Investigation into the Effects of Selenium on Mitochondrial Monoamine Oxidase in Rat Liver

Transcription

1 J. Clin. Biochem. Nutr., 17, 11-17, 1994 Investigation into the Effects of Selenium on Mitochondrial Monoamine Oxidase in Rat Liver Gao-Feng FAN1,* and Lan-Hua CHOU2 Laboratory of Molecular Biology, Institute of Biophysics, Academia Sinica, Beijing , P.R. China Department of Biochemistry, Xian Medical University, Xian , P.R. China (Received February 1, 1994) Summary The structural and functional changes of mitochondrial monoamine oxidase (MAO) in livers of rats fed a selenium (Se)-deficient diet from an endemic area of Keshan disease were investigated. The results indicate that after intake of the Se-deficient diet for weeks, MAO activity and lipid fluidity in the mitochondrial membranes were significantly lower than those of the control rats; on the contrary, MAO degradation and lipid peroxides were significantly higher. Concomitantly, Se content and glutathione peroxidase (GPX) activity in hepatic homogenates of Se-deficient rats markedly decreased. These changes could be prevented or returned to the control levels when the rats were fed the same diet but one supplemented with 0.5 mg/kg N a2 SeO3. In vitro, we also showed that adding 0.5 ppm Na2SeO3 in hepatic submitochondria could significantly decrease MAO degradation induced by the intermediates of lipid peroxidation generated through radiolysis of submitochondria with a 6 Co source. Our results suggest that Se may be important for preventing lipid peroxidation in hepatic mitochondrial membranes, thus maintaining structural and functional integrity of MAO. Key Words: selenium, monoamine oxidase, glutathine peroxidase, lipid peroxidation, Keshan disease Selenium (Se) is an essential trace element in humans and animals. As an important component of glutathione peroxidase (GPX), Se takes part in scavenging hydrogen peroxide and organic peroxides in the body and keeps some free radicals from forming [1]. Se deficiency may impair normal development and physiological function of the target tissues, such as heart, liver, and muscle, in * To whom correspondence should be addressed. 11

2 12 G.-F. FAN and L.-H. CHOU animals [2]. Keshan disease is an endemic cardiomyopathy characterized by local myocardial necrosis, destruction of myocardial membranes, and impairment of oxidative phosphorylation. Se deficiency has been considered to be its major factor and sodium selenite supplementation to the diet has been claimed to be effective for prevention of the disease [3, 4]. A lot of research has shown that the pathomorphological characteristics of Keshan disease are similar to those of the experimental myocardial necrosis induced by catecholamine and that the catecholamine content in the sera and urine of Keshan disease patients is significantly higher than normal [5, 6]. Monoamine oxidase (MAO), a membrane-bound enzyme located in the outer membrane of hepatic mitochondria, is one of the important degradative enzymes of catecholamine. It is well known that Se deficiency can lead to the oxidative destruction of the polyunsaturated fatty acids of the membrane in the well-documented process termed lipid peroxidation, which causes changes in lipid fluidity of membranes and in the activity of membrane-bound enzymes [3, 7, 8]. However, mechanisms underlying the changes in the enzyme activities associated with Se deficiency still remain to be clarified. Therefore, research into the relationship between selenium and MAO in the liver is of great significance for both exploration of the pathogenesis of Keshan disease and elucidation of the mechanisms responsible for the effects of Se on membrane-bound enzymes. In the present study, we undertook investigations into the structural and functional changes in mitochondrial MAO in the liver of Se-deficient and selenium-supplemented rats. MATERIALS AND METHODS Chemicals. [3H] Pargyline was from New England Nuclear (Boston, MA); and cumene hydroperoxide, from Koch-light (Conlbrook Bucks, UK). Kynuramine, 4-hydroxyuinoline (4-HQ), diphenylhexatriene (DPH), and glutathione reductase were all from Sigma (St. Louis, MO). Other chemicals were of the purest commercial grade available. Animals. Male and female Sprague-Dawley rats, weighing g, were randomly divided into 3 groups. Group A, the Se-deficient group, was given a Se-deficient cereal diet (Se: ppm) from an area in China endemic for Keshan disease; it was composed of 641 corn, soybean, 7.51 wheat, 0.61 salt, 0.41 yeast, and 2 ml/kg cod-liver oil. Group B, the Se-supplemented group, received the same Se-deficient diet except that it was supplemented with 0.5 mg/kg sodium selenite (Se: ppm). Group C, the control group, was fed the stock diet (Se: ppm) commonly used at the animal center of Xian Medical University. All groups were fed ad libitum and had free access to water. Animals were used for the experiment after weeks on their diet. Assay of MAO activity. After overnight fasting, the rats were sacrificed under ether anesthesia. The livers were removed immediately and hepatic mitochondria were isolated according to the procedure described by Gazzoti [9]. MAO J. Clin. Biochem. Nutr.

3 SELENIUM AND MONOAMINE OXIDASE 13 activity was determined by the method of Michael [10], with kynuramine as the substrate. 4-HQ produced in the reaction system was measured with an MPF-66 spectrofluorimeter (Perkin-Elmer Carp., Norwalk, CT). The wavelengths of excitation and emission were 315 and 380 nm, respectively. Determination of lipid fluidity of mitochondrial membranes. Fluidity of mitochondrial membranes was measured by the fluorescence polarization method [11], with DPH as a fluorescence probe. The wavelengths of excitation and emission were 360 and 430 nm, respectively. Radical-generating systems. Oxygen-centered radicals were generated as described by Dean et al. [12] by steady-state radiolysis with a 6 Co source. In the presence of oxygen, submitochondrial particles suspended in 0.1 M potassium phosphate buffer saturated with N20/02 (4: 1) at ph 7.2 were used to generated the OH radical, and air-saturated suspensions containing 10 mm formate at ph 4.0 used to generate the H02' radical. Submitochondrial particles were y- irradiated for 24 min with up to 1,200 Gy at 50 Gy/min. Measurements of MAO degradation. Mitochondrial MAO was selectively labeled in situ with [3H] pargyline, an irreversible inhibitor that covalently binds to the enzyme's active site [13, 14]. Mitochondria containing radioactive MAO were then sonicated to prepare submitochondrial particles, and the latter were sedimented at 27,000 x g for 10 min before use. The pellet was resuspended in 0.1 M potassium phosphate buffer containing 1 mm EDTA (1.5 mg protein/ml) and the suspension divided into six parts. One part was counted for the total radioactivity, another was used to compare the amount of MAO degradation among the three groups of rats after weeks of feeding, and the other four were exposed to the pro-oxidant stresses mentioned above in the presence or absence of 0.5 ppm Na2SeO3. Mitochondrial proteins were then precipitated with ZnSO4/Ba(OH)2 as described by Dean et al. [15], and the supernatant was sampled for the measurement of the low-molecular-weight fragments. Degradation of MAO to [3H]- pargyline-labeled low-molecular-weight fragments was calculated as the supernatant radioactivity as a percentage of the total radioactivity. Other assays. Se content was assayed by 2,3-diamininaphalene microfluorescence method[16], and GPX activity was determined by the coupling method of glutathione reductase, with cumene hydroperoxide as the substrate [17]. Lipid peroxide (LPO) was assayed as described by Yagi [18]. Protein was determined by the method of Hartree [19]. RESULTS AND DISCUSSION Effects of Se on mitochondrial MAO in vivo It was previously documented that the activity of membrane-bound enzymes, such as Na+-K+-ATPase, 5'-nucleotidase, and cytochrome c oxidase in cardiac sarcolemma, decreased when rats were fed a Se-deficient diet from an area endemic for Keshan disease [20-22]. But all studies in the literature mainly emphasized Vol. 17, No. 1, 1994

4 14 G.-F. FAN and L.-H. CHOU that this drop in the activities of membrane-bound enzymes resulted solely from conformational alterations caused by changes in the surrounding membrane phospholipids induced by lipid peroxidation under a low Se status. Although some researchers reported that the intermediates in lipid peroxidation can fragment MAO in the mitochondrial outer membrane [15], there is no report documenting the direct destruction of the membrane enzyme protein due to Se deficiency. The results in Table 1 clearly show that mitochondrial MAO activity from Se-deficient rats was significantly lower (p < 0.001) and the amount of MAO degradation to low-molecular-weight fragments was significantly higher (p< 0.001) than those of the control group. An increase in DPH fluorescence polarization, which is inversely related to the fluidity of membrane lipids, was also observed (p<0.05). These data suggest that both the destruction of MAO enzyme protein and the decrease in lipid fluidity contribute to the functional disorder of MAO in rats fed on Se-deficient diet. This is the first system in vivo in which the effects of Se deficiency on the activity and degradation of MAO has been documented. It was also shown that these changes could be returned to the control levels when the rats were fed with the same Se-deficient diet but one supplemented with 0.5 mg/kg N a2 SeO3. In order to learn the causes for the changes mentioned above, selenium content, GPX activity in homogenate and lipid peroxide (LPO) value in mitochondria were examined as variables to assess Se status and lipid peroxidation in the livers. The data in Table 2 show that both Se content and GPX activity in the Table 1. Changes in MAO activity, lipid fluidity, and MAO degradation(%) in the mitochondrial membranes. Values are mean+sem of S rats per group. agroup A, Se-deficient group; Group B, Se-supplemented group; Group C, control group. bexpressed as the supernatant radioactivity (low-molecular-weight fragments) as a percentage of the total radioactivity. * **p <0.001, *p <0.05, significantly different from correspondings control group value. Table 2. Changes in Se content, GPX activity in homogenate, and LPO value in liver mitochondria. The values and groups are the same as in Table 1. amda, malondialdehyde. * * *p < 0.001, *p<005, significantly different from the corresponding control group value. J. Clin. Biochem. Nutr.

5 SELENIUM AND MONOAMINE OXIDASE 15 hepatic homogenate of Se-deficient rats decreased significantly (p<0.001) and LPO value in the mitochondria increased significantly (p<o.os) when compared with the control levels after weeks of feeding. Again, these changes could be prevented when the rats were fed the Se-supplemented diet. These results indicate that Se and Se-containing GPX are also of great importance in preventing accumulation of LPO in mitochondria, and thus in maintaining the structural and functional integrity of MAO. Se directly decreases MAO degradation by free radicals in vitro Previous studies have shown that a trace amount of Se could obviously prevent the dissociation of spectrin from the erythrocyte membrane and promote the association of spectrin with spectrin-stripped inside-out membrane vesicles [23]. It was suggested that this stabilization effect is based on conformational changes in spectrin induced by the reaction of its SH groups with Na2SeO3 [24-27]. This poses a problem about whether Se has a direct protective action on MAO enzyme protein, one that is independent of GPX. Table 3 shows that in the absence of added Se, mitochondrial MAO from all three groups could be fragmented by OH radicals and H02' radicals as well. But the amount of MAO degradation in Se-deficient rats was much higher than that of the Se-supplemented and control rats (p<0.001), indicating that the MAO enzyme protein from the Se-deficient rats is more susceptible to the destructive effect of free radicals. The addition of 0.5 ppm Na25e03 significantly decreased the percent of MAO degradation of all three groups, especially that of the Se-deficient rats (p<0.001). Since GPX is mainly located in the cytoplasm of liver cells, the protective action of Se on MAO, which is located in the outer membrane of mitochondria, can hardly be explained only in terms of Se-containing GPX. This effect seems to be complicated by the direct antioxidant action of Se, which is of great importance in protecting MAO enzyme protein from the damage of lipid peroxidation. In conclusion, we conclude from the results presented herein that the antioxidant action of Se is important not only for maintaining a proper physical state of mitochondrial membrane lipids but also for defending against MAO degradation Table 3. Degradation (%) of MAO in mitochondrial membranes by free radicals and the protective action of Se in vitro. The values, groups, and MAO degradation are the same as in Table 1. ***p<0.041 **p<001., significantly different from the control group. ap <0.05, by <0.01, Cp <0.001, significantly different from the value for the same group in the absence of additional Naz SeO3 (-Se). Vol. 17, No. 1, 1994

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