CsA-induced Oxidative Stress Research Articles

Stimulation of Epstein-Barr virus-infected human B cell growth by physiological concentrations of 4-hydroxynonenal

We had previously shown that cyclosporin A (CsA) directly promoted the immortalization of Epstein-Barr virus (EBV)-infected human B cells (EBV-B cells) via an oxidative stress mechanism. 4-Hydroxynonenal (HNE) is a reactive end-product of lipid peroxidation. We hypothesized that HNE may mediate a direct oxidative stress-promoting effect of CsA on EBV-B cells. HNE-protein adducts in CsA-treated EBV-B cell extracts were assayed immunochemically using a Slot-Blot method. Cell proliferation was assayed by [(3)H]-thymidine incorporation. EBV oncogene latent membrane protein-1 (LMP1) expression was assayed by using PE-conjugated anti-LMP1 antibody in flow cytometry. We found that CsA at 500 ng ml(-1) and 1000 ng ml(-1) significantly increased the level of HNE-protein adducts in EBV-B cells over the control (arbitrary units +/- SE) by 251.3 +/- 7.5 to 361.3 +/- 9.7 and 342.7 +/- 10.7, respectively (p < 0.05, n = 3). EBV-B cells treated with a physiological concentration of HNE (1 microM) for 0.5 and 1 h and cultured for 2 and 4 weeks showed significantly increased [(3)H]-thymidine incorporation. EBV-B cells treated with HNE (1 microM) for 1 h and subsequently cultured for 2 and 4 weeks had a significantly higher ( > 2.0 times) LMP1-positive cell population over the control. In conclusion, in accordance with our previous findings, we show that CsA treatment of EBV-B cells results in increased production of the lipid peroxidation reactive end-product HNE that directly promotes EBV-B cell proliferation and LMP1 expression. This observation provides evidence for further understanding the mechanism of CsA-induced oxidative stress on EBV-related post-transplant lymphoproliferative disorder (PTLD).

The protective effect of caffeic acid phenethyl ester against cyclosporine A-induced cardiotoxicity in rats

Cyclosporine A (CsA) is the immunosuppressor, which is most frequently used in transplant surgery and in the treatment of autoimmune diseases. Oxidative stress has been considered as one of the possible mechanisms of CsA-induced cardiotoxicity. The present investigation examines the ability of caffeic acid phenethyl ester (CAPE), which is an active component of propolis extracts, as a natural antioxidant to protect against CsA-induced oxidative stress and cardiotoxicity. CsA cardiotoxicity was induced by subcutaneous injection of CsA at a dose of 15 mg/kg/body weight daily for 21 days in rats. Cardiotoxicity was evaluated by morphological and biochemical studies. CsA treated rats showed degenerative changes with cardiac fibrosis localized around the fibers. These latters were disorganised and the network was disappeared. The ROS production was increased whereas cytochrome- c-oxidase decreased. The expression and levels of matrix metalloproteinase 2 (MMP2) were increased whereas those of its inhibitor were downregulated. CAPE subcutaneous administration (15 μmol/kg/day) improved cardiac cytoarchitecture, decreased the levels and the expression of MMP2, and increased those of TIMP2 proteins. Moreover, it increased cytochrome- c-oxidase activity and decreased ROS production. These results suggest that CAPE could have protective effect against CsA-induced cardiotoxicity.

Protective role of melatonin in cyclosporine A-induced oxidative stress in rat liver

Cyclosporine A (CsA) is the most widely used immunosuppressive drug for preventing graft rejection and autoimmune disease. However, the therapeutic treatment induces several side effects such as nephrotoxicity, cardiotoxicity, hypertension and hepatotoxicity. Among possible mechanisms of CsA-induced hepatic damage, oxidative stress has been suggested. Melatonin (Mel) has been successfully used as a potent antioxidant against many pathophysiological states. This experimental study was performed to test, during CsA treatment, the alterations of some heat shock proteins (HSP) and the Mel antioxidant properties against CsA-induced injury. Rats were divided into four groups, which were treated respectively with olive oil, Mel alone, CsA and CsA plus Mel for 30 days. At the end of the treatments, the animals were killed and hepatic tissue was treated for morphological (haematoxylin–eosin), biochemical (reduced glutathione, GSH and malondialdehyde, MDA) and immunohistochemical (HSP60, HSP72, GRP75 and MT) analyses. The results indicate that CsA-induced hepatotoxicity was characterised by morphological alterations in tissue architecture, changes in GSH and MDA levels and increase in stress protein expression. In conclusion, our data suggest that the imbalance between production of free oxygen radicals and antioxidant defence systems, due to CsA administration, is a mechanism responsible for oxidative stress. Moreover, we show that Mel plays a protective action against CsA-induced oxidative stress, as supported by biochemical and immunohistochemical results.

4-Hydroxynonenal mediates cyclosporine promoted EBV-induced B cell transformation

Introduction. We had previously shown that cyclosporine A (CsA) directly promoted Epstein--Barr virus (EBV) infected human B cell (EBV-B cell) immortalization via oxidative stress mechanism. 4-Hydroxynonenal (HNE) is a reactive end product of lipid peroxidation. We hypothesized that HNE mediates a direct oxidative stress promoting the effect of CsA on EBV-B cell transformation. Methods. Human B cells were purified from human spleen and incubated with EBV (B95-8) overnight for infection. To assay HNE production, EBV-B cells were treated with 0, 500, and 1000 ng/ml of CsA or with HNE (positive control) or DMSO (0.1%, vehicle control) for 10 min at 37°C. HNE--Protein adducts in the cell extract was assayed by slot-blot using polyclonal HNE antibody. For 3H-thymidine uptake and LMP1 assay, EBV-B cells were treated with HNE (1 μM) for 0, 0.5, 1, and 4 h at 37°C and cultured for 2 and 4 weeks. Cells were then assayed for expression of EBV oncogene LMP1 by using PE-conjugated anti-LMP1 antibody in flow cytometry and for 3H-thymidine uptake. Results. CsA at 500 and 1000 ng/ml significantly increased the level of HNE-protein adducts in EBV-B cells over the control (arbitrary units ± SE) of 251.3 ± 7.5 to 361.3 ± 9.7 and 342.7 ± 10.7, respectively ( P < 0.05, n = 3). EBV-B cells treated with physiological concentration of HNE (1 μM) for 0.5 h and 1 h and cultured for 2 weeks had significantly increased 3H-thymidine incorporation (cpm ± SE) over a control of 839.2 ± 54 to 1192 ± 41.6 and 1998 ± 24.8, respectively ( P < 0.02, n = 6). EBV-B cells treated with HNE (1 μM) for 0.5 and 1 h and cultured for 4 weeks also had significantly increased 3H-thymidine uptake over a control of 1035 ± 206 to 2022 ± 293 and 1941 ± 229, respectively ( P < 0.03, n = 6). HNE (1 μM) treated for 4 h did not have a significant effect on cell proliferation at 2 or 4 weeks. EBV-B cells treated with HNE (1 μM) for 1 h and subsequently cultured for 2 and 4 weeks had a significantly higher (>2.0 times ) LMP1 positive cell population over the control. Conclusion. This observation provides evidence for further understanding of CsA-induced oxidative stress and direct promotion of EBV related post-transplant lymphoproliferative disorder (PTLD).

The protective effect of taurine against cyclosporine A-induced oxidative stress and hepatotoxicity in rats

Cyclosporine A (CsA) is the immunosuppressor which is most frequently used in transplant surgery and in the treatment of autoimmune diseases. Oxidative stress has been implicated as one of the possible mechanisms of CsA-induced hepatotoxicity. The present investigation examined the ability of taurine as an antioxidant to protect against CsA-induced oxidative stress and hepatotoxicity. CsA hepatotoxicity was induced by subcutaneous injection of CsA at a dose of 20 mg/kg body weight daily for 21 days. Hepatotoxicity was assessed by reduced serum total protein level and increased serum levels of gamma glutamyl transferase (GGT), alanine aminotransferase (ALT), and aspartate aminotransaminase (AST). CsA treatment increased lipid peroxidation measured as thiobarbituric acid reactive substances (TBARS) concentration and decreased reduced glutathione (GSH) content and activities of catalase and glutathione peroxidase (GSH-Px) in the rat liver. Taurine administration (1% in the drinking water) for 3 days before and concurrently during CsA injections improved liver functions, as indicated by decline of serum transaminases and GGT levels and elevation of serum total protein. Moreover, taurine significantly reduced hepatic TBARS and increased GSH content and catalase and GSH-Px activities in the hepatic tissue. These results indicate that taurine has a protective action against CsA hepatotoxicity and suggest that taurine may find clinical application against a variety of toxins where cellular damage is a consequence of reactive oxygen species.

Salvage of Cyclosporine A-Induced Oxidative Stress and Renal Dysfunction by Carvedilol

Background: Cyclosporine A (CsA) is the first-line immunosuppressant employed for the management of solid organ transplantation and autoimmune diseases. Nephrotoxicity is the major limitation of CsA use. Recent evidence suggests that reactive oxygen species (ROS) play an important role in mediating CsA nephrotoxicity. The present study was designed to investigate effects of carvedilol, a third-generation β-blocker with potent free radical-scavenging activity on CsA-induced oxidative stress and resultant renal dysfunction in a rat model of chronic CsA nephrotoxicity. Methods: Carvedilol (2.0 and 4.0 mg/kg i.p.) and propranolol (10 mg/kg i.p.) were administered to separate group of animals 24 h before and concurrently with CsA (20 mg/kg s.c.) for 21 days. Renal function was assessed by estimating plasma creatinine, blood urea nitrogen (BUN), creatinine and urea clearance. Tissue lipid peroxidation was measured as thiobarbituric acid-reacting substances (TBARS). Renal morphological alterations were assessed by histopathological examination of hematoxylin-eosin, PAS and Masson’s trichrome stained sections of the kidneys. Results: CsA (20 mg/kg s.c) administration for 21 days produced elevated levels of TBARS and deteriorated renal function as assessed by increased plasma creatinine, BUN and decreased creatinine and urea clearance as compared to vehicle-treated rats. The kidneys of CsA-treated rats showed severe striped interstitial fibrosis, arteriolopathy, glomerular basement thickening, tubular vacuolization and hyaline casts. Propranolol neither decreased TBARS nor improved the renal dysfunction and morphological changes induced by CsA. Both doses of carvedilol markedly reduced elevated levels of TBARS, whereas the higher dose of carvedilol significantly attenuated renal dysfunction and morphological changes in CsA-treated rats. Conclusion: These data clearly indicate the renoprotective potential of carvedilol in CsA-induced nephrotoxicity and suggest a significant contribution of its antilipoperoxidative property in this beneficial effect.

Apoptosis and adaptive responses to oxidative stress in human endothelial cells exposed to cyclosporin A correlate with BCL‐2 expression levels

Treatment of transplanted patients with cyclosporin A (CSA) may cause adverse effects such as nephrotoxicity and hypertension. As CSA is known to induce oxidative stress in several tissues, it may cause vascular problems by triggering oxidative stress in endothelial cells (EC). However, oxidative stress has been reported for acute exposure to CSA concentrations exceeding its clinical range, whereas immunosuppression requires life-long treatment with therapeutic concentrations. We therefore compared the effects of 21 h pharmacological (200 microM) vs. 8 days clinical (0.5-2.5 microM) doses of CSA on cultured human EC. Pharmacological doses of CSA cause a decrease in cell density via apoptosis and a down-regulation of the antiapoptotic protein Bcl-2. However, these effects are independent of CSA-induced oxidative stress. In contrast, therapeutic concentrations of CSA cause Bcl-2 up-regulation and modification of EC morphology, both effects blocked by antioxidants. Therefore, a low level of oxidants may act in EC as second messengers that up-regulate Bcl-2, thus promoting survival of impaired EC. Our data suggest that the oxidative stress induced by clinical concentrations of CSA may be involved in the adverse effects of the drug on the vascular system of transplanted patients via an adaptive response involving Bcl-2 up-regulation rather than an apoptotic process