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.
Highlights
Microbial insecticides containing d-endotoxins (Cry proteins) from Bacillus thuringiensis (Bt) have been used as an alternative to conventional chemical pesticides in agriculture for almost 60 years and recently as resource for insect-resistant genetically modified (GM) plants [1]
After 48 h the activity of dehydrogenases decreased at 500 nM valinomycin by 63%, the ATP content decreased by 47% and the lactate dehydrogenase (LDH) release increased by 42%, revealing membrane alterations
The increasing presence of GM food and feed on the market has provoked a strong demand for a comprehensive risk assessment by use of 90-day rat feeding studies [28]
Summary
Microbial insecticides containing d-endotoxins (Cry proteins) from Bacillus thuringiensis (Bt) have been used as an alternative to conventional chemical pesticides in agriculture for almost 60 years and recently as resource for insect-resistant genetically modified (GM) plants [1]. More than 90% of the feedstuffs for pigs contain genetic modified compounds [2] and the interest in GM crops is continuously increased because of higher agronomic productivity and more nutritious food without the use of pesticides. Since the introduction of GM crops many feeding trials focussed on issues related to consumer safety have been conducted in various animal species. There is an on-going debate on the risk of GM consumption and a demand for additional evidence of GM food and feed safety [8,9,10,11]
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