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Subchronic feeding toxicity studies of drought-tolerant transgenic wheat MGX11-10 in Wistar Han RCC rats
Food and Chemical Toxicology 137 (2020) 111129
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Subchronic feeding toxicity studies of drought-tolerant transgenic wheat MGX11-10 in Wistar Han RCC rats
T
Yinghua Liu, Shujing Zhang, Qinghong Zhou, Shufei Li, Jing Zhang, Li Zhang, Shuqing Jiang, Qian Zhang, Xiaoli Zhou, Chao Wu, Qing Gu∗, Zhi Yong Qian∗∗ Tianjin Centers for Disease Control and Prevention, Tianjin, 300011, China
A R T I C LE I N FO
A B S T R A C T
Keywords: MGX11-10 Genetically modified wheat Drought-tolerant Subchronic toxicity Rat
A subchronic toxicity study were conducted in Wistar Han RCC rats to evaluate the potential health effects of genetically modified (GM), drought-tolerant wheat MGX11-10. Rats were fed a rodent diet formulated with MGX11-10 and were compared with rats fed a diet formulated with its corresponding non-transgenic control Jimai22 and rats fed a basal diet. MGX11-10 and Jimai22 were ground into flour and formulated into diets at concentrations of 16.25, 32.5, or 65%, w/w% and fed to rats (10/sex/group) for 13 weeks. Compared with rats fed Jimai22 and the basal-diet group, no biologically relevant differences were observed in rats fed the GM diet with respect to body weight/gain, food consumption/efficiency, clinical signs, mortality, ophthalmology, clinical pathology (hematology, prothrombin time, urinalysis, clinical chemistry), organ weights, and gross and microscopic pathology. Under the conditions of this study, the MGX11-10 diets did not cause any treatmentrelated effects in rats following at least 90 days of dietary administration as compared with rats fed diets with the corresponding non-transgenic control diet and the basal-diet group. The MGX11-10 diets are considered equivalent to the diets prepared from conventional comparators. The results demonstrated that MGX11-10 wheat is as safe and wholesome as the corresponding non-transgenic control wheat.
1. Introduction Bread wheat (Triticum aestivum L.) is one of the most important food crops, cultivated on about 220 million ha, providing food to one-third of the global population and providing 20% of the global caloric requirements (Gill BS et al., 2004; Shiferaw B et al., 2013; Rasheed A et al., 2018). The yield of wheat is significantly affected by climatic change and scarcity of water resources in the environment. Drought is one of the environmental stresses that seriously limit crop production in the majority of agriculture fields in the world (Al-Maskri et al., 2016; Lesk, C. et al., 2016; Kulkarni M et al., 2017; Matiu et al., 2017). As a result of climate change, the global frequency and severity of drought events is likely to increase (Shahinnia F et al., 2016). It is estimated that a cereal (maize, rice and wheat) loss of 1820 million Mg has been caused by droughts during the past four decades globally (Leng G, 2019). Drought stress caused a reversible decline in leaf water relations, membrane stability, and photosynthetic activity, leading to increased reactive oxygen species (ROS) generation, lipid peroxidation and membrane injury (Abid M et al., 2018). This phenomenon inhibits
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further nutrient absorption and affects crop growth, gene expression, distribution, yield and quality (Stendle and Peterson, 1998; Guo R et al., 2018). A way to improve the drought tolerance of crops is to discover new genes and alleles that allow plants to continue to grow and maintain or increase grain yield under water-limited growing conditions. With the continuous development of transgenic technology, the generalization and application of transgenic crops is progressing rapidly. The methods of modern agricultural biotechnology have been utilized to transfer genes from one organism to another without sexual reproduction and across species. This process allows targeted alterations to be introduced into plant genomes in a more specific and controlled manner and more rapidly than can be achieved through conventional breeding and selection of crops. Wheat event MGX11-10 was genetically modified to improve drought tolerance. The WK gene was cloned from wheat, and the wheat Jimai 22 was used as the transformation receptor. WK gene is a protein kinase gene, WK protein kinase catalyzes phosphorylation of protein, and protein phosphorylation is involved in the transmission of stress signals and plays an important
Corresponding author. Corresponding author. E-mail addresses: [email protected] (Q. Gu), [email protected] (Z.Y. Qian).
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https://doi.org/10.1016/j.fct.2020.111129 Received 4 September 2019; Received in revised form 18 December 2019; Accepted 10 January 2020 Available online 11 January 2020 0278-6915/ © 2020 Published by Elsevier Ltd.
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role in improving plant drought tolerance (Zhao, 2015). Genetically modified plants expressing drought-tolerance traits offer a new strategy for crop protection, but at the same time, present a challenge in terms of food safety assessment. Because the methods used to produce crops with biotechnology differ from those used in conventional breeding, numerous scientific organizations have published guidelines for assessing their safety (WHO, 1991 and 1995; FAO, 1996; and OECD, 1993 and 1997). The primary goal established in these guidelines is comparison analysis between the particular genetically modified (GM) crop and the corresponding non-transgenic control crop with an established history of safe use. In most cases, the data necessary to demonstrate that a GM crop is “as safe as” the corresponding non-transgenic control comparator can be determined based on compositional analysis, however, a number of 13 week (i.e., subchronic) rodent toxicology studies have also been conducted with food and feed fractions from GM crops that support their safety (Bai H et al., 2015; Sabitha et al. 2017; Zou S et al., 2018; Sabitha et al. 2018; Qian et al., 2018a; Qian et al., 2018b). In the present study, we report the results of a 13 week rodent toxicology study conducted with MGX11-10 wheat. This study was conducted in compliance with Chinese guideline on the performance of safety assessment of genetically modified plant and derived products 90-day feeding test in rats（NY/T 1102–2006）at the Experimental Animal Center of the Tianjin Centers for Disease Control and Prevention (Tianjin, China).
Table 1 Study design – dose groups. Group Number
1 2 3 4 5 6 7
Test Substance
The basal-diet control Jimai 22-Low Jimai 22-Mid Jimai 22-High MGX11-10-Low MGX11-10-Mid MGX11-10-High
% in Diet
0 16.2 32.5 65 16.2 32.5 65
Number of Animals/Group Males
Females
10 10 10 10 10 10 10
10 10 10 10 10 10 10
dietary requirements of the rat strain Wistar. All experimental diets were vacuum-packed and sterilized through irradiating by 60Co. The animals were housed one per cage. Feed and drinking water were provided ad libitum for 90 days. 2.4. Body weight, feed consumption and clinical observations Food consumption and body weights were recorded weekly, cageside examination was conducted at least once a day. Significant clinical abnormalities that were evaluated included, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, occurrence of secretions and excretions, neuromuscular function (convulsion, fasciculation, tremor, twitching), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. All animals were observed for morbidity, mortality, and the availability of feed and water.
2. Materials and methods 2.1. Plant materials The GM wheat, MGX11-10, and the corresponding non-transgenic control wheat, JIMAI 22, were supplied by the Chinese Academy of Agricultural Sciences. The wheat was from plants grown concurrently in the same location. After harvest and a storage period of three months, the mature seeds were milled and processed into flour for diet preparation.
2.5. Urinalysis Urine samples were obtained from all animals the week prior to the scheduled necropsy. Animals were housed in metabolism cages and urine samples were collected for 4 h. Water were available ad libitum during this procedure. Urine samples were inspected for color (COL), and clarity (CLA). The urine was analyzed for specific gravity (SG), pH, urobilinogen (URO), protein (PRO), glucose (GLC), ketones (KET), bilirubin (BIL), occult blood (BLD), leukocytes (LEU), and nitrites (NIT). Analysis was performed using RUIKE (Uritest-150) Urine Chemistry Analyzer (Changchun Ruike Medical Technology Co., Ltd, Changchun, China).
2.2. Animals The feeding studies were conducted at the Specific Pathogen Free animal laboratory of the Testing Center of Genetically Modified Organisms at Tianjin (China) approved by the Ministry of Agriculture of China, with the Certificate No. SYXK (Jin) 2014–0001. A total of 140 male and female Wistar Han RCC rats, 4 weeks old and with a uniform weight ( ± 20% of the mean), purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China) with the Certificate No. SCXK (Jing) 2016–0006 11400700259430. Rats were acclimatized to the study conditions for 5 days before the beginning of the treatment period and fed the standard basal diet. Rats were housed in a polypropylene plastic cage singly with ad libitum access to water and food. The animal room conditions were as follows: 20～25°C temperature, 40～70% relative humidity, 12 h/12 h light/dark cycle, 8–15 cycles/h of filtered, nonrecycled air ventilation.
2.6. Hematology and coagulation Animals were fasted overnight prior to blood collection with water provided ad libitum. Blood samples were collected from the abdominal aorta after rats were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital at the scheduled necropsy. Blood samples were mixed with ethylenediaminetetraacetic acid (EDTA) and assayed using an Sysmex XT-2000i hematology analyzer (Sysmex Company, Japan). The following parameters were tested: the white blood cell count (WBC), red blood cell count (RBC), haemoglobin concentration (HGB), haematocrit (HCT), platelet count (PLT) and differential leucocyte count [percent neutrophil (NEU), lymphocyte (LYMPH), monocyte (MONO), eosinophil (EOS), basophil (BASO)]. Blood samples were collected in sodium citrate tubes, centrifuged and plasma collected and assayed for prothrombin time (PT) and activation of partial thrombin time (APTT) using an Sysmex CS-5100 coagulation analyzer (Sysmex Company, Japan)
2.3. Study design Rats were randomly divided into seven groups by body weight with 10 rats/gender/group. The mean body weights of each group varied within ± 20%. Groups of 10 male and 10 female rats were fed diets containing GM wheat, MGX11-10 and the corresponding non-transgenic control wheat Jimai22 and the basal diet for at least 90 days, respectively. A total of seven diets were prepared by Beijing Keao Xieli Feed Co., Ltd. and met standard GB 14924.3–2010 “Laboratory animals – Mice and rat formula feeds.” Flour from MGX11-10 and Jimai22 was formulated into diets at a percentage of 16.25%, 32.5% and 65% of the total diet, respectively (Table 1). And control groups were fed the standard basal diet. The formulation of the diets was adjusted to the
2.7. Clinical chemistry Animals were fasted 16 h prior to blood collection at the end of the 2
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study. Serum was collected after centrifugation and assayed using an Toshiba TBA-40FR hematology analyzer (Toshiba Co., Japan) for the following parameters: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), urea nitrogen (BUN), creatinine (CREA), glucose (GLU), cholesterol(CHOL), triglyceride(TG), alkaline phosphatase(ALP), lactate dehydrogenase(LDH), glutamine transpeptide enzyme（GGT）. Chloride (Cl−), potassium (K+), and sodium (Na+) were assayed using an MEDICA-Easylute Plus Ion analyzer (Medica company, USA).
2.8. Gross necropsy and histopathology After blood samples were collected, all rats were necropsied by exsanguinations and submitted to a full macroscopic postmortem examination. Necropsy included examination of the external surface, all orifices and the cranial, thoracic, abdominal and pelvic cavities, including viscera. The heart, brain, liver, kidneys, adrenal glands, spleen, thymus, uterus (female), ovaries (female), testes (male) and epididymides (male) were trimmed off extraneous fat and weighed immediately. The ratios of organ weight to terminal body weight were calculated. The tissues from all animals were collected and preserved in neutral, phosphate-buffered 10% formal, trimmed, embedded in paraffin, sectioned, mounted on glass microscope slides, stained with hematoxylin and eosin, and histologically examined under a light microscope. The following tissues were examined microscopically: brain, heart, lungs, liver, kidney, adrenal glands, spleen, stomach, intestine (duodenum, jejunum, and ileum), thymus, thyroid glands, testes (male), epididymis (male), prostate (male), ovaries (female) and uterus (female).
Fig. 1. Mean weekly body weights of male rats. Line plots of mean male body weights (g) per group.
2.9. Statistical analysis Statistical analysis was designed to determine if differences exist at day 90 among the GM wheat group, the corresponding non-transgenic wheat group and the basal-diet group. Data of body weights, food efficiency, organ weight, relative organ weight, urinalysis, clinical chemistry, hematology and coagulation were analyzed. Differences between groups were considered significant if p < 0.05. Food efficiency was calculated as (body weight gain/feed consumption) × 100%. Relative organ weight was calculated as (wet weight of organ/body fasting weight) × 100%. All data were presented as group mean values ± standard deviation (X ± SD), and continuous data were compared by ANOVA using SPSS 16.0 software, and frequency data were compared using a Chi-square test (α = 0.05). Quantitative and categorical data for each gender were analyzed separately. Data from the GM groups were first compared with the basaldiet group; then the values from the GM groups were compared with those in the corresponding non-transgenic control groups. A p value of < 0.05 was considered significant, and the letter “a” represents significant difference between the test groups and the basal-diet group, while the letter “b” represents significant difference between transgenic wheat group and the corresponding non-transgenic control group.
Fig. 2. Mean weekly body weights of female rats. Line plots of mean female body weights (g) per group.
3.2. Body weight and feed consumption There were no statistically identified or treatment-related differences in the mean body weights (Fig. 1, Fig. 2), total body weight gains (Table 2) of male or female rats given GM wheat, MGX11-10 at 16.25%, 32.5% and 65% level as compared with their corresponding nontransgenic control groups and the basal-diet group. Mean feed consumption were statistically lower (p < 0.05), when compared with the corresponding non-transgenic group, for the 16.25% MGX11-10 males during study week 5–6 (Fig. 3, Fig. 4) and for the 32.5% MGX11-10 females during study week 8 (Fig. 5, Fig. 6). Mean feed consumption of the 32.5% MGX11-10 females during study week 5,9 and 11 were statistically lower (p < 0.05) than the basal-diet group (Figs. 5 and 7). Total feed efficiency (Table 2) for males in 32.5% MGX11-10 group were comparatively higher (p < 0.05) than the basal-diet group.
3. Results 3.3. Hematology and coagulation 3.1. Clinical observation The following statistically significant differences were found when the MGX11-10 groups were compared with the corresponding nontransgenic control group: percent NEU results for the 16.25% MGX1110 males were higher (p < 0.05) than for the corresponding nontransgenic control group; percent LYMPH for the 16.25% MGX11-10 males were lower (p < 0.05) than the corresponding non-transgenic control group. The values of MONO and APTT for the 16.25% MGX1110 females were higher (p < 0.05) than for the corresponding non-
No signs of morbidity and mortality were observed during the whole 90 days. All rats survived the duration of the feeding trial. During the study, the animals in each group were active, ate normally. Clinical observation of the treated rats throughout the study indicated that none of them showed signs of toxicity in their skin, fur, eyes, mucus membrane, or behavioral changes, diarrhoea, tremors, salivation, sleep, and coma. 3
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Table 2 Growth performances of rats fed different diets for 90 days. Gender Males The basal-diet control 16.25% Jimai 22 32.5% Jimai 22 65% Jimai 22 16.25% MGX11-10 32.5% MGX11-10 65% MGX11-10 Females The basal-diet control 16.25% Jimai 22 32.5% Jimai 22 65% Jimai 22 16.25% MGX11-10 32.5% MGX11-10 65% MGX11-10
Total body weight gain (g)
Total food consumption (g)
Food efficiency (%)
406.1 414.0 421.4 424.5 404.0 434.8 402.0
± ± ± ± ± ± ±
55.0 48.3 34.0 36.4 30.1 48.3 45.6
2572.9 2588.5 2520.9 2478.8 2495.9 2568.3 2450.7
± ± ± ± ± ± ±
236.5 232.9 130.3 123.0 89.6 225.4 160.1
15.8 16.0 16.7 17.1 16.2 17.0 16.4
± ± ± ± ± ± ±
1.3 1.2 0.9 0.8a 1.0 1.5a 0.9
244.0 235.5 232.0 246.5 246.8 233.2 242.9
± ± ± ± ± ± ±
40.8 31.0 32.8 23.4 44.3 28.7 30.7
2063.3 2033.3 2003.8 1917.0 2001.9 1928.2 1930.8
± ± ± ± ± ± ±
234.9 166.6 148.8 87.2 133.6 164.4 201.3
11.8 11.6 11.6 12.9 12.3 12.1 12.6
± ± ± ± ± ± ±
0.9 1.0 1.2 1.0 1.9 0.9 1.5
Data are reported as mean ± SD, n = 10. a: p < 0.05 Significantly different from the basal-diet control group. b: p < 0.05 Significantly different from the corresponding non-transgenic control group.
Fig. 5. Mean weekly feed consumption of female rats. Line plots of mean feed consumption per group (g) and per week in the case of female.
Fig. 3. Mean weekly feed consumption of male rats. Line plots of mean feed consumption per group (g) and per week in the case of male.
Fig. 6. Mean weekly feed consumption of 32.5% MGX11-10 and 32.5% Jimai22 females in the eighth week. Values represent Mean ± SEM. n = 10.*p < 0.05 Significantly different from the corresponding non-transgenic control group.
Fig. 4. Mean weekly feed consumption of 16.25% MGX11-10 and 16.25% Jimai22 males in the fifth and sixth week. Values represent Mean ± SEM. n = 10.*p < 0.05 Significantly different from the corresponding non-transgenic control group.
transgenic control group (Table 3). 3.4. Clinical chemistry The clinical chemistry data (Table 4) illustrated that there were no statistically identified or treatment-related differences in the values of 4
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The value of chloride in the 32.5% MGX11-10 group was lower (p < 0.05) than in the 32.5% Jimai22 group, but the value of chloride in 65% MGX11-10 group was higher (p < 0.05) when compared to 65% Jimai22 group and the basal-diet group. The values of sodium, potassium and chloride for the 16.25% MGX11-10 females were lower (p < 0.05) than for the 16.25% Jimai22 females. The values of sodium, potassium and chloride for the 65% MGX11-10 females were higher (p < 0.05) than for the 65% Jimai22 females. The values of sodium and chloride for the 32.5% and 65% MGX1110 males were higher (p < 0.05) than for the basal-diet group. The values of sodium and chloride for the 65% MGX11-10 females were higher (p < 0.05) than for the basal-diet group. The values of sodium and chloride for the 32.5% MGX11-10 females were lower (p < 0.05) than for the basal-diet group. 3.5. Urinalysis There were no statistically significant differences or treatment-related alterations in any of the urinalysis parameters for male and female rats in GM groups as compared with their respective non-transgenic and the basal-diet controls (Table 5).
Fig. 7. Mean weekly feed consumption of 32.5% MGX11-10 and the basal-diet control females in the fifth, ninth and eleventh week. Values represent Mean ± SEM. n = 10. *p < 0.05 Significantly different from the basal-diet group.
3.6. Organ weights and relative organ weights
ALT, AST, BUN, CHO, TG, GLU, TP, ALB, CRE, ALP, LDH and GGT of male or female rats in 16.25%,32.5% and 65% MGX11-10 groups compared with their corresponding non-transgenic control groups and the basal-diet group. In males, the value of sodium in the 32.5% MGX11-10 group was lower (p < 0.05) than in the 32.5% Jimai22 group, no difference was seen between the 65% MGX11-10 group and 65% Jimai22 group or the 16.25% MGX11-10 group and 16.25% Jimai22 group. The value of sodium in 32.5% and 65% MGX11-10 group were higher (p < 0.05) than in the basal-diet group. In females, the value of sodium in the 16.25% MGX11-10 group was lower (p < 0.05) than in the 16.25% Jimai22 group, the value of sodium in the 65% MGX11-10 group was higher (p < 0.05) than in the 65% Jimai22 group.
There were no statistically identified differences in any of the organ weights and the relative organ weights in males and females given MGX11-10 at 16.25%, 32.5% and 65% level as compared with their corresponding non-transgenic control groups and the basal-diet group (Table 6). 3.7. Histopathological examination The following histopathological changes were observed: a few small round inflammatory cells in portal area were observed in the liver (2 in the basal-diet group, 2 in 65% MGX11-10 group, 1 in 65% Jimai22 group), infiltration of interstitial inflammatory cell were observed in the kidneys (1 in the basal-diet group, 2 in 65% MGX11-10 group),
Table 3 Hematological parameters of rats fed different diets for 90 days. Gender Males WBC(109/L) RBC(1012/L) HGB(g/dL) PLT(109/L) HCT(%) Neu(%) Lymph(%) Mono(%) Eos(%) Baso(%) PT APTT Females WBC(109/L) RBC(1012/L) HGB(g/dL) PLT(109/L) HCT(%) Neu(%) Lymph(%) Mono(%) Eos(%) Baso(%) PT APTT
The basal-diet control
16.25% Jimai 22
32.5% Jimai 22
65%Jimai 22
16.25% MGX11-10
32.5% MGX11-10
65% MGX11-10
2.95 ± 0.66 7.46 ± 0.18 134.2 ± 4.5 1172.2 ± 138.5 36.8 ± 1.9 25.10 ± 7.46 69.86 ± 8.65 2.73 ± 1.14 2.31 ± 0.92 0.00 ± 0.00 8.8 ± 0.3 11.3 ± 2.4
2.98 ± 1.00 7.66 ± 0.23 134.9 ± 6.5 1185.4 ± 138.8 36.9 ± 2.2 23.96 ± 5.90 71.27 ± 6.39 2.71 ± 0.89 2.06 ± 0.73 0.00 ± 0.00 8.7 ± 0.3 11.1 ± 2.7
3.13 ± 0.94 7.68 ± 0.26 137.8 ± 4.0 1202.0 ± 133.9 37.1 ± 0.9 25.02 ± 5.42 68.53 ± 5.88 3.71 ± 0.77 2.74 ± 0.82 0.00 ± 0.00 8.6 ± 0.3 11.2 ± 2.7
2.50 ± 0.59 7.69 ± 0.30 137.3 ± 3.6 1143.8 ± 96.0 37.1 ± 1.1 22.92 ± 2.36 71.30 ± 2.89 3.47 ± 1.26 2.31 ± 0.81 0.00 ± 0.00 8.7 ± 0.1 11.6 ± 2.1
2.79 ± 0.79 7.60 ± 0.32 136.8 ± 6.1 1210.6 ± 123.7 37.4 ± 1.3 30.21 ± 4.08b 64.41 ± 4.05b 3.25 ± 0.95 2.13 ± 0.75 0.00 ± 0.00 8.6 ± 0.3 11.6 ± 2.6
2.91 ± 0.81 7.71 ± 0.29 137.4 ± 5.0 1142.6 ± 146.5 37.6 ± 1.8 27.12 ± 7.44 66.98 ± 8.28 3.41 ± 1.08 2.49 ± 0.47 0.00 ± 0.00 8.5 ± 0.3 10.6 ± 3.8
3.06 ± 0.76 7.48 ± 0.31 137.0 ± 5.2 1148.2 ± 84.0 37.5 ± 1.5 22.81 ± 6.62 71.37 ± 7.48 3.69 ± 1.52 2.13 ± 0.88 0.00 ± 0.03 8.7 ± 0.4 12.1 ± 1.9
1.82 ± 0.38 6.84 ± 0.20 131.0 ± 4.2 1077.0 ± 141.9 36.6 ± 1.5 19.82 ± 2.70 74.90 ± 2.77 2.40 ± 1.04 2.88 ± 0.68 0.00 ± 0.00 8.5 ± 0.4 13.2 ± 1.1
1.67 ± 0.50 6.86 ± 0.34 130.0 ± 5.3 1032.6 ± 171.0 36.1 ± 1.3 19.66 ± 6.69 75.38 ± 7.02 2.12 ± 0.90 2.84 ± 0.78 0.00 ± 0.00 8.2 ± 0.3 11.5 ± 1.9
1.36 ± 0.24a 6.87 ± 0.21 130.1 ± 4.1 1035.4 ± 117.1 36.4 ± 1.9 20.43 ± 4.52 75.43 ± 4.98 1.51 ± 0.83 2.63 ± 0.97 0.00 ± 0.00 8.4 ± 0.4 13.2 ± 2.8
1.50 ± 0.34 6.77 ± 0.22 129.4 ± 5.0 988.0 ± 153.2 35.5 ± 1.2 24.78 ± 5.50 70.05 ± 5.55 2.40 ± 1.30 2.77 ± 1.42 0.00 ± 0.00 8.5 ± 0.3 13.1 ± 1.6
1.63 ± 0.75 6.87 ± 0.18 131.7 ± 4.7 1088.9 ± 78.1 36.3 ± 1.5 22.23 ± 4.21 71.04 ± 4.71 3.26 ± 1.30b 3.47 ± 1.15 0.00 ± 0.00 8.5 ± 0.3 13.2 ± 2.9b
1.66 ± 0.49 6.80 ± 0.27 128.2 ± 5.6 1088.5 ± 100.1 35.9 ± 1.9 23.50 ± 6.44 72.05 ± 6.49 2.11 ± 1.07 2.34 ± 0.82 0.00 ± 0.00 8.5 ± 0.3 13.2 ± 2.2
1.44 ± 0.34 6.90 ± 0.34 130.5 ± 6.2 1057.3 ± 169.5 35.8 ± 1.5 23.69 ± 8.25 70.64 ± 9.70 2.02 ± 1.54 3.65 ± 1.66 0.00 ± 0.00 8.4 ± 0.4 13.4 ± 3.2
Data are reported as mean ± SD, n = 10. a: p < 0.05 Significantly different from the basal-diet control group. b: p < 0.05 Significantly different from the corresponding non-transgenic control group. 5
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Table 4 Clinical biochemistry parameters of rats fed different diets for 90 days. Gender Males ALT(U/L) AST(U/L) TP(g/L) ALB(g/L) GGT(U/L) BUN(mmol/L) GLU(mmol/L) CRE(μmol/L) CHO(mmol/L) TG(mmol/L) ALP(U/L) LDH (U/L) Na+(mmol/L) K+(mmol/L) Cl−(mmol/L) Females ALT(U/L) AST(U/L) TP(g/L) ALB(g/L) GGT(U/L) BUN(mmol/L) GLU(mmol/L) CRE(μmol/L) CHO(mmol/L) TG(mmol/L) ALP(U/L) LDH (U/L) Na+(mmol/L) K+(mmol/L) Cl−(mmol/L)
The basal-diet control
16.25% Jimai 22
32.5% Jimai 22
65%Jimai 22
16.25% MGX11-10
32.5% MGX11-10
65% MGX11-10
45.1 ± 5.4 104.0 ± 6.0 65.4 ± 2.6 37.2 ± 1.3 1.09 ± 0.63 6.46 ± 0.67 8.29 ± 0.49 35.4 ± 3.4 2.35 ± 0.34 0.40 ± 0.13 80.7 ± 10.6 1504.1 ± 339.2 143.7 ± 0.9 4.66 ± 0.27 110.8 ± 0.92
45.9 ± 6.3 99.5 ± 7.3 64.0 ± 2.4 36.5 ± 1.4 1.11 ± 0.40 6.22 ± 0.43 8.24 ± 0.64 33.7 ± 4.5 2.41 ± 0.35 0.45 ± 0.13 89.1 ± 18.3 1356.3 ± 264.6 145.4 ± 5.5 4.78 ± 0.26 112.4 ± 4.59
42.9 ± 7.4 10.3.0 ± 9.9 65.4 ± 2.1 37.1 ± 1.1 1.29 ± 0.34 6.63 ± 0.70 8.61 ± 0.41 34.8 ± 2.9 2.48 ± 0.27 0.40 ± 0.12 92.5 ± 16.7 1413.7 ± 355.4 152.2 ± 1.0a 4.83 ± 0.32 119.9 ± 1.03a
42.4 ± 5.7 100.3 ± 11.6 65.3 ± 3.1 36.9 ± 1.7 1.05 ± 0.37 6.03 ± 0.71 8.72 ± 0.73 35.7 ± 3.3 2.54 ± 0.47 0.43 ± 0.13 89.7 ± 16.7 1444.9 ± 305.3 144.3 ± 1.1 4.62 ± 0.31 112.2 ± 1.31
42.8 ± 5.6 110.4 ± 17.3 64.3 ± 2.1 36.6 ± 1.6 1.10 ± 0.32 6.97 ± 0.82 8.13 ± 0.65 36.4 ± 2.5 2.60 ± 0.36 0.38 ± 0.14 81.7 ± 16.3 1579.3 ± 306.9 146.0 ± 1.6 4.67 ± 0.34 112.2 ± 1.31
41.6 ± 7.7 101.5 ± 7.7 65.3 ± 2.2 37.4 ± 1.5 1.04 ± 0.43 7.20 ± 0.97 7.95 ± 0.61 34.2 ± 2.5 2.51 ± 0.35 0.49 ± 0.16 84.0 ± 10.4 1447.2 ± 246.2 147.4 ± 1.2ab 4.81 ± 0.29 113.6 ± 1.35 ab
46.9 ± 5.7 100.4 ± 6.9 64.2 ± 2.0 36.5 ± 1.2 1.07 ± 0.28 7.05 ± 0.87 7.97 ± 0.81 32.8 ± 2.8 2.43 ± 0.36 0.41 ± 0.21 91.9 ± 11.7 1458.8 ± 235.4 146.6 ± 3.9a 4.83 ± 0.37 113.2 ± 3.46 ab
33.2 ± 5.1 94.6 ± 24.5 64.9 ± 2.3 38.7 ± 1.5 1.35 ± 0.38 6.39 ± 0.79 7.75 ± 1.15 41.9 ± 2.2 2.02 ± 0.61 0.49 ± 0.15 57.4 ± 13.7 1440.5 ± 451.0 152.1 ± 1.1 4.26 ± 0.19 119.1 ± 1.45
36.5 ± 3.8 92.1 ± 19.3 63.6 ± 3.1 38.1 ± 2.0 1.26 ± 0.27 6.42 ± 1.10 7.99 ± 0.47 39.5 ± 2.3 2.21 ± 0.49 0.48 ± 0.13 52.2 ± 10.2 1239.7 ± 363.3 157.1 ± 5.9a 4.54 ± 0.33a 124.7 ± 3.54a
38.4 ± 7.0 92.1 ± 9.3 65.7 ± 3.1 39.3 ± 2.2 1.24 ± 0.34 6.16 ± 0.72 8.44 ± 0.52 39.7 ± 2.8 2.19 ± 0.62 0.42 ± 0.05 53.2 ± 8.8 1245.5 ± 320.0 144.3 ± 0.9a 4.02 ± 0.18 112.5 ± 1.54a
35.9 ± 5.4 92.6 ± 10.5 66.0 ± 3.3 39.7 ± 1.8 1.27 ± 0.38 6.73 ± 1.19 8.37 ± 0.47 39.4 ± 2.6 2.26 ± 0.61 0.44 ± 0.16 53.7 ± 11.0 1135.2 ± 288.3 146.8 ± 3.5a 4.21 ± 0.34 114.3 ± 2.23a
38.2 ± 5.7 96.2 ± 14.6 65.4 ± 3.0 40.0 ± 2.0 1.12 ± 0.29 6.82 ± 0.77 8.55 ± 0.46 39.5 ± 2.4 2.30 ± 0.55 0.43 ± 0.10 53.7 ± 16.0 1129.5 ± 347.6 152.3 ± 1.2b 4.26 ± 0.23b 120.7 ± 1.48b
37.2 ± 8.0 94.4 ± 15.2 63.7 ± 2.9 38.3 ± 1.2 1.21 ± 0.26 6.51 ± 0.46 8.23 ± 0.54 38.2 ± 2.4 2.18 ± 0.53 0.49 ± 0.16 54.4 ± 7.6 1328.9 ± 251.0 145.3 ± 1.3a 4.20 ± 0.35 113.8 ± 1.24a
35.6 ± 6.5 98.6 ± 15.0 62.8 ± 4.4 38.8 ± 3.1 1.29 ± 0.36 6.58 ± 0.91 8.05 ± 0.70 40.8 ± 8.7 2.08 ± 0.37 0.48 ± 0.09 52.8 ± 12.6 1137.6 ± 304.2 156.8 ± 4.1 ab 4.64 ± 0.38 ab 121.3 ± 2.92 ab
Data are reported as mean ± SD, n = 10. a: p < 0.05 Significantly different from the basal-diet control group. b: p < 0.05 Significantly different from the corresponding non-transgenic control group. Table 5 Urinalysis of rats fed different diets for 90 days. Gender Males Urobilinogen Gravity PH Females Urobilinogen Gravity PH
The basal-diet control
16.25% Jimai 22
32.5% Jimai 22
65%Jimai 22
16.25% MGX11-10
32.5% MGX11-10
65% MGX11-10
6.12 ± 5.73 1.024 ± 0.005 7.70 ± 0.63
6.12 ± 5.73 1.029 ± 0.003 6.90 ± 0.46
8.84 ± 7.02 1.050 ± 0.088 7.65 ± 0.94
7.48 ± 6.57 1.023 ± 0.005 7.65 ± 0.94
7.48 ± 6.57 1.023 ± 0.005 7.70 ± 1.03
4.76 ± 4.30 1.022 ± 0.005 7.60 ± 0.91
4.76 ± 4.30 1.024 ± 0.005 7.55 ± 0.86
7.48 ± 6.57 1.021 ± 0.008 7.50 ± 0.82
6.12 ± 5.73 1.020 ± 0.005 7.95 ± 0.60
7.48 ± 6.57 1.018 ± 0.004 8.00 ± 0.67
8.84 ± 7.02 1.023 + 0.005 7.65 ± 0.82
10.20 ± 7.17 1.025 ± 0.005 7.00 ± 0.82
8.84 ± 7.02 1.022 ± 0.009 6.80 ± 0.86
7.48 ± 6.57 1.021 ± 0.006 7.45 ± 0.72
Data are reported as mean ± SD, n = 10.
transferred genes or modifications in the expression of endogenous genes (Schreier et al., 1985; Willmitzer, 1988; Ilyas, 2017; Duan C et al., 2019). Transgenic technology is widely used in medicine, agriculture, research, and industry. The benefits of GM crops include an overall increase in yield of almost 22%, a 36.9% reduction in pesticide use and an increase in farmer profit slightly over 68% (Catherine Kramer et al., 2016). Despite a number of studies demonstrated the assessed genetically modified plants would be as safe as the parental species of these plants (Domingo JL, 2016), the claim of the safety of this feed is still controversial in many countries. The concern about the safety of GM products arises with the increased prevalence of GM products (Raman R. et al., 2017; Tsatsakis AM et al., 2017). The concern about the safety of GM products would require spelling out the values and assumptions (for example, the sufficient evidence for safety) behind risk assessment (Martinelli L. et al., 2013). Therefore, GM products should undergo a strict evaluation before they are allowed to enter the market. A 30- or 90-day feeding study is traditionally used to assess the safety of a GM
slight interstitial pneumonia or focal pneumonia and perivascular inflammatory cell infiltration were observed in the lung and trachea (3 in the basal-diet group, 3 in 65% MGX11-10 group, 1 in 65% Jimai22 group) and slight gland interstitial inflammatory cell infiltration were observed in the prostate (1 in 65% MGX11-10 group). The brain, heart, adrenal gland, spleen, gastrointestinal (duodenum, jejunum and ileum), thymus, thyroid, testis, epididymis, ovary, or uterus tissues did not show any histopathological lesions.
4. Discussion Farmer has used traditional breeding techniques for many years to select desired crop traits. Since the development of advanced genetic technologies over the past 20 years, breeders and scientists were allowed to bring specific desired genetic changes into the plants in a more precise and controlled manner. It is possible to transfer a gene from one organism to another without sexual reproduction. Plants have been engineered using a variety of techniques including expression of 6
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Table 6 Organ/body weight ratios of rats fed with different diets for 90 days. Gender Males Brain Heart Thymus Liver Spleen Kidney Adrenal Testis Epididymis Females Brain Heart Thymus Liver Spleen Kidney Adrenal Uterus Ovary
The basal-diet control
16.25% Jimai 22
32.5% Jimai 22
65%Jimai 22
16.25% MGX11-10
32.5% MGX11-10
65% MGX11-10
0.42 ± 0.03 0.29 ± 0.02 0.11 ± 0.04 2.80 ± 0.15 0.21 ± 0.03 0.72 ± 0.05 0.018 ± 0.003 0.83 ± 0.10 0.371 ± 0.054
0.42 ± 0.04 0.30 ± 0.06 0.10 ± 0.02 2.96 ± 0.24 0.22 ± 0.02 0.76 ± 0.05 0.016 ± 0.003 0.71 ± 0.20 0.337 ± 0.097
0.41 ± 0.03 0.28 ± 0.03 0.11 ± 0.01 2.84 ± 0.18 0.20 ± 0.02 0.71 ± 0.07 0.015 ± 0.002 0.76 ± 0.06 0.330 ± 0.046
0.41 ± 0.03 0.28 ± 0.04 0.11 ± 0.02 2.74 ± 0.13 0.20 ± 0.01 0.73 ± 0.06 0.017 ± 0.003 0.75 ± 0.07 0.326 ± 0.042
0.43 ± 0.03 0.29 ± 0.03 0.10 ± 0.03 2.80 ± 0.14 0.21 ± 0.02 0.74 ± 0.05 0.016 ± 0.004 0.80 ± 0.07 0.354 ± 0.056
0.40 ± 0.04 0.30 ± 0.03 0.10 ± 0.02 2.95 ± 0.24 0.22 ± 0.02 0.76 ± 0.06 0.016 ± 0.005 0.78 ± 0.10 0.323 ± 0.036
0.43 ± 0.04 0.29 ± 0.03 0.10 ± 0.03 2.67 ± 0.34 0.20 ± 0.03 0.75 ± 0.06 0.018 ± 0.007 0.79 ± 0.08 0.345 ± 0.059
0.61 ± 0.11 0.33 ± 0.03 0.16 ± 0.03 2.97 ± 0.26 0.24 ± 0.03 0.72 ± 0.05 0.030 ± 0.008 0.18 ± 0.04 0.072 ± 0.016
0.65 ± 0.07 0.36 ± 0.07 0.13 ± 0.04 3.04 ± 0.56 0.24 ± 0.04 0.80 ± 0.14a 0.032 ± 0.007 0.19 ± 0.05 0.074 ± 0.016
0.65 ± 0.07 0.35 ± 0.04 0.13 ± 0.02 2.91 ± 0.14 0.24 ± 0.04 0.76 ± 0.07 0.034 ± 0.007 0.22 ± 0.05 0.074 ± 0.012
0.61 ± 0.07 0.34 ± 0.06 0.14 ± 0.05 2.79 ± 0.26 0.23 ± 0.03 0.71 ± 0.08 0.033 ± 0.007 0.18 ± 0.05 0.068 ± 0.017
0.63 ± 0.08 0.34 ± 0.06 0.15 ± 0.04 2.93 ± 0.20 0.22 ± 0.05 0.74 ± 0.07 0.033 ± 0.008 0.17 ± 0.03 0.061 ± 0.011
0.64 ± 0.08 0.33 ± 0.03 0.13 ± 0.03 2.92 ± 0.16 0.23 ± 0.02 0.73 ± 0.05 0.034 ± 0.007 0.22 ± 0.07 0.067 ± 0.007
0.64 ± 0.07 0.35 ± 0.04 0.14 ± 0.03 2.88 ± 0.39 0.24 ± 0.01 0.76 ± 0.08 0.031 ± 0.006 0.20 ± 0.03 0.071 ± 0.023
Data are reported as mean ± SD, n = 10. a: p < 0.05 Significantly different from the basal-diet control group. b: p < 0.05 Significantly different from the corresponding non-transgenic control group.
Clinical biochemistry parameters in this study were similar between the groups fed GM wheat and the corresponding non-transgenic control group or the basal-diet control group of male and female rats with the exceptions, as described below. Results of serum electrolyte showed that there were statistically significant differences in some individual parameters between the transgenic wheat groups and their corresponding non-transgenic wheat groups or basal-diet group (Table 4). But none displayed a dose-response. Histopathology examination revealed no biological relevant histopathological change. Urine specific gravity, pH, protein, and leucocyte level were in the normal range in all treated groups (Table 5). Organ weight changes are often associated with treatment-related effects. The choice of appropriate organs to weigh in toxicology studies involves understanding the test article's mechanism of action, metabolism and toxicokinetics; the physiology of the test species (Sellers, R. S. et al., 2007). Organ coefficient is also an important index for toxicological study because it can reduce the variability of data and increase the chances of detecting treatment effects. The results of this study revealed that all the absolute and relative weights of the liver, kidneys, adrenals, spleen, heart, thymus, brain, testes, and epididymides of rats in the GM groups, were comparable to those of the corresponding non-transgenic control groups and the basal-diet control group (Table 6). A low number of histological changes, mostly inflammatory reactions, were observed in basal-diet control group, GM group and the corresponding non-transgenic control group, but there was no difference among basal-diet control group, GM group and the corresponding non-transgenic control group. The changes were found in a similar range in both groups, and incidences of these findings were all within the historical control ranges of our laboratory, all the pathological observations were interpreted to be spontaneous alterations, unassociated with exposure to the genetically modified wheat. Based on the results of this study, there were no treatment related effects in clinical signs, ophthalmic examinations, body weights, feed consumption, hematology, prothrombin time, activation of partial thrombin time, clinical chemistry, urinalysis parameters, organ weights, organ coefficient and gross or histopathological observations.
product. MGX11-10 is a genetically modified wheat which expresses drought tolerant proteins. To assess the safety of MGX11-10, a 90-day rat feeding study was conducted according to the Chinese guideline on the performance of safety assessment of genetically modified plant and derived products 90-day feeding test in rats（NY/T 1102–2006）. In this study, we conducted a feeding study to evaluate the presence of any unintended adverse effects on rats associated with the consumption of diets containing GM wheat at different doses. The results showed there were no significant differences between the diet nutrient composition of GM wheat, the corresponding non-transgenic control group and the basal-diet group. The animals in each group were active with normal shiny coats, and there was no abnormal discharge from the nose, eyes, or mouth during the study. The mean body weights (Figs. 1 and 2), total body weight gains （Table 2) of each group were normal and no statistical differences were observed between groups. A lower mean feed consumption for 16.25% MGX11-10 males during study week 5–6 (Figs. 3 and 4) and for 32.5% MGX11-10 females during study week 8 (Figs. 5 and 6) than the corresponding non-transgenic control group were found. Mean feed consumption of the 32.5% MGX11-10 females during study week 5,9 and 11 were statistically lower (p < 0.05) than the basal-diet group (Figs. 5 and 7). And the total feed efficiency of 32.5% MGX11-10 males were comparatively higher than the basal-diet group (Table 2). But the changes were not dose responsive, transient and within the normal range. Some differences disappeared in the final week of the test. Hematological analyses in humans and animals are a sensitive indicator of drug and chemical toxicity. In analyses of blood indices, no abnormalities in several measurements in the GM-treated animals (white blood cell count, red blood cell count, platelet count, hemoglobin, and neutrophil count) were found, but the percentage of NEU (males in 16.25% MGX11-10 group), the percentage of MONO and APTT (females in 16.25% MGX11-10 group) were significantly increased when compared to the basal-diet control group, and the percent LYMPH for the 16.25% MGX11-10 males were statistically significant compared to the corresponding non-transgenic control group (Table 3). We suspect these differences were of no clinical significance, as the values were similar to the corresponding non-transgenic control group or the basal-diet group, or the change was not dose responsive and the observed differences in the above parameters were all within normal reference intervals for rats of this breed and age in our laboratory.
5. Conclusions The results of this study did not show any adverse effects in rats 7
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following 90 days of dietary exposure to genetically modified wheat MGX11-10. Therefore results support the overall weight of evidence that demonstrates that MGX11-10 wheat is as safe as wheat from the corresponding non-transgenic control group wheat Jimai22.
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CRediT authorship contribution statement Yinghua Liu: Methodology, Project administration, Investigation, Formal analysis, Writing - original draft. Shujing Zhang: Investigation, Data curation, Software. Qinghong Zhou: Investigation, Resources. Shufei Li: Investigation, Resources. Jing Zhang: Investigation. Li Zhang: Investigation. Shuqing Jiang: Supervision, Investigation. Qian Zhang: Software. Xiaoli Zhou: Investigation. Chao Wu: Visualization. Qing Gu: Funding acquisition, Writing - review & editing. Zhi Yong Qian: Conceptualization, Writing - review & editing. Declaration of competing interest The authors declare that they have no known competing financialinterestsor personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by Major science and technology project to create new crop varieties using gene transfer technology (2016zx08011005-009). The authors state that they have no conflict of interest. References Abid, M., Ali, S., Qi, L.K., et al., 2018. Physiological and biochemical changes during drought and recovery periods at tillering and jointing stages in wheat (Triticum aestivum L.). Sci. Rep. 8 (1), 4615. https://doi.org/10.1038/s41598-018-21441-7. Al-Maskri, Ahmed, al-busaidi, Waleed, Al-Nadabi, et al., 2016. Effects of drought stress on wheat (Triticum aestivum L.) cv. Coolly. In: International Conference on Agricultural, Food, Biological and Health Sciences (AFBHS-16), https://doi.org/10.17758/EAP. EAP816233. August 22-24, 2016 Kuala Lumpur (Malaysia). Bai, H., Wang, Z., Hu, R., et al., 2015. A 90-day toxicology study of meat from genetically modified sheep overexpressing TLR4 in Sprague-Dawley rats. PLoS One 10 (4), e0121636. https://doi.org/10.1371/journal.pone.0121636. Chinese Standard NY/T1102-2006, 2006. Safety Assessment of Genetically Modified Plant and Derived Products 90-day Feeding Test in Rats. Standards Press of China, Beijing, China. Domingo, J.L., 2016. Safety assessment of GM plants: an updated review of the scientific literature. Food Chem. Toxicol. 95, 12–18. https://doi.org/10.1016/j.fct.2016.06. 013. Duan, C.L., Mao, T.T., Sun, S.Q., et al., 2019. Constitutive expression of GmF6′H1 from soybean improves salt tolerance in transgenic Arabidopsis. Plant Physiol. Biochem. 141, 446–455. https://doi.org/10.1016/j.plaphy.2019.06.027. FAO, 1996. Biotechnology and Food Safety. Report of a Joint FAO/WHO Consultation 30 September-4 October. FAO Food and Nutrition Paper 61. Food and Agriculture Organization of the United Nations, Rome, Italy. Gill, B.S., Appels, R., Botha-Oberholster, A.M., et al., 2004. A workshop report on wheat genome sequencing: international Genome Research on Wheat Consortium. Genetics 168 (2), 1087–1096. https://doi.org/10.1534/genetics.104.034769. Guo, R., Shi, L., Jiao, Y., et al., 2018. Metabolic responses to drought stress in the tissues of drought-tolerant and drought-sensitive wheat genotype seedlings. AoB Plants 10 (2). https://doi.org/10.1093/aobpla/ply016. ply016. Ilyas, H., Datta, A., Bhunia, A., 2017. An approach towards structure based antimicrobial peptide design for use in development of transgenic plants: a strategy for plant Disease management. Curr. Med. Chem. 24 (13), 1350–1364. https://doi.org/10. 2174/0929867324666170116124558. Kramer, C., Brune, P., McDonald, J., et al., 2016. Evolution of risk assessment strategies
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Subchronic feeding toxicity studies of drought-tolerant transgenic wheat MGX11-10 in Wistar Han RCC rats
T
Yinghua Liu, Shujing Zhang, Qinghong Zhou, Shufei Li, Jing Zhang, Li Zhang, Shuqing Jiang, Qian Zhang, Xiaoli Zhou, Chao Wu, Qing Gu∗, Zhi Yong Qian∗∗ Tianjin Centers for Disease Control and Prevention, Tianjin, 300011, China
A R T I C LE I N FO
A B S T R A C T
Keywords: MGX11-10 Genetically modified wheat Drought-tolerant Subchronic toxicity Rat
A subchronic toxicity study were conducted in Wistar Han RCC rats to evaluate the potential health effects of genetically modified (GM), drought-tolerant wheat MGX11-10. Rats were fed a rodent diet formulated with MGX11-10 and were compared with rats fed a diet formulated with its corresponding non-transgenic control Jimai22 and rats fed a basal diet. MGX11-10 and Jimai22 were ground into flour and formulated into diets at concentrations of 16.25, 32.5, or 65%, w/w% and fed to rats (10/sex/group) for 13 weeks. Compared with rats fed Jimai22 and the basal-diet group, no biologically relevant differences were observed in rats fed the GM diet with respect to body weight/gain, food consumption/efficiency, clinical signs, mortality, ophthalmology, clinical pathology (hematology, prothrombin time, urinalysis, clinical chemistry), organ weights, and gross and microscopic pathology. Under the conditions of this study, the MGX11-10 diets did not cause any treatmentrelated effects in rats following at least 90 days of dietary administration as compared with rats fed diets with the corresponding non-transgenic control diet and the basal-diet group. The MGX11-10 diets are considered equivalent to the diets prepared from conventional comparators. The results demonstrated that MGX11-10 wheat is as safe and wholesome as the corresponding non-transgenic control wheat.
1. Introduction Bread wheat (Triticum aestivum L.) is one of the most important food crops, cultivated on about 220 million ha, providing food to one-third of the global population and providing 20% of the global caloric requirements (Gill BS et al., 2004; Shiferaw B et al., 2013; Rasheed A et al., 2018). The yield of wheat is significantly affected by climatic change and scarcity of water resources in the environment. Drought is one of the environmental stresses that seriously limit crop production in the majority of agriculture fields in the world (Al-Maskri et al., 2016; Lesk, C. et al., 2016; Kulkarni M et al., 2017; Matiu et al., 2017). As a result of climate change, the global frequency and severity of drought events is likely to increase (Shahinnia F et al., 2016). It is estimated that a cereal (maize, rice and wheat) loss of 1820 million Mg has been caused by droughts during the past four decades globally (Leng G, 2019). Drought stress caused a reversible decline in leaf water relations, membrane stability, and photosynthetic activity, leading to increased reactive oxygen species (ROS) generation, lipid peroxidation and membrane injury (Abid M et al., 2018). This phenomenon inhibits
∗
further nutrient absorption and affects crop growth, gene expression, distribution, yield and quality (Stendle and Peterson, 1998; Guo R et al., 2018). A way to improve the drought tolerance of crops is to discover new genes and alleles that allow plants to continue to grow and maintain or increase grain yield under water-limited growing conditions. With the continuous development of transgenic technology, the generalization and application of transgenic crops is progressing rapidly. The methods of modern agricultural biotechnology have been utilized to transfer genes from one organism to another without sexual reproduction and across species. This process allows targeted alterations to be introduced into plant genomes in a more specific and controlled manner and more rapidly than can be achieved through conventional breeding and selection of crops. Wheat event MGX11-10 was genetically modified to improve drought tolerance. The WK gene was cloned from wheat, and the wheat Jimai 22 was used as the transformation receptor. WK gene is a protein kinase gene, WK protein kinase catalyzes phosphorylation of protein, and protein phosphorylation is involved in the transmission of stress signals and plays an important
Corresponding author. Corresponding author. E-mail addresses: [email protected] (Q. Gu), [email protected] (Z.Y. Qian).
∗∗
https://doi.org/10.1016/j.fct.2020.111129 Received 4 September 2019; Received in revised form 18 December 2019; Accepted 10 January 2020 Available online 11 January 2020 0278-6915/ © 2020 Published by Elsevier Ltd.
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role in improving plant drought tolerance (Zhao, 2015). Genetically modified plants expressing drought-tolerance traits offer a new strategy for crop protection, but at the same time, present a challenge in terms of food safety assessment. Because the methods used to produce crops with biotechnology differ from those used in conventional breeding, numerous scientific organizations have published guidelines for assessing their safety (WHO, 1991 and 1995; FAO, 1996; and OECD, 1993 and 1997). The primary goal established in these guidelines is comparison analysis between the particular genetically modified (GM) crop and the corresponding non-transgenic control crop with an established history of safe use. In most cases, the data necessary to demonstrate that a GM crop is “as safe as” the corresponding non-transgenic control comparator can be determined based on compositional analysis, however, a number of 13 week (i.e., subchronic) rodent toxicology studies have also been conducted with food and feed fractions from GM crops that support their safety (Bai H et al., 2015; Sabitha et al. 2017; Zou S et al., 2018; Sabitha et al. 2018; Qian et al., 2018a; Qian et al., 2018b). In the present study, we report the results of a 13 week rodent toxicology study conducted with MGX11-10 wheat. This study was conducted in compliance with Chinese guideline on the performance of safety assessment of genetically modified plant and derived products 90-day feeding test in rats（NY/T 1102–2006）at the Experimental Animal Center of the Tianjin Centers for Disease Control and Prevention (Tianjin, China).
Table 1 Study design – dose groups. Group Number
1 2 3 4 5 6 7
Test Substance
The basal-diet control Jimai 22-Low Jimai 22-Mid Jimai 22-High MGX11-10-Low MGX11-10-Mid MGX11-10-High
% in Diet
0 16.2 32.5 65 16.2 32.5 65
Number of Animals/Group Males
Females
10 10 10 10 10 10 10
10 10 10 10 10 10 10
dietary requirements of the rat strain Wistar. All experimental diets were vacuum-packed and sterilized through irradiating by 60Co. The animals were housed one per cage. Feed and drinking water were provided ad libitum for 90 days. 2.4. Body weight, feed consumption and clinical observations Food consumption and body weights were recorded weekly, cageside examination was conducted at least once a day. Significant clinical abnormalities that were evaluated included, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, occurrence of secretions and excretions, neuromuscular function (convulsion, fasciculation, tremor, twitching), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. All animals were observed for morbidity, mortality, and the availability of feed and water.
2. Materials and methods 2.1. Plant materials The GM wheat, MGX11-10, and the corresponding non-transgenic control wheat, JIMAI 22, were supplied by the Chinese Academy of Agricultural Sciences. The wheat was from plants grown concurrently in the same location. After harvest and a storage period of three months, the mature seeds were milled and processed into flour for diet preparation.
2.5. Urinalysis Urine samples were obtained from all animals the week prior to the scheduled necropsy. Animals were housed in metabolism cages and urine samples were collected for 4 h. Water were available ad libitum during this procedure. Urine samples were inspected for color (COL), and clarity (CLA). The urine was analyzed for specific gravity (SG), pH, urobilinogen (URO), protein (PRO), glucose (GLC), ketones (KET), bilirubin (BIL), occult blood (BLD), leukocytes (LEU), and nitrites (NIT). Analysis was performed using RUIKE (Uritest-150) Urine Chemistry Analyzer (Changchun Ruike Medical Technology Co., Ltd, Changchun, China).
2.2. Animals The feeding studies were conducted at the Specific Pathogen Free animal laboratory of the Testing Center of Genetically Modified Organisms at Tianjin (China) approved by the Ministry of Agriculture of China, with the Certificate No. SYXK (Jin) 2014–0001. A total of 140 male and female Wistar Han RCC rats, 4 weeks old and with a uniform weight ( ± 20% of the mean), purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China) with the Certificate No. SCXK (Jing) 2016–0006 11400700259430. Rats were acclimatized to the study conditions for 5 days before the beginning of the treatment period and fed the standard basal diet. Rats were housed in a polypropylene plastic cage singly with ad libitum access to water and food. The animal room conditions were as follows: 20～25°C temperature, 40～70% relative humidity, 12 h/12 h light/dark cycle, 8–15 cycles/h of filtered, nonrecycled air ventilation.
2.6. Hematology and coagulation Animals were fasted overnight prior to blood collection with water provided ad libitum. Blood samples were collected from the abdominal aorta after rats were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital at the scheduled necropsy. Blood samples were mixed with ethylenediaminetetraacetic acid (EDTA) and assayed using an Sysmex XT-2000i hematology analyzer (Sysmex Company, Japan). The following parameters were tested: the white blood cell count (WBC), red blood cell count (RBC), haemoglobin concentration (HGB), haematocrit (HCT), platelet count (PLT) and differential leucocyte count [percent neutrophil (NEU), lymphocyte (LYMPH), monocyte (MONO), eosinophil (EOS), basophil (BASO)]. Blood samples were collected in sodium citrate tubes, centrifuged and plasma collected and assayed for prothrombin time (PT) and activation of partial thrombin time (APTT) using an Sysmex CS-5100 coagulation analyzer (Sysmex Company, Japan)
2.3. Study design Rats were randomly divided into seven groups by body weight with 10 rats/gender/group. The mean body weights of each group varied within ± 20%. Groups of 10 male and 10 female rats were fed diets containing GM wheat, MGX11-10 and the corresponding non-transgenic control wheat Jimai22 and the basal diet for at least 90 days, respectively. A total of seven diets were prepared by Beijing Keao Xieli Feed Co., Ltd. and met standard GB 14924.3–2010 “Laboratory animals – Mice and rat formula feeds.” Flour from MGX11-10 and Jimai22 was formulated into diets at a percentage of 16.25%, 32.5% and 65% of the total diet, respectively (Table 1). And control groups were fed the standard basal diet. The formulation of the diets was adjusted to the
2.7. Clinical chemistry Animals were fasted 16 h prior to blood collection at the end of the 2
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study. Serum was collected after centrifugation and assayed using an Toshiba TBA-40FR hematology analyzer (Toshiba Co., Japan) for the following parameters: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), urea nitrogen (BUN), creatinine (CREA), glucose (GLU), cholesterol(CHOL), triglyceride(TG), alkaline phosphatase(ALP), lactate dehydrogenase(LDH), glutamine transpeptide enzyme（GGT）. Chloride (Cl−), potassium (K+), and sodium (Na+) were assayed using an MEDICA-Easylute Plus Ion analyzer (Medica company, USA).
2.8. Gross necropsy and histopathology After blood samples were collected, all rats were necropsied by exsanguinations and submitted to a full macroscopic postmortem examination. Necropsy included examination of the external surface, all orifices and the cranial, thoracic, abdominal and pelvic cavities, including viscera. The heart, brain, liver, kidneys, adrenal glands, spleen, thymus, uterus (female), ovaries (female), testes (male) and epididymides (male) were trimmed off extraneous fat and weighed immediately. The ratios of organ weight to terminal body weight were calculated. The tissues from all animals were collected and preserved in neutral, phosphate-buffered 10% formal, trimmed, embedded in paraffin, sectioned, mounted on glass microscope slides, stained with hematoxylin and eosin, and histologically examined under a light microscope. The following tissues were examined microscopically: brain, heart, lungs, liver, kidney, adrenal glands, spleen, stomach, intestine (duodenum, jejunum, and ileum), thymus, thyroid glands, testes (male), epididymis (male), prostate (male), ovaries (female) and uterus (female).
Fig. 1. Mean weekly body weights of male rats. Line plots of mean male body weights (g) per group.
2.9. Statistical analysis Statistical analysis was designed to determine if differences exist at day 90 among the GM wheat group, the corresponding non-transgenic wheat group and the basal-diet group. Data of body weights, food efficiency, organ weight, relative organ weight, urinalysis, clinical chemistry, hematology and coagulation were analyzed. Differences between groups were considered significant if p < 0.05. Food efficiency was calculated as (body weight gain/feed consumption) × 100%. Relative organ weight was calculated as (wet weight of organ/body fasting weight) × 100%. All data were presented as group mean values ± standard deviation (X ± SD), and continuous data were compared by ANOVA using SPSS 16.0 software, and frequency data were compared using a Chi-square test (α = 0.05). Quantitative and categorical data for each gender were analyzed separately. Data from the GM groups were first compared with the basaldiet group; then the values from the GM groups were compared with those in the corresponding non-transgenic control groups. A p value of < 0.05 was considered significant, and the letter “a” represents significant difference between the test groups and the basal-diet group, while the letter “b” represents significant difference between transgenic wheat group and the corresponding non-transgenic control group.
Fig. 2. Mean weekly body weights of female rats. Line plots of mean female body weights (g) per group.
3.2. Body weight and feed consumption There were no statistically identified or treatment-related differences in the mean body weights (Fig. 1, Fig. 2), total body weight gains (Table 2) of male or female rats given GM wheat, MGX11-10 at 16.25%, 32.5% and 65% level as compared with their corresponding nontransgenic control groups and the basal-diet group. Mean feed consumption were statistically lower (p < 0.05), when compared with the corresponding non-transgenic group, for the 16.25% MGX11-10 males during study week 5–6 (Fig. 3, Fig. 4) and for the 32.5% MGX11-10 females during study week 8 (Fig. 5, Fig. 6). Mean feed consumption of the 32.5% MGX11-10 females during study week 5,9 and 11 were statistically lower (p < 0.05) than the basal-diet group (Figs. 5 and 7). Total feed efficiency (Table 2) for males in 32.5% MGX11-10 group were comparatively higher (p < 0.05) than the basal-diet group.
3. Results 3.3. Hematology and coagulation 3.1. Clinical observation The following statistically significant differences were found when the MGX11-10 groups were compared with the corresponding nontransgenic control group: percent NEU results for the 16.25% MGX1110 males were higher (p < 0.05) than for the corresponding nontransgenic control group; percent LYMPH for the 16.25% MGX11-10 males were lower (p < 0.05) than the corresponding non-transgenic control group. The values of MONO and APTT for the 16.25% MGX1110 females were higher (p < 0.05) than for the corresponding non-
No signs of morbidity and mortality were observed during the whole 90 days. All rats survived the duration of the feeding trial. During the study, the animals in each group were active, ate normally. Clinical observation of the treated rats throughout the study indicated that none of them showed signs of toxicity in their skin, fur, eyes, mucus membrane, or behavioral changes, diarrhoea, tremors, salivation, sleep, and coma. 3
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Table 2 Growth performances of rats fed different diets for 90 days. Gender Males The basal-diet control 16.25% Jimai 22 32.5% Jimai 22 65% Jimai 22 16.25% MGX11-10 32.5% MGX11-10 65% MGX11-10 Females The basal-diet control 16.25% Jimai 22 32.5% Jimai 22 65% Jimai 22 16.25% MGX11-10 32.5% MGX11-10 65% MGX11-10
Total body weight gain (g)
Total food consumption (g)
Food efficiency (%)
406.1 414.0 421.4 424.5 404.0 434.8 402.0
± ± ± ± ± ± ±
55.0 48.3 34.0 36.4 30.1 48.3 45.6
2572.9 2588.5 2520.9 2478.8 2495.9 2568.3 2450.7
± ± ± ± ± ± ±
236.5 232.9 130.3 123.0 89.6 225.4 160.1
15.8 16.0 16.7 17.1 16.2 17.0 16.4
± ± ± ± ± ± ±
1.3 1.2 0.9 0.8a 1.0 1.5a 0.9
244.0 235.5 232.0 246.5 246.8 233.2 242.9
± ± ± ± ± ± ±
40.8 31.0 32.8 23.4 44.3 28.7 30.7
2063.3 2033.3 2003.8 1917.0 2001.9 1928.2 1930.8
± ± ± ± ± ± ±
234.9 166.6 148.8 87.2 133.6 164.4 201.3
11.8 11.6 11.6 12.9 12.3 12.1 12.6
± ± ± ± ± ± ±
0.9 1.0 1.2 1.0 1.9 0.9 1.5
Data are reported as mean ± SD, n = 10. a: p < 0.05 Significantly different from the basal-diet control group. b: p < 0.05 Significantly different from the corresponding non-transgenic control group.
Fig. 5. Mean weekly feed consumption of female rats. Line plots of mean feed consumption per group (g) and per week in the case of female.
Fig. 3. Mean weekly feed consumption of male rats. Line plots of mean feed consumption per group (g) and per week in the case of male.
Fig. 6. Mean weekly feed consumption of 32.5% MGX11-10 and 32.5% Jimai22 females in the eighth week. Values represent Mean ± SEM. n = 10.*p < 0.05 Significantly different from the corresponding non-transgenic control group.
Fig. 4. Mean weekly feed consumption of 16.25% MGX11-10 and 16.25% Jimai22 males in the fifth and sixth week. Values represent Mean ± SEM. n = 10.*p < 0.05 Significantly different from the corresponding non-transgenic control group.
transgenic control group (Table 3). 3.4. Clinical chemistry The clinical chemistry data (Table 4) illustrated that there were no statistically identified or treatment-related differences in the values of 4
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The value of chloride in the 32.5% MGX11-10 group was lower (p < 0.05) than in the 32.5% Jimai22 group, but the value of chloride in 65% MGX11-10 group was higher (p < 0.05) when compared to 65% Jimai22 group and the basal-diet group. The values of sodium, potassium and chloride for the 16.25% MGX11-10 females were lower (p < 0.05) than for the 16.25% Jimai22 females. The values of sodium, potassium and chloride for the 65% MGX11-10 females were higher (p < 0.05) than for the 65% Jimai22 females. The values of sodium and chloride for the 32.5% and 65% MGX1110 males were higher (p < 0.05) than for the basal-diet group. The values of sodium and chloride for the 65% MGX11-10 females were higher (p < 0.05) than for the basal-diet group. The values of sodium and chloride for the 32.5% MGX11-10 females were lower (p < 0.05) than for the basal-diet group. 3.5. Urinalysis There were no statistically significant differences or treatment-related alterations in any of the urinalysis parameters for male and female rats in GM groups as compared with their respective non-transgenic and the basal-diet controls (Table 5).
Fig. 7. Mean weekly feed consumption of 32.5% MGX11-10 and the basal-diet control females in the fifth, ninth and eleventh week. Values represent Mean ± SEM. n = 10. *p < 0.05 Significantly different from the basal-diet group.
3.6. Organ weights and relative organ weights
ALT, AST, BUN, CHO, TG, GLU, TP, ALB, CRE, ALP, LDH and GGT of male or female rats in 16.25%,32.5% and 65% MGX11-10 groups compared with their corresponding non-transgenic control groups and the basal-diet group. In males, the value of sodium in the 32.5% MGX11-10 group was lower (p < 0.05) than in the 32.5% Jimai22 group, no difference was seen between the 65% MGX11-10 group and 65% Jimai22 group or the 16.25% MGX11-10 group and 16.25% Jimai22 group. The value of sodium in 32.5% and 65% MGX11-10 group were higher (p < 0.05) than in the basal-diet group. In females, the value of sodium in the 16.25% MGX11-10 group was lower (p < 0.05) than in the 16.25% Jimai22 group, the value of sodium in the 65% MGX11-10 group was higher (p < 0.05) than in the 65% Jimai22 group.
There were no statistically identified differences in any of the organ weights and the relative organ weights in males and females given MGX11-10 at 16.25%, 32.5% and 65% level as compared with their corresponding non-transgenic control groups and the basal-diet group (Table 6). 3.7. Histopathological examination The following histopathological changes were observed: a few small round inflammatory cells in portal area were observed in the liver (2 in the basal-diet group, 2 in 65% MGX11-10 group, 1 in 65% Jimai22 group), infiltration of interstitial inflammatory cell were observed in the kidneys (1 in the basal-diet group, 2 in 65% MGX11-10 group),
Table 3 Hematological parameters of rats fed different diets for 90 days. Gender Males WBC(109/L) RBC(1012/L) HGB(g/dL) PLT(109/L) HCT(%) Neu(%) Lymph(%) Mono(%) Eos(%) Baso(%) PT APTT Females WBC(109/L) RBC(1012/L) HGB(g/dL) PLT(109/L) HCT(%) Neu(%) Lymph(%) Mono(%) Eos(%) Baso(%) PT APTT
The basal-diet control
16.25% Jimai 22
32.5% Jimai 22
65%Jimai 22
16.25% MGX11-10
32.5% MGX11-10
65% MGX11-10
2.95 ± 0.66 7.46 ± 0.18 134.2 ± 4.5 1172.2 ± 138.5 36.8 ± 1.9 25.10 ± 7.46 69.86 ± 8.65 2.73 ± 1.14 2.31 ± 0.92 0.00 ± 0.00 8.8 ± 0.3 11.3 ± 2.4
2.98 ± 1.00 7.66 ± 0.23 134.9 ± 6.5 1185.4 ± 138.8 36.9 ± 2.2 23.96 ± 5.90 71.27 ± 6.39 2.71 ± 0.89 2.06 ± 0.73 0.00 ± 0.00 8.7 ± 0.3 11.1 ± 2.7
3.13 ± 0.94 7.68 ± 0.26 137.8 ± 4.0 1202.0 ± 133.9 37.1 ± 0.9 25.02 ± 5.42 68.53 ± 5.88 3.71 ± 0.77 2.74 ± 0.82 0.00 ± 0.00 8.6 ± 0.3 11.2 ± 2.7
2.50 ± 0.59 7.69 ± 0.30 137.3 ± 3.6 1143.8 ± 96.0 37.1 ± 1.1 22.92 ± 2.36 71.30 ± 2.89 3.47 ± 1.26 2.31 ± 0.81 0.00 ± 0.00 8.7 ± 0.1 11.6 ± 2.1
2.79 ± 0.79 7.60 ± 0.32 136.8 ± 6.1 1210.6 ± 123.7 37.4 ± 1.3 30.21 ± 4.08b 64.41 ± 4.05b 3.25 ± 0.95 2.13 ± 0.75 0.00 ± 0.00 8.6 ± 0.3 11.6 ± 2.6
2.91 ± 0.81 7.71 ± 0.29 137.4 ± 5.0 1142.6 ± 146.5 37.6 ± 1.8 27.12 ± 7.44 66.98 ± 8.28 3.41 ± 1.08 2.49 ± 0.47 0.00 ± 0.00 8.5 ± 0.3 10.6 ± 3.8
3.06 ± 0.76 7.48 ± 0.31 137.0 ± 5.2 1148.2 ± 84.0 37.5 ± 1.5 22.81 ± 6.62 71.37 ± 7.48 3.69 ± 1.52 2.13 ± 0.88 0.00 ± 0.03 8.7 ± 0.4 12.1 ± 1.9
1.82 ± 0.38 6.84 ± 0.20 131.0 ± 4.2 1077.0 ± 141.9 36.6 ± 1.5 19.82 ± 2.70 74.90 ± 2.77 2.40 ± 1.04 2.88 ± 0.68 0.00 ± 0.00 8.5 ± 0.4 13.2 ± 1.1
1.67 ± 0.50 6.86 ± 0.34 130.0 ± 5.3 1032.6 ± 171.0 36.1 ± 1.3 19.66 ± 6.69 75.38 ± 7.02 2.12 ± 0.90 2.84 ± 0.78 0.00 ± 0.00 8.2 ± 0.3 11.5 ± 1.9
1.36 ± 0.24a 6.87 ± 0.21 130.1 ± 4.1 1035.4 ± 117.1 36.4 ± 1.9 20.43 ± 4.52 75.43 ± 4.98 1.51 ± 0.83 2.63 ± 0.97 0.00 ± 0.00 8.4 ± 0.4 13.2 ± 2.8
1.50 ± 0.34 6.77 ± 0.22 129.4 ± 5.0 988.0 ± 153.2 35.5 ± 1.2 24.78 ± 5.50 70.05 ± 5.55 2.40 ± 1.30 2.77 ± 1.42 0.00 ± 0.00 8.5 ± 0.3 13.1 ± 1.6
1.63 ± 0.75 6.87 ± 0.18 131.7 ± 4.7 1088.9 ± 78.1 36.3 ± 1.5 22.23 ± 4.21 71.04 ± 4.71 3.26 ± 1.30b 3.47 ± 1.15 0.00 ± 0.00 8.5 ± 0.3 13.2 ± 2.9b
1.66 ± 0.49 6.80 ± 0.27 128.2 ± 5.6 1088.5 ± 100.1 35.9 ± 1.9 23.50 ± 6.44 72.05 ± 6.49 2.11 ± 1.07 2.34 ± 0.82 0.00 ± 0.00 8.5 ± 0.3 13.2 ± 2.2
1.44 ± 0.34 6.90 ± 0.34 130.5 ± 6.2 1057.3 ± 169.5 35.8 ± 1.5 23.69 ± 8.25 70.64 ± 9.70 2.02 ± 1.54 3.65 ± 1.66 0.00 ± 0.00 8.4 ± 0.4 13.4 ± 3.2
Data are reported as mean ± SD, n = 10. a: p < 0.05 Significantly different from the basal-diet control group. b: p < 0.05 Significantly different from the corresponding non-transgenic control group. 5
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Table 4 Clinical biochemistry parameters of rats fed different diets for 90 days. Gender Males ALT(U/L) AST(U/L) TP(g/L) ALB(g/L) GGT(U/L) BUN(mmol/L) GLU(mmol/L) CRE(μmol/L) CHO(mmol/L) TG(mmol/L) ALP(U/L) LDH (U/L) Na+(mmol/L) K+(mmol/L) Cl−(mmol/L) Females ALT(U/L) AST(U/L) TP(g/L) ALB(g/L) GGT(U/L) BUN(mmol/L) GLU(mmol/L) CRE(μmol/L) CHO(mmol/L) TG(mmol/L) ALP(U/L) LDH (U/L) Na+(mmol/L) K+(mmol/L) Cl−(mmol/L)
The basal-diet control
16.25% Jimai 22
32.5% Jimai 22
65%Jimai 22
16.25% MGX11-10
32.5% MGX11-10
65% MGX11-10
45.1 ± 5.4 104.0 ± 6.0 65.4 ± 2.6 37.2 ± 1.3 1.09 ± 0.63 6.46 ± 0.67 8.29 ± 0.49 35.4 ± 3.4 2.35 ± 0.34 0.40 ± 0.13 80.7 ± 10.6 1504.1 ± 339.2 143.7 ± 0.9 4.66 ± 0.27 110.8 ± 0.92
45.9 ± 6.3 99.5 ± 7.3 64.0 ± 2.4 36.5 ± 1.4 1.11 ± 0.40 6.22 ± 0.43 8.24 ± 0.64 33.7 ± 4.5 2.41 ± 0.35 0.45 ± 0.13 89.1 ± 18.3 1356.3 ± 264.6 145.4 ± 5.5 4.78 ± 0.26 112.4 ± 4.59
42.9 ± 7.4 10.3.0 ± 9.9 65.4 ± 2.1 37.1 ± 1.1 1.29 ± 0.34 6.63 ± 0.70 8.61 ± 0.41 34.8 ± 2.9 2.48 ± 0.27 0.40 ± 0.12 92.5 ± 16.7 1413.7 ± 355.4 152.2 ± 1.0a 4.83 ± 0.32 119.9 ± 1.03a
42.4 ± 5.7 100.3 ± 11.6 65.3 ± 3.1 36.9 ± 1.7 1.05 ± 0.37 6.03 ± 0.71 8.72 ± 0.73 35.7 ± 3.3 2.54 ± 0.47 0.43 ± 0.13 89.7 ± 16.7 1444.9 ± 305.3 144.3 ± 1.1 4.62 ± 0.31 112.2 ± 1.31
42.8 ± 5.6 110.4 ± 17.3 64.3 ± 2.1 36.6 ± 1.6 1.10 ± 0.32 6.97 ± 0.82 8.13 ± 0.65 36.4 ± 2.5 2.60 ± 0.36 0.38 ± 0.14 81.7 ± 16.3 1579.3 ± 306.9 146.0 ± 1.6 4.67 ± 0.34 112.2 ± 1.31
41.6 ± 7.7 101.5 ± 7.7 65.3 ± 2.2 37.4 ± 1.5 1.04 ± 0.43 7.20 ± 0.97 7.95 ± 0.61 34.2 ± 2.5 2.51 ± 0.35 0.49 ± 0.16 84.0 ± 10.4 1447.2 ± 246.2 147.4 ± 1.2ab 4.81 ± 0.29 113.6 ± 1.35 ab
46.9 ± 5.7 100.4 ± 6.9 64.2 ± 2.0 36.5 ± 1.2 1.07 ± 0.28 7.05 ± 0.87 7.97 ± 0.81 32.8 ± 2.8 2.43 ± 0.36 0.41 ± 0.21 91.9 ± 11.7 1458.8 ± 235.4 146.6 ± 3.9a 4.83 ± 0.37 113.2 ± 3.46 ab
33.2 ± 5.1 94.6 ± 24.5 64.9 ± 2.3 38.7 ± 1.5 1.35 ± 0.38 6.39 ± 0.79 7.75 ± 1.15 41.9 ± 2.2 2.02 ± 0.61 0.49 ± 0.15 57.4 ± 13.7 1440.5 ± 451.0 152.1 ± 1.1 4.26 ± 0.19 119.1 ± 1.45
36.5 ± 3.8 92.1 ± 19.3 63.6 ± 3.1 38.1 ± 2.0 1.26 ± 0.27 6.42 ± 1.10 7.99 ± 0.47 39.5 ± 2.3 2.21 ± 0.49 0.48 ± 0.13 52.2 ± 10.2 1239.7 ± 363.3 157.1 ± 5.9a 4.54 ± 0.33a 124.7 ± 3.54a
38.4 ± 7.0 92.1 ± 9.3 65.7 ± 3.1 39.3 ± 2.2 1.24 ± 0.34 6.16 ± 0.72 8.44 ± 0.52 39.7 ± 2.8 2.19 ± 0.62 0.42 ± 0.05 53.2 ± 8.8 1245.5 ± 320.0 144.3 ± 0.9a 4.02 ± 0.18 112.5 ± 1.54a
35.9 ± 5.4 92.6 ± 10.5 66.0 ± 3.3 39.7 ± 1.8 1.27 ± 0.38 6.73 ± 1.19 8.37 ± 0.47 39.4 ± 2.6 2.26 ± 0.61 0.44 ± 0.16 53.7 ± 11.0 1135.2 ± 288.3 146.8 ± 3.5a 4.21 ± 0.34 114.3 ± 2.23a
38.2 ± 5.7 96.2 ± 14.6 65.4 ± 3.0 40.0 ± 2.0 1.12 ± 0.29 6.82 ± 0.77 8.55 ± 0.46 39.5 ± 2.4 2.30 ± 0.55 0.43 ± 0.10 53.7 ± 16.0 1129.5 ± 347.6 152.3 ± 1.2b 4.26 ± 0.23b 120.7 ± 1.48b
37.2 ± 8.0 94.4 ± 15.2 63.7 ± 2.9 38.3 ± 1.2 1.21 ± 0.26 6.51 ± 0.46 8.23 ± 0.54 38.2 ± 2.4 2.18 ± 0.53 0.49 ± 0.16 54.4 ± 7.6 1328.9 ± 251.0 145.3 ± 1.3a 4.20 ± 0.35 113.8 ± 1.24a
35.6 ± 6.5 98.6 ± 15.0 62.8 ± 4.4 38.8 ± 3.1 1.29 ± 0.36 6.58 ± 0.91 8.05 ± 0.70 40.8 ± 8.7 2.08 ± 0.37 0.48 ± 0.09 52.8 ± 12.6 1137.6 ± 304.2 156.8 ± 4.1 ab 4.64 ± 0.38 ab 121.3 ± 2.92 ab
Data are reported as mean ± SD, n = 10. a: p < 0.05 Significantly different from the basal-diet control group. b: p < 0.05 Significantly different from the corresponding non-transgenic control group. Table 5 Urinalysis of rats fed different diets for 90 days. Gender Males Urobilinogen Gravity PH Females Urobilinogen Gravity PH
The basal-diet control
16.25% Jimai 22
32.5% Jimai 22
65%Jimai 22
16.25% MGX11-10
32.5% MGX11-10
65% MGX11-10
6.12 ± 5.73 1.024 ± 0.005 7.70 ± 0.63
6.12 ± 5.73 1.029 ± 0.003 6.90 ± 0.46
8.84 ± 7.02 1.050 ± 0.088 7.65 ± 0.94
7.48 ± 6.57 1.023 ± 0.005 7.65 ± 0.94
7.48 ± 6.57 1.023 ± 0.005 7.70 ± 1.03
4.76 ± 4.30 1.022 ± 0.005 7.60 ± 0.91
4.76 ± 4.30 1.024 ± 0.005 7.55 ± 0.86
7.48 ± 6.57 1.021 ± 0.008 7.50 ± 0.82
6.12 ± 5.73 1.020 ± 0.005 7.95 ± 0.60
7.48 ± 6.57 1.018 ± 0.004 8.00 ± 0.67
8.84 ± 7.02 1.023 + 0.005 7.65 ± 0.82
10.20 ± 7.17 1.025 ± 0.005 7.00 ± 0.82
8.84 ± 7.02 1.022 ± 0.009 6.80 ± 0.86
7.48 ± 6.57 1.021 ± 0.006 7.45 ± 0.72
Data are reported as mean ± SD, n = 10.
transferred genes or modifications in the expression of endogenous genes (Schreier et al., 1985; Willmitzer, 1988; Ilyas, 2017; Duan C et al., 2019). Transgenic technology is widely used in medicine, agriculture, research, and industry. The benefits of GM crops include an overall increase in yield of almost 22%, a 36.9% reduction in pesticide use and an increase in farmer profit slightly over 68% (Catherine Kramer et al., 2016). Despite a number of studies demonstrated the assessed genetically modified plants would be as safe as the parental species of these plants (Domingo JL, 2016), the claim of the safety of this feed is still controversial in many countries. The concern about the safety of GM products arises with the increased prevalence of GM products (Raman R. et al., 2017; Tsatsakis AM et al., 2017). The concern about the safety of GM products would require spelling out the values and assumptions (for example, the sufficient evidence for safety) behind risk assessment (Martinelli L. et al., 2013). Therefore, GM products should undergo a strict evaluation before they are allowed to enter the market. A 30- or 90-day feeding study is traditionally used to assess the safety of a GM
slight interstitial pneumonia or focal pneumonia and perivascular inflammatory cell infiltration were observed in the lung and trachea (3 in the basal-diet group, 3 in 65% MGX11-10 group, 1 in 65% Jimai22 group) and slight gland interstitial inflammatory cell infiltration were observed in the prostate (1 in 65% MGX11-10 group). The brain, heart, adrenal gland, spleen, gastrointestinal (duodenum, jejunum and ileum), thymus, thyroid, testis, epididymis, ovary, or uterus tissues did not show any histopathological lesions.
4. Discussion Farmer has used traditional breeding techniques for many years to select desired crop traits. Since the development of advanced genetic technologies over the past 20 years, breeders and scientists were allowed to bring specific desired genetic changes into the plants in a more precise and controlled manner. It is possible to transfer a gene from one organism to another without sexual reproduction. Plants have been engineered using a variety of techniques including expression of 6
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Table 6 Organ/body weight ratios of rats fed with different diets for 90 days. Gender Males Brain Heart Thymus Liver Spleen Kidney Adrenal Testis Epididymis Females Brain Heart Thymus Liver Spleen Kidney Adrenal Uterus Ovary
The basal-diet control
16.25% Jimai 22
32.5% Jimai 22
65%Jimai 22
16.25% MGX11-10
32.5% MGX11-10
65% MGX11-10
0.42 ± 0.03 0.29 ± 0.02 0.11 ± 0.04 2.80 ± 0.15 0.21 ± 0.03 0.72 ± 0.05 0.018 ± 0.003 0.83 ± 0.10 0.371 ± 0.054
0.42 ± 0.04 0.30 ± 0.06 0.10 ± 0.02 2.96 ± 0.24 0.22 ± 0.02 0.76 ± 0.05 0.016 ± 0.003 0.71 ± 0.20 0.337 ± 0.097
0.41 ± 0.03 0.28 ± 0.03 0.11 ± 0.01 2.84 ± 0.18 0.20 ± 0.02 0.71 ± 0.07 0.015 ± 0.002 0.76 ± 0.06 0.330 ± 0.046
0.41 ± 0.03 0.28 ± 0.04 0.11 ± 0.02 2.74 ± 0.13 0.20 ± 0.01 0.73 ± 0.06 0.017 ± 0.003 0.75 ± 0.07 0.326 ± 0.042
0.43 ± 0.03 0.29 ± 0.03 0.10 ± 0.03 2.80 ± 0.14 0.21 ± 0.02 0.74 ± 0.05 0.016 ± 0.004 0.80 ± 0.07 0.354 ± 0.056
0.40 ± 0.04 0.30 ± 0.03 0.10 ± 0.02 2.95 ± 0.24 0.22 ± 0.02 0.76 ± 0.06 0.016 ± 0.005 0.78 ± 0.10 0.323 ± 0.036
0.43 ± 0.04 0.29 ± 0.03 0.10 ± 0.03 2.67 ± 0.34 0.20 ± 0.03 0.75 ± 0.06 0.018 ± 0.007 0.79 ± 0.08 0.345 ± 0.059
0.61 ± 0.11 0.33 ± 0.03 0.16 ± 0.03 2.97 ± 0.26 0.24 ± 0.03 0.72 ± 0.05 0.030 ± 0.008 0.18 ± 0.04 0.072 ± 0.016
0.65 ± 0.07 0.36 ± 0.07 0.13 ± 0.04 3.04 ± 0.56 0.24 ± 0.04 0.80 ± 0.14a 0.032 ± 0.007 0.19 ± 0.05 0.074 ± 0.016
0.65 ± 0.07 0.35 ± 0.04 0.13 ± 0.02 2.91 ± 0.14 0.24 ± 0.04 0.76 ± 0.07 0.034 ± 0.007 0.22 ± 0.05 0.074 ± 0.012
0.61 ± 0.07 0.34 ± 0.06 0.14 ± 0.05 2.79 ± 0.26 0.23 ± 0.03 0.71 ± 0.08 0.033 ± 0.007 0.18 ± 0.05 0.068 ± 0.017
0.63 ± 0.08 0.34 ± 0.06 0.15 ± 0.04 2.93 ± 0.20 0.22 ± 0.05 0.74 ± 0.07 0.033 ± 0.008 0.17 ± 0.03 0.061 ± 0.011
0.64 ± 0.08 0.33 ± 0.03 0.13 ± 0.03 2.92 ± 0.16 0.23 ± 0.02 0.73 ± 0.05 0.034 ± 0.007 0.22 ± 0.07 0.067 ± 0.007
0.64 ± 0.07 0.35 ± 0.04 0.14 ± 0.03 2.88 ± 0.39 0.24 ± 0.01 0.76 ± 0.08 0.031 ± 0.006 0.20 ± 0.03 0.071 ± 0.023
Data are reported as mean ± SD, n = 10. a: p < 0.05 Significantly different from the basal-diet control group. b: p < 0.05 Significantly different from the corresponding non-transgenic control group.
Clinical biochemistry parameters in this study were similar between the groups fed GM wheat and the corresponding non-transgenic control group or the basal-diet control group of male and female rats with the exceptions, as described below. Results of serum electrolyte showed that there were statistically significant differences in some individual parameters between the transgenic wheat groups and their corresponding non-transgenic wheat groups or basal-diet group (Table 4). But none displayed a dose-response. Histopathology examination revealed no biological relevant histopathological change. Urine specific gravity, pH, protein, and leucocyte level were in the normal range in all treated groups (Table 5). Organ weight changes are often associated with treatment-related effects. The choice of appropriate organs to weigh in toxicology studies involves understanding the test article's mechanism of action, metabolism and toxicokinetics; the physiology of the test species (Sellers, R. S. et al., 2007). Organ coefficient is also an important index for toxicological study because it can reduce the variability of data and increase the chances of detecting treatment effects. The results of this study revealed that all the absolute and relative weights of the liver, kidneys, adrenals, spleen, heart, thymus, brain, testes, and epididymides of rats in the GM groups, were comparable to those of the corresponding non-transgenic control groups and the basal-diet control group (Table 6). A low number of histological changes, mostly inflammatory reactions, were observed in basal-diet control group, GM group and the corresponding non-transgenic control group, but there was no difference among basal-diet control group, GM group and the corresponding non-transgenic control group. The changes were found in a similar range in both groups, and incidences of these findings were all within the historical control ranges of our laboratory, all the pathological observations were interpreted to be spontaneous alterations, unassociated with exposure to the genetically modified wheat. Based on the results of this study, there were no treatment related effects in clinical signs, ophthalmic examinations, body weights, feed consumption, hematology, prothrombin time, activation of partial thrombin time, clinical chemistry, urinalysis parameters, organ weights, organ coefficient and gross or histopathological observations.
product. MGX11-10 is a genetically modified wheat which expresses drought tolerant proteins. To assess the safety of MGX11-10, a 90-day rat feeding study was conducted according to the Chinese guideline on the performance of safety assessment of genetically modified plant and derived products 90-day feeding test in rats（NY/T 1102–2006）. In this study, we conducted a feeding study to evaluate the presence of any unintended adverse effects on rats associated with the consumption of diets containing GM wheat at different doses. The results showed there were no significant differences between the diet nutrient composition of GM wheat, the corresponding non-transgenic control group and the basal-diet group. The animals in each group were active with normal shiny coats, and there was no abnormal discharge from the nose, eyes, or mouth during the study. The mean body weights (Figs. 1 and 2), total body weight gains （Table 2) of each group were normal and no statistical differences were observed between groups. A lower mean feed consumption for 16.25% MGX11-10 males during study week 5–6 (Figs. 3 and 4) and for 32.5% MGX11-10 females during study week 8 (Figs. 5 and 6) than the corresponding non-transgenic control group were found. Mean feed consumption of the 32.5% MGX11-10 females during study week 5,9 and 11 were statistically lower (p < 0.05) than the basal-diet group (Figs. 5 and 7). And the total feed efficiency of 32.5% MGX11-10 males were comparatively higher than the basal-diet group (Table 2). But the changes were not dose responsive, transient and within the normal range. Some differences disappeared in the final week of the test. Hematological analyses in humans and animals are a sensitive indicator of drug and chemical toxicity. In analyses of blood indices, no abnormalities in several measurements in the GM-treated animals (white blood cell count, red blood cell count, platelet count, hemoglobin, and neutrophil count) were found, but the percentage of NEU (males in 16.25% MGX11-10 group), the percentage of MONO and APTT (females in 16.25% MGX11-10 group) were significantly increased when compared to the basal-diet control group, and the percent LYMPH for the 16.25% MGX11-10 males were statistically significant compared to the corresponding non-transgenic control group (Table 3). We suspect these differences were of no clinical significance, as the values were similar to the corresponding non-transgenic control group or the basal-diet group, or the change was not dose responsive and the observed differences in the above parameters were all within normal reference intervals for rats of this breed and age in our laboratory.
5. Conclusions The results of this study did not show any adverse effects in rats 7
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following 90 days of dietary exposure to genetically modified wheat MGX11-10. Therefore results support the overall weight of evidence that demonstrates that MGX11-10 wheat is as safe as wheat from the corresponding non-transgenic control group wheat Jimai22.
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CRediT authorship contribution statement Yinghua Liu: Methodology, Project administration, Investigation, Formal analysis, Writing - original draft. Shujing Zhang: Investigation, Data curation, Software. Qinghong Zhou: Investigation, Resources. Shufei Li: Investigation, Resources. Jing Zhang: Investigation. Li Zhang: Investigation. Shuqing Jiang: Supervision, Investigation. Qian Zhang: Software. Xiaoli Zhou: Investigation. Chao Wu: Visualization. Qing Gu: Funding acquisition, Writing - review & editing. Zhi Yong Qian: Conceptualization, Writing - review & editing. Declaration of competing interest The authors declare that they have no known competing financialinterestsor personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by Major science and technology project to create new crop varieties using gene transfer technology (2016zx08011005-009). The authors state that they have no conflict of interest. References Abid, M., Ali, S., Qi, L.K., et al., 2018. Physiological and biochemical changes during drought and recovery periods at tillering and jointing stages in wheat (Triticum aestivum L.). Sci. Rep. 8 (1), 4615. https://doi.org/10.1038/s41598-018-21441-7. Al-Maskri, Ahmed, al-busaidi, Waleed, Al-Nadabi, et al., 2016. Effects of drought stress on wheat (Triticum aestivum L.) cv. Coolly. In: International Conference on Agricultural, Food, Biological and Health Sciences (AFBHS-16), https://doi.org/10.17758/EAP. EAP816233. August 22-24, 2016 Kuala Lumpur (Malaysia). Bai, H., Wang, Z., Hu, R., et al., 2015. A 90-day toxicology study of meat from genetically modified sheep overexpressing TLR4 in Sprague-Dawley rats. PLoS One 10 (4), e0121636. https://doi.org/10.1371/journal.pone.0121636. Chinese Standard NY/T1102-2006, 2006. Safety Assessment of Genetically Modified Plant and Derived Products 90-day Feeding Test in Rats. Standards Press of China, Beijing, China. Domingo, J.L., 2016. Safety assessment of GM plants: an updated review of the scientific literature. Food Chem. Toxicol. 95, 12–18. https://doi.org/10.1016/j.fct.2016.06. 013. Duan, C.L., Mao, T.T., Sun, S.Q., et al., 2019. Constitutive expression of GmF6′H1 from soybean improves salt tolerance in transgenic Arabidopsis. Plant Physiol. Biochem. 141, 446–455. https://doi.org/10.1016/j.plaphy.2019.06.027. FAO, 1996. Biotechnology and Food Safety. Report of a Joint FAO/WHO Consultation 30 September-4 October. FAO Food and Nutrition Paper 61. Food and Agriculture Organization of the United Nations, Rome, Italy. Gill, B.S., Appels, R., Botha-Oberholster, A.M., et al., 2004. A workshop report on wheat genome sequencing: international Genome Research on Wheat Consortium. Genetics 168 (2), 1087–1096. https://doi.org/10.1534/genetics.104.034769. Guo, R., Shi, L., Jiao, Y., et al., 2018. Metabolic responses to drought stress in the tissues of drought-tolerant and drought-sensitive wheat genotype seedlings. AoB Plants 10 (2). https://doi.org/10.1093/aobpla/ply016. ply016. Ilyas, H., Datta, A., Bhunia, A., 2017. An approach towards structure based antimicrobial peptide design for use in development of transgenic plants: a strategy for plant Disease management. Curr. Med. Chem. 24 (13), 1350–1364. https://doi.org/10. 2174/0929867324666170116124558. Kramer, C., Brune, P., McDonald, J., et al., 2016. Evolution of risk assessment strategies
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