A proteomics-based approach to studying the impact of transgenic maize (MON810) in rats as a model

Abstract

Transgenic maize MON810 is a maize variety that has been genetically modified to express Cry1Ab isolated from the soil bacterium Bacillus thuringiensis (Bt) to produce a natural insecticide (Bt toxin) which kills larvae of the European corn borer (ECB), a major pest of maize. The mode of action of Bt toxin in ECB is through binding to specific receptors on the epithelial cells of the highly alkaline midgut of the insect, resulting in pore-formation, osmotic imbalance, cell lysis and subsequent death of the insect. In contrast, this Bt toxin is considered to be harmless or nontoxic to mammals due to acidified gut pepsinolysis and the lack of Cry protein binding-sites on the mammalian gut epithelial cells. However, to date, no studies have investigated the cellular effects of these Cry proteins at the proteome level. The aim of this study was therefor to investigate the in vivo and in vitro effects of MON810 maize expressing the δ-insecticidal protein Cry1Ab from Bacillus thuringiensis, on differential gene expression at the proteome level in the epithelial cells of the small intestine of the rat as a model for mammals. Proteomic profiling techniques were included for the in vivo and in vitro studies to obtain a better understanding of the underlying molecular responses in rat to MON810. Transgenic Bt maize (MON810), the corresponding parental non-transgenic maize (MON CONV CORN), and 3 other maize varieties, MON Garst 8450, MON Gold HVST H8920 and MCert Rod commercial control (used as internal controls), were provided by Monsanto, USA. All diets were formulated by TestDiet and contained approximately 33% (w/w) corn grain; other diet components were adjusted to provide approximately equal levels of protein, calories and nutrients. Different reference varieties were used in this study to determine whether the changes that may occur with the consumption of MON810 maize lay within the expected range for several different unmodified reference varieties. Two rats feeding trials (7-day and 28-day) were conducted to assess the safety of MON810 maize using forty immature male Wistar rats (rats were between 6 to 7 weeks of age at the beginning of the study). Rats were assigned to the above 5 experimental groups based on body weight means. No adverse behavioural effects on rats were observed and there were no significant differences in absolute body weights, body weight gains, food consumption and feed conversion efficiency between rats fed MON810 in the diet when compared to rats fed diets containing grain from the other maize varieties. Thus the transgenic variety MON810 had no adverse effects on these parameters. Following these feeding studies, rats were sacrificed and the total proteins extracted from the small intestinal epithelial cells were separated by 2D gel electrophoresis. Differentially expressed proteins were identified using SameSpot Progenesis software followed by liquid chromatography–mass spectrometry (LC-MS/MS); the mass spectrometry data were analysed by Global Proteome Machine (GPM) search engine for protein identification. The same proteomic profiling techniques were used for the in vitro approaches for MON810 maize safety evaluation where primary intestinal epithelial cells and HCT116 cell line were used. In vivo effects of these different maize varieties on the proteome of the epithelial cells of the small intestine when all five groups were compared showed that there were 5, 4, 3, 0, 0, differentially expresed protein spots for Mcert, Mon Conv Corn, MON810, Mon Garst and MON Gold, respectively for the 7- day trial. Two stress-related proteins (LDLR chaperone MESD precursor and peroxiredoxin-6) were up-regulated (2.4 fold) in the MON810 group and 1 stress-related protein (thioredoxindependent peroxide reductase) was up-regulated (2.6 fold) in the Mcert group. For the 28-day trial, only 2 proteins spots (representing 6 proteins) were up-regulated in the small intestinal epithelial cells of rats fed MON810, one of which was a stress-related protein (stress-induced phosphoprotein 1; 3.1 fold). In vitro effects of MON810 and its near isogenic line on the proteome of the epithelial cell lines were negligible, with only 4 protein spots (5 stress-related protein) being up-regulated in the small intestinal primary epithelial cells (IE cells) when exposed to Bt maize extracts and 2 protein spots (1 stress-related protein) being up-regulated when exposed to non-Bt maize extracts. There were no differentially expressed spots between the HCT116 cell lines. The findings from the in vivo and in vitro studies both suggest that MON810 has negligible effects on rats at the cellular level. They also confirm the lack of mammalian toxicity when using rats as a model system.