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Insight in the in vitro biodegradibility and cytotoxicity of GNA lectin, PHA-E lectin and Bt toxin

This result comprised a number of tightly linked studies which were addressing aspects such as the in vitro biodegradability and cytotoxicity of the recombinant lectins and Bt toxin. The information and material generated in these studies were used for subsequent experiments to study the effect of these proteins on gene expression in intestinal epithelial cell lines derived from human (Caco-2 cells) and rat (IEC-6 cells).

The biodegradability, i.e. proteolytic degradation, of PHA-E, GNA and Bt toxin was analysed by incubation in simulated gastric fluid (SGF, containing pepsin, pH2.0) and subsequent incubation in simulated intestinal fluid (SIF, containing trypsin, pH7.0). Two protocols were used which differed in the ratio of protein to digestion enzyme (low in the one, high in the other). It could be concluded from these experiments that in SGF the Bt toxin was degraded more rapidly than PHA-E and GNA, that further treatment in SIF resulted in more degradation only in the case of PHA, and that degradation was increased at the low ratio of protein to enzyme.

The cytotoxicity of the lectins, Bt toxin, and peptic-tryptic (PT) digests of these proteins has been determined in order to define the appropriate protein/peptide concentration to be applied for gene expression profiling. Dose-response experiments were performed using Caco-2 cells (both differentiated and undifferentiated) and IEC-6 cells. LDH leakage and conversion of MTT were the cytological parameters examined. Only digests generated according to the high ratio of protein to enzyme method were found to be suitable for further experimentation. The high concentration of digestion enzymes in the other method appeared to be detrimental for the cells.

It could be concluded from these experiments that, irrespective of the type of protein or digest under investigation, 100microgram/ml was the non-cytotoxic concentration to be used for gene expression profiling experiments in Caco-2. With respect to IEC-6, it was concluded that 50microgram/ml of GNA /Cry1Ab protein or PT digest, was the concentration to be used for gene expression studies. Compared to these latter proteins, PHA-E appeared to be more toxic for IEC-6 cells and 20microgram/ml was chosen as the concentration for the gene expression profiling experiments.

More information on the project can be found at: http://www.entransfood.com/RTDprojects/SAFOTEST/safotest.html

Contact

Ad PEIJNENBURG, (Project leader)
Tel.: +31-31-7475462
Fax: +31-31-7417717
E-mail
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