Mohameda, Doha S.; Abol Hagagb, Kaled E.
Lead is a naturally occurring element that has been used since the beginning of civilization. The major environmental sources of metallic lead and its salts are paint, auto exhaust, food, water, and folk remedies such as the use of kohl [1–3]. Lead exposure may occur during the manufacture of batteries, painting, printing, pottery glazing, and lead smelting processes.
Exposure may also occur during the construction of tank linings, piping, and other equipment that carry corrosive gases and liquids, superconductors, and fiber optic technologies, during magnetic resonance imaging and nuclear medicine. All sources of lead contribute to an increase in the permissible exposure limit that has been set by the WHO and other health organizations for metallic lead, lead oxide, and lead salts [4,5].
Lead is absorbed through the digestive system, respiratory tracts, and skin. After absorption into the blood, 99% of lead is bound to erythrocytes and the remaining 1% is present in plasma to be carried to other tissues. Serum lead half-life is around 25 days . Most of the lead absorbed into the body is excreted either by the kidney or through biliary clearance. Adults may ultimately retain only 1% of absorbed lead, but children tend to retain lead more than adults. The three major compartments for the distribution of lead are blood, soft tissue, and bone.
Evidence indicates that lead is a toxic agent with multiple target organs such as the gastrointestinal tract, the hematopoietic system, the immune system, the kidneys, the endocrine system, the reproductive system, and the nervous system . The symptoms of chronic lead poisoning include neurological problems, such as reduced cognitive abilities, nausea, abdominal pain, irritability, insomnia, metal taste in the oral cavity, excess lethargy or hyperactivity, headache and, in extreme cases, seizure and coma. Other associated effects are anemia, kidney disorders, and reproductive problems.
The pathogenesis of lead toxicity is multifactorial, as lead directly interrupts enzyme activation, competitively inhibits trace mineral absorption, binds to sulfhydryl proteins (interrupting structural protein synthesis), alters calcium homeostasis, and lowers the level of available sulfhydryl antioxidant reserves in the body .
Vitamin E is a potent fat-soluble antioxidant in humans. The antioxidant effects of vitamin E have been demonstrated in many experiments in vitro [7,8]. The adrenal gland is an important site for the regulation of fluid homeostasis and sympathetic function by angiotensin II . It is one of the soft tissue organs that can be affected by lead precipitation.
This study was conducted to investigate the toxic effect of lead acetate on the adrenal cortex of adult male albino rats after chronic lead intoxication during a 12-week period and to determine the possible protective effect of vitamin E on these changes.
Materials and methods
Lead acetate and vitamin E.
Preparation of materials
Lead acetate solution (Merck KGaA, Darmstadt, Germany) was prepared in saline and replaced daily to minimize lead precipitates. D-Lα-tocopherol (Vitamin E) was obtained from Pharco Pharmaceuticals (Cairo, Egypt).
In this study, 50 male albino rats of average weight (150–200 g) were used. The experimental protocol was approved by the Ethics Committee for Scientific Research, Faculty of Medicine Sohag University, Sohag, Egypt. The animals were housed at the Animal Facility, Faculty of Medicine. They were kept under conditions of adequate ventilation and temperature, received a standard pallet diet and were allowed water ad libitum. Animals were divided into three groups.
Group I (10 animals): This group was used as a control;
Group II (20 animals): The animals were intraperitoneally injected with a daily dose of lead acetate at a dose of 10 mg/kg body weight for 3 months ;
Group III (20 animals): The animals were administered an intramuscular injection of vitamin E at a dose of 100 mg/kg body weight 6 h before lead acetate injections at the same previous dose and duration  At the end of the experiment, rats were killed and the adrenal glands were extracted. For semithin sections, small pieces of fresh adrenal glands were prefixed in a 1 : 1 mixture of 2% paraformaldhyde and 2.5% gluteraldehyde in phosphate buffer, postfixed in 1% osmium tetroxide for 1 h and washed in a buffer solution. They were washed in 30% alcohol and dehydrated in a graded series of ethanol. Specimens were embedded in epon after treatment in propylene oxide and sectioned in an ultratome using glass knives. Semithin sections were stained with toluidine blue and photographed with a light microscope. Ultrathin sections were stained with uranyl acetate for 5 min and lead citrate for 5 min . They were viewed under a Jeol 1010 transmission electron microscope in Sohag University (Sohag, Egypt).
The percentages of both light and electron microscopic results were calculated for each group and are presented in tables.
In semithin sections, cells were arranged in the form of glomeruli. The glomeruli were separated by sinusoidal capillaries. The cells of the zona glomerulosa were polyhedral in shape and the cytoplasm was mildly vacuolated. The nuclei were large and vesicular (Fig. 1). Using electron microscope (EM), numerous mitochondria, profiles of smooth endoplasmic reticulum (SER), and a few lipid droplets could be observed. The nucleus was euchromatic with peripheral heterochromatin (Fig. 2) (Tables 1 and 2).
Group treated with lead acetate
In semithin sections, the zona glomerulosa showed loss of architecture. Marked destruction of most of the cells was observed. Some cells appeared with a highly vacuolated cytoplasm compared with the control group. Some cells were lost. Some of the nuclei were pyknotic, whereas other cells appeared vesicular (Fig. 3). Using EM, it could be observed that the cytoplasm was filled with large vacuoles and degenerated mitochondria.
The nucleus appeared with an irregular envelope (Fig. 4).
Group pretreated with vitamin E
In semithin sections, most of the cells that lined the glomeruli had a granular cytoplasm. The nuclei were vesicular. There was an apparent decrease in cell number compared with the control. The sinusoidal capillaries were narrowed (Fig. 5). Using EM, it could be observed that the cytoplasm had vacuoles of variable sizes. The mitochondria appeared more or less as the control, and the nucleus was euchromatic with peripheral clumps of heterochromatin (Fig. 6).
In semithin sections, cells appeared highly vacuolated and arranged in cords separated by sinusoidal capillaries. The nuclei were vesicular, with prominent nucleoli. Some cells were binucleated (Fig. 7). Using EM, it could be observed that the cytoplasm contained many mitochondria with vesicular cristae. There were many profiles of SER. Lipid droplets appeared in their cytoplasm. The nucleus was euchromatic with peripheral clumps of heterochromatin (Fig. 8) (Tables 3 and 4).
Group treated with lead acetate
In semithin sections, loss of architecture was observed. Some cells had a highly vacuolated cytoplasm. Some of the nuclei were pyknotic and hyperchromatic. An apparent decrease in cell number was observed compared with the control (Fig. 9). Using EM, it could be observed that the cytoplasm contained vacuoles of variable sizes and confluent lipid droplets. The mitochondria were degenerated and the nucleus had dispersed chromatin (Fig. 10).
In the group pretreated with vitamin E, the cells regained their architecture and were filled with fused vacuoles. The nuclei were vesicular (Fig. 11). Using EM, it could be observed that the mitochondria appeared ballooned with destroyed cristae. The SER was dilated. Many lipid droplets were observed in the cytoplasm. The nucleus was shrunken, hyperchromatic with an irregular outline (Fig. 12).
In semithin sections, the cells were arranged in branched cords separated by sinusoidal capillaries. The cells were polygonal in shape with vesicular nucleus (Fig. 13). Using EM, it could be observed that the cytoplasm contained many mitochondria, some of them large in size, and abundant SER. The nucleus was euchromatic, with peripheral clumps of heterochromatin (Fig. 14) (Tables 5 and 6).
Group treated with lead acetate
In semithin sections, the cells had a vacuolated cytoplasm. The vacuoles were of variable sizes. Some cells appeared hypertrophied and degenerated, with a pale cytoplasm. Wide lumens of sinusoidal capillaries were observed (Fig. 15). Using EM, it could be observed that the cytoplasm of the cells contained large vacuoles of variable sizes. The cytoplasmic organelles were not defined. Lipofuscin pigments were observed in the cytoplasm. The nucleus was small and hyperchromatic compared to the control group (Fig. 16).
Group pretreated with vitamin E
In semithin sections, the cytoplasm was highly vacuolated, some nuclei were vesicular and others were deeply stained. An apparent decrease in cell number was observed compared with the control (Fig. 17).
Using EM, it could be observed that the cytoplasm contained lipid droplets. The cell organelles were not defined and the cytoplasm was studded with heterogenous electron-dense material. The nucleus appeared heterochromatic with an irregular nuclear envelope (Fig. 18).
Chronic exposure to low levels of this agent is of public concern in many countries .
The results of this study revealed significant changes in the histological structure of the different zones of the adrenal cortex in lead-treated rats versus those of the control rats but these changes were not completely reversed with the use of vitamin E before lead acetate.
Using an EM, we observed increased vacuolization in most cells of different zones of the adrenal cortex in lead-treated animals. These vacuoles might be because of the accumulation of lipid droplets in these cells as a result of failure of their release because of the decreased use of cholesterol in aldosteron biosynthesis or as a result of dilatation of SER or swelling of the mitochondria. In groups treated with lead in both the zona glomerulosa and fasiculata, we observed marked degeneration of mitochondria; this might be because of the oxidative stress of lead and a sign of cell injury, whereas disruption of the inner mitochondrial membrane might increase the permeability and allow the solute to enter inside the matrix and lead to its swelling, followed by rupture of the outer mitochondrial membrane and release of the preapoptotic proteins . Similar findings were reported by Sundaram and Witorsch  in testes of rat after exposure to variable toxins.
Destruction of mitochondrial cristae in cells of the zona fasiculata persisted in the group pretreated with vitamin E.
In the zona reticularis of the lead-treated group, we observed the presence of lipofuscie pigments. They appeared to be the product of oxidation of unsaturated fatty acids, and may be a sign of membrane damage of the mitochondria and lysosomes. Lead readily distributes to all tissues and alters the metabolism and physiology of the cells and causes morphological changes that can remain even after lead levels have decreased. Thus, lead toxicity has long-term structural effects .
The pathogenesis of lead toxicity is multifactorial, as lead directly interrupts enzyme activation, competitively inhibits trace mineral absorption, binds to sulfhydryl proteins (interrupting structural protein synthesis), alters calcium homeostasis, and decreases the level of available sulfhydryl antioxidant reserves in the body . Studies suggest that some lead-induced toxic effects may occur through free radical production and oxidative stress .
Lead-induced oxidative stress contributes to the pathogenesis of lead poisoning by disrupting the delicate prooxidant/antioxidant balance that exists within mammalian cells . In in-vitro studies, the production of reactive oxygen species is increased after lead treatment [17,18]. In-vivo studies suggest that lead exposure causes the generation of reactive oxygen species and alteration of antioxidant defense systems in animals and occupationally exposed workers .
It was speculated that lead-induced oxidative damage may result from (a) the inhibition of 5-aminolevulinic acid dehydrogenase by lead, leading to the accumulation of aminolevulinic acid, (b) direct interaction of lead with biological membranes, inducing lipid peroxidation, a potential endogenous source of free radicals, (c) an increase in metabolism, and (d) lead-induced decrease in free radical metabolizing enzymes, glutathione levels [20,21] and intracellular levels of calcium, impairing mitochondrial function.
Vitamin E has also been shown to play a role in immune function, DNA repair, and other metabolic processes [7,22,23]. In semithin sections of the zona glomerulosa of the group that received combined treatment, we observed numerous basophilic granules in the cytoplasm; these granules were most probably free ribosomes as the remaining cells of this zone accumulate ribosomes to initiate division for regeneration of the adrenal cortex as the zona glomerulosa is considered a proliferative zone of the adrenal cortex .
In this study, we observed that the preliminary use of vitamin E before lead injection could not completely reverse lead toxicity but attenuated some lead-induced histological changes. Vitamin E is a fat-soluble vitamin that exists in eight different forms. Each form has its own biological activity, which is a measure of potency or functional use in the body . Antioxidants such as vitamin E act to protect the cells against the effects of free radicals, which are potentially damaging by-products of energy.
Lead exerted a marked toxic effect in the three zones of the suprarenal cortex. This effect was observed at both light and electron microscopic levels. The use of vitamin E cannot reverse most of these changes and so it may have little beneficial effect for protection of the suprarenal cortex against lead acetate toxicity.
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1. Abdel Maaboud RM, Abdel Moneim WM, Fathy HM, Abdel Hadi RH. Analysis of some traditional eye cosmetics by scanning electron microscope E.D.X. and the posibility of systemic absorption after topical application J Egypt Soc Toxicol. 2000;Z24:35–38
2. Rosner D and Markowitz G. Lead: the relevance of history. Mealey's Litigation Report, Lexis, Nexis. 2001; 800:227–4908.
3. Abdel Maaboud RM, Shehata MM, Abdel Maksoud SA. Histological changes in the kidney and liver of the rabbit as a result of the use of kohl Alazhar Assiut Med J. 2003;1:9–24
4. Patel AB, Williams SV, Frumkin H, Kondawar VK, Glick H, Ganju AK. Blood lead in children and its determinants in Nagpur, India Int J Occup Environ Health. 2001;7:119–126
5. Mugahi MN, Heidari Z, Sagheb HM, Barbarestani M. Effects of chronic lead acetate intoxication on blood indices of male adult rat Daru. 2003;11:147–151
6. Ercal N, Gurer Orhan H, Aykin Burns N. Toxic metals and oxidative stress part I: mechanisms involved in metal-induced oxidative damage Curr Top Med Chem. 2001;1:529–539
7. Farrell P, Roberts RShils M, Olson JA, Shike M. Vitamin E Modern nutrition in health and disease. 19948th ed Philadelphia, PA Lea and Febiger:326–341 . In: , editors. . pp.
8. Herbig U, Ferreira M, Condel L, Carey D, Sedivy JM. George Science. 2006;311:1257
9. Biswas NM, Ghosh P. Effect of lead on male gonadal activity in albino rats Kathmandu Univ Med J. 2004;1:43–46
10. Flora SJS, Pande M, Mehta A. Beneficial effect of combined administration of some naturally occurring antioxidants (vitamins) and thiol chelators in the treatment of chronic lead intoxication Chem Biol Interact. 2003;145:267–280
11. Drury RAB, Wallington EA Carleton's histological techniques. 19805th ed Oxford Oxford University Press
12. Loeffler M, Kroemer G. The mitochondrion in cell death control: certainties and incognita Exp Cell Res. 2000;256:19–26
13. Sundaram K, Witorsch RWitorsch R. Toxic effects on the testes Reproductive toxicology. 1995 New York Raven Press:99–122 . In: , editor. . pp.
14. Nehru B, Sidhu P. Behavior and neurotoxic consequences of lead on rat brain followed by recovery Biol Trace Elem Res. 2001;84:113–121
15. Soltaninejad K, Kebriaeezadeh A, Minaiee B, Ostad SN, Hosseini R, Azizi E, Abdollahi M. Biochemical and ultrastructural evidences for toxicity of lead through free radicals in rat brain Hum Exp Toxicol. 2003;22:417–423
16. Hsu PC, Guo YL. Antioxidant nutrients and lead toxicity Toxicology. 2002;180:33–44
17. Hermes Lima M, Pereira B, Bechara EJH. Are free radicals poisoning? Xenobiotica. 1991;21:1085–1090
18. Sandhir R, Julka D, Gill KD. Lipoperoxidative damage on lead exposure in rat brain and its implications on membrane bound enzymes Pharmacol Toxicol. 1994;74:66–71
19. Abdollahi M. Protection by selenium of lead-acetate-induced alterations on rat submandibular gland function Hum Exp Toxicol. 2001;20:28–33
20. Beconi MT, Affranchino MA, Schang LM, Beorlegui NB. Influence of antioxidants on SOD activity in bovine sperm Biochem Int. 1991;23:545–553
21. Sies H, Stahl W. Vitamins E and C, β-carotene and other carotenoids as antioxidants Am J Clin Nutr. 1995;62(6 Suppl):1315S–1321S
22. Packer L. Protective role of vitamin E in biological systems Am J Clin Nutr. 1991;53(4 Suppl):1050S–1055S
23. Patra RC, Swarup D, Dwivedi SK. Antioxidant effects of α tocopherol, ascorbic acid and L-methionine on lead induced oxidative stress to the liver, kidney and brain in rats Toxicology. 2001;162:81–88
24. Engeland WC, Ennen WB, Elayaperumal A, Durand DA, Levay Young BK. Zone-specific cell proliferation during compensatory adrenal growth in rats Am J Physiol Endocrinol Metab. 2005;288:E298–E306