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Role of reactive oxygen intermediates in Japanese encephalitis virus infection in murine neuroblastoma cells
Neuroscience Letters 315 (2001) 9–12 www.elsevier.com/locate/neulet
Role of reactive oxygen intermediates in Japanese encephalitis virus infection in murine neuroblastoma cells Shue-Ling Raung a, Ming-Der Kuo b, Yu-Ming Wang b, Chun-Jung Chen a,* a
Department of Education and Research, Taichung Veterans General Hospital, Taichung 407, Taiwan, ROC b Institute of Preventive Medicine, National Defense Medical Center, Taipei 237, Taiwan, ROC Received 18 August 2001; received in revised form 11 September 2001; accepted 13 September 2001
Abstract Infection with Japanese encephalitis virus (JEV), a mosquito-borne, neurotropic flavivirus, may cause acute encephalitis in humans and induce severe cytopathic effects in various types of cultured cells. This study attempted to determine whether JEV infection induces free radical generation and whether oxidative stress contributes to virus-induced cell death in neuroblastoma cells. A rise in the intracellular level of free radicals indicated by the 2 0 ,7 0 -dichlorofluorescein fluorescence was observed in N18 cells following JEV infection. Cellular flavon-containing enzymes were involved in JEV-induced fluorescent change. Cells were moderately protected from JEV-induced death by diphenyleneiodonium, a flavon-containing enzyme inhibitor, whereas common antioxidants such as N-acetylcysteine, pyrrolidine dithiocarbamate, Tiron, and Trolox turned out to be ineffective. These results suggest that the direct antioxidant action is not helpful in prevention of JEV-induced neuronal cell death. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Antioxidant; Apoptosis; Japanese encephalitis virus; Neuroblastoma; Neurotropism; Oxidative stress
Oxidative stress is defined as any imbalance between oxidants and antioxidants in favor of the oxidants. Oxidants such as reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) are key elements in anti-microbial and anti-tumoral defense, but also contribute to aging and the pathogenesis of a wide array of infectious and non-infectious diseases [6,7]. Oxidative stress has been suggested to be a mediator of cell death induced by a variety of triggers, including viral infection [13,14]. Evidence regarding the role of ROS in the replication and pathogenesis of human immunodeficiency virus (HIV) has led to an increased interest in the role of oxidative stress in other virus infections [13,14]. Many viral infections including those of HIV, Sendai virus, influenza virus, paramyxovirus, hepatitis B virus, bovine viral diarrhea virus, and cytomegalovirus generate ROS and induce oxidative stress [13–16]. The resultant elevated ROS may contribute to viral pathogenesis, regulation of viral replication, host defense, and modulation of cellular responses [13–16]. Japanese encephalitis virus (JEV), a neurotropic virus, is * Corresponding author. Department of Education and Research, Taichung Veterans General Hospital, No. 160, Section 3, Taichung-Gang Road, Taichung 40705, Taiwan, ROC. Tel.: 1886-4-2359-2525 ext. 4022; fax: 1886-4-2359-2705. E-mail address: [email protected] (C.-J. Chen).
one of the major causes of acute encephalitis in humans [2]. In humans, the primary sites for JEV multiplication are most likely in myeloid and lymphoid cells or in vascular endothelial cells [12]. Several lines of evidence suggest that the principal target cells for JEV in the central nervous system (CNS) are neurons [8]. Although the mechanism of neurotropism is yet to be investigated, massive neuronal dysfunction and/or destruction is proposed to be responsible for the manifestations of encephalitis. A wide variety of continuous cell lines can support the productive growth of JEV. Apoptosis and necrosis are proposed to be mechanisms by which JEV killed its infected cell [9]. In general, necrosis and apoptosis are two distinct forms of cell death, the former being a passive process typified by early leakage of the plasma membrane and spillage of the intracellular content, and the latter being a highly regulated process that avoids inflammation and damage to the surrounding tissue. Recently, stimulation of neutrophil respiratory burst and generation of toxic oxygen species following JEV infection have been documented [17]. However, the accumulation of ROS and the role of oxidative stress in the pathogenesis of JEV infection are mostly unclear. Therefore, the purpose of this study was to investigate the effects of the generation and resultant oxidative
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 30 0- X
10
S.-L. Raung et al. / Neuroscience Letters 315 (2001) 9–12
burden following JEV infection on cultured murine neuroblastoma cells. NT113, a Taiwanese JEV strain isolated from mosquito [4], was used throughout this study. The virus was propagated in C6/36 cells. N18 (a murine neuroblastoma) cells were used as source of virus infection. Levels of intracellular free radical were analyzed by the fluorescent signal after the oxidation of a non-fluorescent 2 0 ,7 0 -dichlorofluorescein (Molecular Probe) by ROS, as described previously [20]. The fluorescent signal of oxidized 2 0 ,7 0 -dichlorofluorescein was measured by fluorometer (Fluoroskan Ascent, Labsystems, excitation 488 nm and emission 510 nm). Apoptosis-induced DNA strand breaks were end labeled with dUTP by use of terminal deoxynucleotidyltransferase (TdT) with a commercial kit (In Situ Cell Death Detection Kit, BM) according to the manufacturer’s instructions. Cytotoxicities, as indicated by cell membrane integrities, were assessed by measuring the activity of lactate dehydrogenase (LDH) in the cultured media by the colorimetric detection of formazan, using an LDH diagnostic kit (Promega, WI, USA). To determine virus titers, culture media were harvested for regular plaque-forming assay in BHK21 cells. An increase in fluorescence signal was visualized timedependently in N18 cells after JEV infection (MOI 5) indicating the generation of free radicals. In contrast, mock infection or UV- or boiled-inactivated JEV were unable to cause the fluorescence change (Fig. 1A). The increase in fluorescence signal following JEV infection was prevented by the presence of general antioxidants such as N-acetylcysteine (NAC, 1 mM), pyrrolidine dithiocarbamate (PDTC, 0.025 mM), sodium 1,2-dihydroxybenzene-3,5-disulfonate (Tiron, 0.5 mM), or Trolox (0.4 mM) (Fig. 1B). In addition, the addition of diphenyleneiodonium (DPI, 1 mM), an inhibitor of flavon-containing enzymes such as NADH/nicotinamide adenine dinucleotide phosphate oxidase, attenuated JEV-induced fluorescence change (Fig. 1B). Apoptotic and necrotic cell death are proposed to be mechanisms by which JEV kills its infected cell [9]. We next determined the effects of antioxidants and pharmacological agents on JEV-induced cell death. N18 cells showed increased efflux of LDH into the bathing medium upon JEV infection (Fig. 2A). The additions of above antioxidants all failed to prevent JEV-induced LDH efflux (Fig. 2B). However, JEV-induced LDH efflux was decreased following DPI treatment (Fig. 2B). After 32 h, JEV induced apoptotic cell death (37~45%) in N18 cells as indicated by TUNEL staining (Fig. 3B). Both UV- and boiled-inactivated viruses lost their effect to induce cell apoptosis (Fig. 3C,D). The presence of NAC (Fig. 3E, 35~45%), PDTC (Fig. 3F, 38~46%), Tiron (Fig. 3G, 34~42%), and Trolox (Fig. 3H, 32~49%) failed to abolish the action of JEV to induce cell apoptosis. JEV-induced apoptosis was decreased following DPI (Fig. 3I, 21~29%) treatment. Furthermore, antioxidants did not affect, whereas DPI decreased infectious viral particle release (Table 1).
Fig. 1. Intracellular redox potential change after JEV infection. (A) N18 cells were infected with mock, UV-inactivated, Boiledinactivated, or wild type JEV at 5 MOI. The resulting fluorescence, indicative of ROS generation, was assessed after the addition of 2 0 ,7 0 -dichlorofluorescein (5 mM) for 30 min over times. (B) After 1-h virus adsorption (5 MOI), cells were cultivated further 24 h in the presence of N-acetylcysteine (NAC, 1 mM), pyrrolidine dithiocarbamate (PDTC, 0.025 mM), Tiron (0.5 mM), Trolox (0.4 mM), or DPI (1 mM). Intracellular redox change was indicated by the fluorescent signals of 2 0 ,7 0 -dichlorofluorescein (5 mM) oxidation. The fluorescent intensity of the control was defined as 100%. Values are means ^ SEM (n ¼ 3), **P , 0:01 compared with the control as determined by Student’s t-test following oneway ANOVA.
In this study, infection by JEV triggering generation of free radicals, and apoptotic/necrotic cell death in neuroblastoma cells was demonstrated. Viral amplification was essential to the production of free radicals following JEV infection. The concomitant elevation in free radicals was not the major causative factor in apoptotic or necrotic cell death after JEV infection. Moreover, the flavon-containing enzymes played roles in JEV-induced free radical generation, viral amplification, and cytopathic effect. A wide variety of primary and continuous cell cultures from different origins can support the productive growth of JEV. Nevertheless, why JEV infection is selectively cytolytic to certain cells but non-lytic to others, and the exact mechanism by which JEV induces the death of infected cells, remain largely unknown. Here, we revealed that DPI-sensitive flavon-containing enzymes attributed partially to the JEV-induced cell death (Figs. 2 and 3). The partial suppression of cell death indicated that the signaling pathways of JEV-mediated cell death were not completely blocked by DPI. Other possibilities for JEVinduced cell death include non-specific cell damage, such
S.-L. Raung et al. / Neuroscience Letters 315 (2001) 9–12
Fig. 2. Effects of agents on JEV-induced LDH efflux. (A) N18 cells were infected with mock, UV-inactivated, Boiled-inactivated, or wild type JEV at 5 MOI. Cell damage was assessed by measurement of LDH efflux 40 h after infection. (B) After 1-h virus adsorption (5 MOI), cells were cultivated further 40 h in the presence of N-acetylcysteine (NAC, 1 mM), pyrrolidine dithiocarbamate (PDTC, 0.025 mM), Tiron (0.5 mM), Trolox (0.4 mM), or DPI (1 mM). Cell damage was assessed by measurement of LDH efflux. Values are means ^ SEM (n ¼ 3). **P , 0:01 compared with the control as determined by Student’s t-test following one-way ANOVA.
as changes in membrane permeability, relatively non-specific biochemical mechanisms due to a general down-regulation of host protein synthesis, specific interference with regulator of cell death, and autocrine effects of either ROS or cytokines. Each of these possibilities warrants investigation. Recently, Chang et al. [3] revealed that the cytocidal effect of JEV might be derived partly from increased membrane permeability through the action of viral small hydrophobic non-structural proteins. Oxidative stress has been suggested to be a mediator of apoptosis induced by a variety of triggers, including virus infections [13,14]. This was frequently shown either by the addition of ROS or by the anti-apoptotic effect of certain antioxidants. Some of antioxidants are known to inhibit cell death in viral systems [5,11,19]. Whereas, in our study, none of the antioxidants tested (Figs. 2 and 3) protected N18 cells from JEV-induced apoptotic and necrotic cell death. Although antioxidants that failed to protect against JEVinduced cell death (Figs. 2 and 3), they were able to change the intracellular redox status as indicated by 2 0 ,7 0 -dichlorofluorescein fluorescence (Fig. 1). These results imply that antioxidant therapy is ineffective in JEV infection and that oxidative stress is not the causative agent of JEV-induced cell death.
11
Fig. 3. Effects of agents on JEV-induced apoptosis. After 1-h virus adsorption (5 MOI), cells were cultivated further 32 h in the presence of N-acetylcysteine (NAC, 1 mM, E), pyrrolidine dithiocarbamate (PDTC, 0.025 mM, F), Tiron (0.5 mM, G), Trolox (0.4 mM, H), or DPI (1 mM, I). Apoptotic cell death was examined by TdT-mediated dUTP nick end labeling (TUNEL) and observed under light microscope. Apoptosis was also examined in UVinactivated (C), boiled-inactivated (D), wild type JEV-infected (B) and mock-infected (A) cells. Scale bar ¼ 50 mm.
The detailed mechanisms responsible for the increased level of ROS in JEV-infected cells are largely unknown. Oxidative stress is defined as any imbalance between oxidants and antioxidants in favor of the oxidants. The changes in 2 0 ,7 0 -dichlorofluorescein fluorescence, which is indicative of oxidative stress, may thus be due to increased production of ROS, to a decreased capacity of antioxidants defense mechanisms, or to both. The flavon-containing enzymes seem to be a source of ROS generation in neuronal cells infected by JEV as shown by the inhibition of DPI (Fig. 1). This notion is further supported by the finding that the NADPH oxidase was involved in JEV-induced free radical production in neutrophils [17].
Table 1 Effects of agents on JEV replication a Treatment
Virus titer (PFU/ml)
Medium alone NAC (1 mM) PDTC (25 mM) Tiron (0.5 mM) Trolox (0.4 mM) DPI (1 mM)
1.2 ^ 1.61 £ 10 7 2.4 ^ 1.18 £ 10 7 9.5 ^ 2.72 £ 10 6 8.3 ^ 1.92 £ 10 6 2.9 ^ 1.72 £ 10 7 2.4 ^ 2.14 £ 10 5*
a
N 18 cells were infected with JEV for 24 h in the presence of NAC, PDTC, Tiron, Trolox, or DPI. The JEV titers in the culture supernatants were measured by the plaque assay. Data are expressed as mean ^ SEM, *P , 0:05 (n ¼ 4).
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There is now much evidence to show that oxidants play complex roles in viral diseases, such as influencing host cell metabolism and viral replication, inactivating viruses and causing toxic effects in host tissues [13–16]. Beck et al. [1] demonstrated that the redox state of the host cell may affect the genetic composition of coxsackievirus B3. Hydrogen peroxide promotes replication of HIV, but antioxidants such as N-acetylcysteine have the opposite effect [18]. Recently, the generation of ROS was demonstrated to be critical in the rapid degradation of phagocytosed JE viral protein and nucleic acid [17]. In addition, NO appears to be effective in restricting JE amplification [10]. Although we failed to demonstrate a protective action of antioxidants on JEV-induced neuronal death, these findings suggest that ROS might still play roles in JEV infection. It should be noted that we do not monitor directly individual ROS, such as superoxide anion, hydroxyl radical, or peroxide, so we could not definitively exclude a role for these ROS in regulating JEV-induced neuronal cell death. In conclusion, the common antioxidants such as N-acetylcysteine, pyrrolidine dithiocarbamate, Tiron, and Trolox were ineffective to block JEV-induced neuronal death indicating that oxidative stress is not a mediator of JEV-induced cell death. The signaling pathways of JEV-induced cell death might be partially derived from the involvement of DPI-sensitive flavon-containing enzymes.
[6] [7]
[8]
[9]
[10]
[11]
[12]
[13] [14] [15]
This study was supported by grants TCVGH-897308C and -907307C from the Taichung Veterans General Hospital, Taiwan, ROC. [1] Beck, M.A., Kolbeck, P.C., Rohr, L.H., Shi, Q. and Morris, V.C., Benign human enterovirus becomes virulent in selenium-deficient mice, J. Med. Virol., 43 (1994) 166–170. [2] Chambers, T.J., Hahn, C.S., Galler, R. and Rice, C.M., Flavivirus genome organization, expression, and replication, Annu. Rev. Microbiol., 44 (1990) 649–688. [3] Chang, Y.S., Liao, C.L., Tsao, C.H., Chen, M.C., Liu, C.I., Chen, L.K. and Lin, Y.L., Membrane permeabilization by small hydrophobic nonstructural proteins of Japanese encephalitis virus, J. Virol., 73 (1999) 6257–6264. [4] Chen, C.J., Liao, S.L., Kuo, M.D. and Wang, Y.M., Astrocytic alteration induced by Japanese encephalitis virus infection, NeuroReport, 11 (2000) 1933–1937. [5] Cossarizza, A., Franceschi, C., Monti, D., Salvioli, S., Bellesia, E., Rivabene, R., Biondo, L., Rainaldi, G., Tinari, A. and Malorni, W., Protective effect of N-acetylcysteine in tumor
[16]
[17]
[18]
[19]
[20]
necrosis factor-alpha –induced apoptosis in U937 cells: the role of mitrochondria, Exp. Cell Res., 220 (1995) 232–240. Freeman, B.A. and Crapo, J.D., Biology of disease: free radicals and tissue injury, Lab. Invest., 47 (1982) 412–426. Halliwell, B., Gutteridge, J.M.C. and Cross, C.E., Free radicals. Antioxidants, and human disease – where are we now? J. Lab. Clin. Med., 119 (1992) 598–620. Johnson, R.T., Burke, D.S., Elwell, M., Leake, C.J., Nisalak, A., Hoke, C.H. and Lorsomrudee, W., Japanese encephalitis: immunocytochemical studies of viral antigen and inflammatory cells in fatal cases, Ann. Neurol., 18 (1985) 567–573. Liao, C.L., Lin, Y.L., Wang, J.J., Huang, Y.L., Yeh, C.T., Ma, S.H. and Chen, L.K., Effect of enforced expression of human bcl-2 on Japanese encephalitis virus-induced apoptosis in cultured cells, J. Virol., 71 (1997) 5963–5971. Lin, Y.L., Huang, Y.L., Ma, S.H., Yeh, C.T., Chiou, S.Y., Chen, L.K. and Liao, C.L., Inhibition of Japanese encephalitis virus infection by nitric oxide: antiviral effect of nitric oxide on RNA virus replication, J. Virol., 71 (1997) 5227–5235. Lowy, R.J. and Dimitrov, D.S., Characterization of influenza virus-induced death of J774.1 macrophages, Exp. Cell Res., 234 (1997) 249–258. Mathur, A., Bharadwaj, M., Kulshreshtha, R., Rawat, S., Jain, A. and Chaturvedi, U.C., Immunopathological study of spleen during Japanese encephalitis virus infection in mice, Br. J. Exp. Pathol., 69 (1988) 423–432. Peterhans, E., Reactive oxygen species and nitric oxide in viral diseases, Biol. Trace Elem. Res., 56 (1997) 107–116. Schwarz, K.B., Oxidative stress during viral infection: a review, Free Radical Bio. Med., 21 (1996) 641–649. Schweizer, M. and Peterhans, E., Oxidative stress in cells infected with bovine viral diarrhoea virus: a crucial step in the induction of apoptosis, J. Gen. Virol., 80 (1999) 1147– 1155. Speir, E., Yu, Z.X., Ferrans, V.J., Huang, E.S. and Epstein, S.E., Aspirin attenuates cytomegalovirus infectivity and gene expression mediated by cyclooxygenase-2 in coronary artery smooth muscle cells, Circ. Res., 83 (1998) 210– 216. Srivastava, S., Khanna, N., Saxena, S.K., Singh, A., Mathur, A. and Dhole, T.N., Degradation of Japanese encephalitis virus by neutrophils, Int. J. Exp. Pathol., 80 (1999) 17–24. Staal, J.T., Roederer, M., Herzenberg, L.A. and Herzenberg, L.A., Intracellular thiols regulate activation of nuclear factor B and transcription of human immunodeficiency virus, Proc. Natl. Acad. Sci. USA, 87 (1990) 9943–9947. Verhaegen, S., McGowan, A.J., Brophy, A.R., Fernandes, R.S. and Cotter, T.G., Inhibition of apoptosis by antioxidants in the human HL-60 leukemia cell line, Biochem. Pharmacol., 50 (1995) 1021–1029. Zhu, H., Bannenberg, G.L., Moldeus, P. and Shertzer, H.G., Oxidation pathways for the intracellular probe 2 0 , 7 0 dichlorofluorescein, Arch. Toxicol., 68 (1994) 582–587.
Role of reactive oxygen intermediates in Japanese encephalitis virus infection in murine neuroblastoma cells Shue-Ling Raung a, Ming-Der Kuo b, Yu-Ming Wang b, Chun-Jung Chen a,* a
Department of Education and Research, Taichung Veterans General Hospital, Taichung 407, Taiwan, ROC b Institute of Preventive Medicine, National Defense Medical Center, Taipei 237, Taiwan, ROC Received 18 August 2001; received in revised form 11 September 2001; accepted 13 September 2001
Abstract Infection with Japanese encephalitis virus (JEV), a mosquito-borne, neurotropic flavivirus, may cause acute encephalitis in humans and induce severe cytopathic effects in various types of cultured cells. This study attempted to determine whether JEV infection induces free radical generation and whether oxidative stress contributes to virus-induced cell death in neuroblastoma cells. A rise in the intracellular level of free radicals indicated by the 2 0 ,7 0 -dichlorofluorescein fluorescence was observed in N18 cells following JEV infection. Cellular flavon-containing enzymes were involved in JEV-induced fluorescent change. Cells were moderately protected from JEV-induced death by diphenyleneiodonium, a flavon-containing enzyme inhibitor, whereas common antioxidants such as N-acetylcysteine, pyrrolidine dithiocarbamate, Tiron, and Trolox turned out to be ineffective. These results suggest that the direct antioxidant action is not helpful in prevention of JEV-induced neuronal cell death. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Antioxidant; Apoptosis; Japanese encephalitis virus; Neuroblastoma; Neurotropism; Oxidative stress
Oxidative stress is defined as any imbalance between oxidants and antioxidants in favor of the oxidants. Oxidants such as reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) are key elements in anti-microbial and anti-tumoral defense, but also contribute to aging and the pathogenesis of a wide array of infectious and non-infectious diseases [6,7]. Oxidative stress has been suggested to be a mediator of cell death induced by a variety of triggers, including viral infection [13,14]. Evidence regarding the role of ROS in the replication and pathogenesis of human immunodeficiency virus (HIV) has led to an increased interest in the role of oxidative stress in other virus infections [13,14]. Many viral infections including those of HIV, Sendai virus, influenza virus, paramyxovirus, hepatitis B virus, bovine viral diarrhea virus, and cytomegalovirus generate ROS and induce oxidative stress [13–16]. The resultant elevated ROS may contribute to viral pathogenesis, regulation of viral replication, host defense, and modulation of cellular responses [13–16]. Japanese encephalitis virus (JEV), a neurotropic virus, is * Corresponding author. Department of Education and Research, Taichung Veterans General Hospital, No. 160, Section 3, Taichung-Gang Road, Taichung 40705, Taiwan, ROC. Tel.: 1886-4-2359-2525 ext. 4022; fax: 1886-4-2359-2705. E-mail address: [email protected] (C.-J. Chen).
one of the major causes of acute encephalitis in humans [2]. In humans, the primary sites for JEV multiplication are most likely in myeloid and lymphoid cells or in vascular endothelial cells [12]. Several lines of evidence suggest that the principal target cells for JEV in the central nervous system (CNS) are neurons [8]. Although the mechanism of neurotropism is yet to be investigated, massive neuronal dysfunction and/or destruction is proposed to be responsible for the manifestations of encephalitis. A wide variety of continuous cell lines can support the productive growth of JEV. Apoptosis and necrosis are proposed to be mechanisms by which JEV killed its infected cell [9]. In general, necrosis and apoptosis are two distinct forms of cell death, the former being a passive process typified by early leakage of the plasma membrane and spillage of the intracellular content, and the latter being a highly regulated process that avoids inflammation and damage to the surrounding tissue. Recently, stimulation of neutrophil respiratory burst and generation of toxic oxygen species following JEV infection have been documented [17]. However, the accumulation of ROS and the role of oxidative stress in the pathogenesis of JEV infection are mostly unclear. Therefore, the purpose of this study was to investigate the effects of the generation and resultant oxidative
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 30 0- X
10
S.-L. Raung et al. / Neuroscience Letters 315 (2001) 9–12
burden following JEV infection on cultured murine neuroblastoma cells. NT113, a Taiwanese JEV strain isolated from mosquito [4], was used throughout this study. The virus was propagated in C6/36 cells. N18 (a murine neuroblastoma) cells were used as source of virus infection. Levels of intracellular free radical were analyzed by the fluorescent signal after the oxidation of a non-fluorescent 2 0 ,7 0 -dichlorofluorescein (Molecular Probe) by ROS, as described previously [20]. The fluorescent signal of oxidized 2 0 ,7 0 -dichlorofluorescein was measured by fluorometer (Fluoroskan Ascent, Labsystems, excitation 488 nm and emission 510 nm). Apoptosis-induced DNA strand breaks were end labeled with dUTP by use of terminal deoxynucleotidyltransferase (TdT) with a commercial kit (In Situ Cell Death Detection Kit, BM) according to the manufacturer’s instructions. Cytotoxicities, as indicated by cell membrane integrities, were assessed by measuring the activity of lactate dehydrogenase (LDH) in the cultured media by the colorimetric detection of formazan, using an LDH diagnostic kit (Promega, WI, USA). To determine virus titers, culture media were harvested for regular plaque-forming assay in BHK21 cells. An increase in fluorescence signal was visualized timedependently in N18 cells after JEV infection (MOI 5) indicating the generation of free radicals. In contrast, mock infection or UV- or boiled-inactivated JEV were unable to cause the fluorescence change (Fig. 1A). The increase in fluorescence signal following JEV infection was prevented by the presence of general antioxidants such as N-acetylcysteine (NAC, 1 mM), pyrrolidine dithiocarbamate (PDTC, 0.025 mM), sodium 1,2-dihydroxybenzene-3,5-disulfonate (Tiron, 0.5 mM), or Trolox (0.4 mM) (Fig. 1B). In addition, the addition of diphenyleneiodonium (DPI, 1 mM), an inhibitor of flavon-containing enzymes such as NADH/nicotinamide adenine dinucleotide phosphate oxidase, attenuated JEV-induced fluorescence change (Fig. 1B). Apoptotic and necrotic cell death are proposed to be mechanisms by which JEV kills its infected cell [9]. We next determined the effects of antioxidants and pharmacological agents on JEV-induced cell death. N18 cells showed increased efflux of LDH into the bathing medium upon JEV infection (Fig. 2A). The additions of above antioxidants all failed to prevent JEV-induced LDH efflux (Fig. 2B). However, JEV-induced LDH efflux was decreased following DPI treatment (Fig. 2B). After 32 h, JEV induced apoptotic cell death (37~45%) in N18 cells as indicated by TUNEL staining (Fig. 3B). Both UV- and boiled-inactivated viruses lost their effect to induce cell apoptosis (Fig. 3C,D). The presence of NAC (Fig. 3E, 35~45%), PDTC (Fig. 3F, 38~46%), Tiron (Fig. 3G, 34~42%), and Trolox (Fig. 3H, 32~49%) failed to abolish the action of JEV to induce cell apoptosis. JEV-induced apoptosis was decreased following DPI (Fig. 3I, 21~29%) treatment. Furthermore, antioxidants did not affect, whereas DPI decreased infectious viral particle release (Table 1).
Fig. 1. Intracellular redox potential change after JEV infection. (A) N18 cells were infected with mock, UV-inactivated, Boiledinactivated, or wild type JEV at 5 MOI. The resulting fluorescence, indicative of ROS generation, was assessed after the addition of 2 0 ,7 0 -dichlorofluorescein (5 mM) for 30 min over times. (B) After 1-h virus adsorption (5 MOI), cells were cultivated further 24 h in the presence of N-acetylcysteine (NAC, 1 mM), pyrrolidine dithiocarbamate (PDTC, 0.025 mM), Tiron (0.5 mM), Trolox (0.4 mM), or DPI (1 mM). Intracellular redox change was indicated by the fluorescent signals of 2 0 ,7 0 -dichlorofluorescein (5 mM) oxidation. The fluorescent intensity of the control was defined as 100%. Values are means ^ SEM (n ¼ 3), **P , 0:01 compared with the control as determined by Student’s t-test following oneway ANOVA.
In this study, infection by JEV triggering generation of free radicals, and apoptotic/necrotic cell death in neuroblastoma cells was demonstrated. Viral amplification was essential to the production of free radicals following JEV infection. The concomitant elevation in free radicals was not the major causative factor in apoptotic or necrotic cell death after JEV infection. Moreover, the flavon-containing enzymes played roles in JEV-induced free radical generation, viral amplification, and cytopathic effect. A wide variety of primary and continuous cell cultures from different origins can support the productive growth of JEV. Nevertheless, why JEV infection is selectively cytolytic to certain cells but non-lytic to others, and the exact mechanism by which JEV induces the death of infected cells, remain largely unknown. Here, we revealed that DPI-sensitive flavon-containing enzymes attributed partially to the JEV-induced cell death (Figs. 2 and 3). The partial suppression of cell death indicated that the signaling pathways of JEV-mediated cell death were not completely blocked by DPI. Other possibilities for JEVinduced cell death include non-specific cell damage, such
S.-L. Raung et al. / Neuroscience Letters 315 (2001) 9–12
Fig. 2. Effects of agents on JEV-induced LDH efflux. (A) N18 cells were infected with mock, UV-inactivated, Boiled-inactivated, or wild type JEV at 5 MOI. Cell damage was assessed by measurement of LDH efflux 40 h after infection. (B) After 1-h virus adsorption (5 MOI), cells were cultivated further 40 h in the presence of N-acetylcysteine (NAC, 1 mM), pyrrolidine dithiocarbamate (PDTC, 0.025 mM), Tiron (0.5 mM), Trolox (0.4 mM), or DPI (1 mM). Cell damage was assessed by measurement of LDH efflux. Values are means ^ SEM (n ¼ 3). **P , 0:01 compared with the control as determined by Student’s t-test following one-way ANOVA.
as changes in membrane permeability, relatively non-specific biochemical mechanisms due to a general down-regulation of host protein synthesis, specific interference with regulator of cell death, and autocrine effects of either ROS or cytokines. Each of these possibilities warrants investigation. Recently, Chang et al. [3] revealed that the cytocidal effect of JEV might be derived partly from increased membrane permeability through the action of viral small hydrophobic non-structural proteins. Oxidative stress has been suggested to be a mediator of apoptosis induced by a variety of triggers, including virus infections [13,14]. This was frequently shown either by the addition of ROS or by the anti-apoptotic effect of certain antioxidants. Some of antioxidants are known to inhibit cell death in viral systems [5,11,19]. Whereas, in our study, none of the antioxidants tested (Figs. 2 and 3) protected N18 cells from JEV-induced apoptotic and necrotic cell death. Although antioxidants that failed to protect against JEVinduced cell death (Figs. 2 and 3), they were able to change the intracellular redox status as indicated by 2 0 ,7 0 -dichlorofluorescein fluorescence (Fig. 1). These results imply that antioxidant therapy is ineffective in JEV infection and that oxidative stress is not the causative agent of JEV-induced cell death.
11
Fig. 3. Effects of agents on JEV-induced apoptosis. After 1-h virus adsorption (5 MOI), cells were cultivated further 32 h in the presence of N-acetylcysteine (NAC, 1 mM, E), pyrrolidine dithiocarbamate (PDTC, 0.025 mM, F), Tiron (0.5 mM, G), Trolox (0.4 mM, H), or DPI (1 mM, I). Apoptotic cell death was examined by TdT-mediated dUTP nick end labeling (TUNEL) and observed under light microscope. Apoptosis was also examined in UVinactivated (C), boiled-inactivated (D), wild type JEV-infected (B) and mock-infected (A) cells. Scale bar ¼ 50 mm.
The detailed mechanisms responsible for the increased level of ROS in JEV-infected cells are largely unknown. Oxidative stress is defined as any imbalance between oxidants and antioxidants in favor of the oxidants. The changes in 2 0 ,7 0 -dichlorofluorescein fluorescence, which is indicative of oxidative stress, may thus be due to increased production of ROS, to a decreased capacity of antioxidants defense mechanisms, or to both. The flavon-containing enzymes seem to be a source of ROS generation in neuronal cells infected by JEV as shown by the inhibition of DPI (Fig. 1). This notion is further supported by the finding that the NADPH oxidase was involved in JEV-induced free radical production in neutrophils [17].
Table 1 Effects of agents on JEV replication a Treatment
Virus titer (PFU/ml)
Medium alone NAC (1 mM) PDTC (25 mM) Tiron (0.5 mM) Trolox (0.4 mM) DPI (1 mM)
1.2 ^ 1.61 £ 10 7 2.4 ^ 1.18 £ 10 7 9.5 ^ 2.72 £ 10 6 8.3 ^ 1.92 £ 10 6 2.9 ^ 1.72 £ 10 7 2.4 ^ 2.14 £ 10 5*
a
N 18 cells were infected with JEV for 24 h in the presence of NAC, PDTC, Tiron, Trolox, or DPI. The JEV titers in the culture supernatants were measured by the plaque assay. Data are expressed as mean ^ SEM, *P , 0:05 (n ¼ 4).
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There is now much evidence to show that oxidants play complex roles in viral diseases, such as influencing host cell metabolism and viral replication, inactivating viruses and causing toxic effects in host tissues [13–16]. Beck et al. [1] demonstrated that the redox state of the host cell may affect the genetic composition of coxsackievirus B3. Hydrogen peroxide promotes replication of HIV, but antioxidants such as N-acetylcysteine have the opposite effect [18]. Recently, the generation of ROS was demonstrated to be critical in the rapid degradation of phagocytosed JE viral protein and nucleic acid [17]. In addition, NO appears to be effective in restricting JE amplification [10]. Although we failed to demonstrate a protective action of antioxidants on JEV-induced neuronal death, these findings suggest that ROS might still play roles in JEV infection. It should be noted that we do not monitor directly individual ROS, such as superoxide anion, hydroxyl radical, or peroxide, so we could not definitively exclude a role for these ROS in regulating JEV-induced neuronal cell death. In conclusion, the common antioxidants such as N-acetylcysteine, pyrrolidine dithiocarbamate, Tiron, and Trolox were ineffective to block JEV-induced neuronal death indicating that oxidative stress is not a mediator of JEV-induced cell death. The signaling pathways of JEV-induced cell death might be partially derived from the involvement of DPI-sensitive flavon-containing enzymes.
[6] [7]
[8]
[9]
[10]
[11]
[12]
[13] [14] [15]
This study was supported by grants TCVGH-897308C and -907307C from the Taichung Veterans General Hospital, Taiwan, ROC. [1] Beck, M.A., Kolbeck, P.C., Rohr, L.H., Shi, Q. and Morris, V.C., Benign human enterovirus becomes virulent in selenium-deficient mice, J. Med. Virol., 43 (1994) 166–170. [2] Chambers, T.J., Hahn, C.S., Galler, R. and Rice, C.M., Flavivirus genome organization, expression, and replication, Annu. Rev. Microbiol., 44 (1990) 649–688. [3] Chang, Y.S., Liao, C.L., Tsao, C.H., Chen, M.C., Liu, C.I., Chen, L.K. and Lin, Y.L., Membrane permeabilization by small hydrophobic nonstructural proteins of Japanese encephalitis virus, J. Virol., 73 (1999) 6257–6264. [4] Chen, C.J., Liao, S.L., Kuo, M.D. and Wang, Y.M., Astrocytic alteration induced by Japanese encephalitis virus infection, NeuroReport, 11 (2000) 1933–1937. [5] Cossarizza, A., Franceschi, C., Monti, D., Salvioli, S., Bellesia, E., Rivabene, R., Biondo, L., Rainaldi, G., Tinari, A. and Malorni, W., Protective effect of N-acetylcysteine in tumor
[16]
[17]
[18]
[19]
[20]
necrosis factor-alpha –induced apoptosis in U937 cells: the role of mitrochondria, Exp. Cell Res., 220 (1995) 232–240. Freeman, B.A. and Crapo, J.D., Biology of disease: free radicals and tissue injury, Lab. Invest., 47 (1982) 412–426. Halliwell, B., Gutteridge, J.M.C. and Cross, C.E., Free radicals. Antioxidants, and human disease – where are we now? J. Lab. Clin. Med., 119 (1992) 598–620. Johnson, R.T., Burke, D.S., Elwell, M., Leake, C.J., Nisalak, A., Hoke, C.H. and Lorsomrudee, W., Japanese encephalitis: immunocytochemical studies of viral antigen and inflammatory cells in fatal cases, Ann. Neurol., 18 (1985) 567–573. Liao, C.L., Lin, Y.L., Wang, J.J., Huang, Y.L., Yeh, C.T., Ma, S.H. and Chen, L.K., Effect of enforced expression of human bcl-2 on Japanese encephalitis virus-induced apoptosis in cultured cells, J. Virol., 71 (1997) 5963–5971. Lin, Y.L., Huang, Y.L., Ma, S.H., Yeh, C.T., Chiou, S.Y., Chen, L.K. and Liao, C.L., Inhibition of Japanese encephalitis virus infection by nitric oxide: antiviral effect of nitric oxide on RNA virus replication, J. Virol., 71 (1997) 5227–5235. Lowy, R.J. and Dimitrov, D.S., Characterization of influenza virus-induced death of J774.1 macrophages, Exp. Cell Res., 234 (1997) 249–258. Mathur, A., Bharadwaj, M., Kulshreshtha, R., Rawat, S., Jain, A. and Chaturvedi, U.C., Immunopathological study of spleen during Japanese encephalitis virus infection in mice, Br. J. Exp. Pathol., 69 (1988) 423–432. Peterhans, E., Reactive oxygen species and nitric oxide in viral diseases, Biol. Trace Elem. Res., 56 (1997) 107–116. Schwarz, K.B., Oxidative stress during viral infection: a review, Free Radical Bio. Med., 21 (1996) 641–649. Schweizer, M. and Peterhans, E., Oxidative stress in cells infected with bovine viral diarrhoea virus: a crucial step in the induction of apoptosis, J. Gen. Virol., 80 (1999) 1147– 1155. Speir, E., Yu, Z.X., Ferrans, V.J., Huang, E.S. and Epstein, S.E., Aspirin attenuates cytomegalovirus infectivity and gene expression mediated by cyclooxygenase-2 in coronary artery smooth muscle cells, Circ. Res., 83 (1998) 210– 216. Srivastava, S., Khanna, N., Saxena, S.K., Singh, A., Mathur, A. and Dhole, T.N., Degradation of Japanese encephalitis virus by neutrophils, Int. J. Exp. Pathol., 80 (1999) 17–24. Staal, J.T., Roederer, M., Herzenberg, L.A. and Herzenberg, L.A., Intracellular thiols regulate activation of nuclear factor B and transcription of human immunodeficiency virus, Proc. Natl. Acad. Sci. USA, 87 (1990) 9943–9947. Verhaegen, S., McGowan, A.J., Brophy, A.R., Fernandes, R.S. and Cotter, T.G., Inhibition of apoptosis by antioxidants in the human HL-60 leukemia cell line, Biochem. Pharmacol., 50 (1995) 1021–1029. Zhu, H., Bannenberg, G.L., Moldeus, P. and Shertzer, H.G., Oxidation pathways for the intracellular probe 2 0 , 7 0 dichlorofluorescein, Arch. Toxicol., 68 (1994) 582–587.