimental Cytology Unit, Medical University of Lublin, Lublin, Poland

ABSTRACT ARTICLE HISTORY

The use of electronic cigarettes increases as a supposedly Received 26 May 2019
healthier form of nicotine consumption, but safety of Accepted 9 December 2019
vaping remains uncertain. In this study, we assessed
KEYWORDS
whether the use of electronic cigarettes increases the risk
Vaping; smoking; electronic
of infertility in male rats. Malformations of sperm morph- cigarettes; cigarettes;
ology were more frequent in nicotine-exposed groups than nicotine; sperm;
in the control group. Vacuolization of seminiferous epithe- testis; epididymis
lium, reduction of spermatogenesis, increased apoptosis of
spermatogonia and spermatocytes, and acceleration of
degeneration of testes were observed. The male reproduct-
ive organs are slightly less affected by vaping than by
smoking. Nevertheless, in consequence both may lead to
the reduction of fertility.

Abbreviations: e-cig: electronic cigarettes; PVC: polyvinyl

chloride; ROS: reactive oxygen species

Introduction
Up to 16.7% of couples from developed countries have infertility prob-
lems, among those approximately half is related to male infertility (Lotti
and Maggi 2018). Moreover, an increasing number of couples require
infertility treatment (Mohamed and Abdelrahman 2018). This problem
concerns both women and men. Infertility may be caused by ontogenetic
factors such as genetic and epigenetic alterations, obesity, hypertension,
lack of physical activity, but also by heavy occupational exertion and
environmental pollution, anti-androgenic toxins, medication, stress, and
addictive substances such as coffee, tobacco, and alcohol (Comhaire,

CONTACT Ewelina Wawryk-Gawda ewelina.wawryk@wp.pl Department of Paediatric Pulmonology

R bali Street
and Rheumatology, University Children Hospital of Lublin, Medical University of Lublin, Prof. A.Ge
6, Lublin, 20-093, Poland
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
2 E. WAWRYK-GAWDA ET AL.

Vandenberghe, and Decleer 2017; Bieniek and Lo 2016; Mima, Greenwald,

and Ohlander 2018; Wyck et al. 2018). Smoking of cigarettes has been
documented to alter the fertility of males and females (Sharma et al.
2016). Smoking couples experience a longer waiting time for pregnancy
than non-smokers (Sapra et al. 2016; Valdes-Socin et al. 2010). Moreover,
fetuses of smokers have higher death risk and health problems (Comhaire,
Vandenberghe, and Decleer 2017; Valdes-Socin et al. 2010). Smoking, like
obesity and stress, has deleterious influence on the neuroendocrine system
regulating the reproductive functions. Nicotine alters the thyroid produc-
tion and increases the risk of Graves’ disease with ophthalmopathy, dereg-
ulates the hypothalamic–pituitary–gonadal axis balance, and decreases
estrogen and progesterone production (Totonchi et al. 2016). Hormonal
changes are associated with disturbances of gonads maturation, leading to
infertility of females and to oligospermia or azoospermia in males, two
major factors determining male infertility (Bieniek and Lo 2016; Sharma
et al. 2016; Smith and Vale 2006; Valdes-Socin et al. 2010).
Nicotine replacement therapy is used in smoking dependence treatment
(Kalkhoran and Glantz 2016; McRobbie et al. 2014). Nicotine is adminis-
trated in the form of chewing gums, patches, tablets, and recently in the
form of aerosol (vapor) produced from liquid by electronic devices such as
electronic cigarettes (e-cig) (Etter and Eissenberg 2015). In recent years, the
e-cig market has developed significantly. However, e-cigs are used not only
by smokers who want to quit but also by adolescents and young adults who
have never smoked conventional cigarettes (Fearon et al. 2018; McMillen
et al. 2018; Zarobkiewicz et al. 2016). Data concerning safety of vaping are
still inconclusive (Liu et al. 2018). Mouth or throat irritation, anxiety,
depressed mood, nausea, headache, choking, dryness of the eyes, and insom-
nia are the most frequently observed adverse effects reported by e-cig users
(Liu et al. 2018; Meo and Al Asiri 2014; Walele et al. 2018). The impact of
e-cig vapor on tissues has not yet been described.
In this study, the increase of infertility in male rats upon exposure to
the smoke of conventional cigarettes compared to that related to the
exposure to e-cig vapor was assessed. The effects of e-cig vapor on sper-
matozoa morphology, spermatogenesis and structure of testes, and epi-
didymis of rats were investigated. The hypothesis of the study was that
e-cig is slightly safer than traditional ones.

Materials and methods

Experimental procedure
The experiment was conducted on 30 male Wistar rats with a mean
body weight of ca 188 ± 12 g. The rats were bred in specific-pathogen-
 TOXICOLOGICAL & ENVIRONMENTAL CHEMISTRY 3

free condition at the Experimental Medicine Centre of the Medical

University of Lublin. The animals were divided into six groups of five
animals each: A1, B1, C1, A2, B2, and C2. The animals in groups A1
and A2 were exposed to the vapor of a scent-free liquid with a total
nicotine content of 7.2 mg per cage per 24 hours. During the 10-minute
exposure, the rats were placed in a polyvinyl chloride (PVC) cage with a
volume of 0.1 m3 to inhale 0.6 mL of liquid containing 12 mg nicotine in
1 mL E liquid neutral base (Inawera, Turka, Poland), consisting of ca
47% propylene glycol, ca 5% water, ca 47% vegetable glycerin, using a
suction and pressure device (electric pump HT-468, Joinco Polska,
Warszawa, Poland). The rats in groups B1 and B2 were also placed in
PVC cage and exposed to smoke from 10 cigarettes (LD, Liggett Ducat,
Japan Tobacco, Stary Gostk
ow, Poland) of the same total nicotine con-
tent as the rats in groups A1 and A2, using a suction and pressure
device. The exposures of both groups were conducted on 5 days per
week over the period of 6 weeks. In total, each group of five animals was
exposed to 210 mg nicotine. The rats in the control groups C1 and C2
were subjected to the same inhalation-related stress by taking them to
PVC cages and exposing to air and noise of the same type of suction
and pressure device for 10 min. The animals of groups A1, B1, and C1
were decapitated 24 hours after the last exposure, those of groups A2, B2,
and C2 two weeks after the last exposure (Figure 1). Testes and epididy-
mis were dissected. The weight of each testis was measured. Spermatozoa
were collected from the heads of the epididymis, and a smear test was
performed. After fixing the testes and epididymis in 10% buffered forma-
lin, they were embedded in paraffin blocks and cut into 5-lm-thick sec-
tions. All experiments were conducted with the formal approval of the
Animal Care Committee of the Natural Science University In Lublin
(approval No 30/2015).

Hematoxylin and eosin (H&E) and Masson’s trichrome staining

For morphological evaluation, spermatozoa smears, and slices of epididy-
mis, and testes were stained with H&E. Slices of testes and epididymis
heads and bodies were also stained according to the Masson’s trichrome
protocol as described by Ozawa and Sakaue (2020). Microscopic analysis
was performed with a light microscope (BX8, Olympus, Tokyo, Japan)
using 10x, 40x, and 100x lenses. Of each animal, 50 fields of view were
analyzed. Evaluation and picture documentation were performed using a
microscope with camera and CellSense software (Olympus). The optical
density of the picture of the trichrome-stained area was outlined and
quantified using ImageJ software and its associated color deconvolution
4 E. WAWRYK-GAWDA ET AL.

A1 B1
- 10 rats in cage
- 10 rats in cage
- inhalaon of smoke
C1
- inhalaon of 0,6ml - 10 rats in cage
of liquid (nicone from 10 convenonal - inhalaon of air
12mg/ml) cigarees(nicone - for 6 weeks
- for 6 weeks 0.7mg/cigaree) (5days/week)
(5days/week) - for 6 weeks
(5days/week)

Decapitaon 5
rats of each
group 24 hours
aer last
inhalaon

2 weeks
observaon
remaining rats

A2 B2 C2
decapitaon decapitaon decapitaon
2weeks aer 2 weeks aer 2 weeks aer
last vapor last smoke last air
inhalaon inhalaon inhalaon

Figure 1. Scheme of experiment.

plugin as described by Varghese et al. (2014) and Hern
andez-Morera

et al. (2016).

Immunohistochemical (IHC) staining

IHC staining was performed as previously described (Wawryk-Gawda
et al. 2018). The exposure of the antigenic sites was performed thermally
by incubation of slides with testes slices in 10 mmol/L citrate buffer, pH
6, in a microwave oven (Amica, Wronki, Poland) at 800 W for three
cycles lasting 5 min each. To inhibit the endogenous peroxidase activity,
0.3% hydrogen peroxide in water (Hydrogen peroxide, Stanlab, Lublin,
Poland) in methanol (Metanol CZDA, Stanlab, Lublin, Poland) was used.
Normal Horse Serum (Abcam, Cambridge, UK) was used to block non-
specific binding of antigen. The tissue slices were incubated overnight at
4
C with a primary antibody of caspase 3 (NovocastraTM, Newcastle
 TOXICOLOGICAL & ENVIRONMENTAL CHEMISTRY 5

upon Tyne, UK, dilution 1:50). To visualize the reaction, diaminobenzi-

dine solution (10 g/L, Sigma-Aldrich, Pozna
n, Poland) and hematoxylin
staining were used. Negative controls were prepared in a similar manner
but omitting the specific primary antibody (Crosby et al. 2014). The
material was evaluated light microscopy using 10 and 40 objectives.
The expression was calculated by counting 50 pictures for each animal.

TUNEL
For identification of apoptotic cells in testes, a Click-iTTM TUNEL
colorimetric IHC detection kit (InvitrogenTM, Life Technologies,
Carlsbad, USA) was used according to the manufacturer’s guide.

Statistical analysis
The test results were subjected to statistical analysis using Statistica 13.0
software (StatSoft, Tulsa, OK, United States). The U Mann–Whitney’s
test was used to calculate statistical significance. A p value of less than
.05 was considered statistically significant. The data were presented
as means ± SD.

Results
E-cig vapor-like cigarette smoke induces significant malformations in
spermatozoa
Malformations of sperm structure were observed in the spermatozoa
smear. Epididymal sperms showed twisted bodies, abnormal irregular
tails, and abnormal heads and tails. Pathological changes were most fre-
quently observed in group B2. In groups B1, A1, and A2, malformations
of spermatozoa structure were also observed more frequently than in the
control groups (Figure 2).

E-cig vapor significantly affects testes structure

To further evaluate the e-cig vapor impact, the histological structure of
testes was analyzed. The weights (1.5 ± 0.5 g) and sizes (2  1 cm) of tes-
tes were similar in all groups (p>.05). In group A1, vacuolization, con-
gested blood vessels, germinal epithelium separated from basement
membrane, and 13 ± 2 invaginations of tunica albuginea in one field of
view were observed (Figure 3). In group B1, such pathological changes
were slightly more intense. Invagination of tunica albuginea (16 ± 3) and
reduction of spermatogonia were observed in this cigarette smoke
exposed group. In group C2 degeneration symptoms were seen.
6 E. WAWRYK-GAWDA ET AL.

Figure 2. Spermatozoa smear. C1 (control group 1): 10 ± 3% of sperm with structure malfor-
mation. Please note the twisted body (arrows); A1 (vapor-exposed group): malformed
44 ± 5% of sperm. Please note the twisted tails, abnormally curled tails (picture 1 at magnifi-
cation 1000), lack of fragment of tails (arrows), fragmentation of sperm (arrow head, and
picture 2 at magnification 1000); B1 (smoke-exposed group): 45 ± 5% of sperm with mal-
formation, twisted or shorten tails (arrows); C2 (control group 2): 15 ± 4% of sperm with mal-
formation, twisted tails (arrows); A2 (vapor-exposed group after 2 weeks break): 30 ± 8%,
twisted body, twisted tails (arrows); B2 (smoke-exposed after 2 weeks break): 60 ± 9% of
sperm with malformation; B2a: abnormal flexion of the tails (arrows), the presence of sper-
matocytes, spermatids (black arrows); B2b: malformation of body’s structure (arrows, and pic-
ture 3 at magnification 1000), spermatids (black arrows). p value in U Test: A1:C1 p¼.0018,
B1:C1 p¼.0033, A1:B1 p¼.64, A2:C2 p¼.005, B2:C2 p¼.005, A2:B2 p¼.005. H&E staining.
Magnification ca 400.

Figure 3. C1 (control group 1): normal structure of seminiferous tubules. A1 (vapor-exposed

group): vacuolization of epithelium, irregular shapes of seminiferous tubules, increased
spaces between tubules; B1 (smoke-exposed group): vacuolization and disturbances of epi-
thelium, note invagination of tunica albuginea; C2 (control group 2): vacuolization of epithe-
lium, separation of epithelium from basement membrane, spermatocytes in lumen of
seminiferous tubules; A2 (vapor-exposed group after 2 weeks break): note deep invagination
of tunica albuginea, vacuolization, destructive changes of epithelium; B2 (smoke-exposed
after 2 weeks break): separation of epithelium from basement membrane, degeneration of
seminiferous tubules. H&E staining. Magnification 100.
 TOXICOLOGICAL & ENVIRONMENTAL CHEMISTRY 7

Increased vacuolization and irregular shape of seminiferous tubules, con-

gested blood vessels, and 7 ± 1 invaginations of tunica albuginea vs. 4 ± 1
in group C1 were observed. The strong vacuolization, decreased sperm-
atogenesis, decreased number of spermatogonia, more frequently con-
gested blood vessels, and 12 ± 2 invaginations of tunica albuginea were
noticed in A2. At the same time, distorted and disorganized seminiferous
tubules, strong vacuolization, germinal epithelium separated from base-
ment membrane, and 17 ± 2 invaginations of tunica albuginea were
observed in B2. All nicotine-exposed rats showed thinner covering of
tunica albuginea of the testes than rats of the control groups
(Figure 4(a)).
The Leydig cells (LC) were slightly bigger in B1 than in groups C1
(p ¼ .18) and A1 (p ¼ .002, Figure 4(b)). In group A1, LC were smaller
than in C1 (p ¼ .004). After 2 weeks of nicotine exposure, LC in
groups A2 and B2 were smaller than in C2 (p < .01). The numbers of
LC were increased in A1 and B1 vs C1 (p < .05, Figure 4(c)). After
the break of nicotine exposure, the numbers of LC were similar in
C2, A2, and B2 (p>.05). In all tested groups, according to Masson’s
trichrome staining the collagen deposition was similar. No fibrosis was
observed (Figure 5). The difference of the optical density score was
statistically insignificant.

Figure 4. Measurements. (a) Thickness of tunica albuginea of testes; (b) area of Leydig cells;
(c) the number of Leydig cells in one field of view at 100 magnification; (d) thickness of
tunica albuginea of epididymis; (e) height of epithelium of epididymis’ head; (f) height of
epithelium of epididymis’ corpus; C1 (control group 1), A1 (vapor-exposed group), B1
(smoke-exposed group), C2 (control group 2), A2 (vapor-exposed group after 2 weeks break),
B2 (smoke exposed after 2 weeks break).
8 E. WAWRYK-GAWDA ET AL.

Figure 5. No significant fibrosis of testes was seen in any group. Congested blood vessels
were observed in both smoke- and vapor-exposed groups. C1 (control group 1); A1 (vapor-
exposed group): note the increased vacuolization; B1 (smoke-exposed group): note con-
gested blood vessels with erythrocytes, separation of epithelium from basement membrane;
C2 (control group 2): degeneration symptoms, low number of spermatids; A2 (vapor-exposed
group after 2 weeks break): congested blood vessels with erythrocytes, separation of epithe-
lium from basement membrane, vacuolization, degeneration of Sertoli cells; B2 (smoke-
exposed after 2 weeks break): strong degeneration, decrease of spermatogenesis. Masson’s
trichrome staining. Magnification ca 400.

E-cig vapor and cigarette smoke increase the apoptosis in testes

Expression of caspase 3 was increased in the testes of the nicotine-
exposed groups (Figure 5). Apoptotic bodies in A1 (9 ± 4/field of view)
and B1 (11 ± 4/field of view) were more frequent than in C1 (3 ± 2/field
of view). In TUNEL staining, positive cells were observed most fre-
quently in B1 and B2, but in A1 and A2 they were also more frequent
than in C1 and C2 (Figure 6).

E-cig vapor and cigarette smoke induce noticeable degeneration

in epididymis
In A1 and B1, some tubules of the head and corpus of epididymis were
empty and distorted, and the sperm was more condensed than in the
control group. Moreover, vacuoles were visible in the epithelium, and
spaces between tubules were wider than in the control group. Stereocilia
were frequently long in the control groups (Figure 7). In C2, slight
symptoms of degeneration were seen. Degeneration, empty or just acido-
philic secretion in tubules, low epithelium of epididymal duct, less fre-
quent stereocilia, and more collagen fibers between tubules and initial
fibrosis were noted in A2 and B2 (Figure 7). The epithelium of
 TOXICOLOGICAL & ENVIRONMENTAL CHEMISTRY 9

Figure 6. Expression of caspase 3 (upper panels) and cell apoptosis with positive reaction in
TUNEL (lower panels) was increased in both smoke- and vapor-exposed groups. Note apop-
totic bodies (arrows). C1 (control group 1); A1 (vapor-exposed group); B1 (smoke-exposed
group); C2 (control group 2); A2 (vapor-exposed group after 2 weeks break); B2 (smoke-
exposed after 2 weeks break). Upper panels: IHC staining against caspase 3.
Immunoperoxidase reaction with DAB chromogen and hematoxylin counterstain. Magn ca
400x, lower panels: TUNEL with DAB chromogen and methyl green counterstain,
Magnification ca 100.

epididymis heads was higher in A1 (p ¼ .0019) than in C1 and in A2

(p ¼ .04) than in C2 (Figure 4(e)). In B1 and B2, the height of the epithe-
lium of the epididymal head was similar to those in C1 and C2. The epi-
thelium of epididymal corpus was lower in B2 than in C2 (Figure 4(f)).
The tunica albuginea of epididymis in A1, A2, and B2 was thinner than
in the control groups and thicker and dissected in B1 (Figures 4(d) and
7). Infiltrations by mono-nucleated cells and macrophages were observed
in groups B1, A2, and B2.
10 E. WAWRYK-GAWDA ET AL.

Figure 7. C1 (control group 1):normal epididymal structure; A1 (vapor-exposed group):

empty and distorted tubules, wide space between tubules ca be seen; B1 (smoke-exposed
group): empty tubule, condensed sperm (picture 1: mononucleated cells infiltration); in
Trichrome Masson’s staining thick dissected tunica albuginea; C2 (control group 2): degener-
ation symptoms, numerous collagen fibers between tubules; A2 (vapor-exposed group after
2 weeks break): note empty tubules (picture 2: macrophages); B2 (smoke-exposed after
2 weeks break): degeneration symptoms, numerous collagen fibers between tubules (picture
3: mononucleated cells, macrophages, eosinophil). Upper panels H&E staining. Lower panels:
Masson’s trichrome staining. Magnification ca 400 and ca 1000.
 TOXICOLOGICAL & ENVIRONMENTAL CHEMISTRY 11

Discussion
This study reveals that the inhaled cigarette smoke or e-cig aerosol may
damage the structure of testes and epididymis and disturb spermatogen-
esis. Nicotine is one of the factors that alter the male reproductive func-
tion. In addition to nicotine, cigarette smoke contains combustion
products that are toxic to the reproductive system (Cui et al. 2016).
Whereas devoid of combustion products, the main ingredient of vapor
produced in e-cig is nicotine and propylene glycol (Long 2014; Tayyarah
and Long 2014). Currently, the e-cig usage increases, especially by young
people at the reproductive age; therefore, the impact on fertility is in the
focus of numerous studies (McMillen et al. 2018; Condorelli et al. 2017;
Richmond et al. 2018; Zarobkiewicz et al. 2016). Here, an increase in
abnormal sperm count in the groups exposed to smoke or vapor was
observed. The pathological changes were most frequent in rats exposed
to smoke (group B2), but also in both groups exposed to vapor, numer-
ous abnormal spermatozoa were observed. This is in line with other
studies suggesting that nicotine is responsible for disturbances in sperm
formation (Budin et al. 2017; Condorelli et al. 2018). Abnormal head
rates in heavy smoking groups have been seen by Cui et al. (2016).
Budin et al. (2017) proved that nicotine at low doses (0.6 mg/kg body
weight) administrated intraperitoneally each day for 28 consecutive days
affects sperm count, motility, viability, and morphology. In human
males, structural defects in heads and cytoplasmic residues have been
observed in spermatozoa of tobacco chewers more frequently than in
non-chewers. Mohamed and Abdelrahman (2018) showed that nicotine
administrated orally to male rats at a daily dose of 1 mg/kg body weight
for 30 days causes testicular and epididymal structural changes and mor-
phological abnormalities of sperm. In this study, blood vessels congested
with erythrocytes, separation of epithelium from basement membrane,
and vacuolization of seminiferous epithelium were observed in the
experimental groups. Malformations of tunica albuginea of testes and
epididymis were seen in all rats exposed to smoke or aerosol vapor.
Degeneration of testes and epididymis was noticed in the experimental
groups two weeks after cessation of exposure which confirms the role of
the inhaled substances in the degeneration processes. Still, detailed
pathogenesis of these malformations remains to be elucidated.
Condorelli et al. (2017) suggested that nicotinic acetylcholine receptors
located on the spermatozoa play a role in the alteration of sperm param-
eters such as acrosomal reaction, mitochondrial function, apoptosis, and
chromatin and DNA integrity caused by nicotine exposure. Numerous
other studies indicated that malformations of sperm may be a conse-
quence of the activation of inflammatory reaction and increased
12 E. WAWRYK-GAWDA ET AL.

oxidative stress and increased level of reactive oxygen species (ROS)

caused by nicotine (Meo and Al Asiri 2014; Oyeyipo, Raji, and
Bolarinwa 2014; Sabeti et al. 2016). Nicotine decreases the number of
factors responsible for ROS neutralization (Budin et al. 2017; Ghaffari
and Rostami 2012; Sabeti et al. 2016). Moreover, it also decreases testicu-
lar enzymes such as glutathione peroxidase, glutathione reductase, cata-
lase, and superoxide dismutase, and increases testicular lipid
peroxidation and nitric oxide level (Budin et al. 2017; Oyeyipo, Raji, and
Bolarinwa 2014). Elevated oxidative stress has deleterious effects, induc-
ing double-stranded DNA breaks in the sperm nuclei and apoptosis. It
also affects energy production by the mitochondria situated at the mid-
piece which results in poor sperm motility (asthenozoospermia)
(Comhaire, Vandenberghe, and Decleer 2017). This type of inflammatory
response is likely to be activated by tissue irritation and toxicity caused
also by other substances contained in smoke and e-cig aerosol such as
trace metals and metalloids (Pappas 2012).
In this study, leukocyte and macrophage infiltration was observed in
the epididymis of groups B1, A2, and B2. These cells are responsible for
cytokine production as well as the activation of inflammatory response.
They are also associated with the increase of endogenous ROS produc-
tion. In this study, we also noticed an increase of spermatogonia and
spermatocyte apoptosis and significant differences in the size and num-
ber of LC. Moreover, nicotine may have anti- or pro-apoptotic effects on
tissues. Jalili et al. argued that nicotine activates the apoptosis in the
heart and lungs, but decreases the apoptosis in the kidneys and liver
(Jalili et al. 2017). On the other hand, nicotine given intraperitoneally
increases expression of p53 and caspase 3 in testicular tissue and dimin-
ishes expression of anti-apoptotic protein, Bcl-2, and reduces spermato-
genesis (Mosadegh, Hasanzadeh, and Razi 2017).
The majority of previous studies indicated that nicotine decreases the
testosterone, prolactine, and follicular-stimulating hormone production,
increases or decreases luteinizing hormone, and reduces libido
(Mohamed and Abdelrahman 2018; Mosadegh, Hasanzadeh, and Razi
2017; Oyeyipo, Raji, and Bolarinwa 2013, 2014). In 22 studies including
13,317 men aged from 18 to 61, smokers had higher mean testosterone
levels than nonsmokers (Zhao et al. 2016). Moreover, nicotine leads to
disorganization of peritubular extracellular matrix and myoid cells and
increase of tunica albuginea. The peritubular structures have been proven
to play a role in the modulation of morphology and function of Sertoli
cells, so consequently their pathological changes may disturb spermato-
genesis and conceiving (Aydos et al. 2001). The early exposure to toxins
such as nicotine may cause DNA damage in the mechanism of epigenetic
 TOXICOLOGICAL & ENVIRONMENTAL CHEMISTRY 13

reprogramming. Therefore, it may harm the paternal genetic and the epi-
genetic contribution to the developing embryo and affect the embryo
development (Comhaire, Vandenberghe, and Decleer 2017; Wyck et al.
2018). Thus, vaping should not be used as antismoking treatment of
men in the procreative age.

Conclusion
Substances inhaled from traditional and e-cig lead to both sperm malfor-
mation and spermatogenesis reduction. Vaping-like smoking accelerates
the degeneration of testes and reduces the duration of male fertility. The
pathological changes induced by cigarette smoke are more intense but
the use of e-cig is not negligible.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The study was funded by grant of Minister of Science and Higher Education [No
MNmb 246].

ORCID
Ewelina Wawryk-Gawda http://orcid.org/0000-0001-6914-6735

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