Nova Biotechnol Chim (2020) 19(1): 19-29 DOI 10.36547/nbc.v19i1.574  Corresponding author: hassanienmohamed@hotmail.com, mhassanien@uqu.edu.sa Nova Biotechnologica et Chimica Biosafety of genetically modified wheat (Triticum aestivum) Hi-line 111 Mohamed Abd-Elfattah Mohamed Hassan1, Rafaat Mohamed Elsanhoty2, Mohamed Fawzy Ramadan3,4, and Mohyeldeen Ali Osman5 1Food Technology Research Institute, Agriculture Research Center, 9 ALgamaa Street, Giza, Egypt 2Department of Industrial Biotechnology, Institute of Genetic Engineering and Biotechnology, Sadat City University, Egypt 3Department of Agricultural Biochemistry, Faculty of Agriculture, Zagazig University, Zagazig 44519, Egypt 4Deanship of Scientific Research, Umm Al-Qura University, Makkah, Saudi Arabia 5Department of Biochemistry, Faculty of Agriculture, Cairo University, Egypt Article info Article history: Received: 8th February 2020 Accepted: 13th April 2020 Keywords: Genetic modification GMO Histopathology rats Safety assessment Serum biochemistry Transgenic wheat Abstract Genetically modified (GM) crops were approved for edible use in several countries but their biosafety for organisms remains to be crucial. The objectives of this work were to compare GM wheat (Triticum aestivum) Hi-line 111 (GMW) with native non-GMW wheat (NGMW) to find the differences, if any, in their biosafety. Three groups of albino rats (Rattus norvegicus) were used to study the biosafety of GMW for 30 days. Group 1 was fed on a basal diet (control), and group 2 on a control diet with 30 % replacement of starch with NGMW, while group 3 was fed on the control diet with 30 % replacement of starch with GMW. There were no significant signs of adverse impacts noted in the clinical appearance of animals fed on GMW in terms of initial body weight, absolute or relative organ weights and serum profile in comparison with the control group. However, slight histopathological changes were observed in the organs of animals fed on GMW. Though our results demonstrate GMW biosafety regarding its biochemical parameters, however, detailed description of submucosal edema and further studies on allergenic potential with long feeding periods should be performed to conclude its impacts on health.  University of SS. Cyril and Methodius in Trnava Introduction Agricultural biotechnology and genetic engineering have opened an avenue in the development of genetically modified (GM) plants with improved abiotic stress tolerance, herbicide or insect resistance, and nutritional value (Sthrestha et al. 2008; Raybould 2012; Elfattah et al. 2016; Sun et al. 2019). With the international commercialization of GM organisms (GMOs), GM crops were engineered to solve problems such as weed management, disease, improving functional properties and enhancing production efficiency (Shin et al. 2013; Klymiuk et al. 2018). Genetic modification introduces new genetic information, new genes, and new compounds in the cells of food-producing organisms. They might alter the cellular metabolism of the food- producing organisms in unanticipated ways. These new proteins might also be toxic or cause allergy (Taylor 1997). In addition, the food-producing organism could fail to produce some nutrients or vitamins. Thus, GM foodstuffs could lack some nutrients that are present in the corresponding non- GM foods. Genetic engineering could cause unintended adverse changes in the food chemical composition or food characteristics, thereby rendering foodstuffs to be unsafe (Elsanhoty mailto:hassanienmohamed@hotmail.com mailto:mhassanien@uqu.edu.sa Nova Biotechnol Chim (2020) 19(1): 19-29 20 et al. 2004, 2013; Herman et al. 2009). Based on the criteria for safety, some GM crops have been approved for food or feed uses in several countries (Oguchi et al. 2010). Consumer concerns regarding GM products related to unexpected health impacts that might arise from GM products consumption (Dona and Arvanitoyannis 2009; Martinez-Povida et al. 2009). As recommended by food safety agencies, biosafety and nutritional assessment of GM crops is an important aspect (Chassy 2010). Pressure from public demand and consumers has led some countries to label GMO in foods (Ramadan and Elsanhoty 2012; Elsanhoty et al. 2013; Castigliego et al. 2015; Turkec et al. 2016). Many countries have imposed biosafety laws and surveillance programs to regulate GMOs uses (De Jong 2010). Within EU countries, regulations and laws on the traceability and labeling of GMOs require mandatory labeling of food and/or feed containing GM ingredients above > 0.9 % (The Commission of the European Communities 2003a, b). EU has adopted a zero-tolerance policy towards the low-level presence of unauthorized GMOs in foods, with a 0.1 % threshold for the permissible presence of unauthorized GMO in the feed (The Commission of the European Communities 2011; Turkec et al. 2016). Many studies were performed to investigate the safety of GM food and feed (Elsanhoty et al. 2004, 2006; He et al. 2008; Magaña-Gómez and de la Barca 2009; Appenzeller et al. 2009a,b; Snell et al. 2012). Feeding trials in which rats were fed GM foods for prolonged periods were reported (Brake and Evenson 2004; Brake et al. 2004). Some experiments indicated histopathological abnormalities in organs or tissues and clinical impacts (Appenzeller et al. 2009a,b; Snell et al. 2012; Kaspareit and Rittinghausen 2013; Abdo et al. 2014). Some reports assessed the safety of GM plants and reported changes in the serum parameters of animals treated with GM foodstuffs (Elsanhoty et al. 2004, 2006; Hammond et al. 2006). They noted some differences between animals fed on GM potato Spunta and its control as well as GM corn (MON 810) and its control. However, monitoring of biosafety and nutritional quality for GM food and/or feed require detailed knowledge of the plant composition. Wheat (Triticum aestivum L.) is one of the most important cereals that is globally cultivated (Pigolev et al. 2018; Sun et al. 2019). The GM wheat Hi-Line 111 (GMW) was transformed with plasmid pAB1 harboring the full-length HVA1 cDNA to transform immature GMW Hi-Line 111 cv. Hi-Line embryos (Hong et al. 1988; Lanning et al. 1992; Sivamani et al. 2000). The chemical composition of GMW and non-GMW (NGMW) line as well as the physical and rheological characteristics of the dough were studied (Elfattah et al. 2016). The GMW under study was modified by introducing of barley HVA1 gene, encoding for a member of the group 3 late embryogene sis abundant (LEA) proteins to increase drought tolerance, biomass production and water use efficiency (Sivamani et al. 2000; Elfattah et al. 2016). Our work is aimed to assess the safety of GMW in comparison with the conventional wheat line or NGMW upon animals feeding study. Experimental Materials GMW Hi-Line 111 and its conventional NGMO control line were cultivated and harvested under the same conditions in the Agricultural Genetic Engineering Research Institute (AGERI, Giza, Egypt). The HVA1 expression cassette, containing the HVA1 gene under control of the maize ubi promoter was introduced into T. aestivum L. cv. Hi-Line genome via particle bombardment of immature embryos (Elfattah et al. 2016). Grain samples were freeze-dried prior to mixing to feed in further investigations. The genetic elements in GMW were reported (Elfattah et al. 2016). Non-GMW contained 10.7 moisture (%), 15.6 total protein, 1.61 ash (%), 2.13 lipids (%), 2.63 crude fiber (%), and 69.9 total carbohydrates (%). GMW contained 10.1 moisture (%), 15.9 total protein, 1.61 ash (%), 2.73 lipids (%), 2.71 crude fiber (%), and 69.5 total carbohydrates (%). Experimental conditions Experimental procedures involving the animals and their care were carried out according to the Guidelines for Care and Use of Laboratory Nova Biotechnol Chim (2020) 19(1): 19-29 21 Table 1. The composition of the basal and experimental diet (g.100 g-1). Ingredient CDa [g.100 g-1 diet] Experimental diet Group 2 Group 3 Caseinb 20.0 20.0 20.0 Corn oil 5.0 5.0 5.0 Cellulose 5.0 5.0 5.0 Salt mixture 3.50 3.50 3.50 Vitamin mixture 1.0 1.0 1.0 Starch 55.0 25.0 25.0 Cholin bitartarate 0.2 0.2 0.2 DL methionine 0.3 0.3 0.3 Sucrose 10.0 10.0 10.0 NGMW 30.0 GMW 30.0 a CD means control diet according to Reeves et al. (1993). b Casein contained ≥ 80 % protein. Animals in Biomedical Research as promulgated by the WHO. Fifteen weaning male albino rats (average weight was 65 g) were provided from the Institute of Ophthalmology Research (Giza, Egypt). The animal experiment was ethically approved before collecting the data by the Animal Subjects Committee of the Sadat City University (Egypt). The animals were housed individually in well-aerated cages with screen bottoms and fed on a basal diet as described by Reeves et al. (1993). Salt and vitamins mixtures were prepared according to AOAC (2000). Humidity and temperature were maintained at 60 % and 25 °C, respectively. The animals were randomly sorted into three groups each group contains 5 animals. Group 1 was fed on control diet according to Reeves et al. (1993), and group 2 was fed on the control diet with a replacement of 30 % from starch by NGMW, while group 3 was fed on the control diet with a replacement of 30 % from starch by GMW. Table 1 presents the diet composition and animal groups. During the experiment (30 days), the diet consumed and the body weight were measured every day. The body weight and the rest of the feed were measured to compare some performance parameters such as food intake, body gain, daily body weight gain, final body weight, and feed efficiency. Blood biochemistry and serum analysis At the end of the experiment (30 days), blood samples were collected from treated groups from the orbital venous plexuses using a capillary tube. Blood was centrifuged for 15 min at 3,000 rpm. to recover the serum, which kept at -18 °C until analysis. Serum was analyzed according to Schermer (1967). Kidney function parameters (urea, and creatinine) and liver function enzymes (ALT, and AST) were analyzed using kits obtained from Bio-diagnostic (Cairo, Egypt). Blood glucose was measured according to Trinder (1969). Serum total cholesterol (TC) was determined according to Thomas (1992). Glutamate-pyruvate aminotransferase (GPT), and glutamate oxaloacetate aminotransferase (GOT) activates were measured at 540 nm (Reitman and Frankel 1957). Serum urea was determined at 580 nm according to Patton and Crouch (1977). Total lipids (TL) content was measured by the reaction of TL with the sulfophosphovanillic mixture (Coudon and Bouige 1973). Low-density lipoprotein (LDL)- cholesterol was calculated as the difference between TC and the high-density lipoprotein (HDL)-cholesterol content of the supernatant after precipitation of the LDL by the polyvinyl sulphate in the presence of polyethylene glycol monomethyl ether (Demacker et al. 1984). Triacylglycerols (TAG) content was analyzed as described by Fossati and Prencipe (1982). Organ weights, gross necropsy, and histopathology At the end of the experimental period (30 days), the rats were slaughtered. The organs of each animal (kidney, liver, heart, spleen and testes) were weighed. Kidneys, liver, spleen, and stomach tissues from those organs as well as macroscopically evident lesions were fixed in 4 % buffered formaldehyde for the histological test. Tissues were embedded in paraffin and sections, 4 – 6 lm thick, stained by the standard hematoxylin-eosin (H & E) for light microscopy. The intact small intestines were flushed with a 0.9 % NaCl solution and the length was recorded (Drury and Willington 1980). Statistical analysis Results are presented as means ± confidence intervals (p < 0.05). Means were considered as significantly different, if their confidence Nova Biotechnol Chim (2020) 19(1): 19-29 22 Table 2. Changes in the body weights of animal groups at the end of the experimental period (30 days)a. Parameter Animal group Control group NGMW b group GMWc group Initial body weight [g] 65.2 ± 4.64a 65.6 ± 4.87a 65.8 ± 4.44a Final body weight [g] 108.4 ± 4.74b 147 ± 4.64a 142.6 ± 7.16a Body weight gain [g] 43.2 ± 4.69b 81.4 ± 4.75a 76.8 ± 5.8a The same letter in the same row is not significantly different (p > 0.05). amean ± standard error (n = 5) bNGMW = non genetically modified wheat cGMW = genetically modified wheat intervals were not overlapping. Results from the animal studies were presented as mean ± SD where appropriate according to Gomez (1984). Results and Discussion Biosafety monitoring research performed on GM wheat based on the application of the principle of substantial equivalence, which adopted by leading international food and regulatory bodies such as WHO (WHO 1995). A new food derived from GM crop was found to be equivalent to its non-modified counterpart and safe as the food or feed from the non-modified plant varieties. Substantial equivalence might be classified into; substantially equivalent, substantial equivalent expects for some defined traits, and lack of equivalence (Momma et al. 2000). The present experiment was performed to study the nutritional effects and safety of GMW. A rat performance study was conducted to compare GMW with NGMW. Clinical observations and body weight During the experimental period, no treatment- related signs of adverse impacts in the clinical appearance of rats were noted. The body weights of animals were comparable at the beginning of the experiment, while throughout the study period there was an increase in body weight of NGMW and GMW groups (Table 2). This increase may be due to the high nutrient value of wheat that replaced starch in the feeding formula. Differences between groups were tested for statistical significance (p < 0.05). The results revealed no statistically significant differences between groups in the initial body weight, while at the end of the experiment, there were statistically significant differences between control and other groups. The results agreed with previous reports (Elsanhoty et al. 2004, 2006; Abdo et al. 2014), where no statistically significant differences between groups observed in the initial body weight of rats during feeding of GM potato Spunta, and Bt-corn (MON810: Ajeeb YG) during feeding study. However, the results disagreed with some studies (Zhu et al. 2004; Séralini et al. 2007; Zhu et al. 2015), where some GMOs (Roundup tolerant and MON863) increased the body weight. Zhu et al. (2015) found significant differences in organ weights among dietary groups fed on GM rice (expressing Cry1Ab/1Ac protein) and non-GM rice. Séralini et al. (2007) reported significant Table 3. Changes in the relative organ weight (%) of animal groups at the end of the experimental period (30 days) a. Parameter Animal group Control group NGMW b group GMWc group Final body weight [g] 108.4 ± 4.740b 147 ± 4.640a 142.6 ± 7.160a Liver [%] 3.916 ± 0.068a 3.597 ± 0.241a 3.438 ± 0.093a Spleen [%] 0.459 ± 0.032a 0.489 ± 0.045a 0.432 ± 0.051a Kidney [%] 0.718 ± 0.054a 0.737 ± 0.044a 0.759 ± 0.017a Stomach [%] 0.757 ± 0.024a 0.652 ± 0.044b 0.632 ± 0.026b The same letter in the same row is not significantly different (p > 0.05). amean ± standard error (n = 5) bNGMW = non genetically modified wheat cGMW = genetically modified wheat Nova Biotechnol Chim (2020) 19(1): 19-29 23 Table 4. Changes in the serum biochemical values of animal groups at the end of the experimental period (30 days) a. Parameter Animal group Control group NGMW b group GMWc group GOT [U.L-1] 10.6 ± 0.39a 11.4 ± 0.81a 10.8 ± 0.58a GPT [U.L-1] 11.2 ± 1.53a 10.6 ± 0.51a 10 ± 1.76a Cholesterol [mg.dL-1] 71.2 ± 10.0a 63.2 ± 10.9a 69.2 ± 17.7a HDL cholesterol [mg.dL-1] 64.6 ± 22.7a 43.8 ± 8.87a 57 ± 13.0a Triglyceride [mg.dL-1] 73.8 ± 5.46a 62.6 ± 18.6a 54.6 ± 7.45a Total lipids [mg.dL-1] 517.4 ± 26.0a 379.6 ± 89.6ab 257 ± 72.8b Creatinine [mg.dL-1] 0.396 ± 0.02a 0.396 ± 0.02a 0.506 ± 0.05a Urea [mg.dL-1] 106.2 ± 10.6a 85.8 ± 10.1a 79.2 ± 10.5a The same letter in the same row is not significantly different (p > 0.05). a mean ± standard error (n = 5) b NGMW = non genetically modified wheat c GMW = genetically modified wheat variation in the weights of rats fed on MON 863 compared to the control animals. The findings of the present study showed no significant differences in the absolute body weights of animals fed on GMW in comparison with NGMW and control groups. Relative organ weight The impact of feeding diets containing 30 % GMW and NGMW on the relative weights of some organs (kidney, liver, spleen, and stomach) to the animal body weight is presented in Table 3. The relative ratio of liver to the body weight recorded a value of 3.91 %, 3.59 %, and 3.43 % for control, NGMW group, and GMW group, respectively. The relative ratios of spleen weight for control and NGMW group (0.43 – 0.48 %) were in lower levels as the value resulting from feeding on GMW. In addition, comparable findings were noted for the relative weights of the kidney (Table 3). From these findings, it is clear that there is no significant statistical differences between the studied groups. However, NGMW group, and GMW group showed a significantly lower range of relative ratio for stomach weight (-14 %) and (-16.5 %), respectively. These findings agree with Elsanhoty et al. (2004) and Poulsen et al. (2007) who found an increase in the relative weight of the small intestine (+10 %) in rats fed GM rice, andan increase in the absolute and relative weight of the adrenals. On the other hand, Poulsen et al. (2007) found statistically significantly reduce in the absolute weight of the adrenals (-15 %) in male rats fed on GM rice. Similar results were reported in the previous study on GM potato Spunta (Elsanhoty et al. 2004), wherein no significant differences in the spleen, kidney and heart weights were observed after 1.5 months of feeding. Kilic and Akay (2008) reported that the liver weights were increased in females' rats, but there were no significant differences in males. Séralini et al. (2011) reported that Bt-corn or GM soybean caused significant differences in organs’ weights, especially liver and kidney because of these organs responsible for the detoxification. The obtained results disagree with Abdo et al. (2014) who reported significant differences in all organs weights among different groups after 45 days of feeding study. Effect of GMW on serum parameters The effects of feeding NGMW, and GMW on serum biochemical parameters that reflecting kidney and liver functions are given in Table 4. The results showed no significant differences among the studied parameters in all groups. The health conditions of all groups coincided with normal values of GPT, GOT, cholesterol, HDL- cholesterol, TAG, TL, creatinine, and urea. The results indicated that the kidney and liver functions were in a good state without suffering from acute infections. The total cholesterol was also in the normal physiology level. The results revealed that the general health and metabolic process of the animals were not affected by feeding on GMW, wherein there were no significant differences (p < 0.05) between groups. The exception was in the values of total lipids, Nova Biotechnol Chim (2020) 19(1): 19-29 24 Fig. 1. Liver sections of control (A), NGMW group (B) and GMW group (C). Control group (A) and NGMW group (B) showing the normal histological structure of hepatic lobule, while GMW group (C) showing leucocytes in the hepatic sinusoids (arrows) (H&E X 200). wherein there were significant differences (p < 0.05) between the control group and GMW group. These results agree with Elsanhoty et al. (2004), who reported no significant difference in serum biochemical values between groups fed GM potato Spunta and non-GM potato. Also, the results agreed with Poulsen et al. (2007) who reported that male rats fed on GM rice had a lower plasma concentration of potassium as well as the levels of protein, and albumin. Abdo et al. (2014) reported from subchronic feeding study on rats fed on GM Bt-corn that there were many alternations in the organs weights, hematology, and serum biochemical parameters in Bt-corn group after 45 days but changes were increased after 90 days. The obtained results of lipid parameters Fig. 2. Kidney sections of control (A), NGMW group (B) and GMW group (C). The control group (A) and NGMW group (B) showing normal histological structure, while GMW group (C) showing slight vacuolations of endothelial liming glomerular tufts (arrows) (H&E X 200). disagreed with Gab-Alla et al. (2012) who reported an increase in all lipid parameters (except for HDL- cholesterol) of rats’ blood fed on MON810-Ajeeb YG after 91 days of starting the experiment. Histopathological profile Microscopically examination of liver in the control group exhibited the normal histological structure of hepatic lobule (Fig. 1A). The liver of the rat from the NGMW group showed no histopathological changes (Fig. 1B). However, the liver of rat from group 3 showed the presence of leucocytes in the hepatic sinusoids (Fig. 1C). Concerning kidney, examined sections from control, and non-GMW group revealed B C A B A A C Nova Biotechnol Chim (2020) 19(1): 19-29 25 no histopathological changes (Fig. 2A and B). Meanwhile, kidneys of rat from GMW group showed no changes except slight vacuolations of endothelial lining glomerular tufts (Fig. 2C). Regarding spleen, examined sections from the control group, non-GMW group and GMW group revealed no histopathological changes (Fig. 3A, B and C). Macroscopically examination of the stomach from control revealed the normal histological structure of gastric layers (Fig. 4A), and examined sections from the NGMW group showed no histopathological changes (Fig. 4B). Moreover, the stomach of the rat from GMW group showed submucosal edema associated with few leucocytic cells infiltration (Fig. 4C). These results indicated that the submucosal edema could be caused by GMW. To explain the nontoxicity of GMW, a detailed description of submucosal edema is needed (Fig. 4C). These results agree with Kilic and Akay (2008) and Gab-Alla et al. (2012) who reported minimal histopathological changes in kidney and liver in rats fed on GM corn. The results of histopathological examination agree with the results obtained by Ewen and Pusztai (1999) and Trabalza-Marinucci et al. (2008) who observed no histological differences in pancreas, duodenum, liver, spleen, mesenteric lymph nodes, C B A Fig. 3. Spleen sections of control (A), NGMW group (B) and GMW group (C). Control group (A), NGMW group (B) and GMW group (C) showing normal histological structure (H&E X 200). Nova Biotechnol Chim (2020) 19(1): 19-29 26 cecal appendix, rumen, and abomasum sections between control group and GM maize-fed groups in sheep and lambs. The differences between the groups (individuals) can fall under individual variability or due to the particular location of the individual histological cuts. Conclusions This research aimed to study the biosafety of GMW Hi-line 111. No statistically differences in the biochemical parameters were noted between animals fed on GMW and rats fed on NGMW. When it comes to GMW, which contains a gene from barley, with both the plants belonging to the same genus, we perhaps cannot expect a significant impact on parameters in the serum of the experimental animals compared to commercial wheat of the same strain. Based on the obtained data, it could be concluded that the GMW has no adverse health or toxic effects. However, a detailed description of submucosal edema and further studies on allergenic potential with long feeding periods might be needed. Conflict of Interest The authors declare that they have no conflict of interest. e e C B A Fig. 4. Stomach sections of control (A), NGMW group (B) and GMW group (C). The control group (A) and NGMW group (B) showing normal histological structure, while GMW group (C) showing submucosal edema (e) associated with few leucocytic cells infiltration (arrows) (H&E X 200). Nova Biotechnol Chim (2020) 19(1): 19-29 27 Statements on Compliance with Ethical Standards The procedures followed were in accordance with the institutional and national ethical standards of the responsible committee on animal/human experimentation. References Abdo ME, Barbary OM, El-Sayed Shaltout EO (2014) Feeding study with Bt corn (MON810: Ajeeb YG) on rats: Biochemical analysis and liver histopathology. Food Nutr. Sci. 5: 185-195. AOAC International (2000) Official methods of analysis of AOAC International. 17th Ed. Gaithersburg, MD, USA, Association of Analytical Communities. Appenzeller LM, Malley L, MacKenzie SA, Hoban D, Delaney B (2009a) Subchronic feeding study with genetically modified stacked trait lepidopteran and coleopteran resistant (DAS-Ø15Ø7-1×DAS-59122- 7) maize grain in Sprague-Dawley rats. Food Chem. Toxicol. 47:1512-1520. Appenzeller LM, Munley SM, Hoban D, Sykes GP, Malley LA, Delaney B (2009b) Subchronic feeding study of grain from herbicide-tolerant maize DP-Ø9814Ø-6 in Sprague-Dawley rats. Food Chem. Toxicol. 47: 2269- 2280. Brake DG, Evenson DP (2004) A generational study of glyphosate-tolerant soybeans on mouse fetal, postnatal, pubertal and adult testicular development. Food Chem. Toxicol. 42: 29-36. Brake DG, Thaler R, Evenson DP (2004) Evaluation of Bt (Bacillus thuringiensis) corn on mouse testicular development by dual parameter flow cytometry. J. Agric. Food Chem. 52: 97-102. Castigliego L, Armani A, Tinacci L, Gianfaldoni D, Guidi A (2015) Two alternative multiplex PCRs for the identification of the seven species of anglerfish (Lophius spp.) using an end-point or a melting curve analysis real-time protocol. Food Chem. 166: 1-9. Chassy BM (2010) Food safety risks and consumer health. New Biotechnol. 27: 534-544. Coudon B, Bouige D (1973) Automatic method for determination of total serum lipids, using the sulfophosphovanillic reaction. Ann. Biol. Clin. (Paris) 31: 3-7. De Jong TJ (2010) General surveillance of genetically modified plants in the EC and the need for controls. J.r Verb. Lebensmittel. 5: 181-183. Demacker AG, Hijmans BG, Brenninkmeijer AP, Jansen JS, Van’t Laar A (1984) Five methods for determining low-density lipoprotein cholesterol compared. Clin. Chem. 30: 1797-1800. Dona A, Arvanitoyannis IS (2009) Health risks of genetically modified foods. Crit. Rev. Food Sci. Nutr. 49: 164-175. Drury G, Wallington A (1980) Carletons histological techniques, Oxford University Press. New York, 520 p. Elfattah MA, Elsanhoty RM, Ramadan MF, Osman MO (2016) Composition and rheological properties of flour and dough from genetically modified wheat (Triticum aestivum L.) Hi-Line 111. Cereal Res. Commun. 44: 605-616. Elsanhoty MR, Al-Turki IA, Ramadan FM (2013) Prevalence of genetically modified rice, maize and soy in commercial food products in Kingdom of Saudi Arabia market. Appl. Biochem. Biotechnol. 171: 883-899. Elsanhoty R, Abdelrahman AA, Bögl KW (2004) Quality and safety evaluation of genetically modified potato Spunta with Cry V gene: Chemical composition, determination of some toxins, antinutrients compounds and feeding study on rat. Nahrung/Food 48: 13-18. Elsanhoty RM, Eldesouky AM, Ramadan MF (2006) Safety assessment of genetically modified potato Spunta: Degradation of DNA in gastrointestinal track and carry over to rat organs. J. Food Biochem. 30: 556-578. Ewen SWB, Pusztai A (1999) Effects of diets containing genetically modified potatoes expressing Galanthus nivalis lectin on rat small intestine. Lancet 354: 1353- 1354. Fossati P, Prencipe L (1982) Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clin. Chem. 28: 2077- 2080. Gab-Alla A, Rayan AM, El-Shamei AZS, Shatta AA, Moussa EA (2012) Morphological and biochemical changes in male rats fed on genetically modified corn (Ajeeb YG). J. Amer. Sci. 8: 1117-1123. Gomez KA (1984) Statistical procedures for agricultural research, John Wiley and Sons, New York, p. 139. Hammond B, Lemen J, Dudek R (2006) Results of a 90-day safety assurance study with rats fed grain from corn rootworm-protected corn. Food Chem. Toxicol. 44: 147- 160. He XY, Huang XY, Li KL, Qin W, Delaney B, Luo YB (2008) Comparison of grain from corn rootworm resistant transgenic DAS-59122-7 maize with non transgenic maize grain in a 90-day feeding study in Sprague-Dawley rats. Food Chem. Toxicol. 46: 1994- 2002. Herman RA, Chassy MB, Parrott W (2009) Compositional assessment of transgenic crops: an idea whose time has passed. Trend. Biotechnol. 27: 555-557. Hong B, Uknes SJ, Ho T-HD (1988) Cloning and characterization of a cDNA encoding an mRNA rapidly induced by ABA in barley aleurone layers. Plant Mol. Biol. 11: 495-506. Kaspareit J, Rittinghausen S (2013) Spontaneous neoplastic lesions in Harlan Sprague-Dawley rats. Exp. Toxicol. Pathol. 51: 105-107. Kilic A, Akay TM (2008) A three generation study with genetically modified Bt corn in rats: Biochemical and histopathological investigation. Food Chem. Toxicol. 46: 1164-1170. Nova Biotechnol Chim (2020) 19(1): 19-29 28 Klymiuk V, Yaniv E, Huang L, Raats D, Fatiukha A, Chen S, Feng L, Frenkel Z, Krugman T, Lidzbarsky G, Chang W, Jääskeläinen MJ, Schudoma C, Paulin L, Laine P, Bariana H, Sela H, Saleem K, Sørensen CK, Hovmøller MS, Distelfeld A, Chalhoub B, Dubcovsky J, Korol AB, Schulman AH, Fahima T (2018) Cloning of the wheat Yr15 resistance gene sheds light on the plant tandem kinase-pseudokinase family. Nature Commun. 9: 3735. Lanning SP, Talbert LE, McNeal FH, Alexander WL, McGuire CF, Bowman H, Carlson G, Jackson G, Eckhoff J, Kushnak G, Stewart V, Stallknecht G (1992) Registration of ‘Hi-Line’ wheat. Crop Sci. 32: 283-284. Magaña-Gómez JA, de la Barca AM (2009) Risk assessment of genetically modified crops for nutrition and health. Nutr Rev. 67: 1-16. Martinez-Povida A, Molla-Bauza MB, del Campo Gomis FJ, Martinez LMC (2009) Consumer perceived risk model for the introduction of genetically modified food in Spain. Food Policy 34: 519-528. Momma K, Hashimoto W, Yoon JH, Ozawa S, Fukuda Y, kawai S, Takaiwa F, Utasumi S, Murata K (2000) Safety assessment of rice genetically modified with soybeanglycin by feeding studies in rats. Biosci. Biotechnol. Biochem. 64: 1881-1886. Oguchi T, Onishi M, Mano J, Akiyama H, Teshima R, Futo S, Furui S, Kitta K (2010) Development of multiplex PCR method for simultaneous detection of four events of genetically modified maize: DAS-59122-7, MIR604, MON863 and MON88017. Food Hyg. Safe. Sci. 51: 92- 100. Patton CJ, Crouch SR (1977) Spectrophotometric and kinetics investigation of the Berthelot reaction for the determination of ammonia. Anal. Chem. 49: 464- 469. Pigolev AV, Miroshnichenko DN, Pushin AS, Terentyev VV, Boutanayev AM, Dolgov SV, Savchenko TV (2018) Overexpression of Arabidopsis OPR3 in hexaploid wheat (Triticum aestivum L.) alters plant development and freezing tolerance. Inter. J. Mol. Sci. 19: 3989. Poulsen M, Schrøder M, Wilcks A, Kroghsbo S, Lindecrona RH, Miller A, Frenzel T, Danier J, Rychlik M, Shu Q, Emami K, Taylor M, Gatehouse A, Engel KH, Knudsen I (2007) Safety testing of GM-rice expressing PHA-E lectin using a new animal test design. Food Chem. Toxicol. 45: 364-377. Ramadan MF, Elsanhoty MR (2012) Lipid classes, fatty acids and bioactive lipids of genetically modified potato Spunta with Cry V gene. Food Chem. 133: 1169-1176. Raybould A, Graser G, Hill K, Ward K (2012) Ecological risk assessments for transgenic crops with combined insect-resistance traits: The example of Bt11, MIR604 maize. J. Appl. Entomol. 136: 27-37. Reeves PG, Roossow KL, Landlauf J (1993) Development and testing of the AIN-93 purified diets for rodents: Results on growth, kidney calcification and bone mineralization in rats and mice. J. Nutrition. 123: 1923- 1931. Reitman S, Frankel S (1957) A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 28: 56-63. Schermer S (1967) The blood morphology of laboratory animals, 3rd Ed. F. A. Davis Company, Philadelphia, Pennsylvania, 212 p. Séralini GE, Cellier D, Spiroux J (2007) New analysis of a rat feeding study with a genetically modified maize reveals signs of hepatorenal toxicity. Arch. Environ. Contam. Toxicol. 52: 596-602. Séralini GE, Mesnage R, Clair E, Gress S, Vendômois SJ, Dominique CD (2011) Genetically modified crops safety assessments: present limits and possible improvements. Environ. Sci. Eur. 23: 10. Shin K, Suh S, Lim M, Woo H, Lee JH, Kim H, Cho H (2013) Event-specific detection system of stacked genetically modified maize by using the multiplex-PCR technique. Food Sci. Biotechnol. 22: 1763-1772. Sivamani E, Bahieldin A, Wraith JM, Al-Niemi T, Dyer WE, Ho TD, Qu R (2000). Improved biomass productivity and water use efficiency under water deficit conditions in transgenic wheat constitutively expressing the barley HVA1 gene. Plant Sci. 155: 1-9. Snell C, Bernheim A, Berge JB, Kuntz M, Pascal G, Paris A (2012) Assessment of the health impact of GM plant diets in long-term and multigenerational animal feeding trials: a literature review. Food Chem. Toxicol. 50: 1134-1148. Sthrestha HK, Hwu K, Wang S, Liu L, Chang M (2008) Simultaneous detection of eight genetically modified maize lines using a combination of event- and construct- specific multiplex-PCR technique. J. Agric. Food Chem. 56: 8962-8968. Sun B, Zhao Y, Shi S, Yang M, Xiao K (2019) TaZFP1, a C2H2 type-ZFP gene of T. aestivum, mediates salt stress tolerance of plants by modulating diverse stress- defensive physiological processes. Plant Physiol. Biochem. 136: 127-142. Taylor SL (1997) Food from genetically modified organisms and potential for food allergy. Environ. Toxicol. Pharmacol. 4:121-126. The Commission of the European Communities (2003a) Regulation (EC) No 1829/2003 of the European Parliament. OJEU, L268: 1-23. The Commission of the European Communities. (2003b) Regulation (EC) No 1830/2003 of the European Parliament. OJEU, L268: 24-28. The Commission of the European Communities (2011) Regulation (EU) No 619/2011. OJEU, L166: 9-15. Thomas L (1992) Labor und Diagnose. 4th Ed., Die Medizinische Verlagsgesellschaft, Marburg. Trabalza-Marinucci M, Brandi G, Rondini C, Avellini L, Giammarini C, Costarelli S, Acuti G, Orlandi C, Filippini G, Chiaradia E, Malatesta M, Crotti S, Antonini C, Amagliani G, Manuali E, Mastrogiacomo AR, Moscati L, Haouet MN, Gaiti A, Magnani M (2008) A three-year longitudinal study on the effects of diet containing genetically modified Bt176 maize on the health status and performance of sheep. Livest. Sci. 113: 178-190. http://www.ncbi.nlm.nih.gov/pubmed/17052831 http://www.sciencedirect.com/science?_ob=GatewayURL&_origin=IRSSCONTENT&_method=citationSearch&_piikey=S0308814611009599&_version=1&md5=debefc8e6391fdbb2476d3376e262e63 http://www.sciencedirect.com/science?_ob=GatewayURL&_origin=IRSSCONTENT&_method=citationSearch&_piikey=S0308814611009599&_version=1&md5=debefc8e6391fdbb2476d3376e262e63 http://www.sciencedirect.com/science?_ob=GatewayURL&_origin=IRSSCONTENT&_method=citationSearch&_piikey=S0308814611009599&_version=1&md5=debefc8e6391fdbb2476d3376e262e63 Nova Biotechnol Chim (2020) 19(1): 19-29 29 Trinder P (1969) Determination of glucose in blood using glucose oxidase with an alternative oxygen receptor. Ann. Clin. Biochem. 6: 24-27. Turkec A, Lucas JS, Karlik E (2016) Monitoring the prevalence of genetically modified (GM) soybean in Turkish food and feed products. Food Cont. 59: 766- 772. World Health Organisation (WHO) (1995) Report of a WHO Workshop, World Health Organisation, Food Safety Unit, Geneva, Switzerland. Zhu H, Chen Y, Li Y, Wang J, Ding J, Chen X, Peng Y (2015) A 90 day safety assessment of genetically modified rice expressing Cry1Ab/1Ac protein using an aquatic animal model. J. Agric. Food Chem. 63: 3627-3633. Zhu Y, Li D, Wang F, Yin J, Jin H (2004) Nutritional assessment and fate of DNA of soybean meal from roundup ready or conventional soybeans using rats. Arch. Anim. Nutr. 58: 295-310.