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Effect of prenatal exposure to nicotine/thiocyanate on the pituitary–adrenal axis of 1-month-old rat offspring

Sayed, Manal M.

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The Egyptian Journal of Histology: December 2016 - Volume 39 - Issue 4 - p 307-316
doi: 10.1097/01.EHX.0000512120.79296.60
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Cigarette smoking is a common habit in both developed and developing countries 1. Tobacco smoke contains as many as 4000 chemical constituents. About 60 toxic com- pounds have been identified in tobacco smoke 2,3.

It is well known that tobacco contains numerous compounds that are potentially cytotoxic, such as nicotine, thiocyanate, carbon, and certain gases 4. There is sufficient evidence to prove that tobacco smoking causes endocrine changes in humans. Among all smoking products, nicotine is considered one of the major components that disturb fetal development 5. It has been linked to several deleterious side effects in the offspring of smoking mothers, including impaired development of the sympathoadrenal system, and sudden infant death syndrome 6. In pregnant women who smoke or use nicotine, nicotine crosses the placenta, concentrates in the fetal blood and amniotic fluid, and is detectable in breast milk during lactation 7. Direct or indirect exposure to smoking during pregnancy results in intrauterine growth retardation and low birth weight of the offspring 8.

The hypothalamic–pituitary–adrenal (HPA) axis plays an important role in the regulation of homeostasis and the stress response. HPA axis activity is reflected peripherally by plasma concentrations of adrenocorticotropic hormone (ACTH) and corticosterone released from the anterior pituitary and adrenal cortex, respectively. ACTH release is stimulated by corticotropin-releasing hormone and arginine vasopressin secreted from the paraventricular nuclei of the hypothalamus. The administration of nicotine resulted in increased activity of HPA axis in both adult humans 9 and rodents 10. The initial effects of nicotine in adults are characterized by a marked hypersecretion of ACTH, vasopressin, β-endorphin, prolactin, and luteinizing hormone. Many of these very acute stimulatory effects of nicotine rapidly disappear, probably due to a desensitization of the central nicotinic cholinergic receptors involved 11.

The most important source of thiocyanate is tobacco smoking 12. Thiocyanate crosses the placenta as indicated by similar serum thiocyanate levels in maternal and cord serum 13.

Materials and methods


Nicotine and potassium thiocyanate were purchased from Sigma Chemical Company (St. Louis, MO, USA.) in the form of powder. Nicotine was dissolved in distilled water and was used at 6 mg/kg/day subcutaneously from ges- tation day (4–20) 14. Potassium thiocyanate was given orally at 25 mg/kg/day from gestation day (4–20) 15.


A total of 15 pregnant female albino rats were divided into three groups (five rats each):

  • Group I: this was the control group.
  • Group II: this was the nicotine-treated group. The rats in this group were treated with nicotine at 6 mg/kg/ day subcutaneously from gestation day (4–20) 14.
  • Group III: this was the thiocyanate-treated group.

The rats in this group were treated with potassium thiocyanate orally at 25 mg/kg/day from gestation day (4–20) 15.

The offspring of each group were sacrificed after 1 month. Both adrenal and pituitary glands were immediately removed from the animal. The adrenal glands were dissected free of surrounding fascia and partially bisected in a transverse plane into two equal halves. Small pieces of both the adrenal and pituitary glands were fixed in 2.5% 0.1 mol/l phosphate-buffered glutaraldehyde at 4°C for 2 h, rinsed in 0.1 mol/l PBS, and postfixed in phosphate-buffered 1% osmium tetroxide for 1 h at room temperature and then dehydrated in ascending grades of ethanol. After immersion in propylene oxide, the specimens were embedded in epoxy resin mixture. Semithin sections (about 1 µm thick) were obtained and stained with 1% toluidine blue, examined by means of a light microscope, and photographed.

Ultrathin sections (about 80–90 nm) were stained with uranyl acetate and lead citrate to be examined by a JEOL transmission electron microscope at 80 kV in the Electron Microscopic Unit, Assiut University.


Using computerized assisted image analysis software (Leica Q 500 MCO; Leica, Wetzlar, Germany, connected to a camera attached to a Leica universal microscope at the Histology Department, Faculty of Medicine, Assiut University), the cellular and nuclear areas of the adrenal gland were measured by the arbitrary area method on semithin sections (toluidine blue stain) viewed using ×100 oil immersion lens 16,17.

Statistical analysis

The morphometric data were analyzed using SPSS (SPSS Inc., Chicago, Illinois, USA) to compare various groups with each other. The Mann–Whitney U-test was used to compare the groups and cover a wide range of data.

Data were presented as mean±SD. Differences were considered statistically significant if P value was less than 0.05.

Results Histological results Light microscopic results

Examination of toluidine blue-stained sections from the control group showed a normal histological architecture for the adrenal cortex. The gland is enclosed by a connective tissue capsule. The cells of the zona glomerulosa were arranged in the form of rounded or arched clusters beneath the adrenal gland capsule. The zona glomerulosa is found outermost in the gland with a few lipid droplets (Fig. 1). The cells of the zona fasciculata were large, polyhedral, arranged in straight long cords one or two cells thick, separated by blood sinusoids and run at right angles to the surface of the gland. The cells of the fasciculata have a frothy appearance, or pale vacuolated cytoplasm, as lipid droplets have been dissolved away during histological preparation, and large vesicular rounded nuclei. Lipids are variably distributed throughout the cytoplasm of most cells (Fig. 1).

Figure 1:
A photomicrograph of a semithin section of the adrenal gland from the control group stained with toluidine blue showing the capsule (C), zona glomerulosa (ZG), and zona fasciculata (ZF). The zona glomerulosa cells appear with a few lipid droplets. The zona fasciculata cells are large, polyhedral, with round vesicular nuclei and multiple lipid droplets (↑) mainly in the superficial area and separated by blood sinusoids (◂). ×1000.

In group II, which received nicotine, the zona glomerulosa showed irregular orientation of the cells with marked increase in lipid droplets. Most of the zona fasciculata cells appeared smaller in size with marked increase in cytoplasmic vacuolation (Fig. 2). There was area of focal degeneration in which the cells were highly vacuolated and the nuclei appeared smaller, irregular, and pyknotic. The cells were separated by wide blood sinusoids (Fig. 3).

Figure 2:
A photomicrograph of a semithin section of the adrenal gland from group II stained with toluidine blue showing the capsule (C), zona glomerulosa cells (ZG) with excessive lipid droplets, and zona fascicu- lata cells (ZF) that also appear vacuolated with multiple lipid droplets (↑). Most of the nuclei appear small compared with control. ×1000.
Figure 3:
A photomicrograph of a semithin section of the adrenal gland from group II stained with toluidine blue showing areas of focal degen- eration where most of the nuclei appear shrunken, pyknotic, irregular, and darkly stained (↑) separated by dilated blood capillaries (◂). Note that the cytoplasm is highly vacuolated. ×1000.

In group III, which received thiocyanate, the cells of the zona glomerulosa appeared with little lipid droplets, while the zona fasciculate cells appeared granular with central rounded vesicular nuclei with prominent nucleoli and little lipid content. The cells were separated by wide blood sinusoids (Fig. 4).

Figure 4:
A photomicrograph of a semithin section of the adrenal gland from group III stained with toluidine blue showing the capsule (C), zona glomerulosa (ZG), and zona fasiculata (ZF). The ZG and ZF cells appear with little lipid droplets compared with the nicotine-treated group. Note the presence of blood capillaries (◂). ×1000.

Electron microscopic results

The cells of the zona fasciculata of the control group were characterized by euchromatic rounded nucleus, with prominent nucleolus (Fig. 5). There were numerous characteristic spherical tubular or saccular forms of mitochondria, lipid droplets, and smooth endoplasmic reticulum. The ribosomes lay freely in the cytoplasm (Fig. 6).

Figure 5:
An electron micrograph of a zona fasciculata cell from the control group showing a large rounded euchromatic nucleus (N) with prominent nucleolus, numerous mitochondria (M), smooth endoplasmic reticulum (↑), and several lipid droplets (L). ×5000.
Figure 6:
An electron micrograph of a zona fasciculata cell from the control group showing spherical tubular or saccular forms of mitochon- dria (M) characteristic of zona fasciculata cells. Ribosomes lie freely in the cytoplasm (↑). ×19 000.

In group II, significant ultrastructural alterations within the zona fasciculata cells were apparent as some cells appeared with deeply stained nuclei and irregular nuclear envelope and widened perinuclear space (Fig. 7). Also some cells showed excessive vacuolation of the cytoplasm and an increase in the amount and lipolysis of lipid droplets as some of these lipid were partially dissolved (Fig. 8). Lipid droplets increased significantly in all experimental groups as compared with the control group. Also some cells showed degeneration or destruction of the mitochondria (Figs. 9 and 10) and excessive vacuolation of the cytoplasm (Figs. 11 and 12).

Figure 7:
An electron micrograph of zona fasciculata cells from group II showing darkly stained nuclei (N) with irregular nuclear envelope, wid- ened perinuclear space (↑), and lipid droplets (L) of variable electron density, some of them are partially dissolved (◂). ×3600.
Figure 8:
An electron micrograph of a zona fasciculata cell from group II showing rounded euchromatic nuclei (N), numerous destructed mitochondria (M), and several lipid droplets, some of them partially dissolved (↑). ×3600.
Figure 9:
An electron micrograph of zona fasciculata cells from group II showing darkly stained nuclei (N), numerous destructed mitochondria (M), and several lipid droplets (L). ×3600.
Figure 10:
A magnified part of the previous picture showing dark irregular nucleus (N), numerous destructed mitochondria (M), and lipid droplets (L). ×10 000.
Figure 11:
An electron micrograph of zona fasciculata cells from group II showing excessive vacuolation of the cytoplasm (*) with numerous lipid droplets (L) Nucleus was added (N). ×3600.
Figure 12:
A magnified part of the previous picture showing numer- ous lipid droplets (L), mitochondria (M), nucleus (N,) vacuolation of the cytoplasm (*). ×10 000.

In group III, the cells appeared with large nuclei and well- developed Golgi bodies, and most of the mitochondria appeared normal (Figs. 13 and 14). Lipid droplets were present and some of them appeared dissolved (Fig. 15). The cells were separated by fenestrated blood sinusoids (Fig. 16).

Figure 13:
An electron micrograph of zona fasciculata cells from group III showing normal appearance of cells with rounded central nuclei (N), numerous mitochondria (M), and lipid droplets (L). ×3600.
Figure 14:
A magnified part of the previous picture showing numerous normal mitochondria (M), lipid droplets (L), and Golgi bodies (↑). ×10 000.
Figure 15:
An electron micrograph of zona fasciculate cells from group III showing normal euchromatic nuclei (N), mitochondria (M) appearing more or less like normal, and few lipid droplets (L). Note the fenestrated blood capillary (↑). ×3600.
Figure 16:
A magnified part of the previous picture showing a part of normal euchromatic nucleus (N) and fenestrated blood capillary (↑). ×10 000.

As regards the pituitary gland, the corticotrophs of the control group showed large eccentric rounded nucleus, well-developed Golgi bodies, small mitochondria, and small sparse secretory granules, located at the extreme periphery (Fig. 17).

Figure 17:
An electron micrograph of a corticotroph cell of the pituitary gland from the control group showing large eccentric nucleus with heterochromatin clumps (N), well-developed Golgi (G), and small peripherally located secretory granules (↑). Centrosome (C) appears inside the inset. ×4800.

In the nicotine-treated group, the corticotrophs appeared with dark nuclei, few secretory granules and vacuoles (Figs. 18 and 19). In the thiocyanate-treated group, the corticotrophs appeared more or less similar to those of the control group (Fig. 20).

Figure 18:
An electron micrograph of a corticotroph cell of the pituitary gland from group II showing deeply stained nucleus (N) and a small amount of secretory granules. ×3600.
Figure 19:
A magnified part of the previous picture showing a part of the corticotroph with deeply stained nucleus (N) and a small amount of secretory granules (↑). ×7200.
Figure 20:
An electron micrograph of a corticotroph cell of the pituitary gland from group III, which is similar to normal, with rounded nucleus (N) and secretory granules (↑). ×5800.

Morphometric and statistical results

The mean arbitrary area of the zona fasciculata cells of rats of both the nicotine-treated group and the thiocyanate- treated group was significantly decreased (P<0.05) in comparison with that of the control group. Also the mean arbitrary area of the zona fasciculata cell nuclei of rats of both the nicotine-treated group and the thiocyanate- treated group was significantly decreased (P<0.05) when compared with that of the control group (Tables 1 and 2; Histograms 1 and 2).

Table 1:
Mean arbitrary area of zona fasciculata cells in different groups
Table 2:
Arbitrary area of nucleus in different groups
Histogram 1. Mean arbitrary area of zona fasciculate cells in different groups.
Histogram 2. Arbitrary area of nucleus in different groups.


Smoking has multiple effects on hormone secretion, which is mainly mediated by the pharmacological actions of nicotine and other toxins such as thiocyanate. Smoking affects pituitary, thyroid, adrenal, testicular, and ovarian functions as well as the action of insulin 18. In pregnant women who smoke or use nicotine, nicotine crosses the placenta, concentrates in fetal blood and amniotic fluid, and is detectable in breast milk during lactation 7.

The present study showed that the treatment of pregnant female albino rats with nicotine induced degenerative changes in the adrenal cortex of their offspring in the form of increased vacuolation of the cytoplasm and increased amounts of lipid droplets in all layers. Cells of the zona fasciculate were laden with lipids to the extent that they appeared foamy at low magnification. At the electron microscopic level, destroyed mitochondria, pyknosis, and shrinkage and irregularity of the nuclei were observed. Previous studies confirmed that prenatal nicotinic exposure causes intrauterine growth retardation and HPA-axis-associated neuroendocrine metabolic alterations in the fetal rat 19. It has also been found that perinatal disturbances of the HPA axis could program or imprint the development of tissues and organs, producing later dysfunctions and diseases of the cardiovascular, metabolic, and neuroendocrine systems, which result from impaired adrenocortical function involving glucocorticoid (GC) feedback in the fetus 20.

The changes observed in this study in the adrenal cortex may be due to impaired steroidogenesis 21 or impairments in the synthesis and secretion of corticosterone in the offspring of smoking mothers leading to accumulation of lipid droplets and appearance of cytoplasmic vacuolation, which were observed in the present study. These results are similar to those obtained with prenatal caffeine ingestion at a clinically toxic dose that retards the development of the fetal HPA axis function by directly injuring fetal adrenal steroidogenesis and by fetal overexposure to maternal GC levels 22. This may explain the results seen in the pituitary gland, which showed decreased activity of corticotrophs due to a negative feedback on the pituitary corticotrophs, which revealed a reduction in the number of cytoplasmic secretory granules.

The principle behind the production of GC by zona glomerulosa and zona fasciculata is the ACTH secreted by corticotrophs in the adenohypophysis of the anterior pituitary gland. Negative feedback control normally occurs when the elevated blood levels of cortisol act either on the hypothalamus, anterior pituitary, or both to cause a suppression of ACTH secretion 23.

Prenatal nicotine exposure was demonstrated by Xu et al. 19 to reduce the expression of the placental 11-hydroxysteroid dehydrogenase-2 enzyme responsible for the inactivation of mother-derived GC, which resulted in fetal overexposure to maternal GC; this overproduction may suppress the ACTH production in the offspring.

They added that GC metabolic activation in the fetal hippocampus may account for the inhibition of the fetal HPA axis through negative feedback regulation. This was consistent with the results of some researchers who reported that nicotine administration leads to fetal overexposure to maternal GC, which eventually leads to fetal adrenocortical dysfunction 24.

Previous results demonstrated that prenatal nicotinic exposure can inhibit fetal adrenal steroidogenic acute regulatory (StAR) protein and gene expression in rats. Furthermore, nicotine reduced StAR gene expression and cortisol production in primary human fetal adrenal cortex cells, indicating that prenatal nicotinic exposure could inhibit fetal adrenal steroidogenic synthesis 19.

In this study, some zona fasciculata cells appeared with dark irregular nuclei and widened perinuclear space. These results are supported by Landais et al.25 and Yan et al.21, who found the same results.

The present study demonstrated that the most affected organelle in the adrenal cells in the nicotine-treated group was the mitochondria as they showed marked structural abnormalities and destruction. As a further validation of our results, another experiment proved that cigarette smoking may reduce the capacity of the mitochondria to metabolize cholesterol into pregnenolone 26. This is in agreement with other researchers who decided that there was increased oxidative damage specifically to mitochondrial proteins in the pancreas of nicotine- exposed neonates 27. Moreover, there was increased mitochondrial swelling by EM (electron microscope)in the pancreatic β cells of nicotine-exposed offspring 28. these results is that women who are pregnant should avoid exposure to tobacco smoke.

It is known that nicotine induces oxidative stress both in vitro and in vivo, as indicated by increased free radical production and lipid peroxidation levels in the pancreatic tissue of rats incubated with nicotine 29. Smoking results in an elevation of reactive oxygen species and the depletion of its scavengers in circulating blood. Reactive oxygen species also have been shown to inhibit steroidogenesis at the level of transporting cholesterol into the mitochondria 30.

The action of nicotine was described as follows: (i) it increases oxidative stress; (ii) it activates NF-κB, a redox- sensitive transcription factor; (iii) it activates the 78-kDa glucose-regulated protein promoter, an indication of endoplasmic reticulum stress; (iv) it induces apoptosis; and (v) it increases genotoxic stress 31.

As regards the thiocyanate-treated group, the zona fasciculata cells and corticotrophs appeared less affected compared with the nicotine group. There were few mitochondrial changes. This may be in agreement with Lloyd et al.32, who demonstrated that thiocyanate causes selective damage to mitochondrial membrane protein, resulting in increased permeability and subsequent leakage of cytochrome c into the cytosol.

Cyanide is a potent toxic agent present in cigarette smoke that inhibits the activity of cytochrome oxidase and is metabolized to thiocyanate through sulfuration with thiosulfate by mitochondrial rhodanese enzyme 19. Human exposure to cyanide produces toxic effects by binding to the iron and copper in the active site of cytochrome c oxidase, thereby inhibiting the enzyme. Pituitary–adrenal activity in thiocyanate-treated rats was not changed 33.

Depending on the dose, cyanide can cause histotoxic anoxia and cellular hypoxia 34. Blood thiocyanate is mainly distributed in serum and its presence is regarded as evidence of cyanide detoxification 35.

In this work the effects of nicotine are more apparent or pronounced when compared with those of thiocyanate. This work suggests that nicotine alone may be a key chemical responsible for the effects associated with maternal cigarette smoking on the offspring and that the administration of nicotine, and not other components of tobacco smoke, affects the pituitary adrenal axis in these offspring.


Maternal exposure to cigarette smoke during pregnancy has deleterious effects on the pituitary–adrenal axis of the offspring, and these adverse health outcomes associated with maternal smoking may be attributable, at least in part, to nicotine alone. Therefore, the implication from


Conflicts of interest

There are no conflicts of interest.

No title available.


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adrenal glands; nicotine; pituitary glands; thiocyanate

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