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Copyright: © 2017 Akiibinu OM et al.
Research Article
Journal of Addiction and Preventive Medicine
Open Access
Toxic Effects of Cigarette Smoke and Alcohol on Concept uses of
Exposed Wistar Rats
Moses. O Akiibinu1*, Titiloye J. Oyewumi2, Olutola B. Soile1, Felix A Oyeyiola1, Patrick A. Okarfor3 and Oyetunji T. Kolawole4
1
Department of Biochemistry and Chemistry, College of Pure and Applied Sciences, Caleb University Lagos, Nigeria
2
Immunology Unit, Department of Chemical Pathology, College of Medicine, University of Ibadan, Nigeria
3
Ahmadu Bello University Teaching Hospital, Zaria, Nigeria
4
Department of Pharmacology & Therapeutics, Ladoke Akintola University of Technology, Ogbomoso, Nigeria
Received Date: October 25, 2016, Accepted Date: January 24, 2017, Published Date: January 31, 2017.
*Corresponding author: Department of Biochemistry and Chemistry, College of Pure and Applied Sciences, Caleb University Lagos, Nigeria; E-mail:
[email protected].
Abstract
Introduction: Exposure to certain drugs and environmental
toxicants during pregnancy has been implicated in several metabolic
dysfunctions affecting fetuses and neonates. This study investigated the
effects of cigarette smoke and alcohol on concept uses of the exposed
female Wistar rats.
Materials and Methods: Thirty-two nulliparous female Wistar rats
were used for this study. Twelve (group-1) were exposed to cigarette
smoke, while 10 (group-2) were given alcohol before and during
pregnancy. Another 10 pregnant Wistar rats not exposed to cigarette or
alcohol were used as controls (group-3). The pregnant Wistar rats were
monitored till the day of delivery and number and the body weight of the
pups per litter recorded.
Results: In group-1, gestational age was prolonged significantly (p <
0.01) while the number and body weight of the pups per litter decreased
significantly (p < 0.01) when compared with the controls. Meanwhile, in
group-2, there was no significant change in the gestational age (p > 0.05);
but the number and the body weight of the pups decreased significantly
(p < 0.01) when compared with controls. Wistar rats exposed to
cigarette smoke had significantly (p < 0.01) prolonged gestation when
compared with those exposed to alcohol. The mean number of pups
per litter were not significantly (p > 0.05) different in the two groups
(alcohol and cigarette groups), but the weight of the pups exposed to
alcohol was significantly (p < 0.01) lower when compared with those
exposed to cigarette smoke.
Conclusion: It could be concluded that alcohol and cigarette have
different levels of toxicity in the concept uses. While our findings partly
confirm previous reports on the toxic effects of alcohol and cigarette
smoke on the concept uses of the exposed Wistar rats, the prolonged
gestation in the Wistar rats exposed to cigarette smoke may require
further investigation.
Keywords: Cigarette Smoke; Alcohol; Pregnancy; Litters; Wistar
Rats
consequent compromised lung function [4]. Hemoglobin has
exceptionally high affinity for carbon monoxide content of the
cigarette smoke; forms carboxyhemoglobin, fetal hypoxia and
sudden infant death syndrome [5]. Also, persistent reduction
of uterine blood flow, with fetal and childhood developmental
disorders have been linked to cigarette smoking during pregnancy
[6,7]. In Nigeria, information on the effects of cigarette smoke on
pregnancy outcomes is scarce.
Alcohol is an organic chemical that is rapidly absorbs, circulates,
passes through the placenta to the fetus and diffuses readily
through the lipid bilayer of the cell membrane. It maintains a higher
blood level with lower excretion rate in the fetus when compared
with the mother [8]. It was reported that even low intakes of
alcohol during early pregnancy increased the risk of spontaneous
abortion and fetal death substantially [9]. The rate of stillbirth due
to feto-placental dysfunction increased across alcohol categories
[10]. Kuehn et al. [11] stated that after exposure to heavy alcohol
during pregnancy, 80% of children had one or more abnormalities
characterized by functional neurologic impairment. Polanska et al.
[12] reported that consumption of alcohol during pregnancy may
adversely affect children’s intelligence quotient (IQ), mental health,
memory and verbal or visual performance. The chromosomal
disorders observed in fetus exposed to alcohol showed phenotypic
overlap with fetal alcohol spectrum disorders [13]. A study by
Probyn et al. [14] showed that a liquid diet of 6% ethanol fed to
pregnant dams throughout gestation caused a 3–8% reduction in
fetal weight, fetal growth and brain sparing. The present study was
designed to establish these disorders in Wistar rats exposed to
cigarette smoke or alcohol before and during pregnancy; using the
number of litters produced, period of gestation and body weight of
litters immediately after delivery.
Materials and Methods
Introduction
Animal Materials
Cigarette smoke contains more than 4,000 chemicals including
nicotine, which has its effects mediated predominantly at nicotine
acetylcholine receptors widely distributed in the hypothalamus
and mid-brain. The nicotine facilitates neurotransmitter release
from dopaminergic, cholinergic, glutamergic, serotonergic, and
γ-aminobutyric acid-ergic nerve terminals [1]. Exposure to nicotine
from cigarette smoke can change brain feeding regulation, reduce
appetite and a negative energy state [2]. In addition to the altered
DNA methylation and deregulated expression of micro-RNA,
maternal smoke exposure affects key pathways in fetal growth
and development with [3]. Various studies showed that maternal
nicotine exposure during pregnancy and lactation program, the
offspring to develop lung structural defect later in life with the
Seventy-five matured Wistar rats (30 males, 45 females), of
similar age ranges, each weighing 220-270 g were obtained in
batches for a period of three months, from the central animal house,
College of Medicine, University of Ibadan for this study. They were
on feeds obtained from Ladokun Feeds Ibadan, Nigeria. The male
and female Wistar rats were quarantined in separate cages for seven
days for observation and acclimatization in the animal house. The
female rats were randomly divided into three groups with unique
identification numbers (CG 1–15 for cigarette, AL 1–15 for alcohol, CT
1–15 for controls) and were housed under standard environmental
conditions. They were weighed daily and checked twice a day for
any clinical signs of abnormality, morbidity and mortality until they
were fully mature for mating (five to six weeks old).
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Preparation of the Female Wistar Rats
Results
A total number of 15 female Wistar rats were exposed to
cigarette (London king size, containing 14.9 mg tar and 1.2 mg
nicotine) smoke of four sticks per day in the exposure chamber
over a period of 30 minutes per day for seven days. Since female
Wistar rats go into heat (characterized by bloated vulva with
an open vagina, arching her back and vibrating her ears when
touched) every four to five days, mating was allowed naturally by
introducing two male rats (not exposed to cigarette smoke) into
each cage containing three female rats overnight for three days and
the male rats separated from the female rats. Vaginal wetting or a
waxy plug (mucous plug) in the vaginal of the Wistar rat was used
as evidence of copulation; the date was recorded and the mated
rat moved to the second compartment within the cage. The female
rats were then observed for signs of pregnancy such as cessation of
estrus (heat) cycle, increased appetite, weight gain, bulging out of
nipples and hair loss around nipples. The first 12 Wistar rats to be
pregnant (group 1) were selected for this study. The pregnant rats
were further exposed to cigarette smoke till the day of delivery.
The effects of cigarette on pregnancy outcomes of the Wistar
rats are shown in table 1. The pregnant rats exposed to cigarette
had significantly prolonged gestation (p < 0.01) when compared
with the controls. The mean number of pups per litter and their
weights decreased significantly (p < 0.01) in the cigarette exposed
group when compared with the controls. In pregnant rats on
alcohol (Table 2), the number of pups per litter and their weights
decreased significantly (p < 0.01) when compared with that of
controls. But there was no significant change in the gestational age
(p > 0.05) when compared with the controls. As shown in table 3,
the effects of cigarette smoke and alcohol on the concept uses of the
exposed Wistar rats were compared. Those Wistar rats exposed to
cigarette smoke demonstrated significantly prolonged gestation (p
< 0.01) when compared with those exposed to alcohol. The mean
number of pups per litter were not significantly (p > 0.05) different
in the two groups (alcohol and cigarette groups), but the weight of
the pups exposed to alcohol decreased significantly (p < 0.01) when
compared with those exposed to cigarette smoke.
Another 15 female Wistar rats were given 15 ml of 4%
alcohol daily for seven days. Since female Wistar rats go into heat
(characterized by bloated vulva with an open vagina, arching her
back and vibrating her ears when touched) every four to five days,
mating was later allowed naturally by introducing two male rats
(not exposed to alcohol) into each cage containing three female
rats overnight for three days and the male rats separated from
the female rats. Vaginal wetting or a waxy plug (mucous plug) in
the vaginal of the Wistar rat was used as evidence of copulation;
the date was recorded and the mated rat moved to the second
compartment within the cage. The female rats were then observed
for signs of pregnancy such as cessation of estrus (heat) cycle,
increased appetite, weight gain, bulging out of nipples and hair
loss around nipples. The first 10 Wistar rats to be pregnant (group
2) were selected for this study. The pregnant rats were further
exposed to alcohol till the day of delivery.
A set of 15 female Wistar rats not exposed to either alcohol or
cigarette smoke were allowed to mate naturally by introducing t
male Wistar rats (not exposed to cigarette smoke or alcohol) into
each cage containing three female rats overnight for three days and
the male rats separated from the female rats. Vaginal wetting or a
waxy plug (mucous plug) in the vaginal of the Wistar rat was used
as evidence of copulation; the date was recorded and the mated rat
moved to the second compartment within the cage. The female rats
were then observed for signs of pregnancy; such as cessation of
estrus (heat) cycle, increased appetite, weight gain, bulging out of
nipples and hair loss around nipples. The first 10 Wistar rats to be
pregnant (group-3) were selected and used as controls for groups
1 and 2.
Scoring of Pregnancy Outcomes
Immediately after delivery, the gestational age in each Wistar
rat was recorded and further assessment including weight of
the litters and number of litters per rat were made and recorded
against the corresponding mother.
Statistical analysis
The statistical analysis was performed using Statistical Package
for Social Sciences (SPSS) for windows, version 15.0 (SPSS Inc.
Chicago, USA). The data were expressed as Mean ± SD. Student’s
t-test was used for comparison of data in cigarette smoke / alcohol
exposed Wistar rats and controls. The changes were considered
significant, when p - values were less than 0.05.
Discussion
Gestation is a critical period during which adverse intrauterine
conditions and exposure to certain chemicals or stress can influence
the growth and development of the fetus. Accumulation of products
of cigarette smoke such as cotinine (the metabolite of nicotine),
cadmium (a heavy metal in cigarette smoke), and benzo[a]pyrene (a
polyaromatic hydrocarbon in cigarette smoke) have been detected in
the follicular fluid of women who smoke [15–17]. The four authors
reported that chemicals present in cigarette smoke can accumulate
in the ovary, while other studies showed that the contents of cigarette
smoke cause granulosa cell proliferation defect, increased ovarian cell
apoptosis, and decreased ovarian angiogenesis [18–20]. In the present
study, the reduced number of pups per litter observed is a possible
consequence of ovarian insufficiency induced by the content of
cigarette smoke. It could also be due to fetal hypoxia and intra-uterine
death of the fertilized ova, possibly induced by carbon monoxide in the
Controls
(n = 10)
Cigarette
(n = 12)
p - value
26.0 + 2.5
37 + 4.1
< 0.01*
Gestational age
(days)
Number of litters
Weight of litters (g)
9.0 + 3.2
25.8 + 5.7
Gestational age (days)
Number of litters
Weight of litters (g)
Controls
(n = 10)
26.0 ± 2.5
9.0 ± 3.2
25.8 ± 5.7
4.0 + 1.4
19.1 + 3.5
< 0.01*
< 0.01*
Table 1: Pregnancy outcomes (Mean ± SD) of Wister rats exposed to
cigarette and controls. (*Significantly different from the controls).
Cigarette
(n = 12)
24.7 ± 4.8
3.3 ± 1.3
12.6 ± 4.3
P - value
> 0.05*
< 0.01*
< 0.01*
Table 2: pregnancy outcomes (Mean ± SD) of Wister rats exposed to
alcohol and controls. (*Significantly different from the controls).
Gestational age (days)
Number of pups / litter
Weight of pups (g)
Controls
(n = 12)
37 + 4.1
4.0 ± 1.4
19.1 ± 3.5
Cigarette
(n = 10)
24.7 ± 4.8
3.3 ± 1.3
12.6 ± 4.3
p - value
< 0.01*
> 0.05
< 0.01*
Table 3: Comparison of pregnancy outcomes (Mean ± SD) of Wistar rats
exposed to cigarette smoke and alcohol. (*Significant difference between
effects of cigarette smoke and alcohol).
Citation: Akiibinu MO, Oyewumi TJ, Soile OB, Oyeyiola FA, Okarfor PA, et al. (2017) Toxic Effects of Cigarette Smoke and
Alcohol on Concept uses of Exposed Wistar Rats. J Add Pre Med 1(2): 108.
Page 2 of 4
J Add Pre Med
ISSN: 2474-5049
cigarette smoke. Our finding corroborates the report of Jin et al [21]
which states that in a mother at productive age; 4-vinylcyclohexene
diepoxide (a constituent of cigarette smoke) could cause primary
ovarian insufficiency. Another study linked the impaired fertility in
women smokers with combination of impaired oocyte function and
viability, decreased fertilization rates, altered ovarian steroidogenesis,
depleted ovarian reserves, and increased chromosomal abnormalities
in oocytes [15,22]. Also in a study conducted by Roy et al. [23], was
reported that nicotine reduced the efficiency of the brain and caused
insufficient release of gonadotropic hormones.
The present study showed that the reduced birth weight of pups
born to the rats exposed to cigarette smoke. This could be associated
with possible inhibitory effects of the contents of cigarette smoke
on several metabolic pathways of the concept uses of the exposed
pregnant rats, since high levels of nicotine has been reported
in amniotic fluid, placenta, fetal blood, and also in breast milk
during lactation [6,24,25]. Luck and Nau [24] and Himes et al [26]
attributed the developmental defects in the infants to exposures to
higher levels of nicotine, cotinine and trans-3’-hydroxycotinine in
the meconium during fetal periods. The consequences of exposure
include metabolic defects and reduced body weight in the litters
of the exposed rats [2,26,27]. In the report of Scott-Goodwin et al.
[28], foetal growth defect and other changes observed in prenatal
exposure to tobacco are associated with nicotine and carbon
monoxide. In another study, Kuhnert et al. [29] reported that
cadmium from cigarette smoke accumulates and binds to zinc in the
placenta, lowers the level of zinc available to the fetus, causes defect
in trans-placental availability of iron and low birth weight of the
fetus [30]. Such exposure during pregnancy could cause decreased
pregnancy-associated plasma protein A and insulin growth factors I
and II levels [31], impaired organ development in the offspring and
predisposition of the animal to higher risks later in life [32]. The
reduced birth weight observed in our study therefore confirmed
the intrauterine growth restriction reported by Robinson et al. [33],
England et al. [34] and Himes et al. [26].
This study has revealed a prolonged gestation in Wistar rats
exposed to cigarette smoke. Available reports on neuroimaging
studies suggest that tobacco smoking causes brain atrophy in both
alcoholics and neurologically normal individuals [35]. Therefore,
it could be hypothesized that contents of the cigarette smoke have
inhibitory effects on oxytocin metabolism and on the posterior
pituitary gland of the pregnant Wistar rats used for this study. This
may be justified by the report of Jo et al. [36], that there is a widely
distributed nicotinic acetylcholine receptors in the hypothalamus
and mid-brain which bind nicotine and enhance its accumulation
in the brain. This accumulation of nicotine would cause impaired
brain functions in animals exposed to cigarette smoke [23,37].
Bidwell et al. [38] reported unique risk factor for specific alcohol
initiation reactions during pregnancy. The significantly lower weight
of pups and decreased number of pups per litter born to those Wistar
rats exposed to alcohol in this study could be due to inhibitory
effects of alcohol on essential metabolic pathways in the mother
and fetus. Kumar [39], Thomson [40] and Strandberg-Larsen et al.
[9] had hypothesized that maternal alcohol consumption during
pregnancy would lead to fetal growth retardation, malformations,
developmental defects, and / or spontaneous abortion. This may be
possible because ethanol diffuses easily through the cell membrane
lipid bilayer into the intracellular space where several metabolic
activities may be hindered in the fetus. Previous workers have
reported increased feto-placental dysfunction associated with
alcohol in all categories of pregnant drinkers [3,10]. Other workers
reported that ethanol interferes with micronutrient metabolisms
in the mother and the fetus [41], induces increased hepatic zinc
concentration and decreased plasma zinc [42], impairs folate
transport to the fetus, and lowers folate concentrations within the
Vol. 1. Issue. 2. 2000108
placenta and in the fetus [43]. Our finding agrees with the report of
a study conducted by Probyn et al. [14] on pregnant dams, where
exposure to a liquid diet of 6% ethanol throughout gestation caused
reduction in weight, growth and brain sparing of the fetuses.
Although, a lower concentration of ethanol (15 ml of 4% alcohol)
was administered into our Wistar rats, our finding disagrees with the
reports of Kumar [38] and Thomson [39] who reported prolonged
gestation in Wistar rats exposed to ethanol. It also contradicts the
report of Andersen et al. [9] who stated that even low intakes of
alcohol during early pregnancy increased the risk of spontaneous
abortion. Also, this study does not agree with the report of StrandbergLarsen et al. [9] that alcohol would cause stillbirth or miscarriage.
The authors would like to suggest that, interested workers should
endeavor to determine the concentrations of alcohol that could cause
abortion, miscarriage or prolonged gestation.
Conclusion
It could be concluded that alcohol and cigarette have different levels
of toxicity in the concept uses. While our findings partly confirm previous
reports on the toxic effects of alcohol and cigarette smoke on the concept
uses of the exposed Wistar rats, the prolonged gestation in the Wistar
rats exposed to cigarette smoke may require further investigation.
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Citation: Akiibinu MO, Oyewumi TJ, Soile OB, Oyeyiola FA, Okarfor PA, et al. (2017) Toxic Effects of Cigarette Smoke and
Alcohol on Concept uses of Exposed Wistar Rats. J Add Pre Med 1(2): 108.
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*Corresponding author: Department of Biochemistry and Chemistry, College of Pure and Applied Sciences, Caleb University Lagos, Nigeria; E-mail:
[email protected].
Received Date: October 25, 2016, Accepted Date: January 24, 2017, Published Date: January 31, 2017.
Copyright: © 2017 Akiibinu OM et al. This is an open access article distributed under the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Citation: Akiibinu MO, Oyewumi TJ, Soile OB, Oyeyiola FA, Okarfor PA, et al. (2017) Toxic Effects of Cigarette Smoke and Alcohol on Concept uses of
Exposed Wistar Rats. J Add Pre Med 1(2): 108.
Citation: Akiibinu MO, Oyewumi TJ, Soile OB, Oyeyiola FA, Okarfor PA, et al. (2017) Toxic Effects of Cigarette Smoke and
Alcohol on Concept uses of Exposed Wistar Rats. J Add Pre Med 1(2): 108.
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