Humoral and cellular immune response in Wistar Han RCC rats fed two genetically modified maize MON810 varieties for 90days (EU 7th Framework Programme project GRACE)

Humoral and cellular immune response in Wistar Han RCC rats fed two genetically modified maize... The genetically modified maize event MON810 expresses a Bacillus thuringiensis-derived gene, which encodes the insec- ticidal protein Cry1Ab to control some lepidopteran insect pests such as the European corn borer. It has been claimed that the immune system may be affected following the oral/intragastric administration of the MON810 maize in various differ - ent animal species. In the frame of the EU-funded project GRACE, two 90-day feeding trials, the so-called studies D and E, were performed to analyze the humoral and cellular immune responses of male and female Wistar Han RCC rats fed the MON810 maize. A MON810 maize variety of Monsanto was used in the study D and a MON810 maize variety of Pioneer Hi-Bred was used in the study E. The total as well as the maize protein- and Cry1Ab-serum-specific IgG, IgM, IgA and IgE levels, the proliferative activity of the lymphocytes, the phagocytic activity of the granulocytes and monocytes, the respiratory burst of the phagocytes, a phenotypic analysis of spleen, thymus and lymph node cells as well as the in vitro production of cytokines by spleen cells were analyzed. No specific Cry1Ab immune response was observed in MON810 rats, and anti-maize protein antibody responses were similar in MON810 and control rats. Single parameters were sporadi- cally altered in rats fed the MON810 maize when compared to control rats, but these alterations are considered to be of no immunotoxicological significance. Keywords Acquired immunity analysis · Anti-Cry1Ab antibodies · Anti-maize protein antibodies · Cellular immune response · Cry1Ab · Food allergenicity · Genetically modified maize MON810 · GRACE · Humoral immune response · Immune cell phenotyping · Native immunity analysis · OECD Test Guideline no. 408—repeated dose 90-day oral toxicity study in rodents (1998) Introduction The genetically modified (GM) maize event MON810 expresses a Bacillus thuringiensis-derived gene, namely, a truncated cry1Ab gene encoding an insecticidal protein (δ-endotoxin; Schnepf et al. 1998), to control some lep- Jana Tulinská and Karine Adel-Patient contributed equally to the idopteran insect pests such as the European corn borer study. (Ostrinia nubilalis; Hill et al. 1995). Concerns regarding Electronic supplementary material The online version of this potential adverse health effects following the ingestion article (https ://doi.org/10.1007/s0020 4-018-2230-z) contains of the MON810 maize have been raised, and it has been supplementary material, which is available to authorized users. claimed that the immune system in Atlantic salmon (Sag- stad et al. 2007; Gu et al. 2013), mice (Finamore et al. * Pablo Steinberg pablo.steinberg@mri.bund.de 2008; Adel-Patient et  al. 2011) and pigs (Walsh et  al. 2011) may be affected following the oral/intragastric Extended author information available on the last page of the article Vol.:(0123456789) 1 3 2386 Archives of Toxicology (2018) 92:2385–2399 administration of the MON810 maize. Although feeding Materials and methods Wistar rats with a powder diet containing 60% Bt rice for 90 days did not induce the anti-Cry1Ab IgG and IgE Maize varieties and diets antibody production in the animals, feeding Wistar rats with the powder diet containing 60% Bt rice spiked with The feeding trials D and E performed in the frame of the 0.1% of purified Cry1Ab for 28 days led to the detection GRACE project used the same batch of diets as studies A of low levels of anti-Cry1Ab-specific IgG antibodies, but and B (Zeljenková et al. 2014), respectively, but the diets not to detectable levels of IgE antibodies (Kroghsbo et al. were further stored for 10 months at − 21 °C. The maize 2008). Kroghsbo et  al. (2008) suggested that exposure varieties and the diets used are listed in Table 1. A MON810 via inhalation, not ingestion, induced Cry1Ab-specific maize variety of Monsanto was used in the study D and a immune responses in Wistar rats, since the diet was given MON810 maize variety of Pioneer Hi-Bred was used in the to the animals as a powdered preparation, which can easily study E. The MON810 event content in the diets contain- be inhaled. In this context, Guerrero et al. (2004, 2007) ing 11 and 33% of the GM MON810 maize at the DNA reported immunogenic effects of Cry1Ab applied via the and the protein level as well as the average daily amount of intranasal route. This is in line with a study by Andreassen Cry1Ab ingested by the rats are shown in Table 2. The diets et al. (2015a), which showed that the intranasal adminis- containing the conventional maize varieties PR33W82 and tration of purified Cry1Ab resulted in the production of PR32T83 contained very low levels of the MON810 maize anti-Cry1Ab-specific IgG1 and IgE antibodies in BALB/c event (Table 2), consistent with the detection of MON810 mice. in the maize batches used as input material for these diets A key objective of the GRACE (GMO Risk Assessment (Zeljenková et al. 2014). and Communication of Evidence; http://www .g r ace -fp7. eu) project funded by the European Commission within the Rat feeding trials 7th Framework Programme was to conduct 90-day animal feeding trials, animal studies with an extended time frame The 90-day feeding trials D and E were performed at the as well as analytical, in vitro and in silico studies on GM animal housing facility of the Slovak Medical University maize, to provide recommendations on the appropriate- (Bratislava, Slovakia) by taking into account the guidance ness of these tools for the risk assessment of GM crops by for such studies published by the EFSA Scientific Com- considering the scientific strengths and limitations of the mittee in 2011 (EFSA Scientific Committee 2011) and the different approaches. For this purpose, the GM maize vari- OECD Test Guideline 408 (OECD 1998). For this purpose, ety MON810 was chosen. The authors underline that the 5-week-old male and female Wistar Han RCC rats were pur- GRACE project was not expected to provide data for the chased from Harlan (San Pietro al Natisone, Italy). The study reassessment of the safety profile of the MON810 maize design, the performance and the results of the feeding trials variety, but to explore the value of different approaches including studies on the humoral and cellular immune responses in the context of the EU regulation for the risk Table 1 Maize variety content of the different diets used in the rat assessment of whole GM food/feed. feeding trials D and E In the frame of the GRACE project, two 90-day feed- Diet Maize variety content (%) ing trials, the so-called studies D and E, were performed to Feeding trial D analyze the humoral and cellular immune responses of male and female Wistar Han RCC rats to the MON810 maize,  33% near-isogenic non-GM 33% DKC6666 maize whereby a MON810 maize variety of Monsanto was used  11% GMO 11% DKC6667-YG + 22% in the study D and a MON810 maize variety of Pioneer Hi- DKC6666 Bred was used in the study E.  33% GMO 33% DKC6667-YG In the present study, the total as well as the Cry1Ab-spe- Feeding trial E cific and maize protein serum antibody levels were meas-  33% near-isogenic non-GM 33% PR32T16 ured, thereby allowing to determine whether Cry1Ab could maize potentially elicit an immunogenic and/or adjuvant effect  11% GMO11% PR33D48 + 22% PR32T16 in Wistar rats under the chosen experimental conditions.  33% GMO 33% PR33D48 Moreover, the proliferative activity of the lymphocytes, the Near-isogenic maize variety of DKC6667 YG, from Monsanto phagocytic activity of the granulocytes and monocytes, the Transgenic maize variety (MON 810), from Monsanto respiratory burst of the phagocytes, a phenotypic analysis of spleen, thymus and lymph node cells as well as the in vitro Near-isogenic maize variety of PR33D48, from Pioneer Hi-Bred Transgenic maize variety (MON 810), from Pioneer Hi-Bred production of cytokines by spleen cells were analyzed. 1 3 Archives of Toxicology (2018) 92:2385–2399 2387 Table 2 Cry1Ab levels in the different diets used in the rat feeding trials D and E Study D Control 11% GMO 33% GMO 33% DKC6666 11% DKC6667-YG + 22% DKC6666 33% DKC6667-YG MON810 maize event—genetically modified Detected, n.q. 14.6 50.8 DNA (%) Cry1Ab (ng/mg protein) Not detected 0.77 2.83 Average amount of ingested Cry1Ab (µg/rat/day)  Males – 3 11  Females – 2 8 Study E Control 11% GMO 33% GMO 33% PR32T16 11% PR33D48 + 22% PR32T16 33% PR33D48 MON810 maize event—genetically modified Not detected 18.9 47.6 DNA (%) Cry1Ab (ng/mg protein) Not detected 2.01 5.15 Average amount of ingested Cry1Ab (µg/rat/day)  Males – 7 17  Females – 5 13 n.q. not quantifiable D and E including the periodical health status observations, extract was quantified using the BCA kit and following the the haematology, clinical biochemistry, gross necropsy and instructions of the manufacturer (Pierce, Thermo Scientific, histopathology findings as well as the corresponding statisti- Rockford, IL, USA). Recombinant Cry1Ab protoxin was cal analyses have previously been published (Schmidt et al. produced in Escherichia coli JM103 carrying the expres- 2015, 2016). These data are freely accessible via https :// sion vector pKK223-3:cry1Ab (kindly provided by D. R. www.cadim a.info/. Zeigler, Bacillus Genetic Stock Center, Ohio State Univer- sity, Columbus, OH) and cleaved with trypsin to yield the Assessment of the humoral immune response Cry1Ab toxin (Miranda et al. 2001; Guimaraes et al. 2010). The obtained Cry1Ab toxin was characterized by electropho- The humoral immune response was analyzed at the Labora- resis, mass spectrometry and specific monoclonal antibod- toire d’Immuno-Allergie Alimentaire of the Institut National ies produced in the lab, as previously described (Guimaraes pour la Recherche Agronomique (INRA, Gif sur Yvette et  al. 2010; Adel-Patient et al. 2011). No endotoxin was Cedex, France). detected in protoxin/toxin preparations, as determined by the Lumulus Amebocyte Lysate test (Sigma-Aldrich Chimie, Proteins, antibodies and reagents Lyon, France). To validate the immunoassays and to produce an internal Enzyme immunometric assays were performed in 96-well standard for anti-maize-/anti-Cry1Ab-specific Ab immu- microtiter plates (Immunoplate Maxisorb , Nunc, Roskilde, noassays, plasma from naïve Wistar Han RCC rats as well Denmark) using the AutoPlate Washer and Microfill Micro- as from non-GM maize-, MON810 maize- and Cry1Ab- plate Dispenser equipment from BioTek Instruments (Avan- immunized 6-week-old female Wistar Han RCC rats was tec, Rungis, France). obtained. Rats were purchased from the Harlan Laboratories Maize flour from near-isogenic non-GM maize (Gannat, France) and bred under standard SPF conditions (DKC6666) and MON810 maize (DKC6667-YG) was (autoclaved bedding and sterile water). All animal experi- suspended in 50 mM carbonate buffer (pH 9.6) contain- ments were performed according to European Community ing 0.05% Tween and 0.05% dithiothreitol (DTT). For the rules of animal care and with authorization no. 91-368 of protein extraction, 4 ml of extraction buffer per 500 mg of the French Veterinary Services. All experiments were cov- maize flour were used. After an incubation at 20 °C for 2 h ered by agreement No. 2009-DSV-074 from the Veterinary on a rotational shaker, extracts were centrifuged (1000×g, Inspection Department of Essonne (France). Rats were 15 min, 4 °C). The lipid layer was removed and the super- acclimated for 2 weeks before experimentation and were natants containing the extracted proteins were collected and then kept naïve or were immunized with near-isogenic non- dialyzed against the 50 mM carbonate buffer (pH 9.6) to GM maize (DKC6666), MON810 maize (DKC6667-YG) or remove Tween and DTT. The total protein content of the purified Cry1Ab protoxin (n = 3 rats/group). The Cry1Ab 1 3 2388 Archives of Toxicology (2018) 92:2385–2399 protoxin was previously shown to be highly immunogenic determined by addition of 200 µl/well of Ellman’s reagent (Adel-Patient et al. 2011). Immunization was performed via as an enzyme substrate and the absorbance was measured the i.p. route using 100 µg of purified Cry1Ab or 500 µg of at 414 nm (Pradelles et al. 1985) using automatic reader protein extracted from the maize varieties and alum as adju- plates (MultiskanEx, Thermo Electron Corporation, Vantaa, vant (Alhydrogel 2%, Eurobio, Les Ulis, France). After three Finland). Results are expressed as absorbance unit at 414 nm administrations 2 weeks apart, rats were anesthetized with (mAbs414 ). nm Isoflurane Belamont (Nicholas Piramal Ltd., London, UK) Pre-selected antibodies and concentrations were then and blood obtained via aorta puncture. After a centrifuga- used in sandwich immunoassays. Different anti-isotype anti- tion at 1200×g for 20 min, plasma samples were aliquoted bodies were passively immobilized for 18 h at 4 °C (5 µg/ and kept at − 20 °C until used. A plasma pool per group was ml in 50 mM phosphate buffer pH 7.4, 100 µl/well). After also prepared. washing and saturation in EIA buffer, standard isotypes were added (0–100 ng/ml, in EIA buffer) for 18 h at 4 °C. After Development of immunoassays to measure total IgG, IgE, extensive washing, biotinylated antibodies were added and IgA and IgM levels in rat plasma the binding of the antibodies was evaluated as mentioned before. Specificity/cross-reactivity was assessed by testing Total antibodies were assessed as two-site immunometric the different standard isotypes in each specific assay, while assays using a first specific antibody as capture antibody and intra- and inter-assay variability was assessed by reproduc- a second labelled antibody as tracer. Commercial antibodies ing the same assay on different plates on the same day or on were selected based on availability and literature (Table 3), two separate days (1 week apart). In a final step, to assess the and were tested as capture antibodies or biotinylated to be parallel course of the curves for the standards and the plasma used as tracer antibodies. samples, plasma samples from naïve and immunized rats In a first step, commercial standard isotypes were directly were tested using serial dilutions in parallel to the isotype immobilized to test the specificity/cross-reactivity and func- standard. Nonspecific binding (NSB) was determined using tionality of each biotinylated antibody and to select the anti- dilution buffer instead of standard/serum. bodies to be further used. Various concentrations of standard isotypes (IgA kappa clone IR22, IgE kappa clone IR162, Development of immunoassays to measure maize protein IgM kappa clone IR473 and polyclonal IgG, purified from and Cry1Ab‑specific IgG, IgE, IgA and IgM levels in rat rat sera; all from Bio-Rad AbD Serotec, Oxford, UK) were plasma then passively immobilized onto microtiter plates (0.01 to 1 µg/ml, 50 mM phosphate buffer pH 7.4, 100 µl/well; 18 h at Specific antibodies were assessed on plates coated with + 4 °C). After washing the microtiter plates (washing buffer: maize protein extracts or purified Cry1Ab (5 µg/ml, diluted 0.01 M phosphate buffer, pH 7.4, containing 0.05% Tween in 50 mM phosphate buffer pH 7.4, 100 µl/well). For vali- 20) and saturation in EIA buffer (0.1 M phosphate buffer, dation, serial dilutions of individual plasma samples/pool 0.1% bovine serum albumin, 0.15 M NaCl, 0.01% sodium plasma from naïve or immunized rats were performed in azide), 100 µl of biotinylated antibodies (EZ-Link Sulfo- EIA buffer and applied to coated plates for 18 h at 4 °C. NHS-LC-Biotin; Pierce, Rockford, IL; biotin:antibody molar After washing, biotinylated antibodies were applied for 3 h ratio = 20) were added to the microtiter plates (10–100 ng/ml at room temperature and the binding of the antibodies was in EIA buffer) and incubated for 3 h at 20 °C. After wash- evaluated as mentioned before. No anti-maize-specific IgE ing, acetylcholinesterase (AChE)-labelled streptavidin was or IgA was detected in the plasma from maize-immunized added for 15 min at 20 °C. Microtiter plates were then exten- rats and no anti-Cry1Ab-specific antibodies were detected in sively washed and solid phase-bound AChE activity was rats immunized with MON810 maize. Intra and inter-assay Table 3 Antibodies tested Antibody Origin Type/clone Reference no./source for the development of the immunoassays Anti-rat IgA heavy chain Mouse Monoclonal MARA-1 MCA191/AbD Serotec Anti-rat IgM Mouse Monoclonal MARM-4 MCA189/AbD Serotec Anti-rat IgE Mouse Monoclonal MARE-1 MCA193/AbD Serotec Anti-rat IgE Sheep Polyclonal STAR109/AbD Serotec Anti-rat IgG F(c) Rabbit Polyclonal OARA05389/Aviva Systems Biology Anti-rat IgG Goat Polyclonal STAR71/AbD Serotec Anti-rat κ/λ Mouse Monoclonal MARK-1/MARL-15 SUN202/AbD Serotec 1 3 Archives of Toxicology (2018) 92:2385–2399 2389 variabilities were assessed as mentioned before. Standard measured in duplicates and by making use of forward and curves for anti-maize- or anti-Cry1Ab-specific IgG and side scatter gates. anti-maize- or anti-Cry1Ab-specific IgM were plotted using serial dilutions of the corresponding plasma from immu- Phagocytic activity of granulocytes and monocytes, nized rats (8 points). An arbitrary value of 100 (100 AU) and respiratory burst of phagocytes was assigned to pooled sera from maize protein-/Cry1Ab- immunized rats diluted 1/1000 (IgM) or 1/10,000 (IgG). Thirty microliters of rat heparinized whole blood were pipet- ted into a tube and 10 µl of a working solution of hydro- ethidine (dihydroethidium bromide, HE; Polysciences, War- Assessment of total and specific antibodies in plasma rington, PA) were then added. The HE working solution was from the maize‑fed rats in trials D and E prepared by adding 10 µl of HE stock solution (15.75 mg HE in 5 ml dimethylformamide; Merck, Kenilworth, NJ) to After optimization, validation and preliminary experi- 1 ml Medium 199 (Gibco, Invitrogen, Paisley, UK). Samples ments with randomly selected maize-fed rat plasma sam- were incubated for 15 min at 37 °C. Three microliters of ples (n = 10), immunoassays for total and specific antibodies fluorescein-labelled Staphylococcus aureus bacteria (Molec- were performed with the plasma samples from the different ular Probes, Eugene, OR) was added to each of the “test” rat groups in trials D and E. Standard curves were included tubes (1.4 × 10 bacteria per test). All tubes were incubated in each microtiter plate and all samples were analyzed in a for another 15 min at 37 °C. Samples were put on ice and randomized and blinded manner. All analyses were repeated 700 µl of the cold lysis solution described above were added once. For total antibodies, internal controls (IC, pool of sera for 10 min to lyse red blood cells. In the case of the “control” from naive/immunized rats) were also included in each micr- tubes, the Staphylococcus aureus bacteria were added after otiter plate. the lysis solution. Samples were analyzed using an EPICS XL flow cytometer (Beckman Coulter). The percentage of Assessment of the cellular immune response phagocytic monocytes and phagocytic granulocytes (i.e. those that had phagocytised the fluorescein-labelled Staphy - The cellular immune response was analyzed at the Slovak lococcus aureus bacteria) and the percentage of granulocytes Medical University (Bratislava, Slovakia). with respiratory burst (i.e., those in which dihydroethidine was converted to a fluorescent metabolite by reactive oxygen Phenotypic analysis of spleen, mesenteric lymph nodes, species) were measured in duplicates and by making use of bone marrow and thymus forward and side scatter gates. Nine microliters of a mixture of labelled monoclonal Proliferative activity of lymphocytes antibodies were added to 90  µl of a spleen, mesenteric lymph node, bone marrow and thymus cell suspension The spleen was removed using sterile instruments and placed (2 × 10  cells/ml) and incubated for 30 min at 4 °C in the in sterile RPMI 1640 culture medium with L-glutamine dark. The following antibodies, all purchased from eBiosci- and HEPES buffer (Sigma-Aldrich, St. Louis, MO) sup- ence (San Diego, CA), were used to stain the cells: Anti-Rat plemented with 5 IU heparin/ml (Zentiva, Prague, Czech CD3 FITC, Anti-Rat CD4 PE, Anti-Rat CD8a PerCP-eFluor Republic) and 12 µg gentamycin/ml (Sandoz, Basel, Swit- 710, Anti-Rat CD45R PE and Anti-Rat CD161 PerCP- zerland). Spleen cells were obtained under sterile conditions eFluor 710. Isotype controls (Mouse IgG3 Isotype Control- by washing the spleen with RPMI medium and by making FITC, Mouse IgG2a K Isotype Control-PE, Mouse IgG1 use of a syringe with a needle. The spleen cell suspension K Isotype Control-PerCP-eFluor 710 and Mouse IgG2b was centrifuged at 130×g for 15 min and resuspended in K Isotype Control-PE) were used as negative controls to complete RPMI medium containing 10% foetal calf serum determine background fluorescence. Red blood cells were (FCS, PAA, Linz, Austria). Cell suspensions (2 × 10  cells/ lysed with a lysis solution for 15 min and, n fi ally, phosphate- ml) were dispensed in triplicate wells (150  µl/well) of buffered saline (PBS) was added. The lysis solution was a 96-well microtiter culture plate. The mitogens, all pur- prepared by adding 0.829 g NH Cl (Lachema, Brno, Czech chased from Sigma-Aldrich, were added at the following Republic), 0.1  g KHCO and 0.0037  g Na EDTA (both final concentrations: concanavalin A (Con A; 2.5 µg/ml), 3 2 from Sigma-Aldrich) to 100 ml aqua ad injectionem (Imuna phytohemagglutinin (PHA; 25 µg/ml) and pokeweed mito- Pharm, Šarišské Michaľany, Slovakia). Samples were ana- gen (PWM; 2.5  µg/ml). Moreover, recombinant Cry1Ab lyzed using an EPICS XL flow cytometer (Beckman Coul- at final concentrations of 5, 50 and 500 ng/ml and protein + + + + + ter). The percentage of CD3 , CD3 CD4, CD3 CD8a , extracts from near-isogenic non-GM and MON810 maize − + − + CD3 CD161 and CD3 CD45R cells in each sample was at final concentrations of 50 ng/ml, 500 ng/ml and 5 µg/ml 1 3 2390 Archives of Toxicology (2018) 92:2385–2399 were added to additional cell culture plates. Recombinant based on the interpretation of the calculated SES estimates Cry1Ab as well as the maize protein extracts were obtained (Fig. 1). as described above. The plates with mitogens were incubated for 48 h and those with maize antigens and CryAb toxin for 6 days at 37 °C and 5% C O . Thereafter, each well was Results pulsed with 1 µCi [ H]-thymidine (Moravek Biochemicals, Brea, CA) diluted in 20 µl medium and the plates were incu- Humoral immune response bated at 37 °C for another 24 h. The cell cultures were then harvested on glass filtre papers and the filtres were placed The antibodies selected for the various immunoassays are in scintillation fluid (Perkin Elmer, Waltham, MA). Radio- indicated in Table 4. Typical IgE standard curves showed activity was measured using a Beta Scintillation counter an excellent intra- and inter-assay reproducibility (Supple- Microbeta 2 (Perkin Elmer). Counts per minute (cpm)/cell mentary Material, Fig. 1). Moreover, a good parallel course culture were measured in triplicates for each variable. The and reproducibility of the curves for the standards and index was calculated as the ratio of cpm/cell culture in wells diluted plasma were also observed for total IgM and IgA stimulated with a mitogen/a protein extract and cpm/cell cul- (Fig. 2), demonstrating that the calculated concentrations ture in unstimulated wells. do not depend on the plasma dilution and the experimental day. The limits of detection, determined as the means of In vitro production of cytokines NSB + 3σn − 1, were 98 pg/ml for IgE, 69 pg/ml for IgG and 7.8 ng/ml for IgA and IgM. Specific IgG and IgM immuno- Spleen cells were incubated in complete RPMI medium as assays were further validated and standardized by making described above. One hundred and fifty microliters of the use of a pool of plasma samples from rats immunized with spleen cell suspension (2 × 10  cells/ml) were dispensed in maize or Cry1Ab. The sensitivity and reproducibility of the triplicate wells of a 96-well microtiter culture plate. The corresponding assays were high, as exemplarily shown for mitogen Con A (2.5 µg/ml) or the Cry 1Ab toxin (50 ng/ml) IgG in Fig. 3. Preliminary experiments using some randomly were added in a volume of 50 µl. The plates were incubated selected plasma samples from rats fed MON810 maize were at 37 °C in the presence of 5% C O for 72 or 144 h, respec- performed, thereby allowing to determine the dilutions to be tively. Thereafter, the supernatants were removed and stored used for the analysis of the whole set of samples (Table 5). at − 70 °C. The Pr ocartaPlex Rat Th Complete Panel (14 plex) from eBioscience was used to measure the levels of interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A, interferon-γ (IFN-γ), granulo- cyte-colony-stimulating factor (G-CSF), granulocyte–mac- rophage colony-stimulating factor (GM-CSF) and tumour necrosis factor-α (TNF-α) in spleen cell culture supernatants by following the instructions of the manufacturer and by making use of a Luminex 200 apparatus (Luminex, Madi- son, WI, USA). Fig. 1 Simplified version of a graph allowing visual assessment of Statistics statistical significance as well as the supposed biological and possi- ble toxicological relevance of group comparisons. The standard effect The immunological parameters were analyzed descriptively size point estimate (circle) and the 95% confidence limits (whiskers, bars show confidence interval) illustrate the (standardized) effect (N, mean, median, standard deviation, minimum, maximum, size between two groups. The vertical black line indicates no statisti- 95% confidence interval of the mean). Standardized effect cally significant difference (zero difference), while the vertical grey sizes (SES: difference in means between two groups divided lines indicate the supposed biological and possible toxicological rel- by the pooled standard deviation [SD]) as well as their 95% evance limits (here ± 1.0 SD, according to the study design). If the confidence interval bars cross the zero line but not the grey lines cond fi ence intervals were calculated according to Nakagawa (lie within the ± 1.0 limits), there is evidence for no statistical sig- and Cuthill (2007); for details see also Schmidt et al. (2015). nificance as well as no biological relevance (case a). Two groups are The GMO groups were compared to the control groups: significantly different when the confidence interval bars do not cross GMO11%-control and GMO33%-control. All immunologi- the black vertical line (cases b, c). The effect size between two groups is supposed to be potentially relevant, when the confidence interval cal parameters were displayed in one SES graph and the bars lie outside the ± 1.0 SD limits (case c). Case b indicates statisti- data are expressed as the cage mean ± SD. In this paper, cal significance, but no clear biological relevance. Case d indicates no when comparing the different parameters between a control statistical significance, but no clear negation of biological relevance. and a second group, the wording “significantly different” is This figure is Fig. 1 of the study by Zeljenková et al. (2014) 1 3 Archives of Toxicology (2018) 92:2385–2399 2391 Table 4 Antibodies selected for Isotype Antibodies for the total immunoglobulin immunoassays Antibodies for the specific the total and specific IgE, IgG, immunoglobulin immunoas- IgA and IgM immunoassays says Capture antibody Labelled antibody Labelled antibody IgE STAR109 MARE-1 (100 ng/ml) MARE-1 (100 ng/ml) IgG STAR71 STAR71 (50 ng/ml) STAR71 (50 ng/ml) IgA MARA-1 Anti-rat κ/λ (50 ng/ml) MARA-1 (50 ng/ml) IgM MARM-4 Anti-rat κ/λ (50 ng/ml) MARM-4 (50 ng/ml) Fig. 2 Parallelism of the curves for total IgM (a) and IgA (b) or the dilution factor of the different plasma samples (a dilution fac- obtained with the isotype standard (dark blue) and diluted plasma tor of 1 corresponds to an initial dilution factor of 1/1000 for IgM from experimental rats immunized with GM maize (green), conven- and 1/100 for IgA). mAbs414nm, absorbance unit at 414 nm. (Color tional maize (purple), Cry1Ab protoxin (clear blue) or kept naïve figure online) (red). The x-axis represents the concentration of the isotype standard The amount of total IgG, maize-specific IgG and total IgE corresponding control group, whereas no statistically sig- measured in the plasma of male and female rats in the feed- nificant differences regarding all other cell phenotypes and ing trials D and E are shown in Table 6. In study D, the total lymphoid organs between rats fed the control and the GMO plasma IgG, anti-maize-specific IgG and total IgE levels in diets were observed. The phenotypic analysis of spleen, mes- male rats fed the control or GMO diets were similar (i.e. the enteric lymph node, bone marrow and thymus cells of male differences between the groups were not statistically sig- and female rats in the feeding trial E is shown in Table 8. + + + nificant) and this was also the case of the female animals. In The percentages of CD3 and CD3 CD4 cells in the spleen study E, the anti-maize-specific IgG level in plasma of male of female rats fed the 33% GMO diet were significantly rats fed the 11% GMO diet was significantly higher than that higher and, concomitantly, the percentage of CD3 cells in in plasma of male rats fed the control diet, but this was not the spleen of female rats was significantly lower than that of the case in male rats fed the 33% GMO diet (Table 6). All the control group, whereas no statistically significant differ - other parameters were similar in male and female rats. The ences regarding all other cell phenotypes between rats fed anti-maize protein-specific IgE and IgA antibody levels were the control and the GMO diets were observed. below the detection limit in all experimental groups. No Table  9 lists the percentage of phagocytic monocytes anti-Cry1Ab-specific antibodies were detected in any group. and granulocytes after incubation of the cells with labelled Staphylococcus aureus and the percentage of phagocytes Cellular immune response showing respiratory burst after incubation of the cells with dihydroethidine in the feeding trials D and E. The percentage The phenotypic analysis of spleen, mesenteric lymph of phagocytic granulocytes and the percentage of phago- node, bone marrow and thymus cells of male and female cytes showing a respiratory burst were significantly higher rats in the feeding trial D is shown in Table  7. The per- in female rats fed the 11% GMO diet than in the control + + centage of CD3 CD4 cells in the thymus of male rats fed group in study D, while no other statistically significant dif- the 33% GMO diet was significantly lower than that of the ferences regarding the above-mentioned parameters between 1 3 2392 Archives of Toxicology (2018) 92:2385–2399 Fig. 3 Anti-maize- (a) and anti-Cry1Ab-specific IgG (b) in plasma ferent days (series #1 and #2). The x-axis represents arbitrary units from rats immunized with maize protein extract or Cry1Ab, respec- of specific IgG; a value of 100 arbitrary units was assigned to pooled tively. Assays were performed on the same days on up to eight sepa- plasma from maize-/Cry1Ab-immunized rats diluted 1/10,000. mAb- rate plates. Plates were coated with extracts/protein purified on dif- s414nm, absorbance unit at 414 nm Table 5 Dilutions used for Isotype Dilutions used for the determination of the determination of the total, anti-maize-specific and anti- Total antibody levels Anti-maize-specific Anti-Cry1Ab- Cry1Ab-specific antibody levels antibody levels specific antibody in rat plasma levels IgE 1/20 and 1/40 1/10 1/10 Standard from 400 ng/ml (SDF = 4) 6 7 IgG 1/2 × 10 and 1/10 1/100 and 1/500 1/50 and 1/100 Standard from 30 ng/ml (SDF = 3) IgA 1/100 and 1/500 1/10 1/10 Standard from 1000 ng/ml (SDF = 2) IgM 1/2000 and 1/10,000 1/40 1/40 Standard from 1000 ng/ml (SDF = 2) SDF Serial Dilution Factor (standard curves were performed with 8 concentration points) 1 3 Archives of Toxicology (2018) 92:2385–2399 2393 Table 6 Total IgG, anti-maize protein-specific IgG and total IgE levels in plasma of male and female rats in feeding trials D and E Parameter Male rats Female rats Control 11% GMO 33% GMO Control 11% GMO 33% GMO Trial D  Total IgG level (mg/ml) 2.47 ± 0.73 1.95 ± 0.40 2.56 ± 0.36 3.05 ± 0.50 2.73 ± 0.61 2.77 ± 0.53  Anti-maize protein-specific 985 ± 105 1149 ± 157 971 ± 303 1006 ± 342 879 ± 148 1130 ± 197 IgG level (arbitrary units/ ml)  Total IgE level (ng/ml) 38.07 ± 13.48 42.03 ± 14.76 57.72 ± 33.71 61.66 ± 28.68 47.42 ± 15.89 49.98 ± 29.79 Trial E  Total IgG level (mg/ml) 2.32 ± 1.06 2.42 ± 0.67 2.85 ± 0.27 2.97 ± 0.83 3.27 ± 0.49 2.92 ± 0.46  Anti-maize protein-specific 900 ± 221 1287 ± 150* 1682 ± 1210 1294 ± 283 970 ± 163 1234 ± 399 IgG level (arbitrary units/ ml)  Total IgE level (ng/ml) 38.81 ± 15.07 46.63 ± 26.94 41.16 ± 10.62 66.02 ± 24.06 42.63 ± 18.22 42.37 ± 13.45 The results are expressed as cage mean ± SD (five cages, n = 10 rats) *Statistically significant difference to the control value based on the 95% confidence interval of the SES Table 7 Phenotypic analysis of spleen, lymph node, bone marrow and thymus cells of male and female rats in the feeding trial D Parameter Male rats Female rats Control 11% GMO 33% GMO Control 11% GMO 33% GMO + + Spleen CD3 CD8 cells 40.72 ± 8.26 37.35 ± 4.65 35.94 ± 5.99 42.11 ± 4.80 39.68 ± 5.72 37.87 ± 5.85 Spleen CD3 cells 59.51 ± 8.82 55.97 ± 4.07 56.68 ± 5.83 64.72 ± 3.17 63.39 ± 7.69 60.68 ± 6.23 Spleen CD3 cells 40.50 ± 8.82 44.03 ± 4.07 43.32 ± 5.83 35.29 ± 3.17 36.61 ± 7.69 39.33 ± 6.23 + + Spleen CD3 CD4 cells 40.48 ± 7.16 37.20 ± 3.93 34.09 ± 7.79 42.49 ± 4.52 39.70 ± 6.14 38.05 ± 4.96 − + Spleen CD3 CD45R cells 24.16 ± 4.92 29.96 ± 2.17 28.21 ± 3.50 24.66 ± 2.73 25.40 ± 3.48 26.89 ± 5.03 − + Spleen CD3 CD161 cells 19.62 ± 3.63 24.47 ± 2.62 23.44 ± 2.53 18.97 ± 7.95 18.97 ± 6.89 20.62 ± 7.88 + + Lymph node CD3 CD8 cells 28.87 ± 12.57 29.36 ± 17.70 27.93 ± 13.85 41.73 ± 5.70 40.02 ± 5.87 41.62 ± 6.01 Lymph node CD3 cells 41.89 ± 19.48 42.35 ± 20.81 43.97 ± 19.53 56.51 ± 8.53 53.96 ± 9.41 55.92 ± 8.47 Lymph node CD3 cells 58.12 ± 19.48 57.65 ± 20.81 56.03 ± 19.53 43.49 ± 8.53 46.04 ± 9.41 44.08 ± 8.47 + + Lymph node CD3 CD4 cells 26.22 ± 10.05 26.62 ± 16.46 24.46 ± 12.00 38.88 ± 4.09 37.72 ± 4.75 39.03 ± 4.96 − + Lymph node CD3 CD45R cells 52.81 ± 14.43 53.98 ± 16.52 56.80 ± 16.19 40.96 ± 6.49 44.44 ± 6.11 41.08 ± 7.88 + a Bone marrow CD3 cells 7.75 ± 1.05 7.25 ± 3.11 8.22 ± 2.73 17.44 ± 6.17 15.63 ± 5.71 13.27 ± 2.38 − a Bone marrow CD3 cells 92.26 ± 1.05 92.76 ± 3.11 91.79 ± 2.73 82.57 ± 6.17 84.38 ± 5.71 86.74 ± 2.38 − + a Bone marrow CD3 CD45R cells 71.60 ± 7.46 73.45 ± 8.12 70.37 ± 7.28 64.84 ± 12.22 61.87 ± 16.91 61.91 ± 20.71 − + a Bone marrow CD3 CD161 cells 12.77 ± 6.32 13.43 ± 5.25 12.54 ± 5.77 15.55 ± 4.55 13.19 ± 4.20 14.80 ± 5.09 + + Thymus CD3 CD8 cells 21.37 ± 2.63 22.92 ± 3.95 17.57 ± 4.12 19.46 ± 2.67 20.57 ± 3.20 20.49 ± 3.44 Thymus CD3 cells 25.11 ± 5.60 27.22 ± 7.77 21.42 ± 7.41 21.73 ± 2.73 22.90 ± 3.42 22.81 ± 3.74 Thymus CD3 cells 74.90 ± 5.60 72.78 ± 7.77 78.59 ± 7.41 78.27 ± 2.73 77.11 ± 3.42 77.19 ± 3.74 + + Thymus CD3 CD4 cells 20.04 ± 1.31 21.01 ± 2.42 16.40 ± 2.65* 18.95 ± 2.51 19.96 ± 3.05 19.87 ± 3.29 The table lists the percentage of cells with the indicated phenotype, expressed as cage mean ± SD (five cages; n = 10 rats, if not otherwise stated) *Statistically significant difference to the control value based on the 95% confidence interval of the SES n = 9 rats fed the control and the GMO diets were observed in Cry1Ab, the near-isogenic non-GM maize protein extract both studies. and the GM maize protein extract is shown in Table  10. The proliferative response of spleen cells from male The proliferative response of spleen cells from male rats and female rats of feeding trial D after incubation with fed the 33% GMO diet when incubated with phytohemag- concanavalin A, phytohemagglutinin, pokeweed mitogen, glutinin was significantly lower than that of spleen cells 1 3 2394 Archives of Toxicology (2018) 92:2385–2399 Table 8 Phenotypic analysis of spleen, lymph node, bone marrow and thymus cells of male and female rats in the feeding trial E Parameter Male rats Female rats Control 11% GMO 33% GMO Control 11% GMO 33% GMO + + Spleen CD3 CD8 cells 41.51 ± 6.81 41.41 ± 7.60 37.88 ± 9.13 39.41 ± 3.96 42.92 ± 5.48 46.58 ± 5.59 Spleen CD3 cells 60.86 ± 6.82 60.66 ± 8.39 57.16 ± 11.66 62.73 ± 2.76 65.37 ± 2.10 69.69 ± 4.20* Spleen CD3 cells 39.15 ± 6.82 39.35 ± 8.39 42.84 ± 11.66 37.27 ± 2.76 34.63 ± 2.10 30.32 ± 4.20* + + Spleen CD3 CD4 cells 41.62 ± 5.79 41.00 ± 6.27 37.32 ± 8.68 39.27 ± 3.38 43.36 ± 4.96 46.75 ± 4.68* − + Spleen CD3 CD45R cells 24.56 ± 3.99 25.86 ± 3.83 26.09 ± 5.23 25.46 ± 2.21 24.10 ± 1.62 21.21 ± 3.63 − + Spleen CD3 CD161 cells 19.97 ± 3.00 21.46 ± 2.74 21.39 ± 4.25 19.25 ± 8.71 17.59 ± 7.70 16.26 ± 6.40 + + a Lymph node CD3 CD8 cells 33.47 ± 17.51 27.62 ± 15.23 27.29 ± 13.70 42.50 ± 1.96 42.52 ± 5.80 43.69 ± 12.98 + a Lymph node CD3 cells 45.32 ± 20.51 39.68 ± 19.83 42.93 ± 20.25 57.40 ± 3.39 56.51 ± 7.44 58.01 ± 16.84 − a Lymph node CD3 cells 54.69 ± 20.51 60.33 ± 19.84 57.08 ± 20.25 42.60 ± 3.40 43.50 ± 7.44 41.99 ± 16.84 + + a Lymph node CD3 CD4 cells 31.44 ± 15.77 25.98 ± 12.94 23.91 ± 10.76 40.35 ± 2.16 40.79 ± 5.49 41.15 ± 11.03 − + a Lymph node CD3 CD45R cells 47.83 ± 14.58 52.53 ± 14.22 50.88 ± 13.95 38.49 ± 2.57 39.01 ± 5.15 37.67 ± 11.65 Bone marrow CD3 cells 9.27 ± 1.24 11.93 ± 5.95 9.80 ± 2.63 15.44 ± 3.91 12.30 ± 3.58 17.34 ± 5.72 Bone marrow CD3 cells 90.73 ± 1.24 88.08 ± 5.94 90.21 ± 2.63 84.57 ± 3.91 87.70 ± 3.58 82.67 ± 5.73 − + Bone marrow CD3 CD45R cells 69.67 ± 6.22 65.94 ± 5.58 67.79 ± 10.36 65.92 ± 12.48 70.51 ± 11.47 68.53 ± 7.50 − + Bone marrow CD3 CD161 cells 12.73 ± 7.10 11.95 ± 5.63 11.69 ± 4.43 13.22 ± 3.18 13.49 ± 4.01 13.99 ± 2.73 + + Thymus CD3 CD8 cells 22.71 ± 5.24 24.17 ± 5.89 22.27 ± 4.94 22.80 ± 1.41 20.59 ± 3.26 22.42 ± 0.81 Thymus CD3 cells 26.68 ± 8.54 28.11 ± 8.19 26.32 ± 8.17 25.24 ± 1.43 22.81 ± 3.36 24.82 ± 0.85 Thymus CD3 cells 73.32 ± 8.54 71.90 ± 8.19 73.68 ± 8.17 74.76 ± 1.43 77.20 ± 3.36 75.18 ± 0.85 + + Thymus CD3 CD4 cells 21.39 ± 3.79 22.84 ± 4.88 20.62 ± 3.34 22.39 ± 1.34 20.05 ± 2.97 21.92 ± 0.99 The table lists the percentage of cells with the indicated phenotype, expressed as cage mean ± SD (five cages; n = 10 rats, if not otherwise stated) *Statistically significant difference to the control value based on the 95% confidence interval of the SES n = 9 Table 9 Phagocytic activity of monocytes and granulocytes and respiratory burst in phagocytes of male and female rats in feeding trials D and E Parameter Male rats Female rats Control 11% GMO 33% GMO Control 11% GMO 33% GMO Trial D  Phagocytic activity of monocytes 39.76 ± 11.97 38.83 ± 11.42 32.52 ± 6.33 56.17 ± 10.42 64.08 ± 15.99 48.63 ± 7.13  Phagocytic activity of granulocytes 64.72 ± 6.05 63.10 ± 2.99 59.63 ± 5.05 64.99 ± 5.01 73.37 ± 4.82* 62.93 ± 7.39  Respiratory burst in phagocytes 67.38 ± 5.57 68.39 ± 2.61 62.16 ± 5.04 67.24 ± 6.46 76.25 ± 3.82* 65.80 ± 6.84 Trial E  Phagocytic activity of monocytes 31.14 ± 5.79 38.38 ± 11.63 32.69 ± 8.20 48.18 ± 9.98 52.57 ± 12.66 42.61 ± 10.04  Phagocytic activity of granulocytes 59.47 ± 9.20 63.35 ± 7.48 61.07 ± 6.39 57.91 ± 14.10 68.54 ± 7.14 66.39 ± 7.35  Respiratory burst in phagocytes 61.10 ± 7.69 65.31 ± 7.52 63.73 ± 7.07 62.00 ± 12.30 70.16 ± 8.29 67.93 ± 6.55 The table lists the percentage of phagocytic monocytes and granulocytes after incubation of the cells with labelled Staphylococcus aureus and the percentage of phagocytes showing respiratory burst after incubation of the cells with dihydroethidine, expressed as cage mean ± SD (5 cages; n = 10 rats) *Statistically significant difference to the control value based on the 95% confidence interval of the SES from male rats fed the control diet, whereas all other pro- extract is shown in Table 11. The proliferative response of liferative responses did not differ between the rats fed the spleen cells from male rats fed the 11% GMO diet when GMO diets and those fed the control diet. The proliferative incubated with phytohemagglutinin and when incubated response of spleen cells from male and female rats of feed- with 5 µg/ml GM maize protein as well as the prolifera- ing trial E incubated with concanavalin A, phytohemag- tive response of spleen cells from male rats fed the 33% glutinin, pokeweed mitogen, Cry1Ab, the near-isogenic GMO diet when incubated with 50 ng/ml Cry1Ab were non-GM maize protein extract and the GM maize protein significantly lower than that of spleen cells from male rats 1 3 Archives of Toxicology (2018) 92:2385–2399 2395 Table 10 Proliferative response of spleen cells from male and female rats of feeding trial D incubated with concanavalin A, phytohemagglutinin, pokeweed mitogen, Cry1Ab, the near-isogenic non-GM maize protein extract and the GM maize protein extract Parameter Male rats Female rats Control 11% GMO 33% GMO Control 11% GMO 33% GMO IPR concanavalin A 56.96 ± 10.81 55.99 ± 14.07 50.34 ± 22.91 36.92 ± 13.48 34.94 ± 23.27 29.08 ± 16.32 IPR phytohemagglutinin 22.46 ± 3.90 24.43 ± 7.64 16.42 ± 2.74* 16.83 ± 7.02 13.39 ± 5.60 10.19 ± 3.34 IPR pokeweed mitogen 11.74 ± 1.78 13.42 ± 5.43 9.73 ± 1.99 8.81 ± 3.03 11.78 ± 3.76 7.94 ± 3.35 IPR 5 ng Cry1Ab/ml 1.14 ± 0.16 1.06 ± 0.18 0.93 ± 0.20 0.94 ± 0.12 0.86 ± 0.19 1.19 ± 0.72 IPR 50 ng Cry1Ab/ml 1.09 ± 0.15 1.05 ± 0.10 0.92 ± 0.26 0.91 ± 0.13 0.90 ± 0.13 0.84 ± 0.32 IPR 500 ng Cry1Ab/ml 1.11 ± 0.25 1.11 ± 0.15 0.91 ± 0.16 0.98 ± 0.14 0.99 ± 0.09 0.93 ± 0.05 IPR 50 ng non-GM maize protein/ml 1.02 ± 0.14 1.05 ± 0.33 0.98 ± 0.35 1.01 ± 0.13 0.95 ± 0.16 0.98 ± 0.34 IPR 500 ng non-GM maize protein/ml 0.84 ± 0.09 1.10 ± 0.28 0.92 ± 0.43 0.91 ± 0.10 1.05 ± 0.13 1.01 ± 0.21 IPR 5 µg non-GM maize protein/ml 0.65 ± 0.15 0.74 ± 0.09 0.73 ± 0.37 0.87 ± 0.07 0.87 ± 0.08 1.01 ± 0.26 IPR 50 ng GM maize protein/ml 0.96 ± 0.18 0.93 ± 0.21 0.86 ± 0.19 0.92 ± 0.08 0.93 ± 0.18 1.05 ± 0.12 IPR 500 ng GM maize protein/ml 0.80 ± 0.11 0.93 ± 0.19 0.83 ± 0.29 0.93 ± 0.11 0.90 ± 0.10 0.90 ± 0.17 IPR 5 µg GM maize protein/ml 0.84 ± 0.14 0.87 ± 0.21 0.74 ± 0.28 0.85 ± 0.16 0.83 ± 0.12 0.73 ± 0.10 Spleen cells were incubated for 3  days with concanavalin A, phytohemagglutinin or pokeweed mitogen and for 6  days with Cry1Ab, the near isogenic non-GM maize protein extract or the GM maize protein extract in the given amounts. The table lists the indexed proliferative response (IPR) = proliferative response of stimulated cells/proliferative response of non-stimulated cells, expressed as cage mean ± SD (five cages; n = 10 rats, if not otherwise stated) *Statistically significant difference to the control value based on the 95% confidence interval of the SES n = 9 Table 11 Proliferative response of spleen cells from male and female rats of feeding trial E incubated with concanavalin A, phytohemagglutinin, pokeweed mitogen, Cry1Ab, the near-isogenic non-GM maize protein extract and the GM maize protein extract Parameter Male rats Female rats Control 11% GMO 33% GMO Control 11% GMO 33% GMO IPR concanavalin A 69.72 ± 28.69 52.84 ± 9.36 47.57 ± 11.45 33.80 ± 19.33 34.77 ± 19.62 38.36 ± 21.08 IPR phytohemagglutinin 27.69 ± 3.85 21.53 ± 3.06* 21.85 ± 7.39 15.71 ± 9.41 15.95 ± 11.82 18.15 ± 6.69 IPR pokeweed mitogen 14.15 ± 5.21 13.24 ± 2.75 11.93 ± 3.63 9.44 ± 4.91 9.96 ± 7.22 11.46 ± 3.85 IPR 5 ng Cry1Ab/ml 1.02 ± 0.16 1.09 ± 0.23 0.92 ± 0.19 1.93 ± 2.26 0.73 ± 0.20 0.79 ± 0.20 IPR 50 ng Cry1Ab/ml 1.02 ± 0.13 1.04 ± 0.21 0.80 ± 0.12* 1.50 ± 1.26 0.81 ± 0.21 0.82 ± 0.22 IPR 500 ng Cry1Ab/ml 1.10 ± 0.24 1.01 ± 0.24 0.98 ± 0.15 1.31 ± 0.72 0.88 ± 0.33 0.86 ± 0.23 IPR 50 ng non-GM maize protein/ml 0.93 ± 0.11 0.99 ± 0.06 0.91 ± 0.20 0.96 ± 0.10 0.87 ± 0.23 1.12 ± 0.54 IPR 500 ng non-GM maize protein/ml 0.80 ± 0.06 0.86 ± 0.14 0.77 ± 0.19 0.96 ± 0.18 0.92 ± 0.29 0.78 ± 0.18 IPR 5 µg non-GM maize protein/ml 0.77 ± 0.24 0.68 ± 0.20 0.64 ± 0.12 0.98 ± 0.27 0.86 ± 0.14 0.96 ± 0.40 IPR 50 ng GM maize protein/ml 1.00 ± 0.14 0.90 ± 0.09 0.82 ± 0.13 0.88 ± 0.14 0.83 ± 0.23 0.86 ± 0.10 IPR 500 ng GM maize protein/ml 0.85 ± 0.09 0.92 ± 0.11 0.76 ± 0.14 0.99 ± 0.23 0.84 ± 0.17 0.82 ± 0.20 IPR 5 µg GM maize protein/ml 0.97 ± 0.17 0.70 ± 0.16* 0.75 ± 0.09 0.83 ± 0.22 0.83 ± 0.26 0.93 ± 0.26 Spleen cells were incubated for 3  days with concanavalin A, phytohemagglutinin or pokeweed mitogen and for 6  days with Cry1Ab, the near isogenic non-GM maize protein extract or the GM maize protein extract in the given amounts. The table lists the indexed proliferative response (IPR) = proliferative response of stimulated cells/proliferative response of non-stimulated cells, expressed as cage mean ± SD (five cages; n = 10 rats) *Statistically significant difference to the control value based on the 95% confidence interval of the SES fed the control diet. These differences were not observed The cytokine production by spleen cells from male and in female rats and all other proliferative responses did not female rats of the feeding trials D and E incubated with con- differ between the rats fed the GMO diets and those fed canavalin A or Cry1Ab is shown in Tables 12 and 13, respec- the control diet. tively. IL-2, IL-4, IL-10, IL-17A and TNF-α were detected in 1 3 2396 Archives of Toxicology (2018) 92:2385–2399 Table 12 Cytokine production by spleen cells from male and female rats of feeding trial D incubated with concanavalin A or Cry1Ab Parameter Male rats Female rats Control 11% GMO 33% GMO Control 11% GMO 33% GMO a b Interleukin-2 (pg/ml; Con A) 6339 ± 1364 5759 ± 1921 6635 ± 1015 5549 ± 756 5743 ± 1130 5399 ± 871 Interleukin-4 (pg/ml; Con A) 11.99 ± 4.78 12.13 ± 3.99 12.82 ± 5.15 36.86 ± 59.32 19.62 ± 15.78 18.69 ± 12.20 Interleukin-10 (pg/ml; Con A) 4335 ± 2855 4650 ± 4758 3901 ± 2477 2271 ± 1076 2896 ± 2111 2246 ± 1379 Interleukin-17A (pg/ml; Con A) 224 ± 85 304 ± 236 313 ± 179 265 ± 58 421 ± 328 257 ± 120 Tumour necrosis factor-α (pg/ml Con A) 63.97 ± 15.34 56.20 ± 19.12 53.02 ± 7.50 48.76 ± 7.83 53.81 ± 9.33 44.76 ± 11.42 Interleukin-10 (pg/ml; Cry1Ab) 413 ± 165 219 ± 64 388 ± 130 253 ± 70 308 ± 89 269 ± 143 Spleen cells were incubated for 3 days with concanavalin A (Con A) or for 6 days with Cry1Ab. The table lists the amount of cytokines released into the cell culture medium, expressed as cage mean ± SD (five cages; n = 10 rats, if not otherwise stated) n = 9 n = 8 Table 13 Cytokine production by spleen cells from male and female rats of feeding trial E incubated with concanavalin A or Cry1Ab Parameter Male rats Female rats Control 11% GMO 33% GMO Control 11% GMO 33% GMO c b c a Interleukin-2 (pg/ml; Con A) 7022 ± 1105 6291 ± 1373 6786 ± 1327 5096 ± 982 5809 ± 933 6456 ± 861 Interleukin-4 (pg/ml; Con A) 12.70 ± 4.94 9.64 ± 4.35 7.55 ± 1.06 12.95 ± 5.10 17.31 ± 10.32 42.90 ± 40.33 Interleukin-10 (pg/ml; Con A) 3787 ± 2771 2873 ± 1603 3273 ± 1692 2067 ± 1670 3666 ± 3053 2204 ± 1461 Interleukin-17A (pg/ml; Con A) 268 ± 157 239 ± 96 255 ± 148 311 ± 241 321 ± 159 370 ± 148 Tumour necrosis factor-α (pg/ml Con A) 62.23 ± 14.10 52.51 ± 12.51 69.66 ± 20.32 43.51 ± 10.38 47.69 ± 8.36 57.14 ± 16.68 Interleukin-10 (pg/ml; Cry1Ab) 310 ± 45 278 ± 67 392 ± 129 360 ± 187 329 ± 69 537 ± 159 Spleen cells were incubated for 72 h with concanavalin A (Con A) or for 144 h with Cry1Ab. The table lists the amount of cytokines released into the cell culture medium, expressed as cage mean ± SD (5 cages, n = 10 rats, if not otherwise stated; four cages in the case of n = 7 rats) n = 9 n = 8 n = 7 the supernatant of spleen cells incubated with concanavalin A Discussion and IL-10 was present in the supernatant of spleen cells incu- bated with Cry1Ab, whereby their levels did not significantly In the present study, the impact of feeding MON 810 maize differ between the experimental groups in both feeding trials. on the immune responses of rats was assessed by measur- IL-1α, IL-1β, IL-5, IL-6, IL-12p70, IL-13, G-CSF, GM-CSF ing total and specific antibodies to Cry1Ab and maize pro- and TNF-α were below the detection limit of the correspond- teins, phagocytic activity and responses to mitogenic stim- ing assays. The IFN-γ assay did not deliver biologically con- ulation. Regarding a potential immunogenicity of Cry1Ab sistent results in a first step and could not be repeated, since in the MON810 maize, antibodies against Cry1Ab were no samples were available anymore. not produced in the rats fed the MON810 maize at dietary The SES graphs with the complete set of parameters incorporation levels of 11 or 33%. This was also the case measured in the studies D and E are shown in the Supple- in the preliminary experiments, in which the rats were mentary Material (Figs. 2A–D, 3A–D, respectively). A sum- intraperitoneally immunized with the GM maize to obtain mary of the statistically significant parameter differences antisera to develop and validate the immunoassays. These between control and MON810-fed rats in the trials D and E findings are in accordance with a study by Kroghsbo et al. is shown in Table 14. (2008), in which the authors reported that Cry1Ab induced specific immune responses in Wistar rats depending on the route of exposure, i.e. Cry1Ab induced them when inhaled, but not when ingested. In line with this observa- tion, Andreassen et al. (2015a) showed that the intranasal 1 3 Archives of Toxicology (2018) 92:2385–2399 2397 Table 14 Summary of the statistically significant parameter differences between control and MON810-fed rats in the trials D and E Parameter Study D Study E 11% GMO 33% GMO 11% GMO 33% GMO Male Female Male Female Male Female Male Female Anti-maize-specific IgG level ↑ + + % CD3 CD4 cells in the thymus ↓ + + % CD3 CD4 cells in the spleen ↑ % CD3 cells in the spleen ↑ % CD3 cells in the spleen ↓ Phagocytic activity of granulocytes ↑ Respiratory burst in phagocytes ↑ Proliferative response of spleen cells to phytohemagglutinin ↓ ↓ Proliferative response of spleen cells to MON810 maize protein ↓ Proliferative response of spleen cells to Cry1Ab ↓ instillation of Cry1Ab in BALB/c mice resulted in the pro- maize variety, to laboratory animals. In particular, an duction of Cry 1Ab-specific IgE and IgG1 antibodies. In increased antibody response against an unrelated protein another study, no Cry1Ab-specific immune response was (i.e. ovalbumin) was observed (Vázquez-Padrón et al. 1999; induced after the intragastric administration of Cry1Ab or González-González et al. 2015; Moreno-Fierros et al. 2015). the intragastric sensitization with the MON810 maize vari- In contrast, the adjuvant effect of Cry1Ab on ovalbumin ety DKC6575 in combination with a mucosal Th2 adjuvant was not observed in BALB/c mice after airway exposure in BALB/c mice (Adel-Patient et al. 2011). to extracts of MON810 pollen/leaf or trypsinized Cry1Ab Regarding a potential adjuvant effect of Cry1Ab, i.e. the (Andreassen et al. 2015b). Thus, the issue of adjuvantic- capacity to enhance the immunogenicity of and induce the ity seems to be related to the exposure conditions and, par- sensitisation to an unrelated protein with which it is co- ticularly, to the administered doses, although very little is administered, Guimaraes et al. (2008) showed that the oral known regarding the dose–response relationship to induce administration of Cry1Ab did not increase the sensitization this effect. to peanut proteins but observed a possible impact on the In trial E, the increase in the percentage of CD3 cells + + elicitation of the allergic reaction in the BALB/c mouse and CD3 CD4 cells as well as the decrease in the percent- model. Adel-Patient et al. (2011) observed a significant pro- age of CD3 cells in the spleen were restricted to female duction of IgE and IgG1 antibodies specific to maize pro- rats fed the 33% GMO diet, but not observed in any other teins induced after intragastric sensitization with an extract experimental group, and are not indicative of any patho- of MON810 maize in the presence of a mucosal Th2 adju- physiological process, i.e. are of no toxicological relevance. vant in BALB/c mice when compared to PBS-treated mice. In this context, it should be noted that no histopathological However, there were no differences in the IgE and IgG1 changes were observed in the spleens of the male and female antibody responses to maize proteins between mice treated rats fed the 11% GMO and 33% GMO diets in Trials D and with MON 810 or the conventional maize. In the present E (Schmidt et al. 2017). The possibility that the MON810 study, a statistically significant alteration in the anti-maize maize could affect the phagocytic activity of granulocytes protein antibody response was observed in male rats fed 11% and/or with the respiratory burst of phagocytes after incu- GMO in trial E, but this increase was not observed at the bation with Staphylococcus aureus bacteria was also ana- 33% dietary incorporation level. Hence, the results obtained lyzed. Only the percentage of phagocytic granulocytes and in the feeding trials D and E confirm that Cry1Ab does not the percentage of phagocytes showing a respiratory burst exert an allergenic or an adjuvant activity at the concentra- were statistically higher in female rats fed the 11% GMO diet tions at which it is expressed in the two tested cultivars. than in the control group in study D, but these alterations A systemic and mucosal adjuvant activity was described were not observed when female rats were fed the 33% GMO after the intraperitoneal, intranasal and intragastric adminis- diet and are thus considered of no toxicological significance. tration (in the latter case in the presence of magnesium–alu- Moreover, to determine whether the MON810 maize could minium hydroxide) of a high dose of Cry1Ac, a Bacillus interfere with the ability of spleen cells to undergo a clonal thuringiensis protein that is structurally and functionally proliferation when stimulated in vitro with concanavalin A, similar to Cry1Ab but is not expressed in the MON810 phytohemagglutinin, pokeweed mitogen, Cry1Ab, a near 1 3 2398 Archives of Toxicology (2018) 92:2385–2399 Masako Toda, Zoe Waibler and Stefan Vieths (Paul-Ehrlich-Institut, isogenic non-GM maize protein extract or a GM maize Langen, Germany) for their valuable comments to the manuscript. protein extract, lymphocyte proliferation assays were per- Furthermore, the authors are particularly grateful to a broad range formed. It has to be noted that alterations in the proliferative of stakeholder representatives that attended the GRACE workshops, response of spleen cells were sporadically observed and, engaged in discussions and provided valuable comments in writing on draft study plans as well as on the study results, their draft interpreta- if so, were either not reproduced in both trials, were only tions and conclusions. observed with one out of five stimuli and/or did not occur concentration-dependently, so that they are considered to Compliance with ethical standards be of no toxicological relevance. The only cytokine to be decreased after an incubation of spleen cells from male and Conflict of interest Kerstin Schmidt provides consulting services in female rats with Cry1Ab was interleukin-2 in female rats the field of biostatistics and has advised National and European Au- fed the 11% GMO diet in Trial E. This alteration was not thorities, biotech and pharmaceutical companies as well as research institutions, also in the context of GMO risk assessment. observed in female rats fed the 33% GMO diet in Trial E and not observed at all in the Trial D, so that it is considered to Open Access This article is distributed under the terms of the Crea- be of no toxicological significance. tive Commons Attribution 4.0 International License (http://creat iveco Taken together, only single parameters were sporadically mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- altered in rats fed the MON810 maize when compared to tion, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the control rats, and these alterations are considered to be of no Creative Commons license, and indicate if changes were made. immunotoxicological significance. However, a long-term and continuous airway exposure to small amounts of the Cry1Ab protein could occur in workers handling genetically modified plants expressing this protein References in farms and factories. Information regarding the immu- nological effects of long-term airway exposure to Cry1Ab Adel-Patient K, Guimaraes VD, Paris A, Drumare MF, Ah-Leung is limited. Therefore, it would be advisable to investigate S, Lamourette P, Nevers MC, Canlet C, Molina J, Bernard H, immune responses in workers at a risk of airway exposure Créminon C, Wal JM (2011) Immunological and metabolomic impacts of administration of Cry1Ab protein and MON 810 maize to Cry1Ab protein. in mouse. PLoS ONE 6:e16346 Our data are in accordance with the conclusions and rec- Andreassen M, Rocca E, Bøhn T, Wikmark O-G, van den Berg J, Løvik ommendations provided by the GRACE project (http://www. M, Traavik T, Nygaard UC (2015a) Humoral and cellular immune grace-fp7.eu ), i.e. there is no indication that the performance responses in mice after airway administration of Bacillus thur- ingiensis Cry1Ab and MON810 cry1Ab-transgenic maize. Food of 90-day feeding studies with whole food/feed would pro- Agric Immunol 26:521–537 vide additional information on the safety of the GM maize Andreassen M, Bøhn T, Wikmark O-G, van den Berg J, Løvik M, Traa- MON810 if compared to the compositional analysis of the vik T, Nygaard UC (2015b) Cry1Ab protein from Bacillus thur- GM line and its conventional counterpart (i.e. the genetically ingiensis and MON810 cry1Ab-transgenic maize exerts no adju- vant effect after airway exposure. Scand J Immunol 81:192–200 closest non-GM comparator) in terms of an initial safety EFSA Scientific Committee (2011) EFSA guidance on conducting assessment. repeated-dose 90-day oral toxicity study in rodents on whole food/ In line with the GRACE transparency policy, any inter- feed. EFSA J 9: 2438 ested person will have access to the raw data of studies D Finamore A, Roselli M, Britti S, Monastra G, Ambra R, Turrini A, Mengheri E (2008) Intestinal and peripheral immune response to and E obtained in the frame of the GRACE project through MON810 maize ingestion in weaning and old mice. J Agric Food an internet portal named CADIMA (Central Access Data- Chem 56:11533–11539 base for Impact Assessment of Crop Genetic Improvement González-González E, García-Hernández AL, Flores-Mejía R, López- Technologies; http://www.cadim a.info). Santiago R, Moreno-Fierros L (2015) The protoxin Cry1Ac of Bacillus thuringiensis improves the protection conferred by intranasal immunization with Brucella abortus RB51 in a mouse Acknowledgements This study was carried out as part of the GRACE model. Vet Microbiol 175:382–388 project (“GMO Risk Assessment and Communication of Evidence”), Gu J, Krogdahl A, Sissener NH, Kortner TM, Gelencser E, Hemre G-I, financially supported by the Seventh Framework Programme of the Bakke AM (2013) Effects of oral Bt-maize (MON810) exposure European Community for Research, Technological Development on growth and health parameters in normal and sensitized Atlantic and Demonstration Activities (FP7), Grant agreement no. 311957, salmon, Salmo salar L. Br J Nutr 109:1408–1423 the Dutch Ministry of Economic Affairs and the ITMS Project no. Guerrero GG, Dean DH, Moreno-Fierros L (2004) Structural impli- 26240120033 in the frame of the Operational Research and Devel- cation of the induced immune response by Bacillus thuringien- opment Program of the European Regional Development Fund. The sis Cry proteins: role of the N-terminal region. Mol Immunol analyses of maize and diets were performed by RIKILT Wageningen 41:1177–1183 UR and INRA as partners of the GRACE consortium as well as the Guerrero GG, Russell WM, Moreno-Fierros L (2007) Analysis of the companies Covance and Mucedola contracted by GRACE. We thank cellular immune response induced by Bacillus thuringiensis Cry Helena Nagyova and Edita Mrvikova at the Slovak Medical University for their excellent technical support. The authors would like to thank 1 3 Archives of Toxicology (2018) 92:2385–2399 2399 1A toxins in mice: effect of the hydrophobic motif from diphtheria in Atlantic salmon, Salmo salar L., fed different levels of geneti- toxin. Mol Immunol 44:1209–1217 cally modified maize (Bt maize), compared with its near-iso- Guimaraes V, Drumare MF, Ah-Leung S, Lereclus D, Bernard H, genic parental line and a commercial suprex maize. J Fish Dis Créminon C, Wal JM, Adel-Patient K (2008) Comparative study 30:201–212 of the adjuvanticity of Bacillus thuringiensis Cry1Ab protein and Schmidt K, Schmidtke J, Schmidt P (2015) Statistical analysis report on cholera toxin on allergic sensitisation and elicitation to peanut. a repeated dose 90-oral toxicity/longitudinal study in rodents with Food Agric Immunol 19:325–337 MON810 maize. http://www.cadim a.info. Accessed 19 Jan 2018 Guimaraes V, Drumare MF, Lereclus D, Gohar M, Lamourette P, Schmidt K, Schmidtke J, Kohl C, Wilhelm R, Schiemann J, van der Nevers MC, Vaisanen-Tunkelrott ML, Bernard H, Guillon B, Voet H, Steinberg P (2016) Enhancing the interpretation of sta- Créminon C, Wal JM, Adel-Patient K (2010) In vitro digestion of tistical P values in toxicology studies: implementation of linear Cry1Ab proteins and analysis of the impact on their immunore- mixed models (LMMs) and standardized effect sizes (SESs). Arch activity. J Agric Food Chem 58:3222–3231 Toxicol 90:731–751 Hill M, Launis K, Bowman C, McPherson K, Dawson J, Watkins J, Schmidt K, Schmidtke J, Schmidt P, Kohl C, Wilhelm R, Schiemann Koziel M, Wright MS (1995) Biolistic introduction of a synthetic J, van der Voet H, Steinberg P (2017) Variability of control data Bt gene into elite maize. Euphytica 85:119–123 and relevance of observed group differences in five oral toxicity Kroghsbo SK, Madsen C, Poulsen M, Schrøder M, Kvist PH, Taylor studies with genetically modified maize MON810 in rats. Arch M, Gatehouse A, Shu Q, Knudsen I (2008) Immunotoxicological Toxicol 91:1977–2006 studies of genetically modified rice expressing PHA-E lectin or Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Bt toxin in Wistar rats. Toxicology 245:24–34 Zeigler DR, Dean DH (1998) Bacillus thuringiensis and its pesti- Miranda R, Zamudio FZ, Bravo A (2001) Processing of Cry1Ab delta- cidal crystal proteins. Microbiol Rev 62:775–806 endotoxin from Bacillus thuringiensis by Manduca sexta and Spo- Vázquez-Padrón RI, Moreno-Fierros L, Neri-Bazán L, de la Riva doptera frugiperda midgut proteases: role in protoxin activation GA, López-Revilla R (1999) Intragastric and intraperitoneal and toxin inactivation. Insect Biochem Mol Biol 31:1155–1163 administration of Cry1Ac protoxin from Bacillus thuringiensis Moreno-Fierros L, Verdín-Terán SL, García-Hernández AL (2015) induces systemic and mucosal antibody responses in mice. Life Intraperitoneal immunization with Cry1Ac protoxin from Bacil- Sci 64:1897–1912 lus thuringiensis provokes upregulation of Fc gamma II/and III Walsh MC, Buzoianu SG, Gardiner GE, Rea MC, Gelencsér E, Jánosi receptors associated with IgG in the intestinal epithelium of mice. A, Epstein MM, Ross RP, Lawlor PG (2011) Fate of transgenic Scand J Immunol 82:35–47 DNA from orally administered Bt MON810 maize and effects on Nakagawa S, Cuthill IC (2007) Effect size, confidence interval and immune response and growth in pigs. PLoS ONE 6:e27177 statistical significance: a practical guide for biologists. Biol Rev Zeljenková D, Ambrušová K, Bartušová M, Kebis A, Kovrižnych J, Camb Philos Soc 82:591–605 (see Erratum in Biol Rev Camb Krivošíková Z, Kuricová M, Líšková A, Rollerová E, Spustová Philos Soc 84:515 (2009)) V, Szabová E, Tulinská J, Wimmerová S, Levkut M, Révajová OECD (1998) OECD guidelines for the testing of chemicals, Sect. 4, V, Ševčíková Z, Schmidt K, Schmidtke J, La Paz JL, Corujo M, No. 408: repeated dose 90-day oral toxicity study in rodents. https Pla M, Kleter GA, Kok EJ, Sharbati J, Hanisch C, Einspanier R, ://doi.org/10.1787/97892 64070 707-en Adel-Patient K, Wal J-M, Spök A, Pöting A, Kohl C, Wilhelm R, Pradelles P, Grassi J, Maclouf J (1985) Enzyme immunoassays of Schiemann J, Steinberg P (2014) 90-day oral toxicity studies on eicosanoids using acetylcholine esterase as label: an alternative two genetically modified maize MON810 varieties in Wistar Han to radioimmunoassay. Anal Chem 57:1170–1173 RCC rats (EU 7th Framework Programme project GRACE). Arch Sagstad A, Sanden M, Haugland Ø, Hansen A-C, Olsvik PA, Hemre Toxicol 88:2289–2314 G-I (2007) Evaluation of stress-and immune-response biomarkers Affiliations 1 2 2 1 1 1 Jana Tulinská  · Karine Adel‑Patient  · Hervé Bernard  · Aurélia Líšková  · Miroslava Kuricová  · Silvia Ilavská  · 1 3 3 3 3 2 Mira Horváthová  · Anton Kebis  · Eva Rollerová  · Júlia Babincová  · Radka Aláčová  · Jean‑Michel Wal  · 4 4 4,7 5 5 5 Kerstin Schmidt  · Jörg Schmidtke  · Paul Schmidt  · Christian Kohl  · Ralf Wilhelm  · Joachim Schiemann  · 6,8 Pablo Steinberg 1 5 Faculty of Medicine, Slovak Medical University, Limbová Institute for Biosafety in Plant Biotechnology, Julius 12, 83303 Bratislava, Slovakia Kühn-Institut, Federal Research Centre for Cultivated Plants, Erwin-Baur-Str. 27, 06484 Quedlinburg, Germany Service de Pharmacologie et d’Immunoanalyse, Laboratoire d’Immuno-Allergie Alimentaire (LIAA), INRA, CEA, Institute for Food Toxicology and Analytical Chemistry, Université Paris Saclay, DRF/Institut Joliot/SPI-Bat 136, University of Veterinary Medicine Hannover, Bischofsholer CEA de Saclay, 91191 Gif sur Yvette Cedex, France Damm 15, 30173 Hannover, Germany 3 7 Faculty of Public Health, Slovak Medical University, Present Address: Biostatistics (340c), University Limbová 12, 83303 Bratislava, Slovakia of Hohenheim, Fruwirthstr. 23, 70599 Stuttgart, Germany 4 8 BioMath GmbH, Friedrich-Barnewitz-Str. 8, Present Address: Max Rubner-Institut, Federal Research 18119 Rostock-Warnemünde, Germany Institute of Nutrition and Food, Haid-und-Neu-Str. 9, 76131 Karlsruhe, Germany 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Toxicology Springer Journals
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Abstract

The genetically modified maize event MON810 expresses a Bacillus thuringiensis-derived gene, which encodes the insec- ticidal protein Cry1Ab to control some lepidopteran insect pests such as the European corn borer. It has been claimed that the immune system may be affected following the oral/intragastric administration of the MON810 maize in various differ - ent animal species. In the frame of the EU-funded project GRACE, two 90-day feeding trials, the so-called studies D and E, were performed to analyze the humoral and cellular immune responses of male and female Wistar Han RCC rats fed the MON810 maize. A MON810 maize variety of Monsanto was used in the study D and a MON810 maize variety of Pioneer Hi-Bred was used in the study E. The total as well as the maize protein- and Cry1Ab-serum-specific IgG, IgM, IgA and IgE levels, the proliferative activity of the lymphocytes, the phagocytic activity of the granulocytes and monocytes, the respiratory burst of the phagocytes, a phenotypic analysis of spleen, thymus and lymph node cells as well as the in vitro production of cytokines by spleen cells were analyzed. No specific Cry1Ab immune response was observed in MON810 rats, and anti-maize protein antibody responses were similar in MON810 and control rats. Single parameters were sporadi- cally altered in rats fed the MON810 maize when compared to control rats, but these alterations are considered to be of no immunotoxicological significance. Keywords Acquired immunity analysis · Anti-Cry1Ab antibodies · Anti-maize protein antibodies · Cellular immune response · Cry1Ab · Food allergenicity · Genetically modified maize MON810 · GRACE · Humoral immune response · Immune cell phenotyping · Native immunity analysis · OECD Test Guideline no. 408—repeated dose 90-day oral toxicity study in rodents (1998) Introduction The genetically modified (GM) maize event MON810 expresses a Bacillus thuringiensis-derived gene, namely, a truncated cry1Ab gene encoding an insecticidal protein (δ-endotoxin; Schnepf et al. 1998), to control some lep- Jana Tulinská and Karine Adel-Patient contributed equally to the idopteran insect pests such as the European corn borer study. (Ostrinia nubilalis; Hill et al. 1995). Concerns regarding Electronic supplementary material The online version of this potential adverse health effects following the ingestion article (https ://doi.org/10.1007/s0020 4-018-2230-z) contains of the MON810 maize have been raised, and it has been supplementary material, which is available to authorized users. claimed that the immune system in Atlantic salmon (Sag- stad et al. 2007; Gu et al. 2013), mice (Finamore et al. * Pablo Steinberg pablo.steinberg@mri.bund.de 2008; Adel-Patient et  al. 2011) and pigs (Walsh et  al. 2011) may be affected following the oral/intragastric Extended author information available on the last page of the article Vol.:(0123456789) 1 3 2386 Archives of Toxicology (2018) 92:2385–2399 administration of the MON810 maize. Although feeding Materials and methods Wistar rats with a powder diet containing 60% Bt rice for 90 days did not induce the anti-Cry1Ab IgG and IgE Maize varieties and diets antibody production in the animals, feeding Wistar rats with the powder diet containing 60% Bt rice spiked with The feeding trials D and E performed in the frame of the 0.1% of purified Cry1Ab for 28 days led to the detection GRACE project used the same batch of diets as studies A of low levels of anti-Cry1Ab-specific IgG antibodies, but and B (Zeljenková et al. 2014), respectively, but the diets not to detectable levels of IgE antibodies (Kroghsbo et al. were further stored for 10 months at − 21 °C. The maize 2008). Kroghsbo et  al. (2008) suggested that exposure varieties and the diets used are listed in Table 1. A MON810 via inhalation, not ingestion, induced Cry1Ab-specific maize variety of Monsanto was used in the study D and a immune responses in Wistar rats, since the diet was given MON810 maize variety of Pioneer Hi-Bred was used in the to the animals as a powdered preparation, which can easily study E. The MON810 event content in the diets contain- be inhaled. In this context, Guerrero et al. (2004, 2007) ing 11 and 33% of the GM MON810 maize at the DNA reported immunogenic effects of Cry1Ab applied via the and the protein level as well as the average daily amount of intranasal route. This is in line with a study by Andreassen Cry1Ab ingested by the rats are shown in Table 2. The diets et al. (2015a), which showed that the intranasal adminis- containing the conventional maize varieties PR33W82 and tration of purified Cry1Ab resulted in the production of PR32T83 contained very low levels of the MON810 maize anti-Cry1Ab-specific IgG1 and IgE antibodies in BALB/c event (Table 2), consistent with the detection of MON810 mice. in the maize batches used as input material for these diets A key objective of the GRACE (GMO Risk Assessment (Zeljenková et al. 2014). and Communication of Evidence; http://www .g r ace -fp7. eu) project funded by the European Commission within the Rat feeding trials 7th Framework Programme was to conduct 90-day animal feeding trials, animal studies with an extended time frame The 90-day feeding trials D and E were performed at the as well as analytical, in vitro and in silico studies on GM animal housing facility of the Slovak Medical University maize, to provide recommendations on the appropriate- (Bratislava, Slovakia) by taking into account the guidance ness of these tools for the risk assessment of GM crops by for such studies published by the EFSA Scientific Com- considering the scientific strengths and limitations of the mittee in 2011 (EFSA Scientific Committee 2011) and the different approaches. For this purpose, the GM maize vari- OECD Test Guideline 408 (OECD 1998). For this purpose, ety MON810 was chosen. The authors underline that the 5-week-old male and female Wistar Han RCC rats were pur- GRACE project was not expected to provide data for the chased from Harlan (San Pietro al Natisone, Italy). The study reassessment of the safety profile of the MON810 maize design, the performance and the results of the feeding trials variety, but to explore the value of different approaches including studies on the humoral and cellular immune responses in the context of the EU regulation for the risk Table 1 Maize variety content of the different diets used in the rat assessment of whole GM food/feed. feeding trials D and E In the frame of the GRACE project, two 90-day feed- Diet Maize variety content (%) ing trials, the so-called studies D and E, were performed to Feeding trial D analyze the humoral and cellular immune responses of male and female Wistar Han RCC rats to the MON810 maize,  33% near-isogenic non-GM 33% DKC6666 maize whereby a MON810 maize variety of Monsanto was used  11% GMO 11% DKC6667-YG + 22% in the study D and a MON810 maize variety of Pioneer Hi- DKC6666 Bred was used in the study E.  33% GMO 33% DKC6667-YG In the present study, the total as well as the Cry1Ab-spe- Feeding trial E cific and maize protein serum antibody levels were meas-  33% near-isogenic non-GM 33% PR32T16 ured, thereby allowing to determine whether Cry1Ab could maize potentially elicit an immunogenic and/or adjuvant effect  11% GMO11% PR33D48 + 22% PR32T16 in Wistar rats under the chosen experimental conditions.  33% GMO 33% PR33D48 Moreover, the proliferative activity of the lymphocytes, the Near-isogenic maize variety of DKC6667 YG, from Monsanto phagocytic activity of the granulocytes and monocytes, the Transgenic maize variety (MON 810), from Monsanto respiratory burst of the phagocytes, a phenotypic analysis of spleen, thymus and lymph node cells as well as the in vitro Near-isogenic maize variety of PR33D48, from Pioneer Hi-Bred Transgenic maize variety (MON 810), from Pioneer Hi-Bred production of cytokines by spleen cells were analyzed. 1 3 Archives of Toxicology (2018) 92:2385–2399 2387 Table 2 Cry1Ab levels in the different diets used in the rat feeding trials D and E Study D Control 11% GMO 33% GMO 33% DKC6666 11% DKC6667-YG + 22% DKC6666 33% DKC6667-YG MON810 maize event—genetically modified Detected, n.q. 14.6 50.8 DNA (%) Cry1Ab (ng/mg protein) Not detected 0.77 2.83 Average amount of ingested Cry1Ab (µg/rat/day)  Males – 3 11  Females – 2 8 Study E Control 11% GMO 33% GMO 33% PR32T16 11% PR33D48 + 22% PR32T16 33% PR33D48 MON810 maize event—genetically modified Not detected 18.9 47.6 DNA (%) Cry1Ab (ng/mg protein) Not detected 2.01 5.15 Average amount of ingested Cry1Ab (µg/rat/day)  Males – 7 17  Females – 5 13 n.q. not quantifiable D and E including the periodical health status observations, extract was quantified using the BCA kit and following the the haematology, clinical biochemistry, gross necropsy and instructions of the manufacturer (Pierce, Thermo Scientific, histopathology findings as well as the corresponding statisti- Rockford, IL, USA). Recombinant Cry1Ab protoxin was cal analyses have previously been published (Schmidt et al. produced in Escherichia coli JM103 carrying the expres- 2015, 2016). These data are freely accessible via https :// sion vector pKK223-3:cry1Ab (kindly provided by D. R. www.cadim a.info/. Zeigler, Bacillus Genetic Stock Center, Ohio State Univer- sity, Columbus, OH) and cleaved with trypsin to yield the Assessment of the humoral immune response Cry1Ab toxin (Miranda et al. 2001; Guimaraes et al. 2010). The obtained Cry1Ab toxin was characterized by electropho- The humoral immune response was analyzed at the Labora- resis, mass spectrometry and specific monoclonal antibod- toire d’Immuno-Allergie Alimentaire of the Institut National ies produced in the lab, as previously described (Guimaraes pour la Recherche Agronomique (INRA, Gif sur Yvette et  al. 2010; Adel-Patient et al. 2011). No endotoxin was Cedex, France). detected in protoxin/toxin preparations, as determined by the Lumulus Amebocyte Lysate test (Sigma-Aldrich Chimie, Proteins, antibodies and reagents Lyon, France). To validate the immunoassays and to produce an internal Enzyme immunometric assays were performed in 96-well standard for anti-maize-/anti-Cry1Ab-specific Ab immu- microtiter plates (Immunoplate Maxisorb , Nunc, Roskilde, noassays, plasma from naïve Wistar Han RCC rats as well Denmark) using the AutoPlate Washer and Microfill Micro- as from non-GM maize-, MON810 maize- and Cry1Ab- plate Dispenser equipment from BioTek Instruments (Avan- immunized 6-week-old female Wistar Han RCC rats was tec, Rungis, France). obtained. Rats were purchased from the Harlan Laboratories Maize flour from near-isogenic non-GM maize (Gannat, France) and bred under standard SPF conditions (DKC6666) and MON810 maize (DKC6667-YG) was (autoclaved bedding and sterile water). All animal experi- suspended in 50 mM carbonate buffer (pH 9.6) contain- ments were performed according to European Community ing 0.05% Tween and 0.05% dithiothreitol (DTT). For the rules of animal care and with authorization no. 91-368 of protein extraction, 4 ml of extraction buffer per 500 mg of the French Veterinary Services. All experiments were cov- maize flour were used. After an incubation at 20 °C for 2 h ered by agreement No. 2009-DSV-074 from the Veterinary on a rotational shaker, extracts were centrifuged (1000×g, Inspection Department of Essonne (France). Rats were 15 min, 4 °C). The lipid layer was removed and the super- acclimated for 2 weeks before experimentation and were natants containing the extracted proteins were collected and then kept naïve or were immunized with near-isogenic non- dialyzed against the 50 mM carbonate buffer (pH 9.6) to GM maize (DKC6666), MON810 maize (DKC6667-YG) or remove Tween and DTT. The total protein content of the purified Cry1Ab protoxin (n = 3 rats/group). The Cry1Ab 1 3 2388 Archives of Toxicology (2018) 92:2385–2399 protoxin was previously shown to be highly immunogenic determined by addition of 200 µl/well of Ellman’s reagent (Adel-Patient et al. 2011). Immunization was performed via as an enzyme substrate and the absorbance was measured the i.p. route using 100 µg of purified Cry1Ab or 500 µg of at 414 nm (Pradelles et al. 1985) using automatic reader protein extracted from the maize varieties and alum as adju- plates (MultiskanEx, Thermo Electron Corporation, Vantaa, vant (Alhydrogel 2%, Eurobio, Les Ulis, France). After three Finland). Results are expressed as absorbance unit at 414 nm administrations 2 weeks apart, rats were anesthetized with (mAbs414 ). nm Isoflurane Belamont (Nicholas Piramal Ltd., London, UK) Pre-selected antibodies and concentrations were then and blood obtained via aorta puncture. After a centrifuga- used in sandwich immunoassays. Different anti-isotype anti- tion at 1200×g for 20 min, plasma samples were aliquoted bodies were passively immobilized for 18 h at 4 °C (5 µg/ and kept at − 20 °C until used. A plasma pool per group was ml in 50 mM phosphate buffer pH 7.4, 100 µl/well). After also prepared. washing and saturation in EIA buffer, standard isotypes were added (0–100 ng/ml, in EIA buffer) for 18 h at 4 °C. After Development of immunoassays to measure total IgG, IgE, extensive washing, biotinylated antibodies were added and IgA and IgM levels in rat plasma the binding of the antibodies was evaluated as mentioned before. Specificity/cross-reactivity was assessed by testing Total antibodies were assessed as two-site immunometric the different standard isotypes in each specific assay, while assays using a first specific antibody as capture antibody and intra- and inter-assay variability was assessed by reproduc- a second labelled antibody as tracer. Commercial antibodies ing the same assay on different plates on the same day or on were selected based on availability and literature (Table 3), two separate days (1 week apart). In a final step, to assess the and were tested as capture antibodies or biotinylated to be parallel course of the curves for the standards and the plasma used as tracer antibodies. samples, plasma samples from naïve and immunized rats In a first step, commercial standard isotypes were directly were tested using serial dilutions in parallel to the isotype immobilized to test the specificity/cross-reactivity and func- standard. Nonspecific binding (NSB) was determined using tionality of each biotinylated antibody and to select the anti- dilution buffer instead of standard/serum. bodies to be further used. Various concentrations of standard isotypes (IgA kappa clone IR22, IgE kappa clone IR162, Development of immunoassays to measure maize protein IgM kappa clone IR473 and polyclonal IgG, purified from and Cry1Ab‑specific IgG, IgE, IgA and IgM levels in rat rat sera; all from Bio-Rad AbD Serotec, Oxford, UK) were plasma then passively immobilized onto microtiter plates (0.01 to 1 µg/ml, 50 mM phosphate buffer pH 7.4, 100 µl/well; 18 h at Specific antibodies were assessed on plates coated with + 4 °C). After washing the microtiter plates (washing buffer: maize protein extracts or purified Cry1Ab (5 µg/ml, diluted 0.01 M phosphate buffer, pH 7.4, containing 0.05% Tween in 50 mM phosphate buffer pH 7.4, 100 µl/well). For vali- 20) and saturation in EIA buffer (0.1 M phosphate buffer, dation, serial dilutions of individual plasma samples/pool 0.1% bovine serum albumin, 0.15 M NaCl, 0.01% sodium plasma from naïve or immunized rats were performed in azide), 100 µl of biotinylated antibodies (EZ-Link Sulfo- EIA buffer and applied to coated plates for 18 h at 4 °C. NHS-LC-Biotin; Pierce, Rockford, IL; biotin:antibody molar After washing, biotinylated antibodies were applied for 3 h ratio = 20) were added to the microtiter plates (10–100 ng/ml at room temperature and the binding of the antibodies was in EIA buffer) and incubated for 3 h at 20 °C. After wash- evaluated as mentioned before. No anti-maize-specific IgE ing, acetylcholinesterase (AChE)-labelled streptavidin was or IgA was detected in the plasma from maize-immunized added for 15 min at 20 °C. Microtiter plates were then exten- rats and no anti-Cry1Ab-specific antibodies were detected in sively washed and solid phase-bound AChE activity was rats immunized with MON810 maize. Intra and inter-assay Table 3 Antibodies tested Antibody Origin Type/clone Reference no./source for the development of the immunoassays Anti-rat IgA heavy chain Mouse Monoclonal MARA-1 MCA191/AbD Serotec Anti-rat IgM Mouse Monoclonal MARM-4 MCA189/AbD Serotec Anti-rat IgE Mouse Monoclonal MARE-1 MCA193/AbD Serotec Anti-rat IgE Sheep Polyclonal STAR109/AbD Serotec Anti-rat IgG F(c) Rabbit Polyclonal OARA05389/Aviva Systems Biology Anti-rat IgG Goat Polyclonal STAR71/AbD Serotec Anti-rat κ/λ Mouse Monoclonal MARK-1/MARL-15 SUN202/AbD Serotec 1 3 Archives of Toxicology (2018) 92:2385–2399 2389 variabilities were assessed as mentioned before. Standard measured in duplicates and by making use of forward and curves for anti-maize- or anti-Cry1Ab-specific IgG and side scatter gates. anti-maize- or anti-Cry1Ab-specific IgM were plotted using serial dilutions of the corresponding plasma from immu- Phagocytic activity of granulocytes and monocytes, nized rats (8 points). An arbitrary value of 100 (100 AU) and respiratory burst of phagocytes was assigned to pooled sera from maize protein-/Cry1Ab- immunized rats diluted 1/1000 (IgM) or 1/10,000 (IgG). Thirty microliters of rat heparinized whole blood were pipet- ted into a tube and 10 µl of a working solution of hydro- ethidine (dihydroethidium bromide, HE; Polysciences, War- Assessment of total and specific antibodies in plasma rington, PA) were then added. The HE working solution was from the maize‑fed rats in trials D and E prepared by adding 10 µl of HE stock solution (15.75 mg HE in 5 ml dimethylformamide; Merck, Kenilworth, NJ) to After optimization, validation and preliminary experi- 1 ml Medium 199 (Gibco, Invitrogen, Paisley, UK). Samples ments with randomly selected maize-fed rat plasma sam- were incubated for 15 min at 37 °C. Three microliters of ples (n = 10), immunoassays for total and specific antibodies fluorescein-labelled Staphylococcus aureus bacteria (Molec- were performed with the plasma samples from the different ular Probes, Eugene, OR) was added to each of the “test” rat groups in trials D and E. Standard curves were included tubes (1.4 × 10 bacteria per test). All tubes were incubated in each microtiter plate and all samples were analyzed in a for another 15 min at 37 °C. Samples were put on ice and randomized and blinded manner. All analyses were repeated 700 µl of the cold lysis solution described above were added once. For total antibodies, internal controls (IC, pool of sera for 10 min to lyse red blood cells. In the case of the “control” from naive/immunized rats) were also included in each micr- tubes, the Staphylococcus aureus bacteria were added after otiter plate. the lysis solution. Samples were analyzed using an EPICS XL flow cytometer (Beckman Coulter). The percentage of Assessment of the cellular immune response phagocytic monocytes and phagocytic granulocytes (i.e. those that had phagocytised the fluorescein-labelled Staphy - The cellular immune response was analyzed at the Slovak lococcus aureus bacteria) and the percentage of granulocytes Medical University (Bratislava, Slovakia). with respiratory burst (i.e., those in which dihydroethidine was converted to a fluorescent metabolite by reactive oxygen Phenotypic analysis of spleen, mesenteric lymph nodes, species) were measured in duplicates and by making use of bone marrow and thymus forward and side scatter gates. Nine microliters of a mixture of labelled monoclonal Proliferative activity of lymphocytes antibodies were added to 90  µl of a spleen, mesenteric lymph node, bone marrow and thymus cell suspension The spleen was removed using sterile instruments and placed (2 × 10  cells/ml) and incubated for 30 min at 4 °C in the in sterile RPMI 1640 culture medium with L-glutamine dark. The following antibodies, all purchased from eBiosci- and HEPES buffer (Sigma-Aldrich, St. Louis, MO) sup- ence (San Diego, CA), were used to stain the cells: Anti-Rat plemented with 5 IU heparin/ml (Zentiva, Prague, Czech CD3 FITC, Anti-Rat CD4 PE, Anti-Rat CD8a PerCP-eFluor Republic) and 12 µg gentamycin/ml (Sandoz, Basel, Swit- 710, Anti-Rat CD45R PE and Anti-Rat CD161 PerCP- zerland). Spleen cells were obtained under sterile conditions eFluor 710. Isotype controls (Mouse IgG3 Isotype Control- by washing the spleen with RPMI medium and by making FITC, Mouse IgG2a K Isotype Control-PE, Mouse IgG1 use of a syringe with a needle. The spleen cell suspension K Isotype Control-PerCP-eFluor 710 and Mouse IgG2b was centrifuged at 130×g for 15 min and resuspended in K Isotype Control-PE) were used as negative controls to complete RPMI medium containing 10% foetal calf serum determine background fluorescence. Red blood cells were (FCS, PAA, Linz, Austria). Cell suspensions (2 × 10  cells/ lysed with a lysis solution for 15 min and, n fi ally, phosphate- ml) were dispensed in triplicate wells (150  µl/well) of buffered saline (PBS) was added. The lysis solution was a 96-well microtiter culture plate. The mitogens, all pur- prepared by adding 0.829 g NH Cl (Lachema, Brno, Czech chased from Sigma-Aldrich, were added at the following Republic), 0.1  g KHCO and 0.0037  g Na EDTA (both final concentrations: concanavalin A (Con A; 2.5 µg/ml), 3 2 from Sigma-Aldrich) to 100 ml aqua ad injectionem (Imuna phytohemagglutinin (PHA; 25 µg/ml) and pokeweed mito- Pharm, Šarišské Michaľany, Slovakia). Samples were ana- gen (PWM; 2.5  µg/ml). Moreover, recombinant Cry1Ab lyzed using an EPICS XL flow cytometer (Beckman Coul- at final concentrations of 5, 50 and 500 ng/ml and protein + + + + + ter). The percentage of CD3 , CD3 CD4, CD3 CD8a , extracts from near-isogenic non-GM and MON810 maize − + − + CD3 CD161 and CD3 CD45R cells in each sample was at final concentrations of 50 ng/ml, 500 ng/ml and 5 µg/ml 1 3 2390 Archives of Toxicology (2018) 92:2385–2399 were added to additional cell culture plates. Recombinant based on the interpretation of the calculated SES estimates Cry1Ab as well as the maize protein extracts were obtained (Fig. 1). as described above. The plates with mitogens were incubated for 48 h and those with maize antigens and CryAb toxin for 6 days at 37 °C and 5% C O . Thereafter, each well was Results pulsed with 1 µCi [ H]-thymidine (Moravek Biochemicals, Brea, CA) diluted in 20 µl medium and the plates were incu- Humoral immune response bated at 37 °C for another 24 h. The cell cultures were then harvested on glass filtre papers and the filtres were placed The antibodies selected for the various immunoassays are in scintillation fluid (Perkin Elmer, Waltham, MA). Radio- indicated in Table 4. Typical IgE standard curves showed activity was measured using a Beta Scintillation counter an excellent intra- and inter-assay reproducibility (Supple- Microbeta 2 (Perkin Elmer). Counts per minute (cpm)/cell mentary Material, Fig. 1). Moreover, a good parallel course culture were measured in triplicates for each variable. The and reproducibility of the curves for the standards and index was calculated as the ratio of cpm/cell culture in wells diluted plasma were also observed for total IgM and IgA stimulated with a mitogen/a protein extract and cpm/cell cul- (Fig. 2), demonstrating that the calculated concentrations ture in unstimulated wells. do not depend on the plasma dilution and the experimental day. The limits of detection, determined as the means of In vitro production of cytokines NSB + 3σn − 1, were 98 pg/ml for IgE, 69 pg/ml for IgG and 7.8 ng/ml for IgA and IgM. Specific IgG and IgM immuno- Spleen cells were incubated in complete RPMI medium as assays were further validated and standardized by making described above. One hundred and fifty microliters of the use of a pool of plasma samples from rats immunized with spleen cell suspension (2 × 10  cells/ml) were dispensed in maize or Cry1Ab. The sensitivity and reproducibility of the triplicate wells of a 96-well microtiter culture plate. The corresponding assays were high, as exemplarily shown for mitogen Con A (2.5 µg/ml) or the Cry 1Ab toxin (50 ng/ml) IgG in Fig. 3. Preliminary experiments using some randomly were added in a volume of 50 µl. The plates were incubated selected plasma samples from rats fed MON810 maize were at 37 °C in the presence of 5% C O for 72 or 144 h, respec- performed, thereby allowing to determine the dilutions to be tively. Thereafter, the supernatants were removed and stored used for the analysis of the whole set of samples (Table 5). at − 70 °C. The Pr ocartaPlex Rat Th Complete Panel (14 plex) from eBioscience was used to measure the levels of interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A, interferon-γ (IFN-γ), granulo- cyte-colony-stimulating factor (G-CSF), granulocyte–mac- rophage colony-stimulating factor (GM-CSF) and tumour necrosis factor-α (TNF-α) in spleen cell culture supernatants by following the instructions of the manufacturer and by making use of a Luminex 200 apparatus (Luminex, Madi- son, WI, USA). Fig. 1 Simplified version of a graph allowing visual assessment of Statistics statistical significance as well as the supposed biological and possi- ble toxicological relevance of group comparisons. The standard effect The immunological parameters were analyzed descriptively size point estimate (circle) and the 95% confidence limits (whiskers, bars show confidence interval) illustrate the (standardized) effect (N, mean, median, standard deviation, minimum, maximum, size between two groups. The vertical black line indicates no statisti- 95% confidence interval of the mean). Standardized effect cally significant difference (zero difference), while the vertical grey sizes (SES: difference in means between two groups divided lines indicate the supposed biological and possible toxicological rel- by the pooled standard deviation [SD]) as well as their 95% evance limits (here ± 1.0 SD, according to the study design). If the confidence interval bars cross the zero line but not the grey lines cond fi ence intervals were calculated according to Nakagawa (lie within the ± 1.0 limits), there is evidence for no statistical sig- and Cuthill (2007); for details see also Schmidt et al. (2015). nificance as well as no biological relevance (case a). Two groups are The GMO groups were compared to the control groups: significantly different when the confidence interval bars do not cross GMO11%-control and GMO33%-control. All immunologi- the black vertical line (cases b, c). The effect size between two groups is supposed to be potentially relevant, when the confidence interval cal parameters were displayed in one SES graph and the bars lie outside the ± 1.0 SD limits (case c). Case b indicates statisti- data are expressed as the cage mean ± SD. In this paper, cal significance, but no clear biological relevance. Case d indicates no when comparing the different parameters between a control statistical significance, but no clear negation of biological relevance. and a second group, the wording “significantly different” is This figure is Fig. 1 of the study by Zeljenková et al. (2014) 1 3 Archives of Toxicology (2018) 92:2385–2399 2391 Table 4 Antibodies selected for Isotype Antibodies for the total immunoglobulin immunoassays Antibodies for the specific the total and specific IgE, IgG, immunoglobulin immunoas- IgA and IgM immunoassays says Capture antibody Labelled antibody Labelled antibody IgE STAR109 MARE-1 (100 ng/ml) MARE-1 (100 ng/ml) IgG STAR71 STAR71 (50 ng/ml) STAR71 (50 ng/ml) IgA MARA-1 Anti-rat κ/λ (50 ng/ml) MARA-1 (50 ng/ml) IgM MARM-4 Anti-rat κ/λ (50 ng/ml) MARM-4 (50 ng/ml) Fig. 2 Parallelism of the curves for total IgM (a) and IgA (b) or the dilution factor of the different plasma samples (a dilution fac- obtained with the isotype standard (dark blue) and diluted plasma tor of 1 corresponds to an initial dilution factor of 1/1000 for IgM from experimental rats immunized with GM maize (green), conven- and 1/100 for IgA). mAbs414nm, absorbance unit at 414 nm. (Color tional maize (purple), Cry1Ab protoxin (clear blue) or kept naïve figure online) (red). The x-axis represents the concentration of the isotype standard The amount of total IgG, maize-specific IgG and total IgE corresponding control group, whereas no statistically sig- measured in the plasma of male and female rats in the feed- nificant differences regarding all other cell phenotypes and ing trials D and E are shown in Table 6. In study D, the total lymphoid organs between rats fed the control and the GMO plasma IgG, anti-maize-specific IgG and total IgE levels in diets were observed. The phenotypic analysis of spleen, mes- male rats fed the control or GMO diets were similar (i.e. the enteric lymph node, bone marrow and thymus cells of male differences between the groups were not statistically sig- and female rats in the feeding trial E is shown in Table 8. + + + nificant) and this was also the case of the female animals. In The percentages of CD3 and CD3 CD4 cells in the spleen study E, the anti-maize-specific IgG level in plasma of male of female rats fed the 33% GMO diet were significantly rats fed the 11% GMO diet was significantly higher than that higher and, concomitantly, the percentage of CD3 cells in in plasma of male rats fed the control diet, but this was not the spleen of female rats was significantly lower than that of the case in male rats fed the 33% GMO diet (Table 6). All the control group, whereas no statistically significant differ - other parameters were similar in male and female rats. The ences regarding all other cell phenotypes between rats fed anti-maize protein-specific IgE and IgA antibody levels were the control and the GMO diets were observed. below the detection limit in all experimental groups. No Table  9 lists the percentage of phagocytic monocytes anti-Cry1Ab-specific antibodies were detected in any group. and granulocytes after incubation of the cells with labelled Staphylococcus aureus and the percentage of phagocytes Cellular immune response showing respiratory burst after incubation of the cells with dihydroethidine in the feeding trials D and E. The percentage The phenotypic analysis of spleen, mesenteric lymph of phagocytic granulocytes and the percentage of phago- node, bone marrow and thymus cells of male and female cytes showing a respiratory burst were significantly higher rats in the feeding trial D is shown in Table  7. The per- in female rats fed the 11% GMO diet than in the control + + centage of CD3 CD4 cells in the thymus of male rats fed group in study D, while no other statistically significant dif- the 33% GMO diet was significantly lower than that of the ferences regarding the above-mentioned parameters between 1 3 2392 Archives of Toxicology (2018) 92:2385–2399 Fig. 3 Anti-maize- (a) and anti-Cry1Ab-specific IgG (b) in plasma ferent days (series #1 and #2). The x-axis represents arbitrary units from rats immunized with maize protein extract or Cry1Ab, respec- of specific IgG; a value of 100 arbitrary units was assigned to pooled tively. Assays were performed on the same days on up to eight sepa- plasma from maize-/Cry1Ab-immunized rats diluted 1/10,000. mAb- rate plates. Plates were coated with extracts/protein purified on dif- s414nm, absorbance unit at 414 nm Table 5 Dilutions used for Isotype Dilutions used for the determination of the determination of the total, anti-maize-specific and anti- Total antibody levels Anti-maize-specific Anti-Cry1Ab- Cry1Ab-specific antibody levels antibody levels specific antibody in rat plasma levels IgE 1/20 and 1/40 1/10 1/10 Standard from 400 ng/ml (SDF = 4) 6 7 IgG 1/2 × 10 and 1/10 1/100 and 1/500 1/50 and 1/100 Standard from 30 ng/ml (SDF = 3) IgA 1/100 and 1/500 1/10 1/10 Standard from 1000 ng/ml (SDF = 2) IgM 1/2000 and 1/10,000 1/40 1/40 Standard from 1000 ng/ml (SDF = 2) SDF Serial Dilution Factor (standard curves were performed with 8 concentration points) 1 3 Archives of Toxicology (2018) 92:2385–2399 2393 Table 6 Total IgG, anti-maize protein-specific IgG and total IgE levels in plasma of male and female rats in feeding trials D and E Parameter Male rats Female rats Control 11% GMO 33% GMO Control 11% GMO 33% GMO Trial D  Total IgG level (mg/ml) 2.47 ± 0.73 1.95 ± 0.40 2.56 ± 0.36 3.05 ± 0.50 2.73 ± 0.61 2.77 ± 0.53  Anti-maize protein-specific 985 ± 105 1149 ± 157 971 ± 303 1006 ± 342 879 ± 148 1130 ± 197 IgG level (arbitrary units/ ml)  Total IgE level (ng/ml) 38.07 ± 13.48 42.03 ± 14.76 57.72 ± 33.71 61.66 ± 28.68 47.42 ± 15.89 49.98 ± 29.79 Trial E  Total IgG level (mg/ml) 2.32 ± 1.06 2.42 ± 0.67 2.85 ± 0.27 2.97 ± 0.83 3.27 ± 0.49 2.92 ± 0.46  Anti-maize protein-specific 900 ± 221 1287 ± 150* 1682 ± 1210 1294 ± 283 970 ± 163 1234 ± 399 IgG level (arbitrary units/ ml)  Total IgE level (ng/ml) 38.81 ± 15.07 46.63 ± 26.94 41.16 ± 10.62 66.02 ± 24.06 42.63 ± 18.22 42.37 ± 13.45 The results are expressed as cage mean ± SD (five cages, n = 10 rats) *Statistically significant difference to the control value based on the 95% confidence interval of the SES Table 7 Phenotypic analysis of spleen, lymph node, bone marrow and thymus cells of male and female rats in the feeding trial D Parameter Male rats Female rats Control 11% GMO 33% GMO Control 11% GMO 33% GMO + + Spleen CD3 CD8 cells 40.72 ± 8.26 37.35 ± 4.65 35.94 ± 5.99 42.11 ± 4.80 39.68 ± 5.72 37.87 ± 5.85 Spleen CD3 cells 59.51 ± 8.82 55.97 ± 4.07 56.68 ± 5.83 64.72 ± 3.17 63.39 ± 7.69 60.68 ± 6.23 Spleen CD3 cells 40.50 ± 8.82 44.03 ± 4.07 43.32 ± 5.83 35.29 ± 3.17 36.61 ± 7.69 39.33 ± 6.23 + + Spleen CD3 CD4 cells 40.48 ± 7.16 37.20 ± 3.93 34.09 ± 7.79 42.49 ± 4.52 39.70 ± 6.14 38.05 ± 4.96 − + Spleen CD3 CD45R cells 24.16 ± 4.92 29.96 ± 2.17 28.21 ± 3.50 24.66 ± 2.73 25.40 ± 3.48 26.89 ± 5.03 − + Spleen CD3 CD161 cells 19.62 ± 3.63 24.47 ± 2.62 23.44 ± 2.53 18.97 ± 7.95 18.97 ± 6.89 20.62 ± 7.88 + + Lymph node CD3 CD8 cells 28.87 ± 12.57 29.36 ± 17.70 27.93 ± 13.85 41.73 ± 5.70 40.02 ± 5.87 41.62 ± 6.01 Lymph node CD3 cells 41.89 ± 19.48 42.35 ± 20.81 43.97 ± 19.53 56.51 ± 8.53 53.96 ± 9.41 55.92 ± 8.47 Lymph node CD3 cells 58.12 ± 19.48 57.65 ± 20.81 56.03 ± 19.53 43.49 ± 8.53 46.04 ± 9.41 44.08 ± 8.47 + + Lymph node CD3 CD4 cells 26.22 ± 10.05 26.62 ± 16.46 24.46 ± 12.00 38.88 ± 4.09 37.72 ± 4.75 39.03 ± 4.96 − + Lymph node CD3 CD45R cells 52.81 ± 14.43 53.98 ± 16.52 56.80 ± 16.19 40.96 ± 6.49 44.44 ± 6.11 41.08 ± 7.88 + a Bone marrow CD3 cells 7.75 ± 1.05 7.25 ± 3.11 8.22 ± 2.73 17.44 ± 6.17 15.63 ± 5.71 13.27 ± 2.38 − a Bone marrow CD3 cells 92.26 ± 1.05 92.76 ± 3.11 91.79 ± 2.73 82.57 ± 6.17 84.38 ± 5.71 86.74 ± 2.38 − + a Bone marrow CD3 CD45R cells 71.60 ± 7.46 73.45 ± 8.12 70.37 ± 7.28 64.84 ± 12.22 61.87 ± 16.91 61.91 ± 20.71 − + a Bone marrow CD3 CD161 cells 12.77 ± 6.32 13.43 ± 5.25 12.54 ± 5.77 15.55 ± 4.55 13.19 ± 4.20 14.80 ± 5.09 + + Thymus CD3 CD8 cells 21.37 ± 2.63 22.92 ± 3.95 17.57 ± 4.12 19.46 ± 2.67 20.57 ± 3.20 20.49 ± 3.44 Thymus CD3 cells 25.11 ± 5.60 27.22 ± 7.77 21.42 ± 7.41 21.73 ± 2.73 22.90 ± 3.42 22.81 ± 3.74 Thymus CD3 cells 74.90 ± 5.60 72.78 ± 7.77 78.59 ± 7.41 78.27 ± 2.73 77.11 ± 3.42 77.19 ± 3.74 + + Thymus CD3 CD4 cells 20.04 ± 1.31 21.01 ± 2.42 16.40 ± 2.65* 18.95 ± 2.51 19.96 ± 3.05 19.87 ± 3.29 The table lists the percentage of cells with the indicated phenotype, expressed as cage mean ± SD (five cages; n = 10 rats, if not otherwise stated) *Statistically significant difference to the control value based on the 95% confidence interval of the SES n = 9 rats fed the control and the GMO diets were observed in Cry1Ab, the near-isogenic non-GM maize protein extract both studies. and the GM maize protein extract is shown in Table  10. The proliferative response of spleen cells from male The proliferative response of spleen cells from male rats and female rats of feeding trial D after incubation with fed the 33% GMO diet when incubated with phytohemag- concanavalin A, phytohemagglutinin, pokeweed mitogen, glutinin was significantly lower than that of spleen cells 1 3 2394 Archives of Toxicology (2018) 92:2385–2399 Table 8 Phenotypic analysis of spleen, lymph node, bone marrow and thymus cells of male and female rats in the feeding trial E Parameter Male rats Female rats Control 11% GMO 33% GMO Control 11% GMO 33% GMO + + Spleen CD3 CD8 cells 41.51 ± 6.81 41.41 ± 7.60 37.88 ± 9.13 39.41 ± 3.96 42.92 ± 5.48 46.58 ± 5.59 Spleen CD3 cells 60.86 ± 6.82 60.66 ± 8.39 57.16 ± 11.66 62.73 ± 2.76 65.37 ± 2.10 69.69 ± 4.20* Spleen CD3 cells 39.15 ± 6.82 39.35 ± 8.39 42.84 ± 11.66 37.27 ± 2.76 34.63 ± 2.10 30.32 ± 4.20* + + Spleen CD3 CD4 cells 41.62 ± 5.79 41.00 ± 6.27 37.32 ± 8.68 39.27 ± 3.38 43.36 ± 4.96 46.75 ± 4.68* − + Spleen CD3 CD45R cells 24.56 ± 3.99 25.86 ± 3.83 26.09 ± 5.23 25.46 ± 2.21 24.10 ± 1.62 21.21 ± 3.63 − + Spleen CD3 CD161 cells 19.97 ± 3.00 21.46 ± 2.74 21.39 ± 4.25 19.25 ± 8.71 17.59 ± 7.70 16.26 ± 6.40 + + a Lymph node CD3 CD8 cells 33.47 ± 17.51 27.62 ± 15.23 27.29 ± 13.70 42.50 ± 1.96 42.52 ± 5.80 43.69 ± 12.98 + a Lymph node CD3 cells 45.32 ± 20.51 39.68 ± 19.83 42.93 ± 20.25 57.40 ± 3.39 56.51 ± 7.44 58.01 ± 16.84 − a Lymph node CD3 cells 54.69 ± 20.51 60.33 ± 19.84 57.08 ± 20.25 42.60 ± 3.40 43.50 ± 7.44 41.99 ± 16.84 + + a Lymph node CD3 CD4 cells 31.44 ± 15.77 25.98 ± 12.94 23.91 ± 10.76 40.35 ± 2.16 40.79 ± 5.49 41.15 ± 11.03 − + a Lymph node CD3 CD45R cells 47.83 ± 14.58 52.53 ± 14.22 50.88 ± 13.95 38.49 ± 2.57 39.01 ± 5.15 37.67 ± 11.65 Bone marrow CD3 cells 9.27 ± 1.24 11.93 ± 5.95 9.80 ± 2.63 15.44 ± 3.91 12.30 ± 3.58 17.34 ± 5.72 Bone marrow CD3 cells 90.73 ± 1.24 88.08 ± 5.94 90.21 ± 2.63 84.57 ± 3.91 87.70 ± 3.58 82.67 ± 5.73 − + Bone marrow CD3 CD45R cells 69.67 ± 6.22 65.94 ± 5.58 67.79 ± 10.36 65.92 ± 12.48 70.51 ± 11.47 68.53 ± 7.50 − + Bone marrow CD3 CD161 cells 12.73 ± 7.10 11.95 ± 5.63 11.69 ± 4.43 13.22 ± 3.18 13.49 ± 4.01 13.99 ± 2.73 + + Thymus CD3 CD8 cells 22.71 ± 5.24 24.17 ± 5.89 22.27 ± 4.94 22.80 ± 1.41 20.59 ± 3.26 22.42 ± 0.81 Thymus CD3 cells 26.68 ± 8.54 28.11 ± 8.19 26.32 ± 8.17 25.24 ± 1.43 22.81 ± 3.36 24.82 ± 0.85 Thymus CD3 cells 73.32 ± 8.54 71.90 ± 8.19 73.68 ± 8.17 74.76 ± 1.43 77.20 ± 3.36 75.18 ± 0.85 + + Thymus CD3 CD4 cells 21.39 ± 3.79 22.84 ± 4.88 20.62 ± 3.34 22.39 ± 1.34 20.05 ± 2.97 21.92 ± 0.99 The table lists the percentage of cells with the indicated phenotype, expressed as cage mean ± SD (five cages; n = 10 rats, if not otherwise stated) *Statistically significant difference to the control value based on the 95% confidence interval of the SES n = 9 Table 9 Phagocytic activity of monocytes and granulocytes and respiratory burst in phagocytes of male and female rats in feeding trials D and E Parameter Male rats Female rats Control 11% GMO 33% GMO Control 11% GMO 33% GMO Trial D  Phagocytic activity of monocytes 39.76 ± 11.97 38.83 ± 11.42 32.52 ± 6.33 56.17 ± 10.42 64.08 ± 15.99 48.63 ± 7.13  Phagocytic activity of granulocytes 64.72 ± 6.05 63.10 ± 2.99 59.63 ± 5.05 64.99 ± 5.01 73.37 ± 4.82* 62.93 ± 7.39  Respiratory burst in phagocytes 67.38 ± 5.57 68.39 ± 2.61 62.16 ± 5.04 67.24 ± 6.46 76.25 ± 3.82* 65.80 ± 6.84 Trial E  Phagocytic activity of monocytes 31.14 ± 5.79 38.38 ± 11.63 32.69 ± 8.20 48.18 ± 9.98 52.57 ± 12.66 42.61 ± 10.04  Phagocytic activity of granulocytes 59.47 ± 9.20 63.35 ± 7.48 61.07 ± 6.39 57.91 ± 14.10 68.54 ± 7.14 66.39 ± 7.35  Respiratory burst in phagocytes 61.10 ± 7.69 65.31 ± 7.52 63.73 ± 7.07 62.00 ± 12.30 70.16 ± 8.29 67.93 ± 6.55 The table lists the percentage of phagocytic monocytes and granulocytes after incubation of the cells with labelled Staphylococcus aureus and the percentage of phagocytes showing respiratory burst after incubation of the cells with dihydroethidine, expressed as cage mean ± SD (5 cages; n = 10 rats) *Statistically significant difference to the control value based on the 95% confidence interval of the SES from male rats fed the control diet, whereas all other pro- extract is shown in Table 11. The proliferative response of liferative responses did not differ between the rats fed the spleen cells from male rats fed the 11% GMO diet when GMO diets and those fed the control diet. The proliferative incubated with phytohemagglutinin and when incubated response of spleen cells from male and female rats of feed- with 5 µg/ml GM maize protein as well as the prolifera- ing trial E incubated with concanavalin A, phytohemag- tive response of spleen cells from male rats fed the 33% glutinin, pokeweed mitogen, Cry1Ab, the near-isogenic GMO diet when incubated with 50 ng/ml Cry1Ab were non-GM maize protein extract and the GM maize protein significantly lower than that of spleen cells from male rats 1 3 Archives of Toxicology (2018) 92:2385–2399 2395 Table 10 Proliferative response of spleen cells from male and female rats of feeding trial D incubated with concanavalin A, phytohemagglutinin, pokeweed mitogen, Cry1Ab, the near-isogenic non-GM maize protein extract and the GM maize protein extract Parameter Male rats Female rats Control 11% GMO 33% GMO Control 11% GMO 33% GMO IPR concanavalin A 56.96 ± 10.81 55.99 ± 14.07 50.34 ± 22.91 36.92 ± 13.48 34.94 ± 23.27 29.08 ± 16.32 IPR phytohemagglutinin 22.46 ± 3.90 24.43 ± 7.64 16.42 ± 2.74* 16.83 ± 7.02 13.39 ± 5.60 10.19 ± 3.34 IPR pokeweed mitogen 11.74 ± 1.78 13.42 ± 5.43 9.73 ± 1.99 8.81 ± 3.03 11.78 ± 3.76 7.94 ± 3.35 IPR 5 ng Cry1Ab/ml 1.14 ± 0.16 1.06 ± 0.18 0.93 ± 0.20 0.94 ± 0.12 0.86 ± 0.19 1.19 ± 0.72 IPR 50 ng Cry1Ab/ml 1.09 ± 0.15 1.05 ± 0.10 0.92 ± 0.26 0.91 ± 0.13 0.90 ± 0.13 0.84 ± 0.32 IPR 500 ng Cry1Ab/ml 1.11 ± 0.25 1.11 ± 0.15 0.91 ± 0.16 0.98 ± 0.14 0.99 ± 0.09 0.93 ± 0.05 IPR 50 ng non-GM maize protein/ml 1.02 ± 0.14 1.05 ± 0.33 0.98 ± 0.35 1.01 ± 0.13 0.95 ± 0.16 0.98 ± 0.34 IPR 500 ng non-GM maize protein/ml 0.84 ± 0.09 1.10 ± 0.28 0.92 ± 0.43 0.91 ± 0.10 1.05 ± 0.13 1.01 ± 0.21 IPR 5 µg non-GM maize protein/ml 0.65 ± 0.15 0.74 ± 0.09 0.73 ± 0.37 0.87 ± 0.07 0.87 ± 0.08 1.01 ± 0.26 IPR 50 ng GM maize protein/ml 0.96 ± 0.18 0.93 ± 0.21 0.86 ± 0.19 0.92 ± 0.08 0.93 ± 0.18 1.05 ± 0.12 IPR 500 ng GM maize protein/ml 0.80 ± 0.11 0.93 ± 0.19 0.83 ± 0.29 0.93 ± 0.11 0.90 ± 0.10 0.90 ± 0.17 IPR 5 µg GM maize protein/ml 0.84 ± 0.14 0.87 ± 0.21 0.74 ± 0.28 0.85 ± 0.16 0.83 ± 0.12 0.73 ± 0.10 Spleen cells were incubated for 3  days with concanavalin A, phytohemagglutinin or pokeweed mitogen and for 6  days with Cry1Ab, the near isogenic non-GM maize protein extract or the GM maize protein extract in the given amounts. The table lists the indexed proliferative response (IPR) = proliferative response of stimulated cells/proliferative response of non-stimulated cells, expressed as cage mean ± SD (five cages; n = 10 rats, if not otherwise stated) *Statistically significant difference to the control value based on the 95% confidence interval of the SES n = 9 Table 11 Proliferative response of spleen cells from male and female rats of feeding trial E incubated with concanavalin A, phytohemagglutinin, pokeweed mitogen, Cry1Ab, the near-isogenic non-GM maize protein extract and the GM maize protein extract Parameter Male rats Female rats Control 11% GMO 33% GMO Control 11% GMO 33% GMO IPR concanavalin A 69.72 ± 28.69 52.84 ± 9.36 47.57 ± 11.45 33.80 ± 19.33 34.77 ± 19.62 38.36 ± 21.08 IPR phytohemagglutinin 27.69 ± 3.85 21.53 ± 3.06* 21.85 ± 7.39 15.71 ± 9.41 15.95 ± 11.82 18.15 ± 6.69 IPR pokeweed mitogen 14.15 ± 5.21 13.24 ± 2.75 11.93 ± 3.63 9.44 ± 4.91 9.96 ± 7.22 11.46 ± 3.85 IPR 5 ng Cry1Ab/ml 1.02 ± 0.16 1.09 ± 0.23 0.92 ± 0.19 1.93 ± 2.26 0.73 ± 0.20 0.79 ± 0.20 IPR 50 ng Cry1Ab/ml 1.02 ± 0.13 1.04 ± 0.21 0.80 ± 0.12* 1.50 ± 1.26 0.81 ± 0.21 0.82 ± 0.22 IPR 500 ng Cry1Ab/ml 1.10 ± 0.24 1.01 ± 0.24 0.98 ± 0.15 1.31 ± 0.72 0.88 ± 0.33 0.86 ± 0.23 IPR 50 ng non-GM maize protein/ml 0.93 ± 0.11 0.99 ± 0.06 0.91 ± 0.20 0.96 ± 0.10 0.87 ± 0.23 1.12 ± 0.54 IPR 500 ng non-GM maize protein/ml 0.80 ± 0.06 0.86 ± 0.14 0.77 ± 0.19 0.96 ± 0.18 0.92 ± 0.29 0.78 ± 0.18 IPR 5 µg non-GM maize protein/ml 0.77 ± 0.24 0.68 ± 0.20 0.64 ± 0.12 0.98 ± 0.27 0.86 ± 0.14 0.96 ± 0.40 IPR 50 ng GM maize protein/ml 1.00 ± 0.14 0.90 ± 0.09 0.82 ± 0.13 0.88 ± 0.14 0.83 ± 0.23 0.86 ± 0.10 IPR 500 ng GM maize protein/ml 0.85 ± 0.09 0.92 ± 0.11 0.76 ± 0.14 0.99 ± 0.23 0.84 ± 0.17 0.82 ± 0.20 IPR 5 µg GM maize protein/ml 0.97 ± 0.17 0.70 ± 0.16* 0.75 ± 0.09 0.83 ± 0.22 0.83 ± 0.26 0.93 ± 0.26 Spleen cells were incubated for 3  days with concanavalin A, phytohemagglutinin or pokeweed mitogen and for 6  days with Cry1Ab, the near isogenic non-GM maize protein extract or the GM maize protein extract in the given amounts. The table lists the indexed proliferative response (IPR) = proliferative response of stimulated cells/proliferative response of non-stimulated cells, expressed as cage mean ± SD (five cages; n = 10 rats) *Statistically significant difference to the control value based on the 95% confidence interval of the SES fed the control diet. These differences were not observed The cytokine production by spleen cells from male and in female rats and all other proliferative responses did not female rats of the feeding trials D and E incubated with con- differ between the rats fed the GMO diets and those fed canavalin A or Cry1Ab is shown in Tables 12 and 13, respec- the control diet. tively. IL-2, IL-4, IL-10, IL-17A and TNF-α were detected in 1 3 2396 Archives of Toxicology (2018) 92:2385–2399 Table 12 Cytokine production by spleen cells from male and female rats of feeding trial D incubated with concanavalin A or Cry1Ab Parameter Male rats Female rats Control 11% GMO 33% GMO Control 11% GMO 33% GMO a b Interleukin-2 (pg/ml; Con A) 6339 ± 1364 5759 ± 1921 6635 ± 1015 5549 ± 756 5743 ± 1130 5399 ± 871 Interleukin-4 (pg/ml; Con A) 11.99 ± 4.78 12.13 ± 3.99 12.82 ± 5.15 36.86 ± 59.32 19.62 ± 15.78 18.69 ± 12.20 Interleukin-10 (pg/ml; Con A) 4335 ± 2855 4650 ± 4758 3901 ± 2477 2271 ± 1076 2896 ± 2111 2246 ± 1379 Interleukin-17A (pg/ml; Con A) 224 ± 85 304 ± 236 313 ± 179 265 ± 58 421 ± 328 257 ± 120 Tumour necrosis factor-α (pg/ml Con A) 63.97 ± 15.34 56.20 ± 19.12 53.02 ± 7.50 48.76 ± 7.83 53.81 ± 9.33 44.76 ± 11.42 Interleukin-10 (pg/ml; Cry1Ab) 413 ± 165 219 ± 64 388 ± 130 253 ± 70 308 ± 89 269 ± 143 Spleen cells were incubated for 3 days with concanavalin A (Con A) or for 6 days with Cry1Ab. The table lists the amount of cytokines released into the cell culture medium, expressed as cage mean ± SD (five cages; n = 10 rats, if not otherwise stated) n = 9 n = 8 Table 13 Cytokine production by spleen cells from male and female rats of feeding trial E incubated with concanavalin A or Cry1Ab Parameter Male rats Female rats Control 11% GMO 33% GMO Control 11% GMO 33% GMO c b c a Interleukin-2 (pg/ml; Con A) 7022 ± 1105 6291 ± 1373 6786 ± 1327 5096 ± 982 5809 ± 933 6456 ± 861 Interleukin-4 (pg/ml; Con A) 12.70 ± 4.94 9.64 ± 4.35 7.55 ± 1.06 12.95 ± 5.10 17.31 ± 10.32 42.90 ± 40.33 Interleukin-10 (pg/ml; Con A) 3787 ± 2771 2873 ± 1603 3273 ± 1692 2067 ± 1670 3666 ± 3053 2204 ± 1461 Interleukin-17A (pg/ml; Con A) 268 ± 157 239 ± 96 255 ± 148 311 ± 241 321 ± 159 370 ± 148 Tumour necrosis factor-α (pg/ml Con A) 62.23 ± 14.10 52.51 ± 12.51 69.66 ± 20.32 43.51 ± 10.38 47.69 ± 8.36 57.14 ± 16.68 Interleukin-10 (pg/ml; Cry1Ab) 310 ± 45 278 ± 67 392 ± 129 360 ± 187 329 ± 69 537 ± 159 Spleen cells were incubated for 72 h with concanavalin A (Con A) or for 144 h with Cry1Ab. The table lists the amount of cytokines released into the cell culture medium, expressed as cage mean ± SD (5 cages, n = 10 rats, if not otherwise stated; four cages in the case of n = 7 rats) n = 9 n = 8 n = 7 the supernatant of spleen cells incubated with concanavalin A Discussion and IL-10 was present in the supernatant of spleen cells incu- bated with Cry1Ab, whereby their levels did not significantly In the present study, the impact of feeding MON 810 maize differ between the experimental groups in both feeding trials. on the immune responses of rats was assessed by measur- IL-1α, IL-1β, IL-5, IL-6, IL-12p70, IL-13, G-CSF, GM-CSF ing total and specific antibodies to Cry1Ab and maize pro- and TNF-α were below the detection limit of the correspond- teins, phagocytic activity and responses to mitogenic stim- ing assays. The IFN-γ assay did not deliver biologically con- ulation. Regarding a potential immunogenicity of Cry1Ab sistent results in a first step and could not be repeated, since in the MON810 maize, antibodies against Cry1Ab were no samples were available anymore. not produced in the rats fed the MON810 maize at dietary The SES graphs with the complete set of parameters incorporation levels of 11 or 33%. This was also the case measured in the studies D and E are shown in the Supple- in the preliminary experiments, in which the rats were mentary Material (Figs. 2A–D, 3A–D, respectively). A sum- intraperitoneally immunized with the GM maize to obtain mary of the statistically significant parameter differences antisera to develop and validate the immunoassays. These between control and MON810-fed rats in the trials D and E findings are in accordance with a study by Kroghsbo et al. is shown in Table 14. (2008), in which the authors reported that Cry1Ab induced specific immune responses in Wistar rats depending on the route of exposure, i.e. Cry1Ab induced them when inhaled, but not when ingested. In line with this observa- tion, Andreassen et al. (2015a) showed that the intranasal 1 3 Archives of Toxicology (2018) 92:2385–2399 2397 Table 14 Summary of the statistically significant parameter differences between control and MON810-fed rats in the trials D and E Parameter Study D Study E 11% GMO 33% GMO 11% GMO 33% GMO Male Female Male Female Male Female Male Female Anti-maize-specific IgG level ↑ + + % CD3 CD4 cells in the thymus ↓ + + % CD3 CD4 cells in the spleen ↑ % CD3 cells in the spleen ↑ % CD3 cells in the spleen ↓ Phagocytic activity of granulocytes ↑ Respiratory burst in phagocytes ↑ Proliferative response of spleen cells to phytohemagglutinin ↓ ↓ Proliferative response of spleen cells to MON810 maize protein ↓ Proliferative response of spleen cells to Cry1Ab ↓ instillation of Cry1Ab in BALB/c mice resulted in the pro- maize variety, to laboratory animals. In particular, an duction of Cry 1Ab-specific IgE and IgG1 antibodies. In increased antibody response against an unrelated protein another study, no Cry1Ab-specific immune response was (i.e. ovalbumin) was observed (Vázquez-Padrón et al. 1999; induced after the intragastric administration of Cry1Ab or González-González et al. 2015; Moreno-Fierros et al. 2015). the intragastric sensitization with the MON810 maize vari- In contrast, the adjuvant effect of Cry1Ab on ovalbumin ety DKC6575 in combination with a mucosal Th2 adjuvant was not observed in BALB/c mice after airway exposure in BALB/c mice (Adel-Patient et al. 2011). to extracts of MON810 pollen/leaf or trypsinized Cry1Ab Regarding a potential adjuvant effect of Cry1Ab, i.e. the (Andreassen et al. 2015b). Thus, the issue of adjuvantic- capacity to enhance the immunogenicity of and induce the ity seems to be related to the exposure conditions and, par- sensitisation to an unrelated protein with which it is co- ticularly, to the administered doses, although very little is administered, Guimaraes et al. (2008) showed that the oral known regarding the dose–response relationship to induce administration of Cry1Ab did not increase the sensitization this effect. to peanut proteins but observed a possible impact on the In trial E, the increase in the percentage of CD3 cells + + elicitation of the allergic reaction in the BALB/c mouse and CD3 CD4 cells as well as the decrease in the percent- model. Adel-Patient et al. (2011) observed a significant pro- age of CD3 cells in the spleen were restricted to female duction of IgE and IgG1 antibodies specific to maize pro- rats fed the 33% GMO diet, but not observed in any other teins induced after intragastric sensitization with an extract experimental group, and are not indicative of any patho- of MON810 maize in the presence of a mucosal Th2 adju- physiological process, i.e. are of no toxicological relevance. vant in BALB/c mice when compared to PBS-treated mice. In this context, it should be noted that no histopathological However, there were no differences in the IgE and IgG1 changes were observed in the spleens of the male and female antibody responses to maize proteins between mice treated rats fed the 11% GMO and 33% GMO diets in Trials D and with MON 810 or the conventional maize. In the present E (Schmidt et al. 2017). The possibility that the MON810 study, a statistically significant alteration in the anti-maize maize could affect the phagocytic activity of granulocytes protein antibody response was observed in male rats fed 11% and/or with the respiratory burst of phagocytes after incu- GMO in trial E, but this increase was not observed at the bation with Staphylococcus aureus bacteria was also ana- 33% dietary incorporation level. Hence, the results obtained lyzed. Only the percentage of phagocytic granulocytes and in the feeding trials D and E confirm that Cry1Ab does not the percentage of phagocytes showing a respiratory burst exert an allergenic or an adjuvant activity at the concentra- were statistically higher in female rats fed the 11% GMO diet tions at which it is expressed in the two tested cultivars. than in the control group in study D, but these alterations A systemic and mucosal adjuvant activity was described were not observed when female rats were fed the 33% GMO after the intraperitoneal, intranasal and intragastric adminis- diet and are thus considered of no toxicological significance. tration (in the latter case in the presence of magnesium–alu- Moreover, to determine whether the MON810 maize could minium hydroxide) of a high dose of Cry1Ac, a Bacillus interfere with the ability of spleen cells to undergo a clonal thuringiensis protein that is structurally and functionally proliferation when stimulated in vitro with concanavalin A, similar to Cry1Ab but is not expressed in the MON810 phytohemagglutinin, pokeweed mitogen, Cry1Ab, a near 1 3 2398 Archives of Toxicology (2018) 92:2385–2399 Masako Toda, Zoe Waibler and Stefan Vieths (Paul-Ehrlich-Institut, isogenic non-GM maize protein extract or a GM maize Langen, Germany) for their valuable comments to the manuscript. protein extract, lymphocyte proliferation assays were per- Furthermore, the authors are particularly grateful to a broad range formed. It has to be noted that alterations in the proliferative of stakeholder representatives that attended the GRACE workshops, response of spleen cells were sporadically observed and, engaged in discussions and provided valuable comments in writing on draft study plans as well as on the study results, their draft interpreta- if so, were either not reproduced in both trials, were only tions and conclusions. observed with one out of five stimuli and/or did not occur concentration-dependently, so that they are considered to Compliance with ethical standards be of no toxicological relevance. The only cytokine to be decreased after an incubation of spleen cells from male and Conflict of interest Kerstin Schmidt provides consulting services in female rats with Cry1Ab was interleukin-2 in female rats the field of biostatistics and has advised National and European Au- fed the 11% GMO diet in Trial E. This alteration was not thorities, biotech and pharmaceutical companies as well as research institutions, also in the context of GMO risk assessment. observed in female rats fed the 33% GMO diet in Trial E and not observed at all in the Trial D, so that it is considered to Open Access This article is distributed under the terms of the Crea- be of no toxicological significance. tive Commons Attribution 4.0 International License (http://creat iveco Taken together, only single parameters were sporadically mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- altered in rats fed the MON810 maize when compared to tion, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the control rats, and these alterations are considered to be of no Creative Commons license, and indicate if changes were made. immunotoxicological significance. However, a long-term and continuous airway exposure to small amounts of the Cry1Ab protein could occur in workers handling genetically modified plants expressing this protein References in farms and factories. Information regarding the immu- nological effects of long-term airway exposure to Cry1Ab Adel-Patient K, Guimaraes VD, Paris A, Drumare MF, Ah-Leung is limited. Therefore, it would be advisable to investigate S, Lamourette P, Nevers MC, Canlet C, Molina J, Bernard H, immune responses in workers at a risk of airway exposure Créminon C, Wal JM (2011) Immunological and metabolomic impacts of administration of Cry1Ab protein and MON 810 maize to Cry1Ab protein. in mouse. PLoS ONE 6:e16346 Our data are in accordance with the conclusions and rec- Andreassen M, Rocca E, Bøhn T, Wikmark O-G, van den Berg J, Løvik ommendations provided by the GRACE project (http://www. M, Traavik T, Nygaard UC (2015a) Humoral and cellular immune grace-fp7.eu ), i.e. there is no indication that the performance responses in mice after airway administration of Bacillus thur- ingiensis Cry1Ab and MON810 cry1Ab-transgenic maize. Food of 90-day feeding studies with whole food/feed would pro- Agric Immunol 26:521–537 vide additional information on the safety of the GM maize Andreassen M, Bøhn T, Wikmark O-G, van den Berg J, Løvik M, Traa- MON810 if compared to the compositional analysis of the vik T, Nygaard UC (2015b) Cry1Ab protein from Bacillus thur- GM line and its conventional counterpart (i.e. the genetically ingiensis and MON810 cry1Ab-transgenic maize exerts no adju- vant effect after airway exposure. Scand J Immunol 81:192–200 closest non-GM comparator) in terms of an initial safety EFSA Scientific Committee (2011) EFSA guidance on conducting assessment. repeated-dose 90-day oral toxicity study in rodents on whole food/ In line with the GRACE transparency policy, any inter- feed. EFSA J 9: 2438 ested person will have access to the raw data of studies D Finamore A, Roselli M, Britti S, Monastra G, Ambra R, Turrini A, Mengheri E (2008) Intestinal and peripheral immune response to and E obtained in the frame of the GRACE project through MON810 maize ingestion in weaning and old mice. J Agric Food an internet portal named CADIMA (Central Access Data- Chem 56:11533–11539 base for Impact Assessment of Crop Genetic Improvement González-González E, García-Hernández AL, Flores-Mejía R, López- Technologies; http://www.cadim a.info). Santiago R, Moreno-Fierros L (2015) The protoxin Cry1Ac of Bacillus thuringiensis improves the protection conferred by intranasal immunization with Brucella abortus RB51 in a mouse Acknowledgements This study was carried out as part of the GRACE model. Vet Microbiol 175:382–388 project (“GMO Risk Assessment and Communication of Evidence”), Gu J, Krogdahl A, Sissener NH, Kortner TM, Gelencser E, Hemre G-I, financially supported by the Seventh Framework Programme of the Bakke AM (2013) Effects of oral Bt-maize (MON810) exposure European Community for Research, Technological Development on growth and health parameters in normal and sensitized Atlantic and Demonstration Activities (FP7), Grant agreement no. 311957, salmon, Salmo salar L. Br J Nutr 109:1408–1423 the Dutch Ministry of Economic Affairs and the ITMS Project no. Guerrero GG, Dean DH, Moreno-Fierros L (2004) Structural impli- 26240120033 in the frame of the Operational Research and Devel- cation of the induced immune response by Bacillus thuringien- opment Program of the European Regional Development Fund. The sis Cry proteins: role of the N-terminal region. Mol Immunol analyses of maize and diets were performed by RIKILT Wageningen 41:1177–1183 UR and INRA as partners of the GRACE consortium as well as the Guerrero GG, Russell WM, Moreno-Fierros L (2007) Analysis of the companies Covance and Mucedola contracted by GRACE. We thank cellular immune response induced by Bacillus thuringiensis Cry Helena Nagyova and Edita Mrvikova at the Slovak Medical University for their excellent technical support. The authors would like to thank 1 3 Archives of Toxicology (2018) 92:2385–2399 2399 1A toxins in mice: effect of the hydrophobic motif from diphtheria in Atlantic salmon, Salmo salar L., fed different levels of geneti- toxin. 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Arch Sagstad A, Sanden M, Haugland Ø, Hansen A-C, Olsvik PA, Hemre Toxicol 88:2289–2314 G-I (2007) Evaluation of stress-and immune-response biomarkers Affiliations 1 2 2 1 1 1 Jana Tulinská  · Karine Adel‑Patient  · Hervé Bernard  · Aurélia Líšková  · Miroslava Kuricová  · Silvia Ilavská  · 1 3 3 3 3 2 Mira Horváthová  · Anton Kebis  · Eva Rollerová  · Júlia Babincová  · Radka Aláčová  · Jean‑Michel Wal  · 4 4 4,7 5 5 5 Kerstin Schmidt  · Jörg Schmidtke  · Paul Schmidt  · Christian Kohl  · Ralf Wilhelm  · Joachim Schiemann  · 6,8 Pablo Steinberg 1 5 Faculty of Medicine, Slovak Medical University, Limbová Institute for Biosafety in Plant Biotechnology, Julius 12, 83303 Bratislava, Slovakia Kühn-Institut, Federal Research Centre for Cultivated Plants, Erwin-Baur-Str. 27, 06484 Quedlinburg, Germany Service de Pharmacologie et d’Immunoanalyse, Laboratoire d’Immuno-Allergie Alimentaire (LIAA), INRA, CEA, Institute for Food Toxicology and Analytical Chemistry, Université Paris Saclay, DRF/Institut Joliot/SPI-Bat 136, University of Veterinary Medicine Hannover, Bischofsholer CEA de Saclay, 91191 Gif sur Yvette Cedex, France Damm 15, 30173 Hannover, Germany 3 7 Faculty of Public Health, Slovak Medical University, Present Address: Biostatistics (340c), University Limbová 12, 83303 Bratislava, Slovakia of Hohenheim, Fruwirthstr. 23, 70599 Stuttgart, Germany 4 8 BioMath GmbH, Friedrich-Barnewitz-Str. 8, Present Address: Max Rubner-Institut, Federal Research 18119 Rostock-Warnemünde, Germany Institute of Nutrition and Food, Haid-und-Neu-Str. 9, 76131 Karlsruhe, Germany 1 3

Journal

Archives of ToxicologySpringer Journals

Published: May 31, 2018

References

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