Impact of Bacillus thuringiensis toxin Cry1Ab on rumen epithelial cells (REC) - a new in vitro model for safety assessment of recombinant food compounds.
The growing use of genetically modified crops necessitates viable screening methods for safety evaluation of recombinant feed, particularly for ruminants. A new sheep rumen epithelial cell culture is introduced as an in vitro cell system for safety evaluation especially focussing on feed and food compounds. We used lactate dehydrogenase (LDH) release, WST-1 conversion, ATP content and caspase 3/7 activity to evaluate cytotoxicity of Cry1Ab, one of the newly expressed Bt-proteins in transgene maize. The results were compared to the effects of valinomycin, a potassium ionophore known to induce cytotoxic effects on a wide range of cells. Whereas no toxicity of Cry1Ab was observed in short as well as in long term experiments, even at non-physiological high concentrations, exposure to valinomycin induced apoptosis and a significant response of all viability parameters after a number of hours. The ATP content and the WST-1 conversion reflecting the energy metabolism of the cells appear to be more sensitive indicators of valinomycin toxicity than the LDH release, a parameter which reflects the membrane integrity. This study presents an in vitro model system, that may be useful as a supplementary tool in toxicity screening before testing substances on animals in vivo.
Bondzio
Angelika
A
Department of Veterinary Biochemistry, Freie Universität Berlin, Oertzenweg 19b, 14163 Berlin, Germany. bondzio@zedat.fu-berlin.de
Stumpff
Friederike
F
Schön
Jennifer
J
Martens
Holger
H
Einspanier
Ralf
R
D000255:Adenosine Triphosphate / Q000378:metabolism; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000633:toxicity; D001413:Bacillus thuringiensis / Q000737:chemistry*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000633:toxicity*; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D004731:Endotoxins / Q000302:isolation & purification / Q000633:toxicity*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects* / Q000473:pathology; D030841:Food, Genetically Modified / Q000633:toxicity*; D006460:Hemolysin Proteins / Q000302:isolation & purification / Q000633:toxicity*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D012417:Rumen / Q000187:drug effects* / Q000473:pathology; D012756:Sheep; D019311:Staurosporine / Q000494:pharmacology; D013778:Tetrazolium Salts / Q000378:metabolism; D014634:Valinomycin / Q000633:toxicity
C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; D000590:Amino Acid Chloromethyl Ketones; D000900:Anti-Bacterial Agents; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D004791:Enzyme Inhibitors; D006460:Hemolysin Proteins; D013778:Tetrazolium Salts; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014634:Valinomycin; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D053148:Caspase 3; D053179:Caspase 7; D019311:Staurosporine