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American Journal of Life Science
Researches
www.worldofresearches.com
293
October, 2015
© 2015, World of Researches Publication
Am. J. Life. Sci. Res.
Vol. 3, Issue 4, 293-303, 2015
ISSN: 2332-0206 (Online) ISSN: 2375-7485 (Print)
Fertility and Reproductive Outcome in Mice Following
Trichloroethane (TCE) Exposure
M. A. Al-Griw1, S. A. Al-Azreg2, E. M. Bennour3, S. A. M. El-Mahgiubi2, A. R. Al-Attar2, N. M.
Salama1, A. Elnfati1
1. Developmental Biology Division, Zoology Department, Faculty of Science, University of Tripoli, Tripoli-
Libya
2. Department of Pathology and Clinical Pathology, Faculty of Veterinary Medicine, University of Tripoli,
Tripoli-Libya
3. Department of Internal Medicine, Faculty of Veterinary Medicine, University of Tripoli, Tripoli-Libya.
*Corresponding author: Abdul Hakim Elnfati
INTRODUCTION
Infertility is a major worldwide public health concern because it affects approximately 10%
of all reproductive-aged couples1. Exposure to chemicals during reproductive developmental
windows may predispose individuals to disease and/or dysfunction later in life. 1,1,1-
trichloroethan (TCE), an ambiguous environmental pollutant, is widely used as an industrial
solvent and a degreasing agent 2-4. It has been shown that TCE is well absorbed by all routes of
exposure. At least by inhalation, the rate of uptake is driven initially by tissue loading and then
by metabolism once steady state conditions have been reached1. Numerous studies with
developmental exposure to lower doses than the “safe” dose suggest that TCE exposure causes
various detrimental defects, such as low fetal weight, birth defects, and developmental
disorders. Recent epidemiological studies have shown that chemical exposure environmentally
or occupationally on a daily basis is associated with increase a woman's risk of spontaneous
abortions, infertility, low fetal weights, birth defects1.
However, to the best of the author's knowledge, it has not been examined whether TCE
exposure, during a critical uterine developmental window, has consequences on reproductive
performance (e.g. fertility, gestation and litter life) in later reproductive life. Thus, this study
was performed to investigate the long-term effects of environmentally relevant low and high
dose of TCE on adult reproductive functions, such as fertility and reproductive outcome.
Abstract: Exposure to trichloroethane (TCE), an industrial solvent, has been shown to be negatively
associated with reproductive performance. The present study was performed to assess the effects of TCE
exposure on the reproductive performance and outcome in mice during a critical developmental window of later
reproductive life. A group of female mice were injected intraperitoneally twice weekly for three weeks with
have also been investigated by histopathology. The results showed that TCE exposure has reduced the number
of F0 fertile females comparing to controls. Moreover, TCE exposure resulted in a decreased pups number and
uterine toxicity appeared as a severe endometrial hyperplasia with squamous cell metaplasia and adenomyosis.
These results indicate that TCE exposure during a critical reproductive developmental window could affect the
fertility and interfere with the reproductive outcome in mice.
Keyword: TCE; fertility; reproductive outcome; uterus; mice.
Al-Griw et al., 2015
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October, 2015
MATERIAL AND METHODS
Animals and housing
A total of thirty six female Swiss Albino mice (F0 generation), with an age range of 4-6
weeks and weight range of 21–24 g, were used in this study. Mice were kept under a constant
light-dark cycle (dark period from 7:00 pm to 7:00 am) at 24 ± 1ºC and 55 ± 5% relative
humidity. Food and drinking water were available ad libitum. All efforts were made to
minimize the pain during animal handling and experimentation and to reduce the number of
animals used.
Study design and treatment regimen
TCE (Baxter International) was suspended in corn oil. Female mice (F0) were divided into
four groups of six mice each. These groups were: the low and high dose TCE-treated (100 or
400 µg/kg) groups and the vehicle and sham control groups. For TCE-treated groups, the
doses were calculated and delivered in 80-100 µl of corn oil based on their body weight5-6 .
TCE doses were selected as they considered safe by Environmental Protection Agency (EPA)7
. Vehicle controls were received an equal volume of corn oil only. The sham controls were not
received any exposure. TCE or vehicle were administered intraperitoneally at a defined time
(10:00 am) every 3rd day. The exposure window was selected because this is the critical
development window in the mouse1.
After treatment, F0 female mice were mated with fertility confirmed control males (2
females:1 male ratio). Mating was confirmed by the presence of vaginal plug. Once the plug
was observed, females were separated from males and individually caged. The day the vaginal
plug detected was defined as the first gestation day (GD1). Dams (F0 generation) were
observed daily and body weight gain was measured daily to further confirm pregnancy and for
any adverse clinical signs or abnormal behavior that may result from toxicity. The dams were
allowed to deliver naturally and the delivery day was designed as postnatal day 0 (PND0).
The study comprehensive teratological parameters for F0 females included body weight,
reproductive performance (fertility index; gestation index, mortality), and gross pathology.
The study parameters for F1 offspring included pup size, average pup weight, sex ratio, pup
mortality and stillbirths as recorded on PND0.
After the two-week mating period, unmated females were singly housed, observed for
estrous cycle for another 10 days, and body weight was monitored for another two weeks. The
females were considered infertile if they did not cycle and/or did not have significant body
weight gain during the entire testing period.
Clinical assessment
The clinical assessment included animal survival, body weight gain and histopathology.
Animal survival
During the course of the exposure period, mice were observed twice per day for any
abnormal clinical signs or behavior that may result from toxicity. Mice were assessed for
morbidity and mortality twice daily, midmorning and late afternoon. Night deaths were
recorded the next morning. Two independent observers confirmed the cause of death to
exclude TCE-nonrelated mortality.
Body weight
Mouse body weight in control and TCE-treated groups was assessed on a weekly basis to
monitor the effect of TCE exposure on body weight.
Histopathological examination
After dissection, 10%-formalin-fixed uteri were processed in a series of graded ethanol
solutions and embedded in paraffin wax. Paraffin sections were cut at 6-8 µm thickness,
deparaffinized, rehydrated, stained with hematoxylin and eosin (H&E) and examined under a
light microscope (Leica, Germany) for histopathology.
Fertility and Reproductive Outcome in Mice Following …
Statistics
Data were expressed as means ± SEM (standard error of the means) from 6 female mice of
each group using SPSS software, version 20. A computerized Kolmogorov-Smirnov test was
used to determine whether the data fitted a normal distribution. One-way ANOVA test
followed by Tukey's post hoc comparisons was used to make multiple comparisons between
treatment groups. Student's t-tests were used to make comparisons between two groups. Mann-
Whitney U-test was used for nonparametric samples. Statistical significance was assigned at P
≤ 0.05.
RESULTS
Effect of TCE on animal survival
No mortality has been recorded among mice in all groups along the course of the experiment
except for one death case out of the six females in 400 µg/kg TCE-treated group recorded four
weeks post TCE exposure.
Effect of TCE on body weight
Previous animal and epidemiological studies have linked developmental chemical/ toxicant
exposure to metabolic disorder and obesity1. Therefore, in this study we decided to investigate
the effect of TCE exposure on body weight of F0 female mice by monitoring the body weight
either pre- and post-treatment.
In this study, TCE exposure have not shown an effect on overall body weight in F0 females
as statistical analysis indicated no significant between treated and non-treated groups (data not
shown). However weight gain in F0 females in 400 µg/kg TCEt-reated group, but not 100
µg/kg TCE-treated group (P = 0.332) was significantly higher (P = 0.024) comparing to
controls (Figure1). This concludes that TCE exposure at the dose of 100µg/kg is a No
Observed Adverse Effective Level (NOAEL) for F0 female mice in this study.
Figure 1. Body weight gain of the F0 females. TCE exposure significantly increased weight gain at a dose of
400 µg/kg. Data represent mean ± SEM of n = 6 animals per groups. # Significantly different from the vehicle
controls (P ≤ 0.05).
Effect of TCE on fertility
To determine the effect of TCE exposure on fertility in later reproductive life and whether
the effects would change with age, a number of fertility and reproductive indicators including
percentage of fertile female has been investigated.
The results showed an effect of TCE exposure on the fertility of F0 females. The analysis
showed a reduction in the percent of fertile females in TCE-treated groups as in 100 µg/kg
TCE-treated group, five females out of six females (~83%) gave birth while in 400 µg/kg
TCE-treated group, only one female out of six females (~17%) gave birth (Figure 2).
0
5
10
15 #
Sham Veh 100 400
Body wieght gain (g)
TCE (g/kg)
Al-Griw et al., 2015
296
October, 2015
Statistically, the fertility was significantly reduced in 400 µg/kg TCE-treated F0 females
comparing to controls (P = 0.047; Figure 2).
Figure 2. Effect of TCE exposure on fertility of F0 females. The fertility was significantly reduced in 400 µg/kg
TCE-treated F0 females. Data represent mean ± SEM of n = 6 animals per groups. # Significantly different from
the vehicle controls (P ≤ 0.05).
Effect of TCE on reproductive outcome
To determine the effect of TCE exposure on reproductive outcome, a number of reproductive
indicators including litter number and mortality, pup size and weight and sex ratio have been
investigated. The results showed also that TCE exposure at a dose of 100 or 400 µg/kg
significantly affected the litter number and decreased the average number of pups, especially
for the TCE 400 µg/kg TCE-treated group compared to controls (Figure 3A). However, TCE
exposure at a dose of 100 or 400µg/kg had no effect on pup size comparing to controls (data
not shown).
For the effect of TCE exposure on average live pup weight, TCE exposure at a dose of 100
µg/kg had no effect on the pup weight (P = 0.388) comparing to controls. However, TCE
exposure at a dose of 400 µg/kg has significantly (P = 0.049) increased the average of live
pups weight comparing to controls (Figure 3B). Furthermore, the results of this study showed
that TCE exposure at either a dose of 100 or 400 µg/kg had no effect on litter mortality when
compared with controls.
0
50
100 P=1
#
Sham Veh 100 400
Fertile females (%)
TCE (g/kg)
Fertility and Reproductive Outcome in Mice Following …
Figure 3. Effect of TCE exposure on reproductive life/ outcome. (A) Quantification of pup number. (B)
Measurement of pub weight. Data represent mean ± SEM of n = 6 animals per groups. # Significantly different
from the vehicle controls (P ≤ 0.05).
Regarding the effect of TCE exposure on the litter sex ratios, TCE exposure had a significant
effect on the male/female ratio at a dose of 400 µg/kg. This dose has significantly changed the
litter sex ratio in favor of F1 males comparing to controls (P < 0.05) (Figure 4). However,
neither 100 µg/kg TCE dose exposure nor the vehicle had effect on the litter sex ratio (Figure
4).
Figure 4. Effect of TCE exposure on reproductive outcome. TCE exposure at a dose of 400 µg/kg has
significantly changed the litter sex ratio in favor of F1 males comparing to controls. Data represent mean ± SEM
of n = 6 animals per groups. # Significantly different from the vehicle controls (P ≤ 0.05).
A
B
0
5
10
15
###
#
Sham Veh 100 400
Pup number
TCE (g/kg)
0
1
2
3
#
Sham Veh 100 400
Pup weight (g)
TCE (g/kg)
B
0
25
50
75
100
#
#
Male Female
Sex ratio
Sham treated
Vehicle treated
TCE 100g/kg treated
TCE 400g/kg treated
Al-Griw et al., 2015
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October, 2015
Gross pathological findings and histopathological changes of uterine tissues
At necropsy, no gross pathological changes have been noticed on control mice. In addition,
no histopathological changes were observed in their uterus (Figure 5A) or the other parts of
reproductive tract. However, abilateral asymmetrical enlargement was seen in the uterine body
of several TCE-treated mice at a dose of 100 and 400 µg/kg. Sections from different parts of
uterus were submitted to histopathological examination.
The histopathological examination of uterine tissues of TCE-treated mice at a dose of100
µg/kg showed a marked increase in the endometrial and myometrial thickness (Figure 5B).
This thickening is revealed as diffuse or focal proliferative reactions with characteristic
hyperplasia of uterine glands with prominent stromal elements and/or formation of
intra-luminal polyp. The glands were large, irregular, highly branched and/or cystic lined by
single or double layer of non-ciliated secretary epithelium (Figure 5C). In some cases, polyps
were appeared as evaginated circumscribed mass covered by columnar epithelium with
underlying tissue formed from endometrial stromal elements (Figure 5D). The myometrium
showed normal smooth muscles fiber and vascular constituent, however, some cases showed
invaginated uterine glands in-between (Adenomyosis) (Figure 5E). These histopathological
changes caused by TCE were remarkably increased in mice which were treated with 400 µg/
kg TCE; the endometrial glands were severely ectatic, lined by flattened epithelial cells and
contain few degenerative heterophils and necrotic cellular debris (Figure 5F) and most
endometrial polyps were composed of abundant amounts of loosely or compactly arranged
spindle-shaped or satellite endometrial glands (Figure 5G). Moreover, there was sever
hyperplastic changes with squamous cell metaplasia in endometrial epithelium (Figure 5H). In
conclusion, the uteri of TCE-treated mice showed a severe endometrial hyperplasia with
squamous cell metaplasia and adenomyosis.
Fertility and Reproductive Outcome in Mice Following …
Figure 5. Representative uterine sections of control and TCE-treated mice (H&E staining). A. Uterine sections of
control mice showing normal tissue architecture at magnification X200. B. An endometrial thickening with more
prominent hyperplasic uterine glands and stromal elements in µg/kg TCE-treated mice. C. TCE-treated mice
endometrium showing hyperplasic glands (cystic endometrial hyperplasia) (asterisk). D. Pedunculated
endometrial polyp with an epithelial covering and stromal hyperplasia in 100 µg/kg TCE-treated mice (asterisk).
E. Endometrial tissue includes glands and stromal elements within the myometrium (arrows) in 100 µg/kg
TCE-treated mice. F. The endometrial glands are severly ectatic, lined by flattened epithelial cells and contain
few degenerative heterophils, necrotic cellular debris and secretory material (asterisk). G. Papillary fibroepithelial
polyp arising from the endometrium with cystic change of uterine glands in 400 µg/kg TCE-treated mice
Al-Griw et al., 2015
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October, 2015
(asterisk). H. Hyperplasia and squamous metaplasia of epithelium of uterine glands in 400 µg/kg TCE-treated
mice (arrows).
DISSCUSSION
The exposure to environmental toxic chemicals causes many harm effects in biological cell
systems2-3,5,8-10. TCE is a non-carcinogenic (group 3) because there is inadequate evidence for
carcinogenicity in both human and animals according to the last update of U.S Environmental
Protection Agency (EPA) and National Toxicology Program (NTP) technical report11-12. In
addition, according to WHO toxicological report, TCE is not considered as toxic, and not
necessary to drive a health based guideline standard13. However, the Agency for Toxic
Substances and Disease Registry (ATSDR) indicated that TCE affects many internal organs,
such as cardiovascular and nervous system14.
Despite the TCE safe profile claimed in the aforementioned reports, TCE reproductive
toxicity was reported by several studies in many animal models by oral, inhalation and dermal
exposure15-18. Moreover, prior animal studies have shown that perinatal exposure to TCE
affects the development of the brain, liver, adipose tissue and reproductive tract and adversely
affects their functions2-3,5,8-10. However, based on the best authors knowledge, no study had
focused on the long-term effect of TCE exposure on the fertility in later reproductive life.
Thus, this study was designed to explore the possible hazard effects of the exposure to
environmentally relevant levels of TCE during an embryonic ovarian developmental window
has long lasting effects on reproductive performance in later reproductive life. The
administration of TCE safe considered doses to female mice was through intra-peritoneal
injection. This mode of exposure has never been reported previously but the advantage of such
exposure is to put the toxic chemical in close contact with the target cells and avoid rapid bio-
elimination of TCE, which is two hours19.
In the present study, TCE exposure impaired the fertility and increased body weight gain in
F0 females. In addition, most interestingly, TCE exposure also affected the reproductive
outcome of F0 females.
The current data showed also that TCE had not resulted in a change in the body weight of F0
female mice. This is consistent with a previous study on rats which reported that no body
weight changes were observed following intermittent or continuous exposure to
trichloroethylene vapors at exposure levels in a range of 400–2,500 ppm for 2–13 weeks20. On
the other hand, another study reported > 20% decreased body weight in male rats exposed to
trichloroethylene vapors at 376 ppm for 4 hours per day, 5 days per week and a period of 12 or
24 weeks21. These findings are not consistent with other studies, which have shown that
developmental TCE exposure disturbs metabolism, interferes with adipocyte proliferation and
differentiation and may be linked to obesity in later life20-21. However, as no significant
differences in body weight have been observed between TCE-treated mice and controls, we
speculate in TCE exposure might reprogram the progenitor adipocytes and increase body
weight significantly later in life.
Although some studies indicated that the exposure to TCE decreases body weight gain17, the
results of this study showed a dose-associated increase in the body weight gain of TCE-treated
F0 females and their neonates. Many studies have found that certain toxic chemicals such as
pesticides, the most environmental pollutants, and plastic products can cause weight gain by
disrupting endocrine system22-26. These environmental chemicals have different mechanism of
action such as increasing the activity of estrogen23,25, abnormal adipogenesis25, increasing
inflammatory cytokine activity, causing oxidative stress, inducing abnormal thyroid function
and impacting energy metabolism27. In addition, inspite of rare information of TCE endocrine
toxicity, one study recorded that acute inhalation of TCE disrupts the concentration of
corticosterone and adrenocorticotropic hormones28. It was also noted that TCE has increased
the weight of internal organs of rats29 and their pups30-31. In this study, it is possible that the
Fertility and Reproductive Outcome in Mice Following …
increase in body weight gain due to TCE exposure has been caused by one or more of above
mentioned mechanisms.
Many toxicological and histopathological examinations in different animal models indicated
that exposure to TCE through inhalation has not adverse effects on female reproductive system
following wide range doses (acute, intermediate and chronic) 15,29,32-35. Similar to TCE
inhalation, oral exposure of 100, 300 and 1000 mg/kg/day in mice [36] and 3 mg/kg in rats37-38
did not show any hazard effects on female body weight, survival, fertility, reproductive
performance and gestation period. In this study, our model (with low doses) showed a
significant decrease in the pub number in TCE-treated female litter. This might be attributed to
the effect of TCE on oogenesis and oocyte quality, fertilization processes, early embryo death
or the changes noticed in endometrium which could affect the proper formation and
implantation of placenta.
Abnormal sex ratio may be induced in human and animals by the exposure to chemicals39-40
but changes in sex ratio related to exposure to TCE have not been documented. This study is
providing the first observation about changed litter sex ratio in mice due to exposure to high
doses of TCE. This could be attributed to an increased sensitivity of sperms and/or embryos of
certain genotype to the exposure to a particular chemical resulting in low fertilization rate or
early embryonic death41.
The uterus is one of the major organs of reproductive activity of mammals as it provides the
place and the environment for gestation from the embryonic implantation and placentation till
parturition. Therefore, the pathological conditions interfering with the normal uterine
physiology might result in impaired reproductive performance. This study showed that TCE
exposure induced a bilateral asymmetrical enlargement in the uterine body. Histpathologically,
TCE exposure was associated with endometrial tissue changes appeared as severe endometrial
hyperplasia and adenomyosis. This might be the responsible, at least in part, to the reduced
fertility in TCE-treated F0 females and their reduced litter number.
In conclusion, the current study indicates that TCE exposure during a critical ovarian
developmental window impairs female reproduction life. Further studies focused on
examining the hormone profiles, ovarian morphology at later reproductive life, and pregnancy
status during mid- and late gestation will be helpful in enhancing our understanding of the
mechanism by which TCE affects female reproduction.
Acknowledgements
This investigation was supported in part by the Division of Developmental Biology, Faculty
of Science, University of Tripoli, Tripoli-Libya. The authors zould like to thank Turkia
Aduma, the technical staff at the Department of Pathology and Clinical Pathology, Faculty of
Veterinary Medicine, University of Tripoli, Tripoli-Libya, for her technical assistance.
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