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Apoptosis in neurotoxicity of fumonisin B1
S232
Abstracts / Toxicology Letters 164S (2006) S1–S324
P16-06 Apoptosis in neurotoxicity of fumonisin B1 Kai Savolainen, Helene Stockmann-Juvala, Jonne Naarala, Jarkko Loikkanen, Kirsi V¨ah¨akangas Finnish Institute of Occupational Health, Helsinki, Finland The mycotoxin fumonisin B1 (FB1) is produced by the fungus Fusarium verticillioides, which commonly infects corn across the world. Fusarium species are also found in moisture-damaged buildings, and airborne exposure to mycotoxins like FB1 may therefore be possible. The aim of this study was to investigate the role of apoptosis in the toxicity of FB1. Four different cell lines, human U-118MG glioblastoma, human SH-SY5Y neuroblastoma, rat C6 glioblastoma, and mouse GT17 hypothalamic cells, were exposed to graded doses (0.1–100 M) of FB1 for 0–144 h. Activation of caspase3 like protease was studied by a fluorometric method. DNA fragmentation was visualised by gel electrophoresis and the expression of p53 and Bcl-2 family proteins (Bax, Bcl-2, Bcl-X, Mcl-1) by immunoblotting. At 12 h, caspase-3 like protease activity increased significantly in U-118MG cells, and in the other cell lines, except SH-SY5Y, at later time points after exposure to the highest dose of FB1 (100 M). The results also showed that internucleosomal DNA fragmentation occurred in all cell lines; in C6 cells already at 24 h. On the contrary, the expression of p53 or pro- or antiapoptotic Bcl-2 family proteins were not affected in any of the cell lines at any time points studied. The results of this study indicate that p53 independent apoptosis plays a role in the neurotoxicity induced by FB1. However, the toxicity varies between different types of cell lines. When comparing human cell lines, our results indicate that cells of glial origin (U-118MG cells) may be more sensitive towards FB1 than those of neural origin (SHSY5Y cells). doi:10.1016/j.toxlet.2006.07.141
P16-07 Effect of ochratoxin A on prothrombotic and proinflammatory activities of stimulated blood mononuclear cells Maria Rosaria Carrat`u, Addolorata Coluccia, Pietro Borracci, Maria Rosaria Rossiello, Mario Colucci, Nicola Semeraro Department of Pharmacology and Human Physiology, Medical School, University of Bari, Italy The mycotoxin ochratoxin A (OTA) is an ubiquitous contaminant of human and animal food products. Apart from its known nephrotoxicity and carcinogenicity, OTA has been shown to variably affect several functions of mononuclear leukocytes. We have studied the effect of OTA on tissue factor (TF) and plasminogen activator inhibitor-2 (PAI-2) production by peripheral blood mononuclear cells (MNC) stimulated with endotoxin (1 g/ml, 3 and 18 h at 37 ◦ C for TF and PAI-2, respectively). TF was measured by functional (one-stage clotting time) and immunological (ELISA) assays, and by RT-PCR whereas PAI-2 was assessed by ELISA in conditioned media. OTA caused a dose-dependent reduction in TF activity and antigen (with more than 90% inhibition at the concentration of 1 g/ml) and also reduced PAI-2 release (80% inhibition at 1 g/ml). Inhibition of TF expression was also observed at mRNA level. The inhibitory effect disappeared if OTA was added to MNC suspensions 20–60 min after endotoxin. Moreover, OTA was much less efficient in reducing TF expression when MNC were suspended in medium containing 40 mg/ml human albumin. TF production was also impaired by OTA (1 g/ml) when MNC were stimulated with 10−9 M PMA (99% inhibition), 10 ng/ml IL-1beta (84%) or 100 ng/ml TNF-alpha (55%). Finally, we determined the effect of OTA on endotoxin-induced cytokine release by MNC and found that OTA inhibited IL-6, but not IL-8 or TNF-alpha production, thus ruling out an unspecific effect of the mycotoxin on protein synthesis. Because of the important role of blood clotting activation and fibrin deposition in cell-mediated immune responses, it is suggested that the inhibitory effect on cell TF and PAI-2 expression might represent one of the mechanisms whereby OTA exerts its immunomodulatory activities. doi:10.1016/j.toxlet.2006.07.142
Abstracts / Toxicology Letters 164S (2006) S1–S324
P16-06 Apoptosis in neurotoxicity of fumonisin B1 Kai Savolainen, Helene Stockmann-Juvala, Jonne Naarala, Jarkko Loikkanen, Kirsi V¨ah¨akangas Finnish Institute of Occupational Health, Helsinki, Finland The mycotoxin fumonisin B1 (FB1) is produced by the fungus Fusarium verticillioides, which commonly infects corn across the world. Fusarium species are also found in moisture-damaged buildings, and airborne exposure to mycotoxins like FB1 may therefore be possible. The aim of this study was to investigate the role of apoptosis in the toxicity of FB1. Four different cell lines, human U-118MG glioblastoma, human SH-SY5Y neuroblastoma, rat C6 glioblastoma, and mouse GT17 hypothalamic cells, were exposed to graded doses (0.1–100 M) of FB1 for 0–144 h. Activation of caspase3 like protease was studied by a fluorometric method. DNA fragmentation was visualised by gel electrophoresis and the expression of p53 and Bcl-2 family proteins (Bax, Bcl-2, Bcl-X, Mcl-1) by immunoblotting. At 12 h, caspase-3 like protease activity increased significantly in U-118MG cells, and in the other cell lines, except SH-SY5Y, at later time points after exposure to the highest dose of FB1 (100 M). The results also showed that internucleosomal DNA fragmentation occurred in all cell lines; in C6 cells already at 24 h. On the contrary, the expression of p53 or pro- or antiapoptotic Bcl-2 family proteins were not affected in any of the cell lines at any time points studied. The results of this study indicate that p53 independent apoptosis plays a role in the neurotoxicity induced by FB1. However, the toxicity varies between different types of cell lines. When comparing human cell lines, our results indicate that cells of glial origin (U-118MG cells) may be more sensitive towards FB1 than those of neural origin (SHSY5Y cells). doi:10.1016/j.toxlet.2006.07.141
P16-07 Effect of ochratoxin A on prothrombotic and proinflammatory activities of stimulated blood mononuclear cells Maria Rosaria Carrat`u, Addolorata Coluccia, Pietro Borracci, Maria Rosaria Rossiello, Mario Colucci, Nicola Semeraro Department of Pharmacology and Human Physiology, Medical School, University of Bari, Italy The mycotoxin ochratoxin A (OTA) is an ubiquitous contaminant of human and animal food products. Apart from its known nephrotoxicity and carcinogenicity, OTA has been shown to variably affect several functions of mononuclear leukocytes. We have studied the effect of OTA on tissue factor (TF) and plasminogen activator inhibitor-2 (PAI-2) production by peripheral blood mononuclear cells (MNC) stimulated with endotoxin (1 g/ml, 3 and 18 h at 37 ◦ C for TF and PAI-2, respectively). TF was measured by functional (one-stage clotting time) and immunological (ELISA) assays, and by RT-PCR whereas PAI-2 was assessed by ELISA in conditioned media. OTA caused a dose-dependent reduction in TF activity and antigen (with more than 90% inhibition at the concentration of 1 g/ml) and also reduced PAI-2 release (80% inhibition at 1 g/ml). Inhibition of TF expression was also observed at mRNA level. The inhibitory effect disappeared if OTA was added to MNC suspensions 20–60 min after endotoxin. Moreover, OTA was much less efficient in reducing TF expression when MNC were suspended in medium containing 40 mg/ml human albumin. TF production was also impaired by OTA (1 g/ml) when MNC were stimulated with 10−9 M PMA (99% inhibition), 10 ng/ml IL-1beta (84%) or 100 ng/ml TNF-alpha (55%). Finally, we determined the effect of OTA on endotoxin-induced cytokine release by MNC and found that OTA inhibited IL-6, but not IL-8 or TNF-alpha production, thus ruling out an unspecific effect of the mycotoxin on protein synthesis. Because of the important role of blood clotting activation and fibrin deposition in cell-mediated immune responses, it is suggested that the inhibitory effect on cell TF and PAI-2 expression might represent one of the mechanisms whereby OTA exerts its immunomodulatory activities. doi:10.1016/j.toxlet.2006.07.142