Partners and International Organizations
(English)
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University of Würzburg (D), Nestlé Research Center (CH), Imperial College of Science, Technology and Medicine, London (UK), ENSAT, Toulouse (F), Istituto Superiore di Sanita, Rome (I), Universita degli Studi della Tuscia, Viterbo (I)
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Abstract
(English)
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The effects of ochratoxin A (OTA) on the expression of specifically thyroid hormone (T3)-regulated genes have been tested in serum-free aggregating brain cell cultures. Nanomolar concentrations of ochratoxin A have been found to decrease the levels of cyclic nucleotide phosphohydrolase (CNP) activity, an oligodendroglia-specific enzyme, and glial fibrillary acidic protein (GFAP), an astrocyte-specific intermediate filament protein. Increasing the levels of T3 during prolonged OTA exposure showed partial protection. Similar results were obtained in experiments with shorter exposure times. However, increased T3 also somewhat raised the basal levels of CNP activity and GFAP content. In subsequent studies, OTA interference with T3-dependent gene expression was examined by real-time RT-PCR using the Taqman system. As preliminary work, primers were designed specifically for the rat genes and synthesized commercially. Optimal conditions for analyses were then determined. By this approach, expression of several T3-dependent genes were examined. A first series of experiments were conducted to examine changes in the expression of GFAP, myelin basic protein (MBP), and Na+,K+-ATPase a subunit genes, after prolonged treatment with ochratoxin A. Three-dimensional brain cell cultures have been treated every two days with different concentrations of ochratoxin A (10 and 20 nM), starting at day in vitro (DIV) 5, or have been cultured in the absence of T3 from DIV 5. Samples have been collected at DIV 9 and DIV 14 for RNA analyses. Results confirm the T3-dependence of the three genes, and show a slight decrease in GFAP and MBP mRNA levels, as well as a slight increase in Na+,K+-ATPase mRNA levels due to OTA. The effects of ochratoxin A on these three genes have then been evaluated after short-term treatment. Three-dimensional brain cell cultures have been treated at DIV 18 with different concentrations of ochratoxin A (10, 15 and 20 nM). Samples have been collected after 12, 24 and 48 hours for RNA analyses. Results show a decrease in GFAP mRNA levels. This decrease is already apparent after 12 hours. At this time-point, the decrease is inversely correlated to the concentration of ochratoxin A. The decrease in GFAP mRNA levels is stronger after 24 hours of treatment but is still inversely correlated to the concentration of ochratoxin A, whereas after 48 hours of treatment, the decrease is concentration-dependent. MBP mRNA levels were not changed after 12 hours of treatment, whereas a slight decrease was observed after 24 and 48 hours of treatment with 20 nM. Na+,K+-ATPase mRNA levels were not modified after 12 hours at any of the concentrations tested, whereas a slight increase was observed after 24 and 48 hours of treatment. The results obtained so far show the emergence of characteristic patterns of responses to OTA at the transcriptional level. In addition, it also appeared that the three-dimensional brain cell cultures offer a robust model for gene expression studies.
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