Comparison of the Effects of Bisphenol A Alone

and in a Combination with X-irradiation on Sperm

Count and Quality in Male Adult and Pubescent
Mice
Małgorzata M. Dobrzyńska,1 Ewa A. Jankowska-Steifer,2 Ewa J. Tyrkiel,1 Aneta Gajowik,1
Joanna Radzikowska,1 Krzysztof A. Pachocki1
1
Department of Radiation Hygiene and Radiobiology, National Institute of Public Health—
National Institute of Hygiene, Warsaw, Poland
2
Department of Histology and Embryology, Warsaw Medical University, Warsaw, Poland

Received 9 July 2012; revised 27 February 2013; accepted 5 March 2013

ABSTRACT: Bisphenol A (BPA) is employed in the manufacturing of epoxy, polyester-styrene, and poly-
carbonate resins, which are used for the production of baby and water bottles and reusable containers,
food and beverage packing, dental fillings and sealants. The study was designed to examine the effects
of 8-week exposure (a full cycle of spermatogenesis) to BPA alone and in a combination with X-irradiation
on the reproductive organs and germ cells of adult and pubescent male mice. Pzh:Sfis male mice were
exposed to BPA (5, 10, and 20 mg/kg) or X-rays (0.05 Gy) or to a combination of both (0.05 Gy 1 5 mg/kg
bw BPA). The following parameters were examined: sperm count, sperm motility, sperm morphology, and
DNA damage in male gametes. Both BPA and X-rays alone diminished sperm quality. BPA exposure sig-
nificantly reduced sperm count in pubescent males compared to adult mice, with degenerative changes
detected in seminiferous epithelium. This may suggest a higher susceptibility of germ cells of younger
males to BPA action. Combined BPA with X-ray treatment enhanced the harmful effect induced by BPA
alone in male germ cells of adult males, whereas low-dose irradiation showed sometimes protective or
additive effects in pubescent mice. # 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1301–1313, 2014.
Keywords: bisphenol A; combined exposure; sperm count and morphology; comet assay; histology

INTRODUCTION especially at early stages of spermatogenesis, are particu-

The male reproductive system is a very sensitive target
organ to chemical and physical agents. Reduced fertility,
embryo loss, birth defects, and childhood cancer are
common outcomes of such exposure. Male germ cells,
Correspondence to: M. M. Dobrzyńska;
e-mail: mdobrzynska@pzh.gov.pl
Contract grant sponsor: Polish Ministry of Science and Higher Educa-
tion, 2008–2011, project No. N N404 029535
Published online 25 April 2013 in Wiley Online Library
(wileyonlinelibrary.com). DOI 10.1002/tox.21861
C 2013 Wiley Periodicals, Inc.
larly sensitive, and associated sperm disorders, including
problems in production and maturation of sperm, may
cause infertility. Potential outcomes include normal sper-
matozoa that are produced in abnormally low numbers or
sperm produced in adequate numbers but are immature,
with poor motility and morphology—all can diminish the
chance for fertilization (Sikka, 1999; Aitken et al., 2004).
Estrogen-like endocrine disruptors (xenoestrogens) are
synthetic chemicals that are structurally similar to, and can
act as, endogenous estrogens. Chemical agents working
through endocrine disruptive mechanism may have adverse
effects on the human health (Diamanti-Kandarakis et al.,

1301
1302 DOBRZYŃSKA ET AL.
2009). They interact with the endocrine system at various
levels and may influence the sexual differentiation, matura-
tion, reproduction, growth, metabolism, digestion, cardio-
vascular function, and excretion. Various abnormalities and
disorders reported in humans and wildlife are related to en-
docrine disruptors (Kavlock et al., 1996). Estrogen activity
of some chemical agents may underlie diminished sperm
quantity and quality, as well as being responsible for
defects in the male reproductive tract. Such events have
been observed with growing frequency in the general popu-
lation (Carlsen et al., 1992; Sharpe, 1998; Sharpe et al.,
1998).
Bisphenol A (2,2-bis(4-hydroxyphenyl)propane (BPA)
was first synthesized by Dianin in 1891 and was later inves-
tigated in the 1930s during the search for synthetic estro-
gens. It is one of the most estrogenic compounds among
compounds found in the food wrap packing. There are
more than 3 billion kg of BPA produced in the world annu-
ally and more than 100 ton released into the atmosphere by
yearly production (Vandenberg et al., 2009). BPA is
employed in the manufacturing of epoxy, polyester-styrene,
and polycarbonate resins, which are used in the production
of baby and water bottles and reusable containers, food and
beverage packing, dental fillings and sealants. Metallic
food and beverage cans are protected from rusting and cor-
rosion by the application of epoxy resins as inner coatings
(Staples et al., 1998; Lyons, 2000). BPA migrates from
dental sealants or polycarbonate and epoxy-linked food and
drink containers, leading to consumer exposure of human
(Tyl et al., 2002). It has been found in food (4–23 lg/can),
beverages (7–58 lg/g), and in saliva of patients after appli-
cation of certain dental sealants (90–913 lg/saliva-1 h)
(Akingbemi et al., 2004).
BPA can bind to androgen receptors and inhibit the
action of androgen (Lee et al, 2003). Several studies have
detected BPA in human tissues in the parts per billion
range (Ikezuki et al., 2002; Schonfelder et al., 2002; Cala-
fat et al., 2005), including in human serum, follicular fluid,
in full-term amniotic fluid, (Ikezuki et al., 2002), in umbil-
ical cord blood (Schonfelder et al., 2002) and in breast
milk (Sun et al., 2004). The half-life of BPA is approxi-
mately 6 h, with nearly complete urinary excretion by 24
h, and BPA is metabolized in the liver, where it is con-
verted to BPA-gluc and is rapidly eliminated in the urine
(Volkel et al., 2002).
This study also examined the effects of X-irradiation in
association with BPA on sperm count and quality of male
adult and pubescent male mice. Several human activities
involve exposure to radiation and radioactive substances, in
addition to exposure from cosmic rays and naturally occur-
ring radioactive substances. The use of radioactive materi-
als in industry, agriculture, and research is expanding, and
people have been harmed by mishandling radiation sources
(UNSCEAR, 2008). Spermatogonial stem cells
and differentiating spermatogonia are highly sensitive to
Environmental Toxicology DOI 10.1002/tox
ionizing radiation, whereas spermatocytes are less sensitive,
and spermatids are rather radio-resistant (Rowley et al.,
1974; Hawkins et al., 1989; Bonde and Giwercman, 1995).
Ionizing radiation and BPA are widely present in the
environment. Thus, people are often exposed to both, and
whereas these are usually at low-dose exposure can be for a
long periods of time or indeed may be constant. Exposure
of humans to BPA is inevitable mainly through the diet,
because of the fact that residues of BPA can migrate from
plastic containers, bottles, and coating into food and drinks,
especially during heating. As mentioned before, BPA was
detected in canned liquids and dishes, including dairy prod-
ucts and baby food (Cao et al., 2011).
This study examined the effect of exposure to BPA
alone and in a combination with X-rays for 8 weeks (i.e., a
full cycle of spermatogenesis) on the male mice reproduc-
tive organs and germ cells. As the greatest effects of xen-
oestrogens may occur during puberty, a period of rapid
physiological changes such as growth, spurt, maturation of
the gonads and brain (Roy et al., 2009), and immature
rodents are more sensitive for gonadal effects induced by
endocrine disruptors than adults (Sjo†berg et al., 1985), we
compare the impact of exposure to these agents in pubes-
cent and mature male mice.
MATERIALS AND METHODS
Animals Husbandry and Administration
Procedure
Pzh:Sfis outbreed male mice were housed in standard
rodent cages in a room designed for control of temperature,
humidity, and light cycle (12 h dark and 12 h light). Tap
water and rodent diet were available ad libitum. After 1
week of acclimatisation, males were assigned randomly to
either control or exposed groups. Eight-week-old (mature)
as well as 4.5-week-old (pubescent) male mice were
exposed to BPA dissolved in small amount (900 lL) of
70% ethyl alcohol and then diluted in drinking water served
in glass bottles (150–200 mL) to obtain the following
doses: 5, 10, and 20 mg/kg bw or irradiated with X-rays
(0.05 Gy) or exposed to a combination of low doses of both
agents (0.05 Gy 1 5 mg/kg bw BPA ). The body weight of
animals was checked weekly, and the amounts of BPA-
containing water were checked daily. It was found that the
animals consumed daily approximately 7 mL of water, con-
taining 5, 10, or 20 mg/kg bw of BPA, respectively. On the
basis of above findings, the solution of BPA in water was
prepared weekly. A therapeutic Roentgen unit Medicor
type THX-250 was used as an X-ray source. It was operated
with the following parameters: 175 kV, 10 mA, added fil-
tration 0.5 mm Cu, and HVL 0.8 mm Cu. Mice were
whole-body irradiated at the dose rate of 0.20 Gy/min.
Males were exposed to X-rays or BPA or to both agents for
 EFFECT OF BISPHENOL A AND X-IRRADIATION ON SPERM COUNT AND QUALITY
8 weeks. Control animals were sham-irradiated and unex-
posed to BPA.
The dose of 0.05 Gy was chosen on the basis of the pre-
vious study as a lowest dose of X-rays that decreased sperm
quality without diminishing sperm count (Dobrzyńska,
2003), whereas the doses of BPA chosen for this study
were based on LOAEL (50 mg/kg bw daily) and NOAEL
(5 mg/kg bw daily) values (EFSA, 2006; US EPA, 2008).
The authors obtained permission from Ethical Commis-
sion for Animal Experiments for conducting this study.
Six to nine males of each experimental and control
groups were weighed and sacrificed at 24 h and 1, 4, and 8
weeks after the last treatment. Both testes and epididymides
were removed from each male and weighed.
Sperm Count, Motility, and Morphology
One epididymis from each animal was macerated in 0.2 mL
of 1% solution of trisodium citrate for 5–8 min, minced,
and mixed to a solution volume of 2 mL. The sperm sus-
pension was diluted 1:1 in 10% buffered formalin. The
spermatozoa were counted using improved Neubauer hae-
mocytometer (Searle and Beechey, 1974; Harrison and
Moore, 1980). The contents of the second epididymis were
placed into 0.2 mL of warm (378C) physiological saline.
An aliquot was placed on warm (378C) microscope slide
and covered with a cover slip. In total, 200 cells per animal
were evaluated for motility within 5 min after the killing of
animal (Working et al., 1985). The remaining sperm were
used for the estimation of morphologically abnormal sper-
matozoa, according to the procedure described by Wyrobek
and Bruce (1975). Smears were prepared on microscope
slides, air dried overnight, and stained with eosin
Y. Spermatozoa (1000 per mouse) were analyzed using a
light microscope and any abnormalities recorded such as:
lacking hook, amorphous, banana-shaped heads, folded
themselves, twin tailed, or having twin-head forms.
Comet Assay
For Comet assay analysis, one testis from each animal was
decapsulated, placed in RMPI 1640 medium, and minced
with scissors to form a single-cell suspension. The basic
technique of Singh et al. (1988) and further described by
Anderson et al. (1994) was followed. Five microliters of
cell suspension was mixed with 75 lL of low-melting-point
agarose (LMPA) for embedding onto glass microscope
slides previously covered with normal melting point agrar-
ose. After solidification of the agarose at 48C, another layer
of LMPA was added and allowed to solidify at 48C again.
The slides were immersed in lysing solution overnight at
48C. Then slides were incubated in the electrophoresis solu-
tion for 20 min to allow the unwinding of DNA. Alkaline
electrophoresis was conducted for 20 min at 48C using 24
1303
V and 300 mA. After neutralization, the slides were stained
with EtBr and examined using fluorescence microscope.
Images of 100 randomly selected haploid germ cells from
each animal were recorded and analyzed using CASP
image-analysis software (Końca et al., 2003). The DNA
Tail Moment and Percentage of DNA in Comet Tail were
chosen as the parameters for further analysis.
Light and Electron Microscopic Histological
Procedure
Light Microscopy
One testis from each mouse was fixed in Bouin’s fluid for
40 h, after shallow piercing of the tunica albuginea with a
21-gauge needle to aid in the penetration of the fixative. Af-
ter fixation, the material was briefly washed in tap water,
and dehydrated with graded ethanol and in n-butanol and
paraffin embedded. Serial sections (7 lm) were cut and
stained with hematoxylin and eosin. Slides were examined
using light microscope.
Transmission Electron Microscopy
Immediately after removing, the testes were placed in a fix-
ative, cut into 1 mm3 fragments, and fixed for 2 h in 2.5%
glutaraldehyde, followed by postfixing for 1 h in 1% OsO4
both in 0.1 M phosphate buffer, pH 7.4. After dehydration
in graded ethanol (50–100%) and in propylene oxide, the
material was embedded in Poly/Bed 812 (Polysciences,
Warrington, PA) and cut with a diamond knife on a RMC
type MTXL ultramicrotome. Ultrathin sections were
contrasted with uranyl acetate and lead citrate and exam-
ined in a JEM 100S (Jeol, Japan) transmission electron
microscope.
Statistical Analysis
One-way analysis of variance (ANOVA) was used to deter-
mine significant differences between results of various
groups. Fisher’s post hoc test was applied to determine sig-
nificant changes between groups. p-Values of \0.05 were
considered significant in both analyses.
RESULTS
The effects of 8-week X-irradiation and BPA exposure on
the reproductive organs of mature male mice are summar-
ized in Table I. The mean testes weights were significantly
decreased at 24 h and 1 week post-0.05 Gy irradiation. Sim-
ilarly, 0.05 Gy with 5 mg/kg bw of BPA significantly
reduced testes weight compared to control and BPA alone
at 24 h and 1 week post combined exposure.
Environmental Toxicology DOI 10.1002/tox
1304 DOBRZYŃSKA ET AL.

TABLE I. The effects of BPA alone and in combination with X-irradiation on the reproductive organs of adult male
mice

Mean Mean Mean

Body Testes Relative Epididymides Relative
Weight Weight Testes Weight Epididymides
Time Doses (g) 6 SD (mg) 6 SD Weight (mg) 6 SD Weight

24 h Control 39.2 6 2.1 239.2 6 32.8 0.61 169.2 6 23.6 0.43

Postexposure 5 mg/kg bw BPA 38.9 6 3.0 250.0 6 36.5 0.64 131.4 6 17.3 0.34
10 mg/kg bw BPA 39.8 6 3.4 250.4 6 57.2 0.63 157.2 6 26.6 0.39
20 mg/kg bw BPA 42.1 6 5.4 253.1 6 37.9 0.60 160.7 6 35.7 0.38
0.05 Gy 39.3 6 4.3 179.0 6 16.4* 0.46 146.4 6 20.6 0.37
0.05 Gy 1 5 mg/kg BPA 38.9 6 4.0 161.0 6 36.6*a 0.41 140.3 6 49.7 0.36
1 Week Control 41.1 6 3.0 241.7 6 37.3 0.59 173.6 6 36.4 0.42
postexposure 5 mg/kg bw BPA 38.1 6 2.1 230.8 6 32.5 0.61 156.8 6 40.7 0.41
10 mg/kg bw BPA 37.6 6 1.9 225.0 6 31.6 0.60 154.1 6 30.8 0.41
20 mg/kg bw BPA 38.7 6 4.5 223.36 49.2 0.58 168.8 6 18.2 0.44
0.05 Gy 37.8 6 3.1 175.0 6 31.5* 0.46 155.0 6 26.0 0.41
0.05 Gy 1 5 mg/kg BPA 39.3 6 4.2 176.8 6 30.7*a 0.45 156.0 6 34.9 0.40
4 Weeks Control 37.9 6 4.9 235.7 6 26.4 0.62 166.4 6 53.9 0.44
postexposure 5 mg/kg bw BPA 39.7 6 3.8 253.6 6 52.9 0.64 189.8 6 53.6 0.48
10 mg/kg bw BPA 38.9 6 6.9 241.5 6 36.9 0.62 161.8 6 56.0 0.42
20 mg/kg bw BPA 37.1 6 3.7 244.4 6 39.9 0.66 181.9 6 51.2 0.49
0.05 Gy 40.1 6 4.6 196.2 6 16.3 0.49 168.4 6 12.3 0.42
0.05 Gy 1 5 mg/kg BPA 39.8 6 2.4 211.0 6 33.7 0.53 157.2 6 8.8 0.39
8 Weeks Control 42.2 6 3.1 260.1 6 29.2 0.62 187.4 6 41.5 0.44
postexposure 5 mg/kg bw BPA 39.2 6 2.8 242.6 6 37.6 0.62 210.1 6 59.4 0.54
10 mg/kg bw BPA 42.5 6 6.3 246.1 6 38.8 0.58 178.1 6 60.3 0.42
20 mg/kg bw BPA 37.5 6 4.5 261.0 6 31.7 0.70 193.4 6 63.8 0.52
0.05 Gy 38.8 6 5.4 217.8 6 41.0 0.56 152.0 6 22.2 0.39
0.05 Gy 1 5 mg/kg BPA 40.6 6 2.4 244.7 6 19.9 0.60 201.8 6 47.6 0.50
*
p 0.05 compared to control by Fisher’s test.
a
p 0.05 compared to 5 mg/kg bw BPA by Fisher’s test.

The effects of 8-week X-irradiation and BPA exposure
on the reproductive organs of pubescent male mice are
summarized in Table II. In brief, 0.05 Gy of X-rays signifi-
cantly reduced mean testes weights at 24 h and 1 week
postexposure, whereas combined exposure to low doses of
both agents significantly reduced mean testes weight com-
pared to control and those mice receiving 5 mg/kg bw
BPA.
Sperm quantity and quality of mature at the beginning
of the experiment mice is summarized in Table III. By 24
h and 1 week after, the sperm counts of male mice treated
for 8 weeks to BPA were markedly, but not significantly
and not dose dependently, reduced. Although the sperm
counts in mice receiving 0.05 Gy were decreased at all
time points, result was statistically different from control
mice only at 4-week postexposure. Sperm counts in mice
exposed to 0.05 Gy 1 5 mg/kg bw BPA were, however,
reduced at all time points, and were significantly different
compared to mice receiving 5 mg/kg bw BPA alone at 4-
week postexposure. Moreover, irradiation with 0.05 Gy
significantly increased the percentage of abnormal sperma-
tozoa compared to control value at both 24 h and 4 weeks
postexposure, as did exposure to BPA at 24 h, and 4 and 8
weeks. The frequency of malformed spermatozoa after
combined exposure to 0.05 Gy 1 5 mg/kg bw BPA was
significantly increased at all time points compared to con-
trol, except at 1-week postexposure. There were no signifi-
cantly differences in the percentage of DNA in Comet Tail
and Comet Tail Moment between control and exposed
males.
Sperm quantity and quality in pubescent at the
beginning of exposure male mice are summarized in Table
IV. Sperm counts were significantly reduced at 24 h and
1-week postirradiation with 0.05 Gy as well as at 1 and 4
weeks after the end of application of 20 mg/kg bw of BPA.
Combined 0.05 Gy 1 5 mg/kg bw BPA significantly
reduced sperm count compared to control and BPA values
alone immediately after the end of combined exposure and
1 week later. The percentage of abnormal spermatozoa was
significantly increased at 24 h and 1-week postexposure for
BPA-treated groups but at 4 and 8 weeks later after applica-
tion of 20 mg/kg bw BPA.

Environmental Toxicology DOI 10.1002/tox

 EFFECT OF BISPHENOL A AND X-IRRADIATION ON SPERM COUNT AND QUALITY 1305

TABLE II. The effects of BPA alone and in combination with X-irradiation exposure on the reproductive organs of pu-
bescent male mice

Mean Mean Mean

body Testes Relative Epididymides Relative
weight Weight Testes Weight Epididymides
Time Doses (g)6 SD (mg) 6 SD Weight (mg) 6 SD Weight

24 h Control 36.77 6 3.89 237.89 6 29.00 0.65 228.33 6 45.24 0.62

Postexposure 5 mg/kg bw BPA 39.82 6 4.89 210.20 6 45.47 0.53 201.00 6 81.33 0.50
10 mg/kg bw BPA 36.3 6 3.02 221.43 6 22.88 0.61 183.33 6 58.82 0.51
20 mg/kg bw BPA 38.97 6 5.98 229.83 6 16.22 0.59 219.80 6 59.20 0.56
0.05 Gy 37.88 6 8.21 186.40 6 39.20* 0.49 203.50 6 30.60 0.54
0.05 Gy 1 5 mg/kg BPA 41.74 6 5.25 192.00 6 27.83*a 0.46 229.40 6 30.56 0.55
1 Week Control 37.11 6 5.79 233.44 6 35.37 0.63 252.78 6 54.39 0.68
postexposure 5 mg/kg bw BPA 37.04 6 3.48 242.71 6 36.56 0.66 207.71 6 49.19 0.56
10 mg/kg bw BPA 37.03 6 3.00 227.14 6 18.20 0.61 214.57 6 45.11 0.58
20 mg/kg bw BPA 35.74 6 2.16 224.71 6 32.36 0.63 242.00 6 42.87 0.68
0.05 Gy 33.96 6 4.96 175.20 6 21.28* 0.52 197.00 6 26.02 0.58
0.05 Gy 1 5 mg/kg BPA 34.74 6 6.2 155.40 6 10.24*,a 0.45 194.60 6 84.20 0.56
4 Weeks Control 35.6 6 6.14 235.33 6 20.74 0.66 207.83 6 43.20 0.58
postexposure 5 mg/kg bw BPA 40.37 6 2.86 220.17 6 28.24 0.55 233.00 6 45.03 0.58
10 mg/kg bw BPA 39.6 6 2.34 235.67 6 24.39 0.60 265.33 6 58.67 0.67
20 mg/kg bw BPA 36.7 6 2.81 218.00 6 44.07 0.59 219.50 6 32.99 0.60
0.05 Gy 35.76 6 4.23 209.14 6 17.03 0.58 177.29 6 19.03 0.50
0.05 Gy 1 5 mg/kg BPA 36.4 6 4.80 205.00 6 25.67 0.56 181.00 6 67.13 0.50
8 Weeks Control 39.63 6 6.05 232.71 6 26.45 0.59 224.67 6 19.76 0.57
postexposure 5 mg/kg bw BPA 40.08 6 3.38 265.83 6 22.78 0.66 230.67 6 43.30 0.58
10 mg/kg bw BPA 36.82 6 3.62 221.17 6 38.93 0.60 181.67 6 36.62* 0.49
20 mg/kg bw BPA 37.18 6 2.67 229.83 6 31.35 0.62 185.83 6 38.98 0.50
0.05 Gy 38.76 6 6.55 233.71 6 37.27 0.60 215.43 6 39.29 0.56
0.05 Gy 1 5 mg/kg BPA 33.51 6 7.21 220.14 6 39.80 0.62 220.43 6 59.44 0.66
*
p 0.05 compared to control by Fisher’s test.
b
p 0.05 compared to 5 mg/kg bw BPA by Fisher’s test.

X-irradiation significantly increased the percentage of
malformed spermatozoa 24 h and 8 weeks postlast irradia-
tion. The frequencies of abnormal spermatozoa after com-
bined BPA and X-irradiation treatment were significantly
different from control at 24 h as well as 4- and 8-week post-
exposure. The frequency of malformed spermatozoa in
combined group was significantly reduced at 1 week after
the last exposure and significantly increased 8 weeks, after
the termination of exposure compared to the effect of BPA
alone. At 4 weeks after the end of combined X-ray–BPA
exposure, the percentage of malformed spermatozoa was
significantly elevated compared to the results of irradiation
alone. The Comet Tail Moment value at 24 h after the end
of exposure to 5 mg/kg bw of BPA was significantly
increased compared to control, whereas the result of
combined exposure was significantly reduced compared to
the above dose of BPA. The percentage of DNA in Comet
Tail of males exposed to X-rays was significantly reduced
compared to control value, whereas the result of combined
exposure was significantly reduced compared to the result
of 5 mg/kg bw BPA alone.
Only slight morphological changes, as viewed by light
microscopy, were visible in the gonads of adult, 8-week-old
animals exposed to BPA at doses of 10 and 20 mg/kg,
either treated with X-irradiation or combined X-irradiation
and BPA. These changes occurred mainly 24 h after the ter-
mination of treatment. In some seminiferous tubules degen-
erating spermatogonia and spermatocytes were present.
Occasionally, intraepithelial vacuoles were observed. No
reduction in thickness of the seminiferous epithelium was
evident. Intercellular vacuoles between Sertoli cells and
spermatogonia were observed using electron microscopy in
animals analyzed 24 h after the termination of exposure to
20 mg/kg BPA, to irradiation, or combination of irradiation
and BPA. Although the continuity of the basement mem-
brane of the seminiferous epithelium was always preserved,
this membrane showed pronounced invaginations in the
above-mentioned groups but not in other groups.
Differences in gonad morphology of experimental ani-
mals, as compared to the control [Fig. 1(A)], were more
prominent in pubescent (4.5 week old) compared to adult
(8 week old) animals. In all experimental groups, the

Environmental Toxicology DOI 10.1002/tox

1306 DOBRZYŃSKA ET AL.

TABLE III. The sperm quantity and quality of adult male mice exposed to BPA alone and in combination with X-
irradiation

Sperm % of Mobile % of Abnormal Comet % Comet

Count 3 106/mL Spermatozoa Spermatozoa Tail Moment Tail DNA
Time Doses 6 SD 6 SD 6 SD 6 SD 6 SD

24 h After the Control 4.48 6 1.29 28.88 6 16.84 10.55 6 3.01 2.75 6 0.76 6.15 6 1.97
end of exposure 5 mg/kg BPA 2.89 6 1.87 9.55 6 5.81 17.38 6 4.48 5.05 6 3.81 8.40 6 6.66
10 mg/kg BPA 2.8661.57 11.38 6 11.55 22.72 6 9.76* 3.09 6 2.38 5.88 6 2.55
20 mg/kg BPA 3.19 6 1.20 20.04 6 26.42 23.88 6 5.21* 5.13 6 4.59 8.65 6 5.62
0.05 Gy 2.80 6 1.15 6.15 6 5.70 21.22 6 5.69* 4.23 6 4.62 6.61 6 5.45
0.05 Gy 1 2.18 6 0.95 8.50 6 8.50 20.92 6 5.84* 5.32 6 5.81 7.66 6 7.84
5 mg/kg BPA
1 Week after the Control 4.20 6 3.39 32.55 6 18.08 11.54 6 2.6 2.13 6 1.15 6.12 6 3.04
end of exposure 5 mg/kg BPA 2.59 6 2.22 20.79 6 10.10 13.43 6 5.77 3.44 6 2.01 6.44 6 2.56
10 mg/kg BPA 3.38 6 1.71 18.04 6 13.49 17.27 6 5.75 4.42 6 3.88 7.81 6 4.14
20 mg/kg BPA 1.72 6 0.62 14.75 6 14.69 19.35 6 10.02 2.74 6 1.93 5.35 6 3.18
0.05 Gy 1.73 6 1.11 29.18 6 18.34 18.56 6 3.52 3.67 6 2.68 7.03 6 4.14
0.05 Gy 1 1.84 6 0.8 19.97 6 15.04 19.00 6 6.15 3.17 6 2.29 5.81 6 3.24
5 mg/kg BPA
4 Weeks after the Control 4.82 6 1.87 32.44 6 16.18 9.18 6 1.79 1.85 6 1.74 4.67 6 3.03
end of exposure 5 mg/kg BPA 5.35 6 1.24 31.63 6 19.2 14.35 6 8.66 3.90 6 2.02 5.37 6 1.25
10 mg/kg BPA 4.17 6 1.53 17.91 6 13.6 14.89 6 4.06 4.26 6 2.79 6.36 6 2.47
20 mg/kg BPA 4.09 6 1.96 16.65 6 15.01 27.68 6 21.57* 3.71 6 1.85 5.69 6 2.78
0.05 Gy 2.29 6 0.89* 27.36 6 16.2 25.86 6 6.55* 5.54 6 2.34 9.79 6 3.47
0.05 Gy 1 3.47 6 1.56a 24.54 6 16.07 28.53 6 5.49* 4.00 6 5.9 7.44 6 9.22
5 mg/kg BPA
8 Weeks after the Control 6.05 6 2.16 27.80 6 12.38 8.68 6 3.43 2.56 6 2.33 7.30 6 7.17
end of exposure 5 mg/kg BPA 4.51 6 2.37 21.80 6 15.75 20.32 6 8.00* 3.63 6 2.22 4.89 6 2.49
10 mg/kg BPA 4.12 6 1.92 20.50 6 15.81 18.27 6 10.30* 2.82 6 2.07 4.73 6 4.73
20 mg/kg BPA 3.76 6 1.77 22.83 6 13.67 24.22 6 8.33* 3.98 6 3.42 4.87 6 3.84
0.05 Gy 4.89 6 1.60 29.25 6 18.72 18.63 6 4.86 5.65 6 3.01 9.27 6 3.75
0.05 Gy 1 3.29 6 1.63 20.96 6 19.95 23.65 6 6.62* 3.37 6 2.83 5.59 6 4.29
5mg/kg BPA
*
p 0.05 compared to control by Fisher’s test.
a
p 0.05 compared to 5 mg/kg bw BPA by Fisher’s test.

gonads of animals treated with BPA, irradiated, or exposed
to combined irradiation and BPA contained degenerating
spermatogonia, and primary as well as secondary spermato-
cytes. Degenerating cells were more commonly observed in
the groups of animals exposed to BPA. The number of
degenerated cells increased with a rising doses of BPA and
was high at 24 h and 1 week after the termination of treat-
ment with BPA [Fig. 1(B)], although at 4- and 8-week post-
BPA treatment end these degenerated cells were observed
less frequently. Occasionally, the multinucleated giant cells
located within the seminiferous epithelium in the testes of
animals treated with BPA at the dose of 20 mg/kg were
observed [Fig. 1(H)].
Single intraepithelial vacuoles were found in animals
treated with 20 mg/kg of BPA, irradiated, or exposed to
combine irradiation and BPA [Fig. 1(C–E)]. Such changes
were observed at all time points after the termination of
treatments. Prominent vacuolization and loss of thickness
of some seminiferous tubules was observed 1- and 4-week
posttermination of combined exposure to irradiation and
BPA [Fig. 1(F)]. Spermatogonia and spermatocytes were
clearly separated from Sertoli cells in gonads assessed 24
h or 1 week postend of irradiation [Fig. 1(G)] although
these changes affected only some of the seminiferous
tubules.
Ultrastructural analysis confirmed the presence of
degenerative changes in seminiferous epithelium, including
nuclei of the spermatogonia showed features of degenera-
tion [Fig. 2(B)] as well as vacuoles and condensed chroma-
tin, in mice treated with 20 mg/kg of BPA. Intercellular
vacuoles of different size located at the borders of Sertoli
cells and spermatogonia as well as spermatogonia and sper-
matocytes were identified [Fig. 2(B–D)] in animals irradi-
ated, treated with the combination of irradiation, and BPA

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 EFFECT OF BISPHENOL A AND X-IRRADIATION ON SPERM COUNT AND QUALITY 1307

TABLE IV. The sperm quantity and quality of pubescent male mice exposed to BPA alone and in combination with X-
irradiation

Sperm
Count % of Mobile % of Abnormal Comet % Comet
3 106/mL Spermatozoa Spermatozoa Tail Moment Tail DNA
Time Dose 6 SD 6 SD 6 SD 6 SD 6 SD

24 h Control 4.49 60.99 28.01 6 11.42 6.17 6 2.57 1.65 6 1.91 6.85 6 5.26
Postexposure 5 mg/kg bw BPA 3.67 61.35 31.28 6 16.43 9.72 6 3.78* 5.44 6 5.38* 9.40 6 7.59
10 mg/kg bw BPA 3.57 61.10 37.42 6 16.91 12.74 6 2.02* 4.41 6 3.79 8.84 6 5.16
20 mg/kg bw BPA 4.01 61.78 28.81 625.10 10.25 6 3.75* 3.90 6 2.75 7.20 6 4.14
0.05 Gy 1.87 60.53* 25.25 615.27 10.02 6 2.65* 0.39 6 0.41 1.21 6 0.64*
0.05 Gy 1 2.16 60.29*a 29.19 610.27 10.22 6 2.06* 0.60 6 0.68a 1.90 6 2.00a
5 mg/kg BPA
1 Week Control 4.63 6 1.47 34.44 6 9.66 6.14 6 2.26 2.26 6 1.23 4.58 6 2.44
postexposure 5 mg/kg bw BPA 4.17 6 1.30 24.57 6 15.80 13.46 6 5.40* 2.60 6 2.44 6.51 6 4.03
10 mg/kg bw BPA 3.46 6 1.03 21.89 6 17.99 16.51 6 7.60* 3.04 6 2.52 7.09 6 4.97
20 mg/kg bw BPA 2.84 6 0.70* 17.19 614.65 11.27 6 3.81* 3.19 6 3.49 7.70 6 5.89
0.05 Gy 2.22 6 1.57* 15.46 610.29 7.79 6 3.47 1.8 6 1.53 3.18 6 3.20
0.05 Gy 1 2.75 6 1.45* 12.55 6 6.14 7.06 6 1.28a 5.07 6 2.9 6.43 6 5.30
5 mg/kg BPA
4 Week Control 4.84 6 1.47 25.82 6 10.78 7.20 6 2.27 2.65 6 1.99 4.97 6 3.80
postexposure 5 mg/kg bw BPA 4.97 6 1.53 33.25 6 18.37 9.68 6 1.11 3.46 6 2.18 8.51 6 4.03
10 mg/kg bw BPA 3.15 6 1.56 25.83 6 11.72 8.58 6 1.04 3.21 6 1.54 7.22 6 5.73
20 mg/kg bw BPA 2.45 6 2.13* 26.21 6 21.07 10.62 6 4.78* 3.45 6 3.00 5.84 6 4.98
0.05 Gy 4.76 6 1.69 18.6 6 16.30 6.53 6 1.47 6.43 6 4.84 7.48 6 6.92
0.05 Gy 1 3.66 6 1.22 17.35 6 12.66 12.78 6 6.44*b 5.76 6 3.77 8.09 6 4.78
5 mg/kg BPA
8 Week Control 4.17 6 2.23 26.04 6 11.46 5.23 6 1.47 2.06 6 2.71 4.80 6 4.53
postexposure 5 mg/kg bw BPA 5.59 6 2.26 30.60 615.57 4.83 6 1.36 4.81 6 3.84 8.19 6 5.90
10 mg/kg bw BPA 4.32 6 1.40 25.79 6 19.85 6.03 6 1.95 2.43 6 2.54 5.62 6 4.38
20 mg/kg bw BPA 3.17 6 1.80 13.32 6 7.20 10.11 6 5.28* 3.60 6 3.70 8.30 6 5.33
0.05 Gy 3.93 6 1.86 11.15 6 6.10 9.46 6 4.92* 1.83 6 1.88 2.93 6 2.84
0.05 Gy 1 4.30 6 1.82 19.20 6 14.56 9.69 6 3.39*a 2.66 6 3.92 4.25 6 4.76
5 mg/kg BPA
*
p 0.05 compared to control by Fisher test.
a
p 0.05 compared to 0.05 Gy by Fisher’s test.
b
p 0.05 compared to 5 mg/kg bw BPA by Fisher’s test.

or with BPA alone at the dose of 20 mg/kg. Intracellular DISCUSSION

vacuoles [Fig. 2(E)] and distended cisterns of smooth endo-
plasmic reticulum occurred in the cytoplasm of spermato-
cytes and Sertoli cells, especially in the groups of animals
evaluated 24 h or 1 week after the end of combined exposi-
tion to irradiation and BPA. Although spermatids in various
stages of development were observed in all treatments
groups, with apparently normal Golgi apparatus, a few
spermatids were binucleated [Fig. 2(F)] or contained double
axonemes. The basement membrane of the seminiferous
epithelium was always preserved and continuous, showing
sometimes a prominent invaginations. Finally, the morphol-
ogy of the stoma of testes and of the Leydig cells appeared
unchanged by the treatments both in pubescent and in adult
animals.
There are some evidences that diminished sperm count
and quality of males in the general population might be
the result of increasing human exposure to endocrine dis-
ruptors (Carlsen et al., 1992; Sharpe and Skakkebaek,
1993). Xenoestrogens affect male reproductive organs of
animals exposed either perinatally and after puberty
(O’Donnell et al., 2001; Sharpe, 2006). Prepubertal expo-
sure to xenoestrogens may influence secondary sexual de-
velopment (Roy et al., 2009), whereas during puberty sex
hormones may influence structural modifications of the
central nervous system and also influence the development
of sexually differentiated behaviors (Pilgrim and Hutchin-
son, 1994).

Environmental Toxicology DOI 10.1002/tox

1308 DOBRZYŃSKA ET AL.

Fig. 1.

Environmental Toxicology DOI 10.1002/tox

 EFFECT OF BISPHENOL A AND X-IRRADIATION ON SPERM COUNT AND QUALITY 1309

Animal and in vitro studies have shown that BPA can
cause testicular toxicity, as well as reduce sperm production
after exposure of neonatal, pubertal, and adult laboratory
rodents (vom Saal et al., 1998; Sakue et al., 2001; Ashby
et al., 2003; Ho et al., 2006; Richter et al., 2007; Wetherill
et al., 2007; Pacchierotti et al., 2008). The sensitivity of pu-
bescent animals is still not well studied; however, Takao et al.
(1999) stated that exposure to BPA around the pubertal period
may directly disrupt the male mouse reproductive tract.
Hereth et al. (2004) observed that BPA significantly
reduced epididymal sperm count in pubertal rats after 5
weeks of treatment by lowering plasma testosterone and
that this might be a reason for the higher susceptibility of
younger animals to the harmful effects of BPA. Sakue et al.
(2001) described a subpopulation of cells impacted by BPA
which resulted in a 40% decrease in daily sperm produc-
tion. Similarly, we observed this reduced sperm count at 1-
and 4-week postend of a 8-week 20 mg/kg bw BPA expo-
sure in pubescent male mice. This harmful effect on germ
cells may be owing to the induction of reactive oxygen spe-
cies by BPA in the epididymal sperm (Kabuto et al., 2004).
Anjum et al. (2011) reported reduced activity of oxidant
enzymes in the animals treated with BPA.
Results presented in this study showed that male game-
tes were more sensitive in younger mice to lethal damage
induced by BPA, and that sperm count was significantly
reduced in pubescent male mice exposed to 20 mg/kg bw
of BPA. This finding was associated with the increased
incidence of degenerating spermatogonia and spermato-
cytes as well as intraepithelial vacuoles and multinucleated
giant cells within seminiferous epithelium. Multinucleated
giant cells having [2 micronuclei were also observed by
Takao et al. (1999) in seminiferous tubes of the testis after
8-week exposure to higher dose of BPA than in study (120
mg/kg/day). Simultaneously, the appearance of multi-
nucleated giant cells and exfoliation of spermatids as well
as incomplete specialization, redundant ectopic specializa-
tion, and aplasia in Sertoli cells and spermatids was noted
by Toyama et al. (2004). Tan et al. (2003) showed that sub-
acute exposure to BPA caused degeneration of the germinal
epithelium of juvenile male rats. Multinucleated giant cells
were present in all animals and indeed some did not show
any form of spermatogenic cycle.
This study also showed that the germ cells of pubescent
males exposed to BPA took longer to recover. An increased
level of abnormal spermatozoa was observed just after the
end of exposure both in adult and in pubescent males, but 1
week later in younger males only. BPA-induced DNA dam-
age in pubescent males was observed just after the termina-
tion of exposure, similarly to the results reported in other
studies (Meeker et al., 2011; Dobrzyńska and Radzikowska,
2013).
As previous studies have shown, targets of BPA within
the testis include Sertoli cells and Leydig cells. Overexpo-
sure to estrogens may inhibit Sertoli cell proliferation,
development, and function (O’Donnell et al., 2001). More-
over, expression of Connexin43, a major gap junction com-
ponent of Sertoli cells that plays an important role during
spermatogenesis (Brehm et al., 2007, Sridharan et al.,
2007), is also affected by BPA (Allard and Colaiacovo,
2011). Leydig cells produce testosterone to support sperma-
togenesis and fertility in adulthood. BPA was found to act
directly in Leydig cells, because it caused testosterone pro-
duction to be decreased in these cells in vitro (Akingbemi
et al., 2004). The proliferation of precursors and adult type
of Leydig cells during a period of pubertal development is
important for the establishment of the adult complement of
Leydig cells (O’Donnell et al., 2001). Germ cell develop-
ment relies on a highly coordinated interaction with the
Sertoli cells. Germ and Sertoli cells communicate directly
via ligand/receptor-mediated interactions or paracrime fac-
tors. The production and secretion of many Sertoli cell

Fig. 1. A: Typical light microscopic features of seminiferous tubules of control mice. Fragments of three tubules with normal
spermatogenic cells closely attached to each other. Measurement of 150 3 115 mm (300 3 300 DPI). B: Typical light micro-
scopic features of seminiferous tubules of experimental mice, 1 week postend of treatment with BPA at 20 mg/kg; degenerat-
ing cells (arrow) with a pycnotic nuclei close to the lumen of seminiferous tubule. Measurement of 150 3 114 mm (300 3 300
DPI). C: Typical light microscopic features of seminiferous tubules of experimental mice, 1 week postend of BPA treatment at
20 mg/kg; single intraepithelial vacuole (arrow) of different sizes and number located between spermatogenic cells. Measure-
ment of 149 3 112 mm (300 3 300 DPI). D: Typical light microscopic features of seminiferous tubules of experimental mice, 1
week postend of treatment with BPA at 20 mg/kg; single intraepithelial vacuole of different sizes and number located between
spermatogenic cells. Measurement of 151 3 115 mm (300 3 300 DPI). E: Typical light microscopic features of seminiferous
tubules of experimental mice, 1 week postend of treatment with BPA at 20 mg/kg; single intraepithelial vacuole of different
sizes and number located between spermatogenic cells. Measurement of 150 3 115 mm (300 x 300 DPI). F: Typical light mi-
croscopic features of seminiferous tubules of experimental mice, 1 week postend combined exposure to irradiation and BPA;
reduced thickness of seminiferous epithelium filled with numerous vacuoles. Measurement of 151 3 115 mm (300 3 300
DPI). G: Typical light microscopic features of seminiferous tubules of experimental mice, 1 week after end of irradiation; sepa-
ration of spermatogonia and spermatocytes from the Sertoli cells (arrow). Measurement of 151 3 115 mm (300 3 300 DPI). H:
Typical light microscopic features of seminiferous tubules of experimental mice, 1 week after the termination of treatment with
BPA at 20 mg/kg; single multinucleated giant cells (arrow) within seminiferous tubule. Measurement of 150 3 115 mm (300 3
300 DPI). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Environmental Toxicology DOI 10.1002/tox

1310 DOBRZYŃSKA ET AL.

Fig. 2. A: Electron micrographs of the fragments of seminiferous tubules of the control mice; spermatogonia (SPG) are closely
attached to each other and to spermatocytes and Sertoli cells (not visible at this picture). Measurement of 93 3 67 mm (300 3
300 DPI). B: Electron micrographs of the fragments of seminiferous tubules of experimental mice; 1 week after the termination of
treatment with BPA at 20 mg/kg; spermatogonium with degenerated nucleus containing vacuoles (arrow heads); intercellular
vacuoles (arrow) are also visible. Measurement of 93 3 67 mm (300 3 300 DPI). C: Electron micrographs of fragments of seminif-
erous tubules of experimental mice; 1 week after the termination of treatment with BPA at 20 mg/kg; group of small intercellular
vacuoles (arrow) located at the border of spermatogonium (SPG) and spermatocyte (SPC). Measurement of 93 3 67 mm (300 3
300 DPI). D: Electron micrographs of the fragments of seminiferous tubules of the experimental mice; 1 week after the termination
of treatment with BPA at 20 mg/kg; large intercellular vacuoles (V) between spermatogonia. Measurement of 93 3 67 mm (300 3
300 DPI). E: Electron micrographs of the fragments of seminiferous tubules of experimental mice; 1 week after the termination of
combined exposure to irradiation and BPA; numerous intracellular vacuoles (asterisks) in spermatocyte. Measurement of 93 3 67
mm (300 3 300 DPI). F: Electron micrographs of the fragments of seminiferous tubules of experimental mice; 1 week after the ter-
mination of combined exposure to irradiation and BPA; binucleated (N) spermatid. Measurement of 93 3 67 mm (300 3 300 DPI).

Environmental Toxicology DOI 10.1002/tox

 EFFECT OF BISPHENOL A AND X-IRRADIATION ON SPERM COUNT AND QUALITY
proteins involved germ cell development occur in a stage-
dependent manner (O’Donnell et al., 2001). Environmental
toxicants are able to increase oxidative stress by downregu-
lating the production of antioxidant enzymes, leading to
male infertility by disrupting the cell junction and adhesion
between Sertoli–Sertoli cells and/or Sertoli–germ cells via
the phosphatidylinositol 3-kinase/c-Src/focal adhesion
kinase signaling pathway (Wong and Cheng, 2011).
Decreased number or dysfunction of Leydig and Sertoli
cells might be a reason of diminished sperm count and
quality in pubescent males.
The response after X-irradiation was also different in
each age group. The diminished sperm count observed in
pubescent males after exposure (to mature spermatozoa and
late spermatids) was correlated with reduced testes weight.
The significantly reduced sperm count observed in males
was irradiated as 4.5 weeks of age was associated with the
occurrence of degenerating cells, which may suggest induc-
tion of apoptosis. The reduced sperm count after irradiation
is mainly attributed to apoptosis of germ cells, with
genomic abnormalities likely initiated by Trp53 (Liu et al.,
2006). On the other hand, reduced DNA damage at 24 h
after the end of irradiation (as well as combined X-ray–
BPA exposure of younger males) may reflect stimulation of
DNA repair after low (0.05 Gy) X-ray dose, as the DNA
damage-control biosystem is stimulated by low-dose radia-
tion (Pollycove, 1998).
The current is the first study to report on the effects
sperm count and quality in adult and pubescent male mice,
by combined X-ray–BPA subchronic, covering the full
spermatogenic cycle, exposure. After 8 weeks of combined
exposure to low doses of both agents, significantly reduced
testes weight and the sperm count, compared to control and
exposed to this dose of BPA, were observed in adult and in
pubescent males. The effect observed in younger animals
was associated with reduced sperm count. The reduced
sperm count and testes weight after combined exposure
were rather caused by X-rays. A previous study reported
significantly diminished sperm quantity and quality after 2
weeks of combined X-rays and BPA treatment compared to
control, but not a significant difference between the results
of combined exposure and each agent alone (Dobrzyńska
and Radzikowska, 2013).
After combined X-ray–BPA exposure, the percentage of
abnormal spermatozoa in adult mice was markedly higher
than post-treatment to the lowest dose of BPA alone. Such
effects were similar in pubescent males, except, here, the
results were noted 1 week after the termination of exposure.
The significant reduction in the percentage of DNA con-
tents in Comet tail compared to controls and BPA alone at
24 h postend of combined exposure was noted in younger
males. However, the similar tendency was also observed in
older mice. This result reflects mainly the influence of X-
rays, because such effects were also present after 8-week
irradiation. Low doses of radiation stimulate physiological
1311
mechanisms which may also helps in discover and repair
damage induced after combined exposure. This finding cor-
related well with the results of our previous 2-week study,
where the level of DNA damage in germ and somatic cells
after combined X-ray–BPA exposure was significantly
lower compared to BPA alone (Dobrzyńska and Radzikow-
ska, 2013).
CONCLUSIONS
In conclusion, our results showed significant differences in
the response of male germ cells to BPA exposure between
adult and pubescent males. These harmful effects by BPA
were more striking in pubescent male gametes, suggesting
higher susceptibility of germ cells of younger mammals.
Combined treatment mainly enhanced the harmful effects
induced by BPA in male germ cells. However, in pubescent
males, the protective effects to DNA of low doses of radiation
in male gametes were observed just after the end of exposure.
Owing to wide consumption, by young people, of food
packed in plastic boxes and drinks packed in bottles con-
taining BPA, limitation of use of BPA, during manufactur-
ing, in such products should be taken into consideration for
the prevention of potentially damaging effects to the repro-
ductive health of men.
The authors acknowledge outstanding technical assistance of
Izabela Remiszewska and Anna Sawicka.
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