Immunological and metabolomic impacts of administration of Cry1Ab protein and MON 810 maize in mouse

We have investigated the immunological and metabolomic impacts of Cry1Ab administration to mice, either as a purified protein or as the Cry1Ab-expressing genetically modified (GM) MON810 maize. Humoral and cellular specific immune responses induced in BALB/cJ mice after intra-gastric (i.g.) or intra-peritoneal (i.p.) administration of purified Cry1Ab were analyzed and compared with those induced by proteins of various immunogenic and allergic potencies. Possible unintended effects of the genetic modification on the pattern of expression of maize natural allergens were studied using IgE-immunoblot and sera from maize-allergic patients. Mice were experimentally sensitized (i.g. or i.p. route) with protein extracts from GM or non-GM maize, and then anti-maize proteins and anti-Cry1Ab-induced immune responses were analyzed. In parallel, longitudinal metabolomic studies were performed on the urine of mice treated via the i.g. route. Weak immune responses were observed after i.g. administration of the different proteins. Using the i.p. route, a clear Th2 response was observed with the known allergenic proteins, whereas a mixed Th1/Th2 immune response was observed with immunogenic protein not known to be allergenic and with Cry1Ab. This then reflects protein immunogenicity in the BALB/c Th2-biased mouse strain rather than allergenicity. No difference in natural maize allergen profiles was evidenced between MON810 and its non-GM comparator. Immune responses against maize proteins were quantitatively equivalent in mice treated with MON810 vs the non-GM counterpart and no anti-Cry1Ab-specific immune response was detected in mice that received MON810. Metabolomic studies showed a slight "cultivar" effect, which represented less than 1% of the initial metabolic information. Our results confirm the immunogenicity of purified Cry1Ab without evidence of allergenic potential. Immunological and metabolomic studies revealed slight differences in mouse metabolic profiles after i.g. administration of MON810 vs its non-GM counterpart, but no significant unintended effect of the genetic modification on immune responses was seen.     

A Comparison of the Effects of Three GM Corn Varieties on Mammalian Health

We present for the first time a comparative analysis of blood and organ system data from trials with rats fed three main commercialized genetically modified (GM) maize (NK 603, MON 810, MON 863), which are present in food and feed in the world. NK 603 has been modified to be tolerant to the broad spectrum herbicide Roundup and thus contains residues of this formulation. MON 810 and MON 863 are engineered to synthesize two different Bt toxins used as insecticides. Approximately 60 different biochemical parameters were classified per organ and measured in serum and urine after 5 and 14 weeks of feeding. GM maize-fed rats were compared first to their respective isogenic or parental non-GM equivalent control groups. This was followed by comparison to six reference groups, which had consumed various other non-GM maize varieties. We applied nonparametric methods, including multiple pairwise comparisons with a False Discovery Rate approach. Principal Component Analysis allowed the investigation of scattering of different factors (sex, weeks of feeding, diet, dose and group). Our analysis clearly reveals for the 3 GMOs new side effects linked with GM maize consumption, which were sex- and often dose-dependent. Effects were mostly associated with the kidney and liver, the dietary detoxifying organs, although different between the 3 GMOs. Other effects were also noticed in the heart, adrenal glands, spleen and haematopoietic system. We conclude that these data highlight signs of hepatorenal toxicity, possibly due to the new pesticides specific to each GM corn. In addition, unintended direct or indirect metabolic consequences of the genetic modification cannot be excluded.     

Assessment of Real-time PCR Based Methods for Quantification of Pollen-mediated Gene Flow from GM to Conventional Maize in a Field Study

Maize is one of the main crops worldwide and an increasing number of genetically modified (GM) maize varieties are cultivated and commercialized in many countries in parallel to conventional crops. Given the labeling rules established e.g. in the European Union and the necessary coexistence between GM and non-GM crops, it is important to determine the extent of pollen dissemination from transgenic maize to other cultivars under field conditions. The most widely used methods for quantitative detection of GMO are based on real-time PCR, which implies the results are expressed in genome percentages (in contrast to seed or grain percentages). Our objective was to assess the accuracy of real-time PCR based assays to accurately quantify the contents of transgenic grains in non-GM fields in comparison with the real cross-fertilization rate as determined by phenotypical analysis. We performed this study in a region where both GM and conventional maize are normally cultivated and used the predominant transgenic maize Mon810 in combination with a conventional maize variety which displays the characteristic of white grains (therefore allowing cross-pollination quantification as percentage of yellow grains). Our results indicated an excellent correlation between real-time PCR results and number of cross-fertilized grains at Mon810 levels of 0.1–10%. In contrast, Mon810 percentage estimated by weight of grains produced less accurate results. Finally, we present and discuss the pattern of pollen-mediated gene flow from GM to conventional maize in an example case under field conditions.     

Maize plants infestation by Fusarium spp. and deoxynivalenol in genetically modified corn hybrid and traditional maize cultivars

The objective of the performed investigations was to isolate pathogenic fungi from contaminated maize cobs, to assess the appearance of maize cob fusariosis and to determine grain contamination with deoxynivalenol in the cultivation of genetically modified maize containing a gene resistance against European corn borer (Ostrinia nubilalis Hbn) as well as selected non-modified cultivars. The plant material comprised the following genetically modified maize cultivar: DKC 3421 YG (MON 810) and non-modified cultivars obtained from Smolice Plant Breeding Ltd., IHAR Group: Junak (FAO 210-220), Prosna (FAO 220), SMH (FAO 230), Baca (FAO 220). Prior to harvesting, the occurrence of maize cob fusariosis was determined in the 89 (BBCH) developmental ripening stage. Microbiological assessment was carried out on grains selected from cobs characterized by various pathological symptoms. In 2008, a total of 133 isolates was obtained from the examined samples of infected maize plants, of which 51 isolates were species-identified, while in 2009, the total of 123 isolates were determined, of which 63 were species-identified. In both experimental years, the majority of isolates contained fungi from the Fusarium genus. The performed analysis of mean levels of cob contamination by fusarioses revealed that DKC 3421 YG (MON 810) and SMH (FAO 230) cultivars showed the smallest levels of contamination as well as the lowest percent of cob contamination per plant, while Junak (FAO 210-220) and Baca (FAO 220) cultivars were characterized by the highest degree of contamination. The lowest deoxynivalenol concentrations were determined in years 2008 and 2009 in the case of the DKC 3421 YG (MON 810) cultivar, whereas Prosna (FAO 220) cultivar was characterized by the highest deoxynivalenol concentration.     

High-throughput sequence-based analysis of the intestinal microbiota of weanling pigs fed genetically modified MON810 maize expressing Bacillus thuringiensis Cry1Ab (Bt maize) for 31 days

The objective of this study was to investigate if feeding genetically modified (GM) MON810 maize expressing the Bacillus thuringiensis insecticidal protein (Bt maize) had any effects on the porcine intestinal microbiota. Eighteen pigs were weaned at ~28 days and, following a 6-day acclimatization period, were assigned to diets containing either GM (Bt MON810) maize or non-GM isogenic parent line maize for 31 days (n = 9/treatment). Effects on the porcine intestinal microbiota were assessed through culture-dependent and -independent approaches. Fecal, cecal, and ileal counts of total anaerobes, Enterobacteriaceae, and Lactobacillus were not significantly different between pigs fed the isogenic or Bt maize-based diets. Furthermore, high-throughput 16S rRNA gene sequencing revealed few differences in the compositions of the cecal microbiotas. The only differences were that pigs fed the Bt maize diet had higher cecal abundance of Enterococcaceae (0.06 versus 0%; P < 0.05), Erysipelotrichaceae (1.28 versus 1.17%; P < 0.05), and Bifidobacterium (0.04 versus 0%; P < 0.05) and lower abundance of Blautia (0.23 versus 0.40%; P < 0.05) than pigs fed the isogenic maize diet. A lower enzyme-resistant starch content in the Bt maize, which is most likely a result of normal variation and not due to the genetic modification, may account for some of the differences observed within the cecal microbiotas. These results indicate that Bt maize is well tolerated by the porcine intestinal microbiota and provide additional data for safety assessment of Bt maize. Furthermore, these data can potentially be extrapolated to humans, considering the suitability of pigs as a human model.     

Detection limits of the strip test and PCR for genetically modified corn in Brazil

Brazilian legislation establishes a labeling limit for products that contain more than 1% material from genetically modified organisms (GMOs). We assessed the sensitivity of the lateral flow strip test in detection of the GMO corn varieties Bt11 and MON810 and the specificity and sensitivity of PCR techniques for their detection. For the strip test, the GMO seeds were mixed with conventional seeds at levels of 0.2, 0.4 and 0.8% for Bt11, and 0.4, 0.8 and 1.6% for MON810. Three different methodologies were assessed and whole seeds, their endosperm and embryonic axis were used. For the PCR technique, the GMO seeds of each of the two varieties were mixed with conventional seeds at levels of 20, 10, 5, 2, 1, and 0.5%. The seeds were ground and the DNA extracted. For detection of the GMO material, specific primers were used for MON810 and Bt11 and maize zein as an endogenous control. The sensitivity of the strip test varied for both maize varieties and methodologies. The test was positive for Bt11 only at 0.8%, in contrast with the detection limit of 0.4% indicated by the manufacturer. In the multiplex PCR, the primers proved to be specific for the different varieties. These varieties were detected in samples with one GMO seed in 100. Thus, this technique proved to be efficient in detecting contaminations equal to or greater than 1%.     

Transgene Expression and Bt Protein Content in Transgenic Bt Maize (MON810) under Optimal and Stressful Environmental Conditions

Bt protein content in transgenic insect resistant (Bt) maize may vary between tissues within plants and between plants growing under different environmental conditions. However, it is unknown whether and how Bt protein content correlates with transgene expression, and whether this relationship is influenced by stressful environmental conditions. Two Bt maize varieties containing the same transgene cassette (MON 810) were grown under optimal and stressful conditions. Before and during stress exposure, the upper leaves were analysed for transgene expression using quantitative RT-PCR and for Bt content using ELISA. Under optimal conditions there was no significant difference in the transgene expression between the two investigated Bt maize varieties whereas Bt protein content differed significantly. Transgene expression was correlated with Bt protein content in only one of the varieties. Under stressful environmental conditions we found similar transgene expressions as under optimal conditions but Bt content responded differently. These results suggest that Bt content is not only controlled by the transgene expression but is also dependent on the genetic background of the maize variety. Under stressful conditions the concentration of Bt protein is even more difficult to predict.     

Rapid quantification methods for genetically modified maize contents using genomic DNAs pretreated by sonication and restriction endonuclease digestion for a capillary-type real-time PCR system with a plasmid reference standard

For rough quantitative analysis of genetically modified maize contents, rapid methods for measurement of the copy numbers of the cauliflower mosaic virus 35S promoter region (P35S) and MON810 construct-specific gene (MON810) using a combination of a capillary-type real-time PCR system with a plasmid DNA were established. To reduce the characteristic differences between the plasmid DNA and genomic DNA, we showed that pretreatment of the extracted genomic DNA by a combination of sonication and restriction endonuclease digestion before measurement is effective. The accuracy and reproducibility of this method for MON810 content (%) at a level of 5.0% MON810 mixed samples were within a range from 4.26 to 5.11% in the P35S copy number quantification. These methods should prove to be a useful tool to roughly quantify GM maize content.     

A Phytotoxin,Betaenone C,and Its Related Metabolites of Phoma betae Fr

For rough quantitative analysis of genetically modified maize contents, rapid methods for measurement of the copy numbers of the cauliflower mosaic virus 35S promoter region (P35S) and MON810 construct-specific gene (MON810) using a combination of a capillary-type real-time PCR system with a plasmid DNA were established. To reduce the characteristic differences between the plasmid DNA and genomic DNA, we showed that pretreatment of the extracted genomic DNA by a combination of sonication and restriction endonuclease digestion before measurement is effective. The accuracy and reproducibility of this method for MON810 content (%) at a level of 5.0% MON810 mixed samples were within a range from 4.26 to 5.11% in the P35S copy number quantification. These methods should prove to be a useful tool to roughly quantify GM maize content.     

Degradation of Cry1Ab protein from genetically modified maize (MON810) in relation to total dietary feed proteins in dairy cow digestion

To investigate the relative degradation and fragmentation pattern of the recombinant Cry1Ab protein from genetically modified (GM) maize MON810 throughout the gastrointestinal tract (GIT) of dairy cows, a 25 months GM maize feeding study was conducted on 36 lactating Bavarian Fleckvieh cows allocated into two groups (18 cows per group) fed diets containing either GM maize MON810 or nearly isogenic non-GM maize as the respective diet components. All cows were fed a partial total mixed ration (pTMR). During the feeding trial, 8 feed (4 transgenic (T) and 4 non-transgenic (NT) pTMR) and 42 feces (26 T and 18 NT) samples from the subset of cows fed T and NT diets, and at the end of the feeding trial, digesta contents of rumen, abomasum, small intestine, large intestine and cecum were collected after the slaughter of six cows of each feeding group. Samples were analyzed for Cry1Ab protein and total protein using Cry1Ab specific ELISA and bicinchoninic acid assay, respectively. Immunoblot analyses were performed to evaluate the integrity of Cry1Ab protein in feed, digesta and feces samples. A decrease to 44% in Cry1Ab protein concentration from T pTMR to the voided feces (9.40 versus 4.18 μg/g of total proteins) was recorded. Concentrations of Cry1Ab protein in GIT digesta of cows fed T diets varied between the lowest 0.38 μg/g of total proteins in abomasum to the highest 3.84 μg/g of total proteins in rumen. Immunoblot analysis revealed the extensive degradation of recombinant Cry1Ab protein into a smaller fragment of around 34 kDa in GIT. The results of the present study indicate that the recombinant Cry1Ab protein from MON810 is increasingly degraded into a small fragment during dairy cow digestion.     

Sensitivity of the cereal leaf beetle Oulema melanopus (Coleoptera: Chrysomelidae) to Bt maize-expressed Cry3Bb1 and Cry1Ab

The sensitivity of the cereal leaf beetle, Oulema melanopus (Coleoptera: Chrysomelidae), to maize-expressed Bacillus thuringiensis (Bt) proteins was investigated in the present study. Neonate larvae of O. melanopus were caged on leaves of Cry3Bb1-expressing (MON88017) or Cry1Ab-expressing (MON810) Bt maize, the corresponding near-isolines, or two non-related, conventional maize varieties. Larval survival was reduced on Cry3Bb1-expressing, but not on Cry1Ab-expressing maize compared with conventional varieties. Differences among conventional varieties were also present. The amount of eaten leaf material, developmental time to prepupal stage, and prepupal weight did not differ between Bt maize varieties and their corresponding near-isolines. In an additional feeding study with newly emerged adults, survival and beetle weight did not differ when leaves of Cry3Bb1-expressing maize or the near-isoline were offered as food over 3 weeks. ELISA measurements revealed that larvae feeding on Bt maize contained rather high Cry3Bb1 or Cry1Ab levels, which were in the same order of magnitude as the leaves. In contrast, concentrations in feces were one order, and concentrations in prepupae and adults two orders of magnitude lower.     

Effect of feeding genetically modified Bt MON810 maize to ∼40-day-old pigs for 110 days on growth and health indicators

A total of 72 male weaned pigs were used in a 110-day study to investigate the effect of feeding genetically modified (GM) Bt MON810 maize on selected growth and health indicators. It was hypothesised that in pigs fed Bt maize, growth and health are not impacted compared with pigs fed isogenic maize-based diets. Following a 12-day basal period, pigs (10.7 ± 1.9 kg body weight (BW); ∼40 days old) were blocked by weight and ancestry and randomly assigned to treatments: (1) non-GM maize diet for 110 days (non-GM), (2) GM maize diet for 110 days (GM), (3) non-GM maize diet for 30 days followed by GM maize diet up to day 110 (non-GM/GM) and (4) GM maize diet for 30 days followed by non-GM maize diet up to day 110 (GM/non-GM). BW and daily feed intake were recorded on days 0, 30, 60 and 110 (n = 15). Body composition was determined by dual energy X-ray absorptiometry (n = 10) on day 80. Following slaughter on day 110, organs and intestines were weighed and sampled for histological analysis and urine was collected for biochemical analysis (n = 10). Serum biochemistry analysis was performed on days 0, 30, 60, 100 and 110. Growth performance and serum biochemistry were analysed as repeated measures with time and treatment as main factors. The slice option of SAS was used to determine treatment differences at individual time points. There was no effect of feeding GM maize on overall growth, body composition, organ and intestinal weight and histology or serum biochemistry on days 60 and 100 and on urine biochemistry on day 110. A treatment × time interaction was observed for serum urea (SU; P < 0.05), creatinine (SC; P < 0.05) and aspartate aminotransferase (AST; P < 0.05). On day 30, SU was lower for the non-GM/GM treatment compared with the non-GM, GM and GM/non-GM treatments (P < 0.05). On day 110, SC was higher for the non-GM/GM and GM/non-GM treatments compared with non-GM and GM treatments (P < 0.05). Overall, serum total protein was lower for the GM/non-GM treatment compared with the non-GM/GM treatment (P < 0.05). The magnitude of change observed in some serum biochemical parameters did not indicate organ dysfunction and the changes were not accompanied by histological lesions. Long-term feeding of GM maize to pigs did not adversely affect growth or the selected health indicators investigated.     

No Adjuvant Effect of Bacillus thuringiensis-Maize on Allergic Responses in Mice

Genetically modified (GM) foods are evaluated carefully for their ability to induce allergic disease. However, few studies have tested the capacity of a GM food to act as an adjuvant, i.e. influencing allergic responses to other unrelated allergens at acute onset and in individuals with pre-existing allergy. We sought to evaluate the effect of short-term feeding of GM Bacillus thuringiensis (Bt)-maize (MON810) on the initiation and relapse of allergic asthma in mice. BALB/c mice were provided a diet containing 33% GM or non-GM maize for up to 34 days either before ovalbumin (OVA)-induced experimental allergic asthma or disease relapse in mice with pre-existing allergy. We observed that GM-maize feeding did not affect OVA-induced eosinophilic airway and lung inflammation, mucus hypersecretion or OVA-specific antibody production at initiation or relapse of allergic asthma. There was no adjuvant effect upon GM-maize consumption on the onset or severity of allergic responses in a mouse model of allergic asthma.     

Effect of Bt maize on the plant-aphid–parasitoid tritrophic relationships

The effect of Bt maize on aphid parasitism and the aphid–parasitoid complex was measured in field conditions on three transgenic varieties, two derived from Event MON810 and one from Bt176, and their near-isogenics in a two-year study. No differences in aphid abundance were found between Bt maize varieties and their near-isogenics. Differences within Bt and within near-isogenic varieties were found, but only in one year. Differences in aphid abundance were probably better accounted for the variety background and year conditions than by the transgenesis or Event. Lysiphlebus testaceipes (Cresson), Lipolexis gracilis Förster (Hymenoptera, Braconidae, Aphidiinae) and Aphelinus sp. (Hymenoptera, Aphelinidae) were the prevalent parasitoids. Bt maize did not alter the aphid–parasitoid associations and had no effect on the aphid parasitism and hyperparasitism rates. The results suggest that Bt maize has no negative impact on second, third and fourth levels of the trophic relationships studied.     

Proteomics as a complementary tool for identifying unintended side effects occurring in transgenic maize seeds as a result of genetic modifications

To improve the probability of detecting unintended side effects during maize gene manipulations by bombardment, proteomics was used as an analytical tool complementary to the existing safety assessment techniques. Since seed proteome is highly dynamic, depending on the species variability and environmental influence, we analyzed the proteomic profiles of one transgenic maize variety (event MON 810) in two subsequent generations (T05 and T06) with their respective isogenic controls (WT05 and WT06). Thus, by comparing the proteomic profiles of WT05 with WT06 we could determine the environmental effects, while the comparison between WT06 and T06 seeds from plants grown under controlled conditions enabled us to investigate the effects of DNA manipulation. Finally, by comparison of T05 with T06 seed proteomes, it was possible to get some indications about similarities and differences between the adaptations of transgenic and isogenic plants to the same strictly controlled growth environment. Approximately 100 total proteins resulted differentially modulated in the expression level as a consequence of the environmental influence (WT06 vs WT05), whereas 43 proteins resulted up- or down-regulated in transgenic seeds with respect to their controls (T06 vs WT06), which could be specifically related to the insertion of a single gene into a maize genome by particle bombardment. Transgenic seeds responded differentially to the same environment as compared to their respective isogenic controls, as a result of the genome rearrangement derived from gene insertion. To conclude, an exhaustive differential proteomic analysis allows to determine similarities and differences between traditional food and new products (substantial equivalence), and a case-by-case assessment of the new food should be carried out in order to have a wide knowledge of its features.     

The relationship of proline content and metabolism on the productivity of maize plants

The free proline content in maize ear-leaves, silk and pollen were analyzed in field grown plants which had matured to the pollination stage. Using maize hybrids PR34F02, PR35P12 and PR36B08 field trials were set up at two locations in eastern Croatia in two different years. Two enzymes of proline metabolism were analyzed in the same leaf samples and specific activities of synthetase (P5CS) and proline dehydrogenase (PDH). Plant productivity was evaluated at harvest by the estimation of total and fully developed grain number per ear and per plant, the mean single grain mass, and the mass of grain per plant. The year in which the plants were grown had a very significant effect on the free proline content in the leaf and pollen, as well as on the enzyme activities assayed. The differences between the plants from the two localities were very significant in all tested parameters of plant grain productivity. There was a significant genotype effect on proline content and P5CS total activity in leaf and on all the productivity parameters. Some of the correlations established suggest that the rate of proline synthesis and degradation in maize ear-leaf at pollination might contribute to the final grain production of the maize plant. Multiple regression analyses was used to further analyze the relationship between proline and grain productivity, but it is clear that future work should include other environmental conditions, plant species and organs such as roots.Key words: maize, maize silk, plant productivity, pollen, proline, proline dehydrogenase, Δ¹-pyrroline-5-carboxylate synthetase, Zea mays L.     

A proteomic-based approach to study underlying molecular responses of the small intestine of Wistar rats to genetically modified corn (MON810)

A genetically modified (GM) commercial corn variety, MON810, resistant to European corn borer, has been shown to be non-toxic to mammals in a number of rodent feeding studies carried out in accordance with OECD Guidelines. Insect resistance results from expression of the Cry1Ab gene encoding an insecticidal Bt protein that causes lysis and cell death in susceptible insect larvae by binding to midgut epithelial cells, which is a key determinant of Cry toxin species specificity. Whilst whole animal studies are still recognised as the ‘gold standard’ for safety assessment, they only provide indirect evidence for changes at the cellular/organ/tissue level. In contrast, omics-based technologies enable mechanistic understanding of toxicological or nutritional events at the cellular/receptor level. To address this important knowledge-gap and to gain insights into the underlying molecular responses in rat to MON810, differential gene expression in the epithelial cells of the small intestine of rats fed formulated diets containing MON810, its near isogenic line, two conventional corn varieties, and a commercial (Purina?) corn-based control diet were investigated using comparative proteomic profiling. Pairwise and five-way comparisons showed that the majority of proteins that were differentially expressed in the small intestine epithelial cells in response to consumption of the different diets in both 7-day and 28-day studies were related to lipid and carbohydrate metabolism and protein biosynthesis. Irrespective of the diet, a limited number of stress-related proteins were shown to be differentially expressed. However these stress-related proteins differed between diets. No adverse clinical or behavioural effects, or biomarkers of adverse health, were observed in rats fed GM corn compared to the other corn diets. These findings suggest that MON810 has negligible effects on the small intestine of rats at the cellular level compared with the well-documented toxicity observed in susceptible insects.