EFFECT OF ORGANIC SELENIUM COMPOUNDS ON THE ACTIVITY OF GLUTATHIONE PEROXIDASE AND SUPEROXIDE DISMUTASE IN SELECTED MOUSE TISSUES

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1 Bull. Vet. Inst. Pulawy 47, , 2003 EFFECT OF ORGANIC SELENIUM COMPOUNDS ON THE ACTIVITY OF GLUTATHIONE PEROXIDASE AND SUPEROXIDE DISMUTASE IN SELECTED MOUSE TISSUES IRENA MUSIK*, (/)%,(7$ 67$526à$:6.$ AND KAZIMIERZ PASTERNAK* * Department of General Chemistry, Medical University, Lublin, Poland ** Lublin Regional Centre of Oncology, Lublin, Poland musik@panaceum.am.lublin.pl Received for publication April 02, The aim of the study was to determine the activity of antioxidative enzymes GPH-Px and SOD as well as selenium concentration and distribution in mice treated with selenium compounds. Organic selenium compounds obtained in our laboratory: 4-(o-tolyl-) selenosemicarbazide of p-chlorobenzoicacid and 3-(p-chlorobenzoylamino-)-2-(o-tolylimino-) - 4-phenyl-4-selenazoline were administrated to mice in a dose of 5x10-4 mg/1 g body weight for 10 d. Results were compared with control group without supplementation and with the group in which sodium selenite was given. A correlation was observed between GPH-Px level and Se distribution in mouse tissues. Our investigations let presume that the obtained organic compounds can be good and assimilable selenium forms. Key words: mice, selenium, supplementation, antioxidative enzymes. Selenium is an indispensable trace element ensuring normal functioning of the organism. Deficiency of this element may lead to disturbances of the immune system functions (8), to acceleration of cardiovascular complications and thyroid function disorders (2). Decreased selenium level is also observed in neoplastic diseases (10) and Alzheimer s disease (5). Selenium deficiency may result from dietary errors, disorder of the synthesis of proteins containing selenocystein and abnormal selenium transport (2). Selenocystein is a basic component of selenoproteins such as: glutathione peroxidase (GPH-Px), 5-tyronine deiodase, selenoprotein P and selenoprotein W (4, 7). Glutathione peroxidase is an essential enzyme of the antioxidative system protecting the body against harmfull effect of oxygen radicals. Free radicals originate in respiratory metabolism occurring in mitochondria and microsomes, enzymatic reactions with involvement of cyclooxygenase, lipooxygenase, metaloproteinases, in the course of glucose autooxidation and in many other processes taking place in the organism (6).

2 568 GPH-Px is an enzyme decomposing hydroxides of fatty acids (ROOH) and hydrogen peroxide (H 2 O 2 ). This enzyme, apart from the presence of Se, requires the presence of reduced glutathione (GSH), which is supplied by glutathione reductase (17). The source of hydrogen for the reduction of oxigentated glutathione (GSSG) is NADPH, hence, the high rate of reproduction of this nucleotide in the liver and erythrocytes affects the reaction efficiency. One of antioxidative enzymes is superoxide dismutase (SOD). It occurs in many kinds of isometric variations: cytoenzymatic (containing Cu, Zn in the catalytic part), mitochondrial (containing Mn) and extracellular. SOD inhibits cytochrome C reduction by the reduction of superoxide anionanionoradical (13). The investigations aimed to define the effect of organic selenium compounds on the activity of antioxidative enzymes in extracts of cerebral, renal, hepatic and adrenal tissues and also selenium distribution in selected mouse tissues. Material and Methods The investigations were carried out on female SWISS mice divided randomly into the following 4 groups: group A control group without Se supplementation group B received sodium selenite dissolved in water group C received 4-(o-tolyl)-selenosemicarbazide of p-chlorobenzoicacid (aliphatic compound) dissolved in an emulsion concocted of olive oil, Arab gum and water in 2:1:1.5 ratio group D supplemented with 3-(p-chlorobenzoylamino-)-2-(otolylimino)-4-phenyl-4-selenazoline annular compound in emulsion prepared like in group C. Each group consisted of 10 mice. The mice received standard feed LSM and drinking distilled water ad libitum. The animals were weighed before and after the experiment. Animals in groups B, C, D were given selenium compounds through gastric probe once a day for ten days in a dose of 5x10-4 mg Se/g body weight. Organic compounds in aliphatic form: 4-(o-tolyl)-selenosemicarbazide of p-chlorobenzoicacid and annular form: 3-(p-chlorobenzoylamino-)-2-(o-tolylimino)-4-phenyl-4-selenazoline were obtained in the Chair and Department of General Chemistry of the University School of Medicine in Lublin (16). After ten days of the experiment the animals were anaesthetized with vet-butal and their brain, kidneys, adrenals and liver were taken for examinations. Then 0.5 g of particular tissue sample was homogenized in 0.1 M Tris HCl buffer (ph 7.4). Because of small masses of the brain, kidneys and adrenals, the organs were pooled (in batches 2-3 organs) to obtain an adequate amount of tissue. The obtained homogenate was centrifuged for 30 min at 5000xg rotations. Protein content was determined in the supernatant with the Paglia and Valentine method (18) and GPH-Px was measured at 340 nm. Superoxide dismutase (SOD) activity was measured at 480 nm according to Misra and Fridovich (9). The selenium content was analysed spectrophotometrically (specord H-40, Carl Zeiss Jena) to determine the toluene solution absorbance at the wave length of 350 nm according to Bem (1). The concentration was calculated against the standard curve plotted from DANB and standard solution of 100 µg Se ml -1 in 0.1 mol 1 in hydrochloric acid solution. Comparisons between control and tested groups were made using the Cochran-Cox test. Values were considered significant at P < 0.05.

3 569 Results The highest and statistically significant GPH-Px activity was demonstrated in extracts of brain and liver tissues as well as increased activity in the kidneys and adrenals in the group supplemented with inorganic Na 2 SeO 3 compared with the control group (A) and with the group that was given 3-(p-chlorobenzoylamino-)-2-(otolylimino)-4-phenyl-4-selenazoline (group D). After supplementation with selenosemicarbazide (group C), however, the highest mean values were found in the brain and showed statistical significance compared with the control group and the group of mice administered with selenazoline (group D). values of GPH-Px activity in kidney tissue in the group administered with selenazoline showed statistical significance compared with all the groups of mice. In the adrenals only after supplementation with sodium selenite a slight increase in GPH-Px activity was observed while in the remaining groups, in which organic selenium compounds were given a statisticaly significant decrease was found compared with the control group. The results are given in Table 1. Table 1 GSH-Px activity in extracts of the examined mouse tissues after supplementation with selenium compounds Brain Kidney Adrenal Live Group ± SD ± SD ± SD ± SD U/mg protein U/mg protein U/mg protein U/mg protein A 0.17± ± ± ± 0.41 B 0.31± 0.07 a 2.58± ± ± 2.56 C 0.29± 0.03 a,b 2.59± ± ± 1.01 a D 0.16± 0.06 b 1.76± 0.39 a 0.02± ± 1.72 SD - standard deviation A - controls group without Se supplementation B - sodium selenite supplementation C-4-(o-tolyl-)-selenosemicarbazide of p-chlorobenzoic acid supplementation D-3-(p-chlorobenzoylamino-)-2-(o-tolylimino-)-4-phenyl-4-selenazoline supplementation a - statistically significant in comparison with control group b - statistically significant in comparison with B group SOD activity decreased in the kidneys after supplementation with all the selenium compounds showing statistical significance in groups B and D compared with the control group A. A statistically significant SOD decrease was also found in the brain after supplementation with Na 2 SeO 3 (group B) and selenosemicarbazide (group C) compared with the other groups i.e. the control (group A) and group D, which received selenazoline. No significant changes were found in the adrenals after supplementation with all the selenium compounds compared with the control group.

4 570 In liver tissue, however, in all cases after supplementation with selenium compounds a significant, almost twofold increase in SOD activity in comparison with the control group was observed. The results are given in Table 2. Table 2 SOD activity in extracts of examined mouse tissues after supplementation with selenium compounds Brain Kidney Adrenal Live Group ± SD ± SD ± SD ± SD U/mg protein U/mg protein U/mg protein U/mg protein A 13.30± ± ± ± 22.8 B 5.90± 2.7 a,c 17.10± 6.2 a,d 2.45± ± a C 9.25± 4.3 a 29.20± ± ± 323.9a D 13.90± 3.5 b,c 6.90± 0.8 a,b,c 1.92± ± a A,B,C,D and a, b - see explanations in Table 1 c - statistically significant in comparison with C group d - statistically significant in comparison with D group No body mass inhibition was observed. Body weights before the experiment ranged from 24 to 27g while after the experiment they amounted to 25.2 ± 1.3 g 25.8 ± 1.7 g in the control group (A) and were 24.8±1.6 g 25.6±0.9 g in group supplemented with Na 2 SO 3, 24.2±2.3 g 24.8±1.6 g in group with selenosemicarbazide (C) and 24.3±2.1 g 24.7±0.9 g in group with selenazoline (D). Examinations of the brain, kidney and liver after supplementation with selenium compounds showed that the highest selenium accumulation was observed after administration of selenosemicarbazide and amounted to ± µg/1 g tissue in the brain, ± 6.57 µg/1 g tissue in the kidney and ± 6.80 µg/1 g tissue in the liver. It was also noticed that after supplementation with selenazoline a slight decrease in selenium concentration occurred in kidney tissue and it was ± 7.53 µg/1 g tissue compared with the control group, whose value amounted to ± 9.32 µg/1 g tissue (Table 3). Discussion Positive results of selenite supplementation in numerous pathologic conditions, sometimes only in selenium deficiencies, encourage to look for a proper selenium form and an adequate administration regimen. In our study we compared the effect of the supplementation with two obtained selenium compounds: 4-(o-tolyl)- selenosemicarbazide of p-chlorobenzoicacid and 3-(p-chlorobenzoylamino-)-2-(otolylimino)-4-phenyl-4-selenazoline and inorganic sodium selenite on the activity of GPH-Px, SOD as well as selenium assimilation and distributive binding in selected mouse tissues. There is a correlation between selenium content and glutathione peroxidase level; the activity of this enzyme increases with selenium increase (3).

5 571 Table 3 The selenium content in mouse organs following the supplementation with selenium compounds Brain Kidney Liver Group organ mass content µg/1 g organ mass content µg/1 g organ mass content µg/1 g ±SD (g) tissue ±SD ±SD (g) tissue ±SD ±SD (g) tissue ±SD A 0.400± ± ± ± ± ± B 0.450± ± ± ± ± ±7.720 a C 0.390± ±9.920 a 0.250± ± ± ±6.800 a D 0.320± ± ± ± ± ±4.930 a a - statistically significant in comparison with control group (A) A,B,C,D - see explanations in Table 1

6 572 Low activity of glutathione peroxidase is connected with symptoms of selenium deficiency. Hence, selenium deficiency in the body decreases activity of the enzyme, which, together with catalase, superoxide dismutase (SOD) and alpha-tocopherol, prevents excessive oxidation of cellular membranes (18). GPH-Px activity after supplementation with the obtained selenium compounds is obviously evident for this correlation. Decreased mean values of SOD activity in the brain after Na 2 SeO 3 and 4- (o-tolyl)-selenosemicarbazide of p-chlorobenzoicacid supplementation may result from two high doses of selenium per 1 g body weight. It turned out favourable, however, that despite a high dose (5 mg Se/kg body weight) the administered compounds did not inhibit body weight gain during the experiment. Sodium selenite given in different doses, mg Se/kg (19, 20), 0.1-1mg Se/kg (12), mg Se/kg (11) and 10 mg Se/kg (14, 15) was a point of reference for newly synthetised compound. Selenium supplied as organic compound is better assymilable than inorganic compounds selenians and selenins administered in diet supplementing preparations (14). The results of our study have confirmed good solubility in the applied emulsion of organic compounds obtained by us, which do not dissolve in water. Examination of Se concentration in individual tissues has shown that selenium is absorbed from the alimentary tract and built into the tissues in varying degrees (14, 15). Partially positive results are encouraging to look for an appropriate therapeutic form of selenium. This is evidenced by our investigations. References 1. Bem E.M.: Simple spectrophotometric method for selenium determination in biological material. Chemia Anal., 1979, 24, Berry T.: A selenium transport protein model of a sub-type of schizophrenia. Med. Hypotheses, 1994, 43, Buckman T.D., King A., Sutphin M.S.: Platelet glutathione peroxidase and monoamine oxidase activity in schizophrenics with CT scan abnormalities: relation to psychosocial variables. Psychiatry Res.,1990, 131, Burk R.F., Hill K.E.: Selenoprotein P a selenium-rich extracellular glycoprotein. J. Nutr., 1994, 124, Cornett C.R., Markesbery W.R., Ehmann W.D.: Imbalances of trace elements related to oxidative damage in Alzheimer ' s disease brain. Neurotoxicology, 1998, 19, Derejczyk J.: Free- radical reactions and DQWLR[LGDQWV3RVW S\1DXN0HG 12, )ORULDF]\N% Proteins participating in selenium metabolism. Ann. Univ. Mariae &XULH6NáRGRZVND0HG 9/10, Forceville X.,Vitoux D., Gauzit R., Combes A., Lahilaire P., Chappuis P.: Selenium, systemic immune response syndrome, sepsis, and outcome in critically ill patients. Crit. Care Med., 1998, 26, Fridovich I.: Biological effects of superoxide radicals. Arch. Biochem. Biophys., 1986, 58, Gerland M., Morris J.S., Meier J.: Prospective study of toenail selenium levels and cancer among women. J. Natl. Cancer Inst.,1995, 87,

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