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Journal of the Anatomical Society of India

Effect of Cadmium On The Morphology Of Adrenal Gland In Mice

Author(s): Al-Motabagani, M.A.H.

Vol. 51, No. 2 (2002-07 - 2002-12)

Department of Anatomy, College of Medicine, King Faisal University, Damman. SAUDI ARABIA

Abstract

Cadmium is a heavy metal with which man has been in contact more and more in recent years. It affects many endocrine glands. To observe its effects on adrenal gland cadmium was administered in single large, and in small daily doses for 7 days to adult male mice. The single dose failed to produce any effect on the gland, while the multiple small doses induced hypertrophy of the zona glomerulosa of the adrenal cortex.

Key words: Long bones; vascularization; nutrient arteries, femoral diaphysis

Introduction:

Increasing environmental concentrations of Cadmium (Cd) has precipitated concern over the toxicological potential to this heavy metal with which man has been brought more and more in contact during recent years. Exposure to cadmium produces diliterous effects on the cellular architecture and metabolism in a variety of body tissues. It causes retardation of growth rates (Hill et al, 1963) testicular atrophy and necrosis (Parizek, 1957) kidney damage (Axelsson & Pistcatar, 1966) impaired hepatic and pancreatic function (Rastogi & Singhal, 1975), it also induces hypertrophy and hyperplasia of the pituitary gonadotrophs in the brown spiny mouse (Madyastha & Dutt, 1979) Some workers demonstrated that cadmium affected the endocrine glands, namely the thyroid, the pituitary, the pancreas and the testes (Nishiyama & Takata, 1982) However, the adrenal glands are found to have more capacity for cadmium accumulation than any other endocrine gland (Killmer, 1980) These reports suggest that the adrenal gland may be a highly susceptible organ to cadmium compared with other endocrine glands (Nishiyama & Takata, 1982).

The present study was designed to investigate the effects of cadmium on the adrenal gland of adult male mice, both morphologically and histochemically, with special reference to the cortical zonation, the size of cortical and meduallary cells, under the light microscope.

Materials and Methods:

Forty eight adult male mice were divided into four groups (A, B, C and D). The animals in group A were used as control and were injected with a single dose of normal saline subcutaneously, whereas those of group B were injected with a single dose of cadmium chloride 6 mg/kg body weight dissolved in the same volume of normal saline. On the other hand, the animals in group C received normal saline s/c daily for 7 days, while those in group D were injected with cadmium chloride 2 mg/kg body weight for 7 consecutive days. (Table 1).

Table 1 : Grouping of Animals:

Group No. of animals Treatment Dose
(mg/kg)(ml/kg)
No. of Injections Duration (days)
A 1 (control) 12 Normal saline 0.9% 5 ml 1 1
B 12 Cadmium chloride 6 mg/kg 1 1
C (control) 12 Normal (saline 0.9%) 5 ml 7 7
D 12 Cadmium chloride 2 mg/kg 7 7

Table 2 : Summary of methods of study :

Specimens Fixative Sectioning Staining and purpose
Right adrenal (X) Buffered neutral formalin 5m thick paraffin sections H & E for different measurements and general morphology
Left adrenal (X) Buffered neutral formalin 10m thick frozen sections Sudan black B for lipids
Right adrenal (Y) Buffered neutral formalin 4m thick paraffin sections Methyl green thionin stain for DNA and RNA
Left adrenal (Y) Regaud& fluid 5m thick paraffin sections Schmorl's ferric chloride ferricyanide test

Animals in group A and B were sacrificed 24 hours after the single injection, while those of group C and D were sacrificed 24 hours after the seventh injection. At autopsy the animals of each group were equally divided into subgroups X and Y, the adrenals removed from each subgroup were processed as shown in Table (2).

Paraffin sections were stained and studied for size of cortical zones, medulla, cell size in cortex and medulla, lipid, DNA and RNA contents in the adrenal cells and chromaffin reaction in adrenomedullary cells.

Observations and Result:

Under the light microscope the capsule in the control as well as in some cadmium treated animals was thin and moderately cellular, however, in some cadmium injected animals a thick capsule with numerous fibroblasts was observed. The cellular arrangement in different zones remained within normal limits in control animals; however, after seven days of cadmium injections the cells in the different zones of the cortex showed changes in their general morphology. The chromaffin cells were surrounded by dilated vessels and the area looked congested. The cortico medullary junction was intensely stained in the treated animals.

The single dose of cadmium produced an insignificant decrease of zona glomerulosa (Table 3). In the animals so treated no other cortical or medullary area showed any appreciable change (Table 4, 5 and 6).

Table 3 : Mean width and cell size of Zona Glomerulosa in different groups :

Group Width of zona glomerulosa (microns) Cell's size (microns)
A 33.51 ± 0.90 8.31 ± 0.37
B 32.96 ± 0.71 8.15 ± 0.40
C 31.21 ± 1.07 7.67 ± 0.31
D 41.29 ± 2.71 8.90 ± 0.23

Mean ± standard error.

Statistical analysis of the difference in mean width and cell size between:

A and B P > 0.05 P > 0.05 C and D P < 0.05 P < 0.05

Table 4 : Mean Width and cell size of Zona Fasciculata in different groups :

Group Width of zona
fasciculata (microns)
Cell's size
(microns)
A 141.35 ± 6.35 12.24 ± 0.29
B 143.14 ± 4.18 12.91 ± 0.32
C 160.04 ± 1.07 13.11 ± 0.38
D 156.07 ± 1.42 12.64 ± 0.32

Mean ± standard error.

Statistical analysis of the difference in mean width and cell size between :

A and B P > 0.05 P > 0.05
C and D P > 0.05 P > 0.05

Table 5 : Mean width and cell size of Zona Reticularis in different groups :

Group Width of zona
reticularis (microns)
Cell&'s size
(microns)
A 33.32 ± 2.47 7.36 ± 0.59
B 32.43 ± 2.39 6.44 ± 0.41
C 33.33 ± 2.93 7.67 ± 0.54
D 35.39 ± 3.06 8.07 ± 0.44

Mean ± standard error.

Statistical analysis of the difference in mean width and cell size between :

A and B P > 0.05 P > 0.05
C and D P > 0.05 P > 0.05

Table 6 : Mean width of Medulla and cell size in different groups :

Group Total width
medulla
(microns)
Size of light
cells (microns)
Size of of
(microns)
A 768.00 ± 16.85 14.26 ± 0.22 13.71 ± 0.18
B 756.00 ± 23.99 14.33 ± 0.27 13.76 ± 0.53
C 826.80 ± 26.28 13.70 ± 0.52 12.97 ± 0.45
D 849.60 ± 37.49 15.64 ± 0.55 13.76 ± 0.29

Mean ± standard error.

Statistical analysis of the difference in mean width and light and dark cell size between :

A and B P > 0.05 P > 0.05 P > 0.05
C and D P > 0.05 P > 0.05 P > 0.05

On the other hand, after 7 days of cadmium injections a significant increase in the total width of zona glomerulosa as well as its cell size was observed (table 3). In these animals, an insignificant decrease was observed in the width of zona fasciculata (Table 4) but reticularis showed no change (Table 5). The adrenal medulla showed an insignificant increase in the size of its light and dark cells (Table 6).

DNA/RNA content:

Depending upon its staining characteristics the cells of the adrenal gland were evaluated for their DNA/RNA content. A marked increase in cytoplasmic RNA was noticed in group D as evidenced by the increased intensity of staining. The increase was more marked in the medulla than in the cortex. No obvious change was seen in group B.

Lipid content:

Seven days injections of cadmium caused moderate lipid depletion in the intracellular content of the zona glomerulosa, and the globules decreased in size as compared to the control group. Fasciculata and reticularis showed no noticeable change. Single dose injection did not show any remarkable change.

Chromaffin reaction :

High intensity reaction was observed among the different cells of the adrenal medulla in group D animals.

Adrenal weight:

A significant increase in the absolute and relative adrenal weight was noted in the animals given cadmium for 7 days, whereas with the single dose injection the increase was not significant (Table 7).

Table 7 : Mean absolute and relative weights of Adrenal in different groups:

Group Absolute weight of
adernal (mg)
Relative weight
of adrenal
(mg/100 gm body weight)
A 5.05 ± 0.28 11.48 ± 0.74
B 5.14 ± 0.27 12.17 ± 0.34
C 5.07 ± 0.22 12.28 ± 0.31
D 6.02 ± 0.12 14.51 ± 0.23

Mean ± standard error.

Statistical analysis of the mean widths and cell sizes between :

Absolute weight Relative weight
A and B P> 0.05 A and B P> 0.05
C and D P< 0.001 C and D P< 0.001

Discussion:

A single cadmium injection resulted in an insignificant increase in adrenal weight, while the 7 days injection showed a significant increase in weight and vascularity of the cortex and medulla. Previous work by Nishiyama and Nakamura (1984) have shown that subacute treatment with cadmium resulted in a significant increase of total protein content in the adrenocortical cells, with associated gland enlargement. In the present study the increased weight of the adrenal gland may be due to increased vascularity as well as hypertrophy of the glandular tissue and the increased protein content in the cells. It was also clear that a single injection with cadmium failed to produce any appreciable change in cell size or lipid content in any zone of adrenal cortex. When injected for 7 days, however, cadmium caused moderate lipid depletion and a decrease in the size of lipid globules with increase in glomerulosal cell size resulting in the hypertrophy of this zone. According to Nishiyama and Nakamura (1984) cadmium stimulates DNA and RNA synthesis in adrenocortical cells, and therefore the total intracellular protein increases and the gland enlarges. ACTH stimulates DNA synthesis in the adrenal gland (Masui & Garsen, 1970). But the zona glomerulosa is only slightly under the control of ACTH (Guyton, 1986). On the other hand, the zona fasciculata showed no hypertrophy although it is controlled by ACTH. Therefore, stimulation of DNA/ RNA seems to have negligible effect in the hypertrophy of the glomerular zone.

Glomerulosal cells secrete aldosterone which is regulated by renin-angiotensin system including water and electrolyte balance. Increase in the size of glomerulosal cells may be the result of some disturbance in renin-angiotensin system or the effect of cadmium on kidney tubules with resultant electrolyte imbalance. Axelsson and Pistcator, 1966 observed functional disorders in the proximal tubules of rabbit kidney after s/c injections of cadmium. It is likely that cadmium produces its effect through the kidneys by decreasing aldosterone production below normal, and to compensate for that the glomerulosal cells undergo hypertrophy. Cadmium produced not much change in the lipid content, but some decrease in cellular size of the zona fasciculata, which though reduced in width was not significant statistically. Reticularis showed no noticeable change. Some workers like Nishiyama, &amp; Nakamura (1984) observed low levels of ACTH in animals given cadmium in same dose over a same period as that in the present study.

Those researchers are in favour of the effect of cadmium on adrenal through pituitary or hypothalamus, and claim that cadmium exerts its effect on pituitary or hypothalamus inhibiting secretion of ACTH. This may be the reason why the cell size of the zona fasciculata, instead of increasing due to stress or stimulation by cadmium, decreased, causing some reduction in the width of this zone. Rastogi and Singhal (1975) found that stress induced by chronic exposure to cadmium results in enhanced increase in the synthesis of adrenal catecholamines. The present study, which is of short duration, revealed that the increased secretary activity was not responsible for any marked change in medullary cell size or number.

References:

  1. Axelsson, B. and Pistcator, M. (1966): Renal damage after prolonged exposure to cadmium. Archives of Environmental Health, 12: 360.
  2. Guyton, A.C. Text book of medical physiology, 7th ed W.B. Saunders, Philadelphia. (1986).
  3. Hill, C.H., Matrone, G., Payne, W.L. and Barher, C.W. (1963): In vivo interactions of cadmium with copper, zinc and iron, Journal of Nutrition 80: 227.
  4. Killmer, W.E. (1980): Uptake and retention of cadmium 109 in the pituitary, the adrenals and the thyroid of the laboratory rat. International Journal of Applied Radio isotopes. 31: 607-09.
  5. Madhyastha, N.A. and Dutt, N.H.G. (1979): Cadmium induced hypertrophy and hyperplasia of pituitary gonadotrophs of the brown spiny mouse. Indian Journal of Experiment Biology. 17: 637-39.
  6. Masui, H. and Garren, L.D. (1970). On the mechanism of action of adrenocoticotropic hormone. Journal of Biology &amp; Chemistry 25: 2627-32.
  7. Nishiyama, S. and Nakamura, K. (1984): Stimulation of adrenal DNA synthesis in cadmium-treated male rats. Toxicology & Applied Pharmacology. 74: 337-44.
  8. Nishiyama, S. and Takata, T. (1982): Effects of cadmium on the level of serum corticosterone and adrenocortical function in male and female rats. Research Communication, Chem. Pathology Pharmacology, 37: 65-80.
  9. Parizek, J. (1957): The destructive effect of cadmium ion on testicular tissue and its prevention by zinc. Journal of Endocrinology 15: 56-63.
  10. Rastogi, R.B. and Singhal, R.L. (1975): Effect of chronic treatment on rat adrenal catecholamines. Endocrinal Research Communication, 2: 87-94.

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Fig. No. 1. Section of adrenal in control male mice showing capsule (C) zona glomerulosa (G), zona fasciculata (F), zona reticularis (R) and adrenal medulla (M), Perinephric fat (PF) is also seen outside the capsule. (Paraffin section, H &amp; E stain. Photomicrograph x160.)

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Fig. No. 2. Section of adrenal cortex in male mice with cadmium treatment (seven days) showing haemorrhage in zona reticularis. (Paraffin section, H &amp; E stain. Photomicrograph x320.)

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Fig. No. 3. Section of adrenal in male mice with cadmium treatment (seven days) showing congestion in medulla (arrow). (Paraffin section, H &amp; E stain. Photomicrograph x160.)

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Fig. No. 4. Section of adrenal in control male mice showing distribution of lipid globules in diferent zones of the cortex. (Frozen section, Sudan Black B stain. Photomicrograph x160.)

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Fig. No. 5. Section of adrenal cortex in male mice with cadmium treatment (seven days) showing moderate decrease in lipid content of the zona glomerulosa as compared to control. (Frozen section, Sudan Black B stain. Photomicrograph x160.)

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Fig. No. 6 Section of adrenal in control male mice showing green stained DNA in the nuclei and purple stained RNA in the cytoplasm. (Paraffin section, Methyl green thionin stain. Photomicrograph x320.)

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Fig. No. 7 Part of the section as shown in figure 6 under higher magnification showing DNA (green) and cytoplasm RNA (purple) (Paraffin section, Methyl green thionin stain. Photomicrograph x1600.)

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Fig. No. 8 : Section of adrenal in male mice with cadmium treatment (seven days) showing high content of RNA in medulla. The picture shows an increase in the intensity of the staining as compared to control. (Paraffin section, Methyl green thionic stain. Photomicrograph x320.)

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Fig. No. 9 : Part of the section as shown in figure 8 under higher magnification showing DNA (green) &amp; RNA (purple) (Paraffin section, Methyl green thionin stain. Photomicrograph x1600.)

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Fig. No. 10 : Section of adrenal in control male mice showing chromaffin reaction in medulla. (Paraffin section, Schmrol&'s ferric chloride ferricyanide test. Photomicrograph x160.)

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Fig. No. 11. Section of adrenal in male mice with cadmium treatment (seven days) showing marked intensity of chromaffin reaction in medulla as compared to control. (Paraffin section, Schmrol&'s ferric chloride ferricyanide test. Photomicrograph x160.)

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