Antioxidant Effect of Selenium-containing Glutathione S-Transferase in Rat Cardiomyocytes

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1 CHEM. RES. CHINESE UNIVERSITIES 2012, 28(3), Antioxidant Effect of Selenium-containing Glutathione S-Transferase in Rat Cardiomyocytes YIN Li 1, HAN Xiao 1, YU Yang 1, GUO Xiao 1, REN Li-qun 1, FANG Jing-qi 1, LIU Zhi-yi 1, YAN Gang-lin 2* and WEI Jing-yan 1* 1. College of Pharmaceutical Science, Jilin University, Changchun , P. R. China; 2. Key Laboratory of Molecular Enzymology and Engineering, Ministry of Education, College of Life Science, Jilin University, Changchun , P. R. China Abstract As one of the most important antioxidant enzymes, glutathione peroxidase(gpx) protects cells and tissues from oxidative damage, and plays an important role in cardiovascular and cerebrovascular injuries induced by oxidative stress. The antioxidant effect of selenium-containing glutathione S-transferase(Se-GST), a mimic of GPX was investigated on rat cardiomyocytes. To explore the protection function of Se-GST in hydrogen peroxide(h 2 O 2 ) challenged rat cardiomyocytes, we examined malondialdehyde(mda), lactate dehydrogenase(ldh), superoxide dismutase(sod) and cell apoptosis. The results demonstrate exposure of rat cardiomyocytes to H 2 O 2 for 6 and 12 h induced the significant increases of MDA, LDH and apoptosis rate of cardiomyocytes, but pretreatment of rat cardiomyocytes with Se-GST at or unit/ml prevents oxidative stress induced by H 2 O 2 with the decreases of cell apoptosis. All the results hint Se-GST has antioxidant activity for oxidative stress challenged rat cardiomyocytes. Keywords Selenium-containing glutathione S-transferase(Se-GST); Cell apoptosis; Cardiac myocyte; Oxidative stress Article ID (2012) Introduction Oxidative stress is considered to be essential for the pathogenesis of aging process and some degenerative diseases(atherosclerosis, myocardial ischemia/reperfusion injury, heart failure, cardiovascular disease, diabetes, pulmonary fibrosis, neurodegenerative disorders, and arthritis) [1,2]. Oxidative stress can induce the apoptosis of cardiomyocytes, directly leading to cardiovascular damage with the abnormal structure and dysfunction of cardiovascular system. Moreover, it could trigger lipid peroxidation. Peroxidation of fatty acyl groups, which occurs mostly in membrane phospholipids, can markedly alter the physicochemical properties of membrane lipid bilayers, and leads to severe cellular dysfunction. Malondialdehyde(MDA) is the main product of lipid peroxidation. The concentration of MDA determines the infection of H 2 O 2 to cell lipid peroxidation. Affected by the lipid peroxidation, the cell membrane is destroyed, leading to cellular leakings. Lactate dehydrogenase(ldh) is considered as a sensitive indicator of cell membrane integrity. Superoxide dismutase(sod), together with catalase and glutathione peroxidase(gpx), comprises the first defense line of oxidative damage. The level of SOD is the marker of the degree of oxidative injury. As one of the most important antioxidant enzymes, GPX protects cells and tissues against oxidative damage by catalyzing the reduction of hydroperoxides with glutathione(gsh) and blocking the radical reaction caused by lipid peroxides, thereby protecting cardiovascular and cerebrovascular systems *Corresponding authors. jingyanwei@yahoo.com.cn; ygl@jlu.edu.cn Received December 26, 2011; accepted February 8, Supported by the National Natural Science Foundation of China(Nos , ). against oxidative damage [3,4]. Mammalian cytosolic glutathione S-transferases(GSTs) are all dimeric with subunits of amino acids in length. Based on amino acid sequence similarities, seven classes of cytosolic GST are recognized in mammalian species, as designated Alpha, Mu, Pi, Sigma, Theta, Omega, and Zeta [5]. Accumulated evidence has suggested that GST has the similar binding site of GSH and relative location of key amino acid reacting with GPX, so it could be chemically modified to a mimic with GPX activity [6]. A common thioredoxin-fold of GST and GPX possibly suggest that GST could be manipulated into selenium-containing glutathione S-transferase (Se-GST), having GPX activity. Based on the assumption, Se-GST was generated by incorporation selenocysteine(sec) into the activity site with hgst Z1-1 as a backbone [7]. The hypothesis Se-GST with high GPX activity has been proved, with chemical analysis, by predecessors of our laboratory. This research aimed at limiting oxidative damage or attenuating its related regulating pathways for therapeutic interventions and cure of cardiovascular and cerebrovascular diseases. Herein lies the antioxidant effect of the GPX mimic on rat cardiomyocytes. 2 Materials and Methods E. coli M15[pREP4] containing pqez1(human GST Zeta 1-1) was kindly provided by Prof. Board P.(Molecular Genetics Group, John Curtin School of Medical Research, Australian National University, Canberra, Australia). Fetal bovine serum

2 No.3 YIN Li et al. 455 (FBS) and trypsin were obtained from Gibco(Rockville, MD, USA). MDA detection reagent kit and LDH colorimetry kit were purchased from Nanjing Jiancheng Research Institution of Biotechnology(Nanjing, China). Culture medium, Dulbecco s modified Eagle e medium(dmem, low glucose) and all other chemicals were purchased from Sigma(St. Louis, MO, USA). 2.1 Generation of Se-GST The human glutathione S-transferase Z1-1 was prepared by gene engineering method as mentioned previously [8], and then modified into Se-GST, a GPX mimetic with high catalytic efficiency, in which the serine residues adjacent to the GSH-binding site can be mutated to Sec as described previously [9]. And the results show that the GPX activity of Se-GSTZ is (8602±32) unit/mol according to ref.[9]. 2.2 Isolation and Culture of Rat Cardiac Myocytes Primary neonatal rat heart cell cultures were prepared from previously described ventricular myocardium with some modification [10]. Briefly, the rats were killed with ethyl ether. The ventricular myocardia were surgically removed, minced into 1 3 mm 3 pieces, and then washed twice with phosphate-buffered saline(pbs). The minced tissues were dissociated by 4 6 repeated exposures to g/ml trypsin solution at 37 C with gentle agitation. Supernatants were collected and centrifuged at 1500 r/min for 10 min. The cell pellets were resuspended in DMEM with 20% FBS, and plated in a culture dish. The cells were incubated for 2 h at 37 ºC in a humidified atmosphere containing 5% CO 2 to promote early adherence of fibroblasts. Subsequently, the floating cardiac myocytes were collected, counted, plated in the 96-well plates or culture dishes and cultured for 3 4 d at 37 ºC in a humidified atmosphere containing 5% CO 2. Further experiments were performed following the culture. 2.3 Measurement of Cardiac Myocytes Viability Rat cardiac myocytes( cells/ml) were seeded in the 96-well plates and cultured for 72 h. The cardiac myocytes were treated with various concentrations of Se-GST(0, , 0.001, 0.005, 0.01, 0.05, 0.1 unit/ml) for a period of time(6.5 and 12.5 h), 3-(4,5)-dimethylthiahiazo(-z-yl)-3,5-di-phenytetrazoliumromide(MTT) test was performed to measure the toxicity of Se-GST. And then cardiac myocytes were incubated with Se-GST(0, , 0.001, 0.005, 0.01, 0.05 unit/ml) for 0.5 h before they were treated with additional 200 µmol/l H 2 O 2 for a period of time(6 and 12 h). MTT test was performed to measure cardiac myocytes viability on an enzyme-linked immunesorbent assay plate reader at 490 nm. 2.4 Assay of Apoptosis Rat cardiac myocytes were treated as described in Section 2.3. The percentage of apoptosis was measured by Annexin V- FITC/PI(FITC=fluorescein isothiocyanate; PI=propidine iodide) staining flow cytometry, and determined by Annexin V-FITC/PI kit. 2.5 Determination of MDA, LDH Released and SOD The cardiac myocytes were preincubated with Se-GST ( and unit/ml) for 0.5 h, and then incubated with additional 200 µmol/l H 2 O 2 for a period of time(6 and 12 h). Lipid peroxidation product MDA was determined via MDA detection reagent kit as described in manufacturer s instructions. The level of MDA was estimated by the absorbance value at 532 nm. The LDH content of medium was assayed via LDH colorimetry kit as described in manufacturer s instructions. The LDH released was estimated by the absorbance value of medium at 510 nm. The SOD content of medium was assayed via SOD colorimetry kit as described in manufacturer s instructions. The SOD was estimated by the absorbance value of medium at 550 nm. 2.6 Statistical Analysis The results are presented as mean±standard deviation. One-way analysis of variance(anova) was used for evaluation differences between various treatment conditions, and least significant different test was used for multiple comparisons within treatment groups. SPSS 11.0 statistical software was used for all the analyses. Difference was regarded as statistically significant when P< Results 3.1 Effects of Se-GST on Cardiac Myocytes Viability To fix the working concentration and action period of H 2 O 2 for establishing a cell modal under oxidative stress, with an approximate death rate of 50% 60% of cardiac myocytes, the experiment of screening different concentrations of H 2 O 2 and action period on the cell viability was performed firstly. And then, we carried out the test for the toxicity of Se-GST and the response of the cells to ascertain the safety dose of Se-GST. Finally, the protecting effect of Se-GST against H 2 O 2 was measured by the comparison between the cell viability of cardiac myocytes pretreated with different concentration of Se-GST for 0.5 h prior to exposing to H 2 O 2 and that of the cells only treated with H 2 O 2. Fig.1 shows that exposure to 200 μmol/l H 2 O 2 for a period time of 6 h or 12 h could make approximately 50% or 60% cardiac myocytes death, respectively. Therefore, all subsequent experiments were performed with 200 μmol/l H 2 O 2. Fig.2 indicates that Se-GST( unit/ml) alone would not trigger cellular damage, and promote cell proliferation in the form of a parabola line. Fig.3 demonstrates that pretreatment with Se-GST could increase the viability of H 2 O 2 -induced oxidative injuried cardiac myocytes. To be concrete, preincubation of cardiac myocytes with U/mL Se-GST before exposure to H 2 O 2 for 6 h and that with U/mL Se-GST for 12 h exhibited a maximal cell viability, compared with those of cardiac myocytes incubated by Se-GST

3 456 CHEM. RES. CHINESE UNIVERSITIES Vol.28 of other concentrations for the same time. Fig.1 Cell viability of cardiac myocytes incubated in H 2 O 2 with indicated concentrations for 4(a), 6(b) and 12 h(c), respectively Fig.3 Cell viability of cardiac myocytes preincubated in Se-GST with different concentration and treated with 200 µmol/l H 2 O 2 for 6 and 12 h, respectively a. Control; b. H 2 O 2 (200 µmol/l); c. Se-GST( U/mL); d. Se-GST ( U/mL); e. Se-GST(0.001 U/mL); f. Se-GST(0.005 U/mL); g. Se-GST(0.01 U/mL); h. Se-GST(0.05 U/mL); ** P<0.01, compared with normal control group(6 and 12 h); ## P<0.01, compared with 200 µmol/l H 2 O 2 group(6 and 12 h). 3.2 Effects of Se-GST on LDH Released, MDA and SOD Fig.2 Cell viability of cardiac myocytes incubated in Se-GST with indicated concentrations for 6.5(a) and 12.5 h(b), respectively Fig.4(A) shows that the concentration of LDH in the medium of oxidative damage group is much higher than that in the medium of normal state(p<0.01), the permeability of LDH through cell membrane is changed and the integrity of the cell membrane is violated after cardiac myocytes were exposed to H 2 O 2 for 6 and 12 h, whereas Se-GST can inhibit the release of LDH, maintain the integrity of cell membrane, and protect Fig.4 Effects of Se-GST on the concentrations of LDH(A), MDA(B) and SOD(C) in rat cardiac myocytes exposed to H 2 O 2 The concentrations of LDH, MDA, SOD were estimated by the absorbance value obtained on a spectrophotometer. ** P<0.01, compared with normal control group(6 and 12 h); ## P<0.01, compared with 200 µmol/l H 2 O 2 group(6 and 12 h). cardiac myocytes against H 2 O 2 -mediated lipid peroxidation. As shown in Fig.4(B), 200 µmol/l H 2 O 2 significantly increased the content of MDA in cardiac myocytes(p<0.01), and MDA production was partly inhibited in the cells surrounded by the medium containing Se-GST, compared with that in the cells only treated with H 2 O 2 (P<0.01). These data indicate that Se-GST has a strong ability to inhibit lipid peroxidation induced by H 2 O 2 in cardiomyocytes. According to the data in Fig.4(C), the levels of SOD were declined in the cells treated with 200 µmol/l H 2 O 2 for 6 and 12 h compared with that in the cells of the control group (P<0.05, P<0.01). However, the expression of intracellular SOD is gradually recovered after the pretreatment of the cells by Se-GST with different concentrations. This demonstrates that Se-GST exerts protect effect against the oxidative injury. 3.3 Effects of Se-GST on Apoptosis When H 2 O 2 was introduced into cardiac myocytes, the imbalance of regional redox occurred, eliciting the cell response to oxidative injury, such as apoptosis. Fig.5 indicates that the exposure of the cardiomyocytes to 200 µmol/l H 2 O 2 for a period time of 6 or 12 h would alter the rate of apoptosis, increasing from 2% to 61.1%, and 0.7% to 71.3%, respectively. Furthermore, different concentrations of Se-GST could reduce the rate of the cell apoptosis.

4 No.3 YIN Li et al. 457 Fig.5 Effects of Se-GST on the rate of apoptosis of rat cardiac myocytes exposed to H 2 O 2 (A) Control(6 h), 0.2%(rate of apoptosis); (B) H 2 O 2 (200 µmol/l, 6 h), 61.1%; (C) Se-GST( U/mL)+H 2 O 2 (200 µmol/l, 6 h), 31.4%; (D) Se-GST(0.001 U/mL)+H 2 O 2 (200 µmol/l, 6 h), 34.2%; (E) control(12 h), 0.7%; (F) H 2 O 2 (200 µmol/l, 12 h), 71.3%; (G) Se-GST( U/mL)+H 2 O 2 (200 µmol/l, 12 h), 52.5%; (H) Se-GST(0.001 U/mL)+H 2 O 2 (200 µmol/l, 12 h), 40.1%. Q 1, Q 3 : Normal cells. Q 2 : late apoptotic and necrotic cells. Q 4 : early apoptotic cells. 4 Discussion Lipid peroxidation is one of the major outcomes of free radical-mediated injury. A variety of lipid byproducts are produced as a result of lipid peroxidation, such as MDA and 4-hydroxynonenal, some of which can exert adverse and/or beneficial biological effects. Among the end-products of lipid peroxidation, GSTs make GSH conjugate with the 2-alkenals acrolein and crotonaldehyde [11,12], as well as 4-hydroxy-2- alkenals of between 6 and 15 carbon atoms in length [13], and the conjugation of GSH with the (S)-enantiomer of 4-hydroxynonenal is favored over that of GSH with the (R)- enantiomer [14]. Further, GSTs catalyze the conjugation of cholesterol-5,6-oxide, epoxyeicosatrienoic acid, and 9,10-epoxystearic acid with GSH [11]. These findings indicate that GST can provide the cell with protection against a range of harmful electrophiles produced during oxidative damage to membranes [12]. In this study, H 2 O 2 increased the LDH release and MDA production significantly, indicating that H 2 O 2 could induce oxidative stress and increase the lipid peroxidation in cardiomyocytes. Futhermore, it shows lower expression of LDH and production of MDA after pretreatment of Se-GST in comparison with only treated with H 2 O 2, demonstrating Se-GST can inhibit lipid peroxidation induced by H 2 O 2, and maintain cell membrane integrity. Those data indicate that Se-GST shows high GST activity in the cardiomyocytes attacked by oxidative stress. GST can be subdivided into two domains, an N-terminal thioredoxin-like domain and a C-terminal all α-helical domain [15]. There has been an opinion confirmed by many experiments which shows thioredoxin peroxidase is a novel inhibitor of apoptosis and may act directly on peroxidase to inhibit the intracellular accumulation of key peroxides in apoptosis [16]. Se-GST employs GST as a backbone, and the structure of the new protein suggests that Se-GST has an N-terminal thioredoxin-like domain and Sec in the binding site of GSH, similar to GPX. As a result, we can draw an assumption that Se-GST should have an anti-apoptosis effect in theory. All the results related to apoptosis in this paper have borne out the correctness of this appraisal. Therefore, this confirmed our proposal that Se-GST has GPX s activity from another angle. Detection of SOD in cardiomyocytes reveals that the concentration of SOD is recovered gradually to almost the normal level after the pretreatment of cardiomyocytes with Se-GST, compared with that of SOD in the cells only treated with H 2 O 2. It is obvious from the data that Se-GST could catalyze the reduction of H 2 O 2 and other kind of intracellular hydroperoxides by GSH, much the same as GPX, and decrease the content of H 2 O 2 and various intracellular hydroperoxides, resulting in the alleviation of cell s damage subjected by H 2 O 2. Collectively, Se-GST, generated by chemical modification, showed an antioxidant effect by uniting the activity of GST with that of GPX. In general, the delicate regional redox balance, in particular the appropriate tone of hydroperoxides, is known to be involved in cellular signaling, evoking several forms of cellular responses, for example, programmed cell death, proliferation, cytokine production, and so on [17]. However, cells would subject to oxidative injury when the imbalance occurs. Oxidative injury elicits a wide spectrum of responses ranging from proliferation to growth arrest, to senescence, to cell death at the cellular level. The particular outcome observed can vary significantly from one cell type to the next, as well as with respect to the agent examined, its dosage and/or duration of treatment. As a member of reactive oxygen species(ros), H 2 O 2 could be delivered into cell via the cell membrane. The redox balance in cardiomyocytes is disturbed when cells exist in the medium containing H 2 O 2 for a period of time, giving rise to oxidative damage, plunge in cell viability, and apoptosis of cardiomyocytes. According to the data in this paper, the cell viability dropped dramatically and the level of oxidative injury aggravated significantly with the increase of the concentration of H 2 O 2 in the medium and the time of cardiomyocytes surrounded by medium containing H 2 O 2. In more specific terms, released LDH in cells treated only with H 2 O 2 for

5 458 CHEM. RES. CHINESE UNIVERSITIES Vol h was higher than that in the cells treated for 6 h. Furthermore, by Annexin V-FITC/PI staining flow cytometry, the rate of apoptotic cells increased along with the increase in the degree of oxidative damage. It demonstrates that cardio myocytes elicit the response of cell apoptosis after damaged by H 2 O 2. These data confirm the argumentation that ROS is a major contributor to myocardial apoptosis [18 20]. When the cells were pretreated with Se-GST, the apoptosis rate showed a decrease trend in our research, which means Se-GST can protect cardio myocytes against apoptosis induced by oxidative stress. In view of the fact that oxidative stress is implicated in a wide variety of diseases, including atherosclerosis, myocardial ischemia/ reperfusion injury, and heart failure, our results show Se-GST, a GPX mimic, has an antioxidant effect in vitro, could provide experimental evidence for the clinical application of GPX mimics. 5 Conclusions In summary, Se-GST combines the activity of GST with that of GPX and plays a key role in protecting cardiac myocytes against oxidative stress. We found the antioxidant effect of GPX mimic, Se-GST in vitro, which provide experimental evidence for the clinical application of GPX mimics. For thoroughly testify the above conclusion in vitro findings, additional in vivo researches referring to Se-GST treatment in animal models and patients with cardiovascular diseases are needed in the future. Reference [1] Gutteridge J. M., Free Radic. Res. Commun., 1993, 19(3), 141 [2] Kehrer J. P., Crit. Rev. Toxicol., 1993, 23(1), 21 [3] Storz P., Front Biosci., 2005, 10, 1881 [4] Lefer D. J., Granger D. N., Am. J. Med., 2000, 109(4), 315 [5] Wu J., Fang Y. Z., Yang S., Lupton J. R., Turner N. D., J. Nutr., 2004, 134(3), 489 [6] Ursini F., Maiorini M., Valente M., Ferri L., Gregolin C., Biochem. Biophys. Acta, 1982, 710(2), 197 [7] Sheehan D., Meade G., Foley V. M., Dowd C. A., Biochem. J., 2001, 360(1), 1 [8] Yu H. J., Liu J. Q., Bock A., Li J., Luo G. M., Shen J. C., J. Biol. Chem., 2005, 280, [9] Zheng K.Y., Board P. G., Fei X. F., Sun Y., Lü S. W., Yan G. L., Liu J. Q., Shen J. C., Luo G. M., Int. J. Biochem. Cell. Biol., 2008, 40(10), 2090 [10] Huo R., Shi Y., Xu J. J., Yan F., Lü S. W., Su J. M., Duan Y. J., Fan J., Ning B., Cong D. L., Yan G. L., Luo G. M., Wei J. Y., Chem. Res. Chinese Universities, 2009, 25(2), 216 [11] Blackburn A. C., Tzeng H. F., Anders M. W., Board P. G., Pharmacogenetics, 2000, 10(1), 49 [12] Hayes J. D., Pulford D. J., Crit. Rev. Biochem. Mol. Biol., 1995, 30(6), 445 [13] Hayes J. D., McLellan L. I., Free Radic. Res., 1999, 31(4), 273 [14] Hubatsch I., Ridderström M., Mannervik B., Biochem. J., 1998, 330(1), 175 [15] Hiratsuka A., Hirose K., Saito H., Watabe T., Biochem. J., 2000, 349(3), 729 [16] Polekhina G., Board P. G., Blackburn A. C., Parker M. W., Biochemistry, 2001, 40(6), 1567 [17] Zhang P., Liu B., Kang S. W., Seo M. S., Rhee S. G., Obeid L. M., J. Biol. Chem., 1997, 272(49), [18] Nakashima I., Takeda K., Kawamoto Y., Okuno Y., Kato M., Suzuki H., Arch. Biochem. Biophys., 2005, 434(1), 3 [19] Galanq N., Sasaki H., Maulik N., Toxicology, 2000, 148(2/3), 111 [20] Clanachan A. S., Jaswal J. S., Gandhi M., Bottorff D. A., Transplantation, 2003, 75(2), 173