Abstract
(Englisch)
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The role of oxidative stress in the toxicity of OTA has been addressed through the analysis of biomarkers of cellular stress response, biomarkers of redox status and chemical markers of oxidative damage. In an earlier study, it was observed that short-term exposure to high doses of OTA produced a significant induction of heat shock protein 32 (HSP32) also named heme oxygenase 1 (HO-1) in rat kidney but not in the liver (Gautier et al., 2001, Free Rad. Biol. Med.). These data further confirmed that oxidative stress is stimulated under OTA treatment and that it may play a role in OTA-mediated toxicity. HO-1 (HSP 32) is considered as a highly sensitive biomarker of cellular response to oxidative stress. It is under the dependence of redox-sensitive transcription factors such as AP1 and NFkappaB. To further investigate the cellular response to OTA, other biomarkers known to be regulated through similar mechanisms (e.g. other HSPs) were investigated in in vitro rat kidney cell systems and in primary rat hepatocytes.
· OTA induces iNOS and HO-1 expression. In vitro, OTA did not influence the expression of HSP25, HSP70 and GRP78. The most striking result obtained was a significant induction of the inducible nitric oxide synthase (iNOS), an enzyme involved in the proinflammatory response.
· A consequence of iNOS induction is the generation of NO (nitric oxide). NO can rapidly interact with superoxide anion to form the strong oxidant peroxynitrite which can bind to proteins (mainly on tyrosine to form nitrotyrosine) and produce toxicity. In addition peroxinitrite may form several DNA adducts which can result in genotoxicity.
· A mass spectrometry-based approach has been developed to accurately analyse soluble and protein bound 3-nitrotyrosine in biological fluids and tissues. Preliminary data show an induction of protein-bound nitrotyrosine in OTA treated NRK cells and primary hepatocytes after 24h of treatment.
· The main detoxification pathway of NO is through conjugation with glutathione (GSH). Therefore a higher production of NO would result into a increased consumption of intracellular GSH. The effects of OTA on intracellular GSH were therefore investigated in cell cultures. OTA reduced significantly the intracellular level of GSH in OTA treated NRK cells and primary hepatocytes after 24h and 48h of treatment.
· NF-kappaB and AP1 are important signaling pathways for the regulation of gene transcription by oxidative stress. They may be part of the mechanism of OTA-mediated iNOS expression since the iNOS promotor present AP-1 and NF-kappa B binding sites. Overexpression of AP-1 has been shown to cause cellular proliferation and transformation. Electrophoretic mobility shift assays, specific for the analysis of the activation of the transcription factors AP1 and NFkappaB, were implemented. Prelimary data obtained in kidney and hepatic cell cultures indicate an OTA-mediated activation of both transcription factors after 24h or 48h of OTA treatment. Dose-responses appear similar to what was found with iNOS induction.
· Impact of OTA on the redox status will be further analysed with improved methods. Effect of OTA on GSH has been described above. In addition, a LC-MS/MS based method was implemented to accurately analyze alpha-tocopherol and alpha-tocopherolquinone (Mottier et al., 2002 Anal. Biochem., 301, 128-135) . Analyses were performed in the plasma, liver and kidney of the in vivo study. No difference was found between OTA-treated and control animals.
· For DNA damage analysis (oxidized DNA bases), a new DNA extraction procedure preventing artifactual production of 8-oxodG formation has been developed and a manuscript is currently in press (Gremaux et al., Carcinognesis). DNA hydrolysis was improved for the detection of other markers such as methyladenine, thymidine diol and thymidine glycol.
· Effects of OTA on chemical markers of lipid damage (malondialdehyde and 4-hydroxynonenal) can now be studied since sensitive methods have been successfully implemented.
· Identification of alternative mechanisms of OTA carcinogenicity is promised by DNA-array approach. Alteration in gene expression is one of the earliest cellular response to hazardous materials. In addition, changes in gene expression induced by a specific chemical are likely to reflect its specific mechanisms of action. Therefore, to study the potential effects of a compound on the expression of a large number of genes is considered as a promising approach to get insight on its mechanism of toxicity. Such an approach has been applied to OTA. In a first set of experiments, a Clontech macro-array containing 1000 different gene sequences was applied to evaluate the effects of OTA on gene expression in primary rat hepatocytes. Confirmatory experiments and comparison with target tissues (kidney and brain) are ongoing. In parallel, the application of a rat microarray system from Affymetric has been initiated with samples from the 21 days and 4 months in vivo studies. The rat microarray from Affimetrix contains more than 5000 genes. The quality control and the statistical power of this new system are significantly improved as compared to the Clontech approach. Experiment has been performed. Data analysis is ongoing.
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