title: Cry1Ab treatment has no effects on viability of cultured porcine intestinal cells, but triggers Hsp70 expression.
abstract: In vitro testing can contribute to reduce the risk that the use of genetically modified (GM) crops and their proteins show unintended toxic effects. Here we introduce a porcine intestinal cell culture (IPEC-J2) as appropriate in vitro model and tested the possible toxic potential of Cry1Ab protein, commonly expressed in GM-maize. For comprehensive risk assessment we used WST-1 conversion and ATP content as metabolic markers for proliferation, lactate dehydrogenase release as indicator for cells with compromised membrane and transepithelial electrical resistance as parameter indicating membrane barrier function. The results were compared to the effects of valinomycin, a potassium ionophore, known to induce cytotoxic effects in most mammalian cell types. Whereas no toxicity was observed after Cry1Ab treatment, valinomycin induced a decrease in IPEC-J2 viability. This was confirmed by dynamic monitoring of cellular responses. Additionally, two dimensional differential in-gel electrophoresis was performed. Only three proteins were differentially expressed. The functions of these proteins were associated with responses to stress. The up-regulation of heat shock protein Hsp70 was verified by Western blotting as well as by enzyme-linked immunosorbent assay and may be related to a protective function. These findings suggest that the combination of in vitro testing and proteomic analysis may serve as a promising tool for mechanism based safety assessment.
authors.affiliation: Institute of Veterinary Biochemistry, Freie Universität Berlin, Berlin, Germany. bondzio@zedat.fu-berlin.de
authors.forename: Angelika
authors.initials: A
authors.lastname: Bondzio
authors.forename: Ulrike
authors.initials: U
authors.lastname: Lodemann
authors.forename: Christoph
authors.initials: C
authors.lastname: Weise
authors.forename: Ralf
authors.initials: R
authors.lastname: Einspanier
mesh_terms: D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000494:pharmacology*; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival / Q000187:drug effects; D017097:Electric Impedance; D004731:Endotoxins / Q000235:genetics / Q000494:pharmacology*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006460:Hemolysin Proteins / Q000235:genetics / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D030821:Plants, Genetically Modified; D013552:Swine; D014634:Valinomycin / Q000494:pharmacology; D003313:Zea mays / Q000737:chemistry / Q000235:genetics
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D018840:HSP70 Heat-Shock Proteins; D006460:Hemolysin Proteins; D007476:Ionophores; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014634:Valinomycin; D007770:L-Lactate Dehydrogenase