Histopathological changes due to the effect of selenium in experimental cockerels

Trace elements are essential for normal human development and functioning of the body1. In biological systems, these trace elements are mostly bound to proteins, forming metalloproteins. Some of the metals in these metalloproteins are part of enzymatic systems, and have structural and storage functions2. Abnormalities in the metabolism of several trace elements have been reported in the pathogenesis of human diseases23. With the lifestyle modifications, many countries are facing increasing rates of cardiovascular diseases (CVD) including heart disease, hypertension, hyperlipidaemia, etc4. Low levels of selenium can contribute to heart failure, and being deficient in selenium seems to make atherosclerosis worse5. But studies have shown that selenium supplements do neither seem to have any effect on the progression of heart disease, nor do they protect against heart attack6. Selenium, supplementation (1 ppm) has been shown to increase LDL receptor activity and m-RNA expression7. Selenium concentrations were found to be inversely associated with the coronary heart disease and dilated cardiomyopathy in observational studies and the evidence from a few randomized studies is still inconclusive89. We have shown that 1mg of selenium supplementation elevated serum lipid profile in experimental rabbits910. Evaluation of these changes at the tissue level may reveal the mechanistic role of metal. Therefore, an attempt was made to study the effect of 0.14mg of selenium supplementation on histopathological changes in various organs in experimental cockerels.

Material & Methods

This study was conducted in the department of Pathology, Sri Venkateswar Institute of Medical Sciences (SVIMS), Tirupati, India during June 2000 to December 2000. The present study was undertaken on 40 male white Leghorn chicks (Gallus domesticus) weighing 674±93.37g. These chicks were procured from commercial poultry farm. The animals were maintained in the laboratory and were divided into four groups:

Group C (control): 10 cockerels were fed with stock diet of 100g per day (diet source: Krishna Poultry Farm, Tirupati, Andra Pradesh, India).

Group CH: 10 cockerels were fed with stock diet of 100 g + 100 mg of cholesterol + 1g butter per day; (cholesterol source: SRL, Mumbai, India and butter source: Amul India).

Group Se: Ten cockerels were fed with stock diet of 100g + 140 μg selenium as selenite per day (sodium selenite soruce: SRL, Mumbai) and

Group CH+Se: Ten cockerels were fed with stock diet of 100 g + 100 mg cholesterol + 1g butter + 140μg selenium as selenite per day.

The study protocol was approved by the Institutional Ethical committee, SVIMS, Tirupati.

These cockerels were fed for six months and no adverse clinical effects were observed during the period. After six months, the animals were sacrificed and organs (liver, kidney, heart, and descending aorta) were isolated. Tissue sections were made from each organ fixed on 10 per cent neutral buffered formalin. The sections were washed and dehydrated with graded ethanol series in the processor. After processing, tissue sections were embedded in paraffin blocks. Sealed sections were made using an automated microtome with a thickness of 4-5μm. Sections were stained with hematoxylin and eosin (H& E), and photographed under light microscope (Nikon, 463170, Japan) with attached camera (Nikon, 1103473, Japan).

Results

No abnormalities were detected in the liver, kidney and blood vessel but mononuclear infiltration in the heart of control group was observed. In liver, xanthochromatic collections in group CH; hydropic degeneration in group Se and lobular disarray, hydropic degeneration and kuppfer cell hyperplasia in group CH+Se were observed (Fig. 1a–d). In kidney, mild mononuclear infiltration in interstitium in groups CH, Se and CH+Se was observed (Fig. 2a–d). Myocyte disruption, and mononuclear infiltration in heart tissue in group CH and CH+Se and disruption of muscle bundles with vascular congestion in group Se were observed (Fig. 3a–d). Smooth muscle proliferation in the media of blood vessel was observed in group CH, Se and CH+Se (Fig. 4a–d).

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Fig. 1

(a). Group C - Liver (Control) (H&E 4×10). (b). Group CH - Liver (H&E 4 × 10) - Xanthochromic collections (arrow). (c). Group Se - Liver (H&E 10 × 10) - Hydropic degeneration. (d). Group CH+Se - Liver (H&E 4 × 10) Lobular disarray, (I^st from the left arrow) hydropic degeneration (II^nd from left arrow) & kupfer cell hyperplasia (III^nd from left arrow).

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Fig. 2

(a). Group C - Kidney (Control) (H&E 10 × 10). (b). Group CH - Kidney (H&E 10 × 10) - Mild mononuclear infiltrate in interstitium. (c). Group Se - Kidney (H&E 10×10) - Mild interstitial inflammation (arrows). (d). Group CH+Se - Kidney (H&E 10×10) - Mild mononuclear infiltrate interstitium.

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Fig. 3

(a). Group C - Heart (Control) (H&E 4 × 10) - Mild mononuclear infiltrate. (b). Group CH - Heart (H&E 10 × 10) - Myocyte disruption (1^st from left arrow), cholesterol clefts, (II^nd from left arrow), and mononuclear infiltration. (III^rd from left arrow), (c). Group Se - Kidney (H&E 10×10) - Disruption of muscle fibres (upper arrow) and mononuclear infiltration (lower arrow). (d). Group CH+Se - Heart (H&E 10×10) - mononuclear infiltration (1^st from left arrow) and Myocyte disruption (IInd from left arrow).

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Fig. 4

(a). Group C - Descending aorta (Control) (H&E 4 × 10). (b). Group CH - Descending aorta (H&E 40 × 10) - Smooth muscle cell proliferation in the media. (c). Group Se - Descending Aorta (H&E 40×10) - Smooth muscle cell proliferation in the media. (d). Group CH+Se - Heart descending aorta (H&E 40×10) - Smooth muscle cell proliferation in the media.

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Discussion

Selenium enters the environment from both geochemical and anthropogenic sources. Much of selenium in the environment comes from selenium dioxide produced by burning of coal and other fossil fuels. Inhalation of selenite and selenium dioxide can produce injury to respiratory tract, the cardiovascular and peripheral vascular system, brain, muscle, kidney and liver11. Acute Se toxicity is indicated by breath with garlic odour, dyspnoea, vomiting, and respiratory failure. Pathological changes in various organs include, congestion in the liver with areas of focal necrosis, congestion in the kidney, endocarditis, myocarditis petechial haemorrhages of the epicardium12. Chronic toxicity is indicated by growth inhibition and mortality, liver damage, splenomegaly and enlarged pancreas13. Food is the main source of Se for man, the dietary intake mainly depends on the region of origin of the food stuffs and the protein content14.

In this study, the effect of selenium was investigated on histopathological changes in four organs namely liver, kidney, heart and blood vessel in control against cholesterol, selenium and selenium with cholesterol fed cockerels. In selenium exposed liver, hydropic degeneration was observed which suggested liver injury induced by the selenium. Hydropic dengeration occurs as a result of ion and fluid homeostatis leading to an increase in intraceullar water15. Mononuclear infiltration in interestititum of kidney in selenium exposed group was observed in the present study. Infiltration of mononuclear cells was observed in innermost cortex of pig kidney in hypercholesterolaemic condition where microvascular density was more16. It is suggested that these cell by secreting cytokines and growth factors induce new vessel growth thereby interfering with the regulation and/or spatial distribution of intrarenal blood flow, resulting in renal disease progression16.

Monocytes disruption in heart tissue as observed in groups Se and CH+Se animals suggested the inflammatory changes in which monocyte chemoattracnt protein-1 (MCP-1) has been implicated to play an important role in many conditions including atherosclerosis17. Under conditions of chronic systemic inflammation, mediators derived from the myocardium may also participate in the pathogenesis of heart disease18. Smooth muscle proliferation in the media of blood vessel in selenium exposed group of animals was observed in the present study. Smooth muscle cells are found in media of the blood vessel with low proliferative index and in the process of atherogenesis, their proliferation is increased in the innermost part of media and intima19. Movement of medial cells into intima is a single, although initiating, step in the series of events which relate arterial smooth muscle to the atherosclerotic process20.

The results of present study show that selenium induces atherogenesis via inflammation. The mechanisms of toxicity of selenium reported in the literature are redox cycling of auto-oxidisable selenium metabolites, glutathione depletion21, protein synthesis inhibition, depletion of S-adenosyl-methionine (cofactor for selenide methylation), general replacement of sulphur and reactions with critical sulphydryl groups of proteins and cofactors19. Observational and randomized clinical trials have also raised concern that high selenium exposure may lead to adverse cardiometabolic effects, at least in selenium replete populations22. The optimum dose of Se is 140 μg, potential toxic dose is 800 μg per day in cockerels23. The safe dietary intake in humans is approximately 800 μg per day and the lethal dose is 5 mg24. The present results showed that the optimum dose of Se (140 μg/day) induced atherogenesis with cockerels with experimentally induced hypercholesterolaemia.