Pharmacological Research, Vol. 35, No.

3, 1997

NICOTINE AND STRESS: EFFECT ON SEX HORMONES AND LIPID PROFILE IN

FEMALE RATS
MANAL M. ABD EL MOHSEN, ATEF T. FAHIM*, TAREK M. K. MOTAWI and NABILA A. ISMAIL
Department of Biochemistry, Faculty of Pharmacy, Kasr El-Aini st., Cairo University, Cairo, Egypt

Accepted 14 November 1996

This work aimed to study the changes in sex hormones and lipid profile in adult female albino
rats subjected to treatment with nicotine (N), immobilization stress (S), or their combinations
(N+S). These treatments were applied either for one day (T1) or daily for 10 days (T10), after
which rats in the estrus stage were used for the determination of plasma corticosterone (CS),
serum sex hormones as progesterone (P), estrogen (E), FSH, LH and serum lipid profile
including total cholesterol (TC), HDL-C, LDL-C, triacylglycerol (TG) and non esterified fatty
acids (NEFA).
It was clear that either N or S raised plasma CS and serum P levels in both the treatment
regimens and that N+S induced a higher level of these hormones compared to each treatment
alone. Serum E level was only elevated during T10 regimen only. An increase in serum LH
level was only observed after a single exposure to either N or S, however their combination
abolished the stimulatory effect induced by each treatment alone. Serum FSH was not altered
by exposure to either N or S alone in both regimens, but in the T10 regimen their combination
significantly lowered FSH level.
Regarding the effect on serum lipid profile, serum TC was increased in all T10 regimen
groups. LDL-C was increased by N+S treatment in both regimens, however no change in
HDL-C level was observed in all groups. Serum NEFA was increased in all the treated groups
during T10 regimen, while in the T1 regimen NEFA level was only elevated by the combination
N+S. Serum TG was insignificantly altered in all the treated groups. The observed changes in
the lipid pattern were attributed to the alterations occurred in CS and female sex hormones that
caused by N, S or their combinations. 1997 The Italian Pharmacological Society

KEY WORDS: nicotine, immobilization stress, corticosterone, progesterone, estrogen, FSH, LH, cholesterol,
lipoproteins, non esterified fatty acids.

INTRODUCTION C) and decreased concentration of the antiatherogenic

It is generally recognized that stress is an inescapable
part of life. In mammals, exposure to stress results in
a wide range of physiological and behavioural
responses including alterations in endocrine and
reproductive functions in many species [1]. Hormones
secreted during stress may directly affect gonadal
secretion of sex steroids, as corticotrophin releasing
hormone (CRH) and corticosteroids secretion induced
by a stressor may affect pituitary responsiveness to
gonadotrophin releasing hormone (GnRH) [2]. Fur-
thermore, while several dietary and genetic factors
contribute to the atherogenic lipoproteins profiles [3],
stress also contributes to unfavorable concentrations
of lipoproteins that may predispose to cardiovascular
diseases [4]. Studies in non-human primates showed
that stress induces increased concentration of the
atherogenic low density lipoprotein cholesterol (LDL-
*To whom correspondence should be addressed.
high density lipoprotein cholesterol (HDL-C) [5].
Tobacco smoking has always been associated with
stress. Smokers increase the intensity of smoking
when confronted with a variety of environmental
stressors. The number of females long-term tobacco
users has been continuously increasing. It has been
implicated that nicotine (N), a main constituent of
tobacco smoke, is responsible for a high incidence of
reproductive disorders in females. Studies showed that
the acute administration of N results in a rise in
plasma glucocorticoid levels [6] via the release of
ACTH from the anterior pituitary [7]. N has been
reported to affect the hypothalamic-pituitary events
that lead to ovulation, mainly the ovulatory surge of
luteinizing hormone (LH) [8]. Eneroth et al. [9]
showed that N can inhibit the secretion of follicle-sti-
mulating hormone (FSH) in ovarectomized female
rats while Blake [10] found no effect of N on plasma
FSH of normal female rats. Increased circulating cate-
colamines and subsequent increased rate of lipolysis

1043–6618/97/030181–07/$25.00/0/fr960115 1997 The Italian Pharmacological Society

182
and high serum non esterified fatty acids (NEFA)
were observed after N injection in animals [11] and
after smoking in humans [12]. It was shown that long-
term consumption of oral N to monkeys induced an
atherogenic lipoprotein profile by enhancing lipolytic
conversion of very low density lipoprotein (VLDL) to
LDL. However, other reports showed that N had no
effect on lipoproteins level and can be therapeutically
used in the antismoking programs [13].
Since it is generally believed that the perception of
emotional stress and smoking of nicotine are closely
associated, and since a possible site for their effects
on reproduction and lipid profile is via changes in the
hormonal pattern, the present study was performed to
investigate the effect of the combination of N with
stress on the level of plasma corticosterone, female
sex hormones as well as lipid profile in female rats.
MATERIALS AND METHODS
Animals
Adult female albino rats (Wistar strain) weighing
150–200 g were used in this study. The rats were
exposed to a constant light-dark cycle to ensure reg-
ular vaginal smear patterns. Experiments were perfor-
med on rats chosen in the estrus stage, as determined
by the examination of their vaginal smears, since it
was observed that the induction of immobilization
stress in rats caused the predominance of this stage.
Treatments
Nicotine (N) free base (Hopkin and Williams, Ltd
England) diluted with saline was injected intraperi-
toneally (i.p.) in a dose of 0.5 mg kg−1 body weight in
a single or repeated injections. This dose was reported
to produce a plasma N level in rats nearly equivalent
to that resulted from the absorbance of N of the smoke
of 20 cigarettes [14]. This dose was also shown to
increase plasma corticosterone and catecolamines in
rats [7].
Immobilization stress (S) was applied according to
the method described by Tache’ et al. [15].
Design of experimental work
Two experimental regimens were performed. A
single treatment regimen (T1) in which rats, chosen in
the estrus stage, were either subjected to S for one
hour (S1), injected with a single dose of N (N1) or
stressed for one hour during which N was injected 20
minutes before killing (N1+S1). The other treatment
regimen lasted for 10 days (T10). In this regimen, a
group of rats was exposed to S for 5 h daily and
injected i.p. with saline (S10), while N was injected
into two other groups either alone (N10) or in combi-
nation with stress (N10+S10), and the animals in the
estrus stage were then killed 20 minutes after the last
injection of saline or N. Another group of animals
were injected daily with N for 10 days and in the last
Pharmacological Research, Vol. 35, No. 3, 1997
day, rats in the estrus stage were stressed for 1 h
before killing (N10+S1). Control untreated groups
receiving saline were run for each treatment regimen.
Methods of analysis
In all experiments, animals were killed by decapi-
tation. The time of decapitation was kept between
12–2 p.m. to minimize the variations in the measured
hormonal level during the day. Blood samples were
collected in heparinized and non-heparinized tubes.
The separated serum samples were used for the esti-
mation of progesterone (P) by RIA method using pro-
gesterone (I125) coated tube kit obtained from Orion
Diagnostics, Finland, while serum levels of estradiol
(E), LH and FSH were determined by RIA method
using double antibody kits obtained from Diagnostic
Products Corporation (DPC), USA. Furthermore,
serum samples were used for the estimation of total
cholesterol (TC) [16], HDL-C [17], triacylglyercol
(TG) [18] and NEFA [19]. Serum LDL-C was calcu-
lated from the equation developed by Friedewald et
al. [20]. Separated plasma was used for the estimation
of corticosterone (CS) level [21].
Statistical analysis of data was carried out using
Student t-test.
RESULTS
Effect on plasma corticosterone level
Data in Fig. 1 showed that plasma CS was increased
by 272% and 217% of the control value in response to
S1 and N1 treatments respectively, while in the case of
S10 and N10 treatments the increase was found to be
161% and 191% respectively. The increase in plasma
CS induced by the combination of N1+S1 treatment
was not significantly different from that produced by
each treatment alone, whereas the combinations
N10+S10 and N10+S1 induced a higher level of the
plasma hormone amounted to 353% and 332% of the
control value respectively.
Effect on serum sex hormones level
Either S1, N1 treatments or their combination caused
a marked increase in serum P level (Fig. 2) reaching
220%, 310% and 345% of the control value respect-
ively. The increase in serum P by these treatments in
T10 regimen was less if compared to the corresponding
treatments in T1 regimen. The combination (N10+S1)
highly raised P level by 303% of control and this is
significantly higher than that induced by N1 treatment
alone. No significant changes in serum E (Fig. 3) were
observed in groups of T1 regimen, while an increase
amounting to 164%, 178% and 189% of the control
was induced by S10, N10 treatments and their combi-
nation respectively. The increase in serum E caused
by the treatment (N10+S1) was not significantly higher
than that caused by N10 treatment alone. Serum LH
(Fig. 4) was slightly elevated by S1 and N1 treatments
Pharmacological Research, Vol. 35, No. 3, 1997 183

*
†
300 *
400 ‡
*
‡
Percentage of control

Percentage of control
*
300 *
* 200 *
* *
*
200
*

100
100

0 0
T1 T10 N10 + S1 T1 T10 N10 + S1

Fig. 1. Effect of nicotine (N), immobilization stress (S) and
their combinations on plasma corticosterone level for one
(T1) or ten (T10) days. (Values are expressed as percentage of
control±SEM) (n=8). h, (S); , (N); , (N+S). Control value
for plasma corticosterone in rats is 18.4±1.56 µg dl−1.
*Significant difference from control group at P<0.05.
†Significant difference of N+S from the corresponding
stressed group at P<0.05. ‡Significant difference of N+S
from the corresponding nicotine-treated group at P<0.05.
Fig. 3. Effect of nicotine (N), immobilization stress (S) and
their combinations on serum estradiol level for one (T1) or
ten (T10) days. (Values are expressed as percentage of control
±SEM) (n=8). h, (S); , (N); , (N+S). Control value for
serum estradiol in rats is 9.37±1.30 pg ml−1. *Significant
difference from control group at P<0.05.

200
Percentage of control

*
400 *
†
* * *
‡ †
Percentage of control

300 100
* *
†
*
200
*

100 0
T1 T10 N10 + S1

0 Fig. 4. Effect of nicotine (N), immobilization stress (S) and

T1 T10 N10 + S1
Fig. 2. Effect of nicotine (N), immobilization stress (S) and
their combinations on serum progesterone level for one (T1)
or ten (T10) days. (Values are expressed as percentage of
control±SEM) (n=8). h, (S); , (N); , (N+S). Control value
for serum progesterone in rats is 10.8±1.26 ng ml−1.
*Significant difference from control group at P<0.05.
†Significant difference of N+S from the corresponding
stressed group at P<0.05. ‡Significant difference of N+S
from the corresponding nicotine-treated group at P<0.05.
while their combination abolished this effect, however
no changes in serum LH were observed during T10
regimen. Serum FSH (Fig. 5) was shown to be slightly
reduced only by the treatment with either N10+S10 or
N10+S1 combination, while no changes in the hormone
level were observed during other treatments.
their combinations on serum LH level for one (T1) or ten
(T10) days. (Values are expressed as percentage of control
±SEM( (n=8). h, (S); , (N); , (N+S). Control value for
serum LH in rats is 2.20±0.13 mIU ml−1. *Significant
difference from control group at P<0.05. †Significant
difference of N+S from the corresponding stressed group at
P<0.05.
Effect on serum lipid profile (Table I)
No changes in the studied lipid parameters were
induced by the treatments S1 or N1, however their
combination increased serum TC, LDL-C and NEFA
by 131%, 179% and 200% of the control value
respectively. In T10 regimen, S, N and their combi-
nation (N10+S10) as well as the combination N10+S1
caused an increase in serum TC reach 132%, 123%,
125% and 114% of the control respectively. Serum
LDL-C was only increased by the S10 treatment alone
or in combination with N10. Serum NEFA was signifi-
184 Pharmacological Research, Vol. 35, No. 3, 1997

120 sequent increase in ACTH secretion from the pitu-

itary, which in turn stimulated steroidogenesis in the
100 target tissue [22]. It was noticeable that the repeated
*
Percentage of control

† exposure to S for 10 days resulted in a less response

80 *
60
40
20
0
T1 T10 N10 + S1
Fig. 5. Effect of nicotine (N), immobilization stress (S),
and their combinations on serum FSH level for one (T1) or
ten (T10) days. (Values are expressed as percentage of control
±SEM) (n=8). h, (S); , (N); , (N+S). Control value for
serum FSH in rats is 2.69±0.14 mIU ml−1. *Significant
difference from control group at P<0.05. †Significant
difference of N+S from the corresponding stressed group at P
<0.05.
cantly highly increased in response to S10, N10 and the
combination of N10 with either S10 or S1 by about
186%, 197%, 173% and 191% of the control value
respectively. No changes in serum HDL-C or TG
levels were observed during our treatments.
DISCUSSION
In the present study, plasma CS level was shown to be
increased by S in both the treatment regimens. This
could be explained on the basis that S induced a state
of stimulation of the hypothalamic-pituitary-adreno-
cortical (HPA) axis. This effect would trigger the
release of CRH from the hypothalamus with a sub-
on CS level compared to that induced by S1. This
could be attributed to the attenuation of the HPA axis
response to that particular stimuli, as after prolonged
exposure to stress, there appeared to be a desensitiz-
ation of CRH neurons to neurotransmitters released
during stress and a decrease in the releasable pool of
CRH [23]. Furthermore, Hauger et al. [24] showed
that the adaptation of ACTH responses to chronic
stress might be in part mediated by the down regu-
lation of anterior pituitary CRH receptors.
Administration of N was also shown to highly elev-
ate plasma CS level compared to control in both the
treatment regimens. It was well documented that N is
a potent stimulus for ACTH secretion. It appears from
previous literature [25] that the response to N was
mediated by central nicotinic receptors present in the
hypothalamus. This activates multiple catecholam-
inergic brain stem regions which act on receptors that
may be located on CRH-containing neurons and regu-
late ACTH secretion [26]. Although the combination
(N1+S1) did not modify the effect produced by either
stimulus alone, the combinations (N10+S10) and
(N10+S1) resulted in a significantly higher level of
plasma CS compared to that produced by either treat-
ments alone. Sakellaris and Vernikos-Danellis [27]
claimed that chronically-stressed animals did not
seem to possess a normal pituitary-adrenal axis, since
they showed a greater sensitivity to a second stimuli
when compared with controls. It was claimed that dur-
ing chronic treatments, the endogenous arginine-vaso-
pressin (AVP) is essential for sustaining the pituitary
adrenal stress response when the pituitary is refractory
to CRH stimulation [28] and that the pituitaries of ani-
mals subjected to chronic stimulus are hypersensitive

Table I
Effect of nicotine (N), immobilization stress (S) and their combinations on serum total cholesterol (TC),
HDL-C, LDL-C, triacylglycerol (TG) and non esterified fatty acids (NEFA) for one (T1) or ten (T10) days.
(Values shown are means±SE for 6–8 animals in each group)
Group TC HDL-C LDL-C TG NEFA
(mg dl−1) (mg dl−1) (mg dl−1) (mg dl −1) (mM l −1)
Control T1 68.6±6.90 41.3±2.33 19.1±3.90 48.7±5.60 1.00±0.14
Control T10 60.0±3.00 33.8±2.90 14.3±2.20 58.7±5.50 0.94±0.11

S1 72.9±6.30 45.9±2.00 23.7±4.30 48.3±6.50 0.86±0.07

S10 79.4±3.40* 42.0±2.50 24.5±2.60* 64.6±3.70 1.75±0.19*

N1 69.2±5.50 43.1±2.10 25.1±4.50 43.3±4.70 1.00±0.07

N10 73.9±1.60* 41.0±2.20 19.7±2.10 65.0±5.00 1.85±0.08*

N1+S1 89.1±5.10*† 49.9±4.70 34.2±3.60* 52.7±2.60 2.00±0.34*†‡

N10+S10 74.9±3.90* 41.4±3.70 22.4±2.20* 55.4±4.00 1.63±0.09*
N10+S1 68.5±2.20* 39.3±1.90 17.9±1.90 56.4±7.00 1.80±0.11*
*Significant difference from control group at P<0.05.
†Significant difference of N+S from the corresponding stressed group at P<0.05.
‡Significant difference of N+S from the corresponding nicotine-treated group at P<0.05.
Pharmacological Research, Vol. 35, No. 3, 1997
to the effect of AVP which controls ACTH secretion
[30]. So when another acute or chronic stimulus was
applied, a higher plasma CS response was observed.
Furthermore, N and stress are postulated to act upon
HPA axis via functionally separate pathways and that
there is little, if any cross adaptation [31].
Another finding of this study was that S in T1 and
T10 regimens produced a significant increase in serum
P level. The exact source of P is likely to be derived
from the adrenal glands, as preclinical studies have
shown that stress increased P in ovarectomized female
animals, as well as in castrated male animals [31].
Moreover, adrenalectomy has shown to abolish stress-
induced P release [32]. The increase in ACTH during
stress is responsible for the increase in P production
from the adrenals [33]. In addition, the increase in cir-
culating prolactin levels during stress might also con-
tribute to the increase in P level since prolactin
potentiated the action of ACTH on the adrenals and
this action was more pronounced on P production
[34]. N administration resulted in increasing serum P
level in both the treatment regimens. This might also
be due to increased ACTH during N treatment. The
changes observed in P level during T 10 regimen in
response to S, N or their combinations are similar to
those of CS. This might indicate a common regulation
through ACTH, which exerts its major action on the
processes which are involved in the transformation of
cholesterol to pregnenolone [35].
From our results, it was shown that serum E level
was only increased during T10 regimen. This increase
is suggested to be mainly of adrenal origin, since this
response was reported to disappear in adrenalecto-
mized rats [36]. Moreover, from our data no corre-
lation was found between serum E and LH in different
groups. It was reported that ACTH increased E level
in rats adrenals [37] as well as in human serum [38].
Since ACTH stimulates CS and P production in the
biosynthetic reactions from cholesterol to pregneno-
lone, it seems reasonable that ACTH would stimulate
adrenal estrogens at the same site.
An increase in serum LH levels was only observed
after a single exposure to either S or N. It was
reported that acute exposure to S [39] or a single N
treatment [40] activates the various hypothalamic
norepinephrine nerve-terminal systems, exerting a
stimulatory effect on GnRH and subsequent LH
release [41]. This effect which is mediated via α-adre-
nergic neurons, appears to depend on the level of the
circulating sex steroids [42]. When N was combined
with S in the T1 regimen, the stimulatory effect on
serum LH level which was induced by each treatment
alone was abolished. Fuxe et al. [43] showed that the
combination of acute stress with acute nicotine
counteracted the action of one another on the hypo-
thalamic norepinephrine nerve terminals. It was also
noticeable that serum LH level returned to the control
value after repeated exposure to either S, N or their
combinations. It has been reported that chronic
185
exposure to a stressful stimulus results in an activation
of central opoid neuronal function with the release of
β-endorphin that exert an inhibitory influence on the
hypothalamo-pituitary-gonadal axis and directly
inhibits GnRH neurons [44].
Our results revealed that serum FSH was not affec-
ted by exposure to either S or N alone in both treat-
ment regimens. Previous reports had described that
the FSH response to either stress or nicotine treat-
ments was less pronounced than the LH response [45].
Generally, a relatively strong neuronal stimulus is
required to produce a change in FSH level. The
potency of GnRH in inducing FSH secretion is only
one fifth that of its LH releasing activity [46]. On the
other hand, the groups (N 10+S10) and (N10+S1) showed
a significant decrease in serum FSH. This was attri-
buted to the elevation of P level in these groups which
cause an inhibition in FSH release. This negative
feedback effect of P is mainly exerted at the hypothal-
amic level where it decreases GnRH pulse frequency
by inducing the release of β-endorphin [47].
It was clear that serum TC was significantly
increased in all T10 regimen groups. An increase in
LDL-C was only induced by the combination of N
with S while no change of HDL-C level was noticed.
It seems likely that the increase in E and P levels
observed in T10 regimen in response to N or S might
be responsible for the changes in serum lipid pattern.
It was reported [48] that estradiol causes an increase
in total, LDL- and HDL-cholesterol when given to
female rats and that the simultaneous P administration
partly reversed the enhancing effect of estradiol on
HDL-C fraction. Estradiol alone or combined with
progesterone increased the synthesis of several apop-
roteins in female rats [49]. Although both apo-B and
VLDL where shown to be increased by estrogen
administration to postmenopausal women [50], how-
ever studies in baboons suggested that increased LDL
apoB in P treated animals resulted from higher con-
version of VLDL to LDL [51]. In addition, while
estrogen induced-increase in HDL appears to be
attributable to increased apoA1 synthesis, the ability
of P to reverse such effect may be due to that P accel-
erate apoA1 catabolism [52]. In addition, Bryant et al.
[53] showed that chronic S, through activation of cen-
tral endogenous opoid systems, causes impaired pro-
tein synthesis which may result in a reduced or defec-
tive production of LDL-receptors or recognition of
apoprotein B of LDL.
Serum NEFA was elevated, in the T1 regimen, only
by the combination [N+S]. It was shown that either
stress or nicotine [54] treatment stimulates the release
of epinephrine and norepinephrine in the circulation
within a few minutes and their combination might
enhance their lipolytic effect. On the other hand,
increased NEFA level observed in all groups during
T10 regimen could be a result of the delayed lipolytic
effect of the elevated corticosterone observed in these
groups. Glucocorticoids promote lipolysis via syn-
186
thesis of a new hormone sensitive lipase protein by a
cAMP-independent pathway [55] and by sensitizing
the adipose tissue to the lipolytic effect of cat-
echolamines [56].
It could be concluded that female sex hormones
were affected by S or N, probably mediating their
effects on reproductive dysfunction. The combination
of N with S produced more pronounced changes in the
levels of P and FSH. The observed disturbances in
serum lipid pattern in response to chronic S or N
could explain the increased incidence of atheroscler-
osis and coronary heart diseases associated with stress
and or cigarette smoking.
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