Induction of Epstein-Barr virus (EBV) lytic cycle in vitro causes oxidative stress in lymphoblastoid B cell lines.

Here, we investigated the effect of induction of the Epstein-Barr virus (EBV) viral lytic cycle on the oxidant/antioxidant balance in three lymphoblastoid cell lines: B95-8, Raji, and LCL C1. The induction of the EBV lytic cycle was done by a non-stressing dose of 12-0-tetradecanoylphorbol-13-acetate (8 nM). Oxidative stress was assessed by measuring malondialdehyde as a parameter of lipid peroxidation, the levels of glutathione, and the activities of three antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase). After 48 h (peak of lytic cycle), a significant decrease in superoxide dismutase activity was observed in B95-8, Raji, and LCL C1 cells (P < 0.05). In addition, in B95-8 cells also a significant decrease of catalase activity was detected (P < 0.05). The glutathione peroxidase activity and the glutathione level were not significantly modified by the induction in any of the cell lines. We found a significant rise in malondialdehyde levels in B95-8, Raji, and LCL C1 cells after the induction of the lytic cycle compared to controls (P < 0.05). In conclusion, induction of EBV lytic cycle in lymphoblastoid cells causes increased oxidative stress in the host cells within 48 h, a process that could be involved in malignant transformations.

High levels of autoantibodies against catalase and superoxide dismutase in nasopharyngeal carcinoma.

INTRODUCTION: Autoantibodies against the antioxidant enzymes have been described in Epstein-Barr virus-associated diseases. Here, we hypothesize that Epstein-Barr virus (EBV), which is associated with nasopharyngeal carcinoma (NPC), induces anticatalase and/or antisuperoxide dismutase autoantibodies that inhibit catalase and/or superoxide dismutase activities and thereby contribute to the oxidative stress status described in this pathology. METHODS: Using a standard enzyme-linked immunosorbent assay (ELISA), the levels of immunoglobulin G (IgG), and M (IgM) directed against catalase and superoxide dismutase in the sera of 30 NPC patients and 30 healthy control individuals were evaluated. The antioxidative profile was tested among the same patients by measuring serum catalase and superoxide dismutase activities. To investigate the implication of EBV in the establishment of autoantibody production in NPC patients, a correlation study between serological testing for EBV viral capsid antigen immunoglobulin G (VCA IgG) and autoantibodies against both enzymes was undertaken. FINDINGS: The levels of IgG against superoxide dismutase and catalase were found to be increased in sera patients compared to controls (P<0.001). NPC patients exhibited decreased catalase (P<0.001) and superoxide dismutase activities (P<0.001) in their sera. However, a positive correlation between superoxide dismutase IgM antibody and IgG antibody titers to VCA (P<0.05, r=0.483, n=21) was found. A positive correlation between catalase (IgM) antibodies and IgG antibody titers to VCA (P<0.05, r=0.546, n=30) was also found. CONCLUSION: High levels of anticatalase and antisuperoxide dismutase antibodies in the sera of NPC patients were found.

Reactive oxygen species production and antioxidant enzyme expression after Epstein-Barr virus lytic cycle induction in Raji cell line.

In a previous study, we have described oxidative stress during Epstein-Barr virus lytic cycle induction. Oxidative stress was evidenced by the observed high MDA levels and the decreased activities of antioxidant enzymes. We hypothesised that the lower activities of the antioxidant enzymes decrease were the result of either the excessive production of reactive oxygen radical species (ROS) or a negative regulation of the antioxidant enzyme gene expressions. In an attempt to clarify this situation, EBV lytic cycle was induced in Raji cell line by a non-stressing dose of 12-0-tetradecanoylphorbol-13-acetate. BZLF-1, superoxide dismutase, and catalase gene expressions were then analysed using semi-quantitative RT-PCR, simultaneously at a kinetic of 6, 12, 24, 36, and 48 h. ROS production was evaluated by chemiluminescence. A study was conducted to establish whether ROS production, BZLF-1, and the expression of antioxidant genes were inter-correlated. Induction of the lytic cycle resulted in increased expressions of the genes of superoxide dismutase and catalase, which began at 24 h (p < 0.05) and reached a peak at 48 h (p < 0.05). Significant increases of the ROS levels were observed in TPA-treated Raji cell line at 12 h, as compared with untreated cells, reaching a peak at 48 h after EBV lytic cycle induction. ROS production correlates positively with BZLF-1, SOD, and CAT gene expressions (p < 0.05; r = 0.913, r = 0.978, and r = 0.955, respectively). A positive correlation was also observed between BZLF-1 and antioxidant gene expressions (p < 0.05; r = 0.961 and r = 0.987, respectively). In conclusion, the observed increases of the SOD and CAT gene expressions eliminate the hypothesis of a repression of the respective genes during the induction of the lytic cycle. On the other hand, the observed direct correlation between the BZLF-1 gene expression and the ROS production is indicative of a role of this gene in oxidative stress.

Transcription of the Epstein-Barr virus lytic cycle activator BZLF-1 during oxidative stress induction.

While latent Epstein-Barr virus infection can be in vitro reactivated by various reagents such as 12-0-tetradecanoylphorbol-13-acetate and calcium ionophore, relatively little is known about in vivo physiological and biochemical factors implicated in this reactivation. Previous studies have described an association between oxidative stress and Epstein-Barr virus infection. In this present study, we investigated the effect of oxidative stress inductors: H2O2 and FeSO4 on reactivation of EBV through BZLF-1 gene expression. Oxidative stress was induced in Raji cell line with 0.2 mM H2O2 or with 0.1 mM FeSO4, and assessed by malondialdehyde level determination, as well as superoxide dismutase and catalase genes expression. Simultaneously, the expression of Epstein-Barr virus immediate-early gene BZLF-1 was analyzed by RT-PCR after 6, 12, 24, 36, and 48 h after H2O2 or FeSO4 treatment. Oxidative stress was evidenced in the Raji cell line by high MDA level as well as superoxide dismutase and catalase genes up-regulation. The transcripts of BZLF-1 were detected from 6 h after 30 min of H2O2 or FeSO4 treatment and maintained until 48 h. These results strongly suggest that oxidative stress contributes to the reactivation of EBV lytic cycle, through induction of BZLF-1 gene expression, a process that may play an important role in the pathogenesis of EBV-associated diseases.

Lipid peroxidation and antioxidant system in the tumor and in the blood of patients with nasopharyngeal carcinoma.

Reactive oxygen species play a key role in cancer development by inducing and maintaining the oncogenic phenotypes of cancer cells. In this study, we examined lipid peroxidation and antioxidant enzymes activities in the blood and in the tumor of nasopharyngeal carcinoma patients. Plasma malondialdehyde, conjugated dienes, erythrocytes catalase, and superoxide dismutase activities have been measured in 30 untreated nasopharyngeal carcinoma patients and 30 controls on one hand. On the other hand, tumor malondialdehyde level, catalase, and superoxide dismutase activities have been measured in five nasopharyngeal carcinoma patients and compared with four controls. The lipid peroxidation was confirmed in the plasma by the high levels of malondialdehyde and conjugated dienes (p<0.001, respectively). Additionally, significantly higher concentrations of malondialdehyde were found in biopsies compared to the control group (p<0.001). In erythrocytes, superoxide dismutase activity was higher in patients than in controls (p<0.05), while it was unchanged in the tumor (p>0.05). Both erythrocytes and tumor catalase activities were significantly lower in patients than in controls (p<0.001, respectively). Statistical studies have shown a positive correlation between malondialdehyde level and IgA antibodies level against Epstein­Barr virus capsid antigen (p<0.05). In conclusion, we reported the presence of an oxidative stress in the blood and in the biopsies of nasopharyngeal carcinoma patients where Epstein­Barr virus seems to play a role.