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Full-Text PDF: 663-670.pdf
Immunotoxicological Evaluation of Wheat Genetically Modified with TaDREB4 Gene on BALB/c Mice
 

Original Article

Immunotoxicological Evaluation of Wheat Genetically Modified with TaDREB4 Gene on BALB/c Mice*

LIANG Chun Lai, ZHANG Xiao Peng, SONG Yan, and JIA Xu Dong#

Key Laboratory of Food Safety Risk Assessment, Ministry of Health (China National Center for Food Safety Risk Assessment) Beijing 100021, China

*This research was supported by the National GMO Cultivation Major Project of New Varieties (2008ZX08011-005, 2011ZX08011-005).

#Correspondence should be addressed to JIA Xu Dong. Tel: 86-10-67770977; E-mail: jiaxudong2004@163.com

Biographical note of the first author: LIANG Chun Lai, female, born in 1982, assistant professor, majoring in food toxicology.

Received: March 20, 2013;             Accepted: April 30, 2013

 

 

*This research was supported by the National GMO Cultivation Major Project of New Varieties (2008ZX08011-005, 2011ZX08011-005).

#Correspondence should be addressed to JIA Xu Dong. Tel: 86-10-67770977; E-mail: jiaxudong2004@163.com

Biographical note of the first author: LIANG Chun Lai, female, born in 1982, assistant professor, majoring in food toxicology.

Received: March 20, 2013;             Accepted: April 30, 2013

 

Abstract

Objective  To evaluate the immunotoxicological effects of genetically modified wheat with TaDREB4 gene in female BALB/c mice.

Methods  Female mice weighing 18-22 g were divided into five groups (10 mice/group), which were set as negative control group, common wheat group, parental wheat group, genetically modified wheat group and cyclophosphamide positive control group, respectively. Mice in negative control group and positive control group were fed with AIN93G diet, mice in common wheat group, non-genetically modified parental wheat group and genetically modified wheat group were fed with feedstuffs added corresponding wheat (the proportion is 76%) for 30 days, then body weight, absolute and relative weight of spleen and thymus, white blood cell count, histological examination of immune organ, peripheral blood lymphocytes phenotyping, serum cytokine, serum immunoglobulin, antibody plaque-forming cell, serum half hemolysis value, mitogen-induced splenocyte proliferation, delayed-type hypersensitivity reaction and phagocytic activities of phagocytes were detected.

Results  No immunotoxicological effects related to the consumption of the genetically modified wheat were observed in BALB/c mice when compared with parental wheat group, common wheat group and negative control group.

Conclusion  From the immunotoxicological point of view, results from this study demonstrate that genetically modified wheat with TaDREB4 gene is as safe as the parental wheat.

Key words: Genetically modified wheat; Immunotoxicological effects; BALB/c mice

Biomed Environ Sci, 2013; 26(8):663-670    doi: 10.3967/0895-3988.2013.08.005     ISSN:0895-3988

www.besjournal.com(full text)            CN: 11-2816/Q          Copyright ©2013 by China CDC

 


INTRODUCTION

G

enetic modification techniques have been progressed in modern agriculture with a fast speed, the global area of commercial cultivation of genetically modified (GM) crops has been increased for 15 consecutive years[1], GM crops expressing desirable traits (i.e. herbicide tolerance, insect resistance) could significantly decrease yield losses and reduce the use of insecticides, but in the meantime raise a controversy against GM food over inherent or potential risks to human health[2]. Safety assessment of GM crops is based on the concept of substantial equivalence which was developed by OECD[3] and further elaborated by FAO/WHO[4], however, whether use of the concept was adequate for safety assessment or has limitations with respect to identification of unknown effects caused extensive debate among scientists[5-7], Millstone et al. suggested that extensive biological, toxicological, and immunological tests should be conducted for GM foods[5].

Wheat is an important source of food, and China is the largest wheat producer in the world, but drought has severely restricted the productivity as the major wheat producing areas in China are arid or semiarid areas. In these two years since the program “Abiotic-tolerant GM Wheat New Variety Development” was launched, progress has been achieved and many drought tolerance GM wheat lines have been developed with prospect to enhance drought-enduring and thus improve the productivity. Dehydration-responsive element-binding (DREB) proteins are important transcription factors in plant responses and signal transduction, they can improve the drought and salt tolerance of plants thus provide an excellent opportunity to develop stress-tolerant GM crops[8], the GM wheat with TaDREB4 gene in this study was produced by Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, the TaDREB4 gene was obtained from xiaobaimai wheat cultivar and introduced into jimai19 wheat cultivar with a biological function of salt-tolerant and drought-tolerant.

Until now, Immunological effects of GM crops were largely focused on their potential allergenicity, while potential immunotoxicological effects of GM foods consumption was rarely concerned. One study by Kroghsbo et al. found a dose-dependent increase in mesenteric lymph node weight and total immunoglobulin A when feeding PHA-E transgenic rice to rats[9], another feeding trial with MON810 Bt maize induced alterations in intestinal and peripheral immune response to weaning and old mice, the author pointed out that the gut and peripheral immune response to GM crop ingestion is important in a safety evaluation[10]. In the present study, we evaluated the immunotoxicological effects of GM wheat consumption. Wheat flour from the GM wheat, parental wheat (jimai19) and common wheat were formulated into balanced basic AIN93G diets[11] at a proportion of 76%, then fed to BALB/c mice, another two groups of mice fed with AIN93G diet were set as negative control and positive control, respectively, 30 days later, a range of immunotoxicological parameters covering immunopathological, humoral immunity, cellular immunity and non-specific immunity were detected.

MATERIALS AND METHODS

Materials

The GM wheat, parental wheat and common wheat were obtained from Institute of Crop Sciences, Chinese Academy of Agricultural Sciences (Beijing, China). Cyclophosphamide (CY) was purchased from Jiangsu Hengrui Medicine Co., Ltd. (Jiangsu, China). Sheep red blood cells (SRBC) were purchased from Beijing Laboratory Biology technology Co., Ltd. (Beijing, China). FITC hamster anti-mouse CD3e, APC rat anti-mouse CD19, PE rat anti-mouse CD49b, APC rat anti-mouse CD4, PE rat anti-mouse CD8a, lysing buffer and Mouse Th1/Th2 cytokine kit were purchased from Becton, Dicknson and Company (Franklin Lakes, NJ, USA). Mouse IgG ELISA, Mouse IgA ELISA and Mouse IgM ELISA kits were purchased from GenWay Biotech, Inc. (San Diego, CA, USA). Concanavalin A (ConA) and Lipopolysaccharide (LPS) were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). Thiazoyl blue tetrazolium bromide (MTT) was from Amresco LLC (Solon, OH, USA). RPMI 1640 medium, Hanks’ balanced salt solution (HBSS), fetal bovine serum, phosphate buffered saline (PBS) and penicillin-streptomycin solution were from Thermo Fisher Scientific Inc (Logan, UT, USA).

Animals and Treatment

Female BALB/c mice, 6-8 weeks old with body weight of 18-22 g were used for the study, guinea pigs (250 g) were used for preparation of complement for plaque-forming cell assay and hemolysis test. The animals were provided by Beijing HFK Bioscience Co., Ltd. (Beijing, China), and were maintained in a controlled environment at a temperature of 20-26 °C, relative humidity of 40%-70%, artificially illuminated (fluorescent lights) with a 12:12 h light/dark cycle and air exchanged at 10-15 times/h. Mice were acclimatized for 5 days with AIN93G diet and then divided into negative control group, common wheat group, parental wheat group, GM wheat group and positive control group randomly with 10 mice/group based on body weight. Mice in the GM wheat, parental wheat and common wheat group were fed with feedstuffs formulated with 76% corresponding wheat, mice in negative control and positive control (administered with 200 mg/kg of CY by intraperitoneal injection  24 h before the termination of the study) group were fed with AIN93G diet, respectively. Mice were housed by group with ad libitum access to water and corresponding diets for 30 days.

Toxicological and Haematological Parameters

Body weight of mice was measured at weekly intervals and at the end of the experiment, the absolute and relative weights (percentage of terminal body weight) of spleen and thymus were determined on day 30, the spleen, thymus, femur and lymph nodes (cervical, axillary, mesenteric lymph nodes and Peyer’s patch) were fixed in 10% formalin for histological examination. For haematological studies, whole blood was collected from the retro-orbital plexus of each mouse in the presence of anticoagulant, then white blood cell count (WBC) was measured with a COULTER Ac·T diff2 Hematology Analyser (Beckman Coulter Inc., USA).

Peripheral Blood Lymphocytes Phenotyping

Whole blood was collected from the retro-orbital plexus of each mouse in the presence of anticoagulant. 50 μL blood cell suspensions were stained with three-color combinations of antibodies of FITC hamster anti-mouse CD3e, APC rat anti-mouse CD19 and PE rat anti-mouse CD49b or antibodies of FITC hamster anti-mouse CD3e, APC rat anti-mouse CD4 and PE rat anti-mouse CD8 for 20 min at room temperature in the dark, then the blood samples were lysed with 2 mL ammonium chloride-based lysing buffer for 20 min at room temperature in the dark and washed with 2 mL PBS. Subsequently, the samples were resuspended in  0.5 mL PBS and analyzed on FACSCalibur flow cytometer using CellQuest software (Becton, Dicknson, and Company, USA). Appropriate isotype controls were used for compensation controls.

Serum Cytokine Quantification

Whole blood was collected from retro-orbital plexus of each mouse without the addition of anticoagulant. Following centrifugation, the sera were obtained and assayed for levels of cytokine including IL-2, IL-4, IFN-γ, and TNF by Mouse Th1/Th2 Cytokine Kit. The assay procedure was in accordance with descriptions in the manufacture’s instruction. Briefly, 50 μL mixed capture beads were added into each assay tube, and 50 μL standards, blank or serum samples were added into the appropriate tubes, then PE detection reagent were added into each tube and incubated for 2 h at room temperature. The samples were washed with 1 mL wash buffer, then resuspended in 300 μL wash buffer and analyzed on FACSCalibur flow cytometer using CellQuest software.

Serum Immunoglobulin Quantification

The sera were obtained as described in the part of Serum Cytokine Quantification and assayed for concentrations of immunoglobulin including IgG, IgM, and IgA by ELISA kits. The assay procedures were in accordance with descriptions in the manufacturer’s instructions. Briefly, 100 μL standards, blank or diluted serum samples were added into predesignated wells of 96-well plates in duplicate, the microtiter plates were covered and incubated at room temperature for 1 h, then washed 4 times with wash solution. 100 μL enzyme-antibody conjugate was added into each well, and the plates were covered and incubated at room temperature for  30 min, then washed 4 times with wash solution. Subsequently, 100 μL TMB substrate solution was added into each well, and incubated at room temperature for 10 min, finally, 100 μL stop solution was added into each well, and the absorbance  (450 nm) of the contents of each well was determined using an ELISA Reader (BioTek., USA).

Preparation of Spleen Cell Suspensions

Single cell splenocyte suspensions were prepared as described in Technical Standards for Testing and Assessment of Health Food (2003) published by the Ministry of Health[12]. The spleens were aseptically removed and transferred to culture dishes containing 2 mL cold HBSS. Forceps and carbasus were used to finely triturate the spleen, then the cell suspensions were transferred into tubes and washed twice in HBSS and centrifuged for 10 min at 1000 rpm at 4 °C. The cell pellet was resuspended in appropriate culture medium for the assay being performed. Spleen cell counts were performed using a hemocytometer.

Plaque-forming Cell (PFC) Assay

The PFC assay was performed by a modified Jerne’s method[12]. Mice were immunized on day 25 with 0.2 mL of 2% (v/v) SRBC suspension in sterile saline via intraperitoneal injection. Five days post-immunization, the mice were sacrificed and spleen cell suspensions were prepared as stated in the part of Preparation of spleen cell suspensions.  25 μL of spleen cell suspension in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution (5×106 cells/mL), 50 μL of 10% (v/v) SRBC in SA buffer solution (0.46 g C4H4N2O3, 0.1 g MgCl2·6H2O, 0.2 g CaCl2·2H2O, 8.38 g NaCl, 0.252 g NaHCO3, and 0.3 g C8H11N2NaO3 solved in 1000 mL stilled water) and 500 μL agar solution (0.5 g/mL in HBSS, pH 7.2-7.4) were mixed in a glass tube, then poured onto slides. The slides were covered on a special frame after the mixtures were solidified, and incubated at 37 °C for 1-1.5 h, then guinea pig complement was added to the slot between the slides and bottom of the frame. The slides were incubated at 37 °C for another 1-1.5 h, then plaque production was enumerated and the results were expressed as the number of PFC per 106 splenocytes.

Hemolysis Test

Mice were immunized on day 25 with 0.2 mL of 2% (v/v) SRBC suspension in sterile saline via intraperitoneal injection. Five days post-immunization, the sera were obtained as described in the part of Serum Cytokine Quantification and assayed for serum half hemolysis value (HC50). 1 mL SA buffer solution, 0.5 mL 10% (v/v) SRBC, 1 mL diluted guinea pig complement (1:8 diluted with SA buffer solution) and 2 μL mouse serum were added into sample tubes. Control tube without mouse serum was set at the same time. All tubes were kept in water bath for 15-30 min at 37 °C, then the tubes were kept in ice bath to terminate the reaction. After centrifugation for 10 min at  2000 rpm, 1 mL supernatant was collected and added into 3 mL drabkin solution (1.0 g NaHCO3, 0.05 g KCN, and 0.2 g K3Fe(CN)6 solved in 1000 mL stilled water), at the same time, 0.25 mL 10% (v/v) SRBC and 3.75 mL drabkin solution were mixed and standing for 10 min as positive control. The absorbance at 540 nm was detected, the HC50 value was got by the equation: HC50 = (OD1/OD2)500 (OD1 = the OD value of sample well subtract that of control well, OD2 = the OD value of positive control well subtract that of control well).

Mitogen-induced Splenocyte Proliferation

The mitogens used in this assay were ConA (100 μg/mL) and LPS (400 μg/mL), dissolved in distilled water. The mice were sacrificed and spleen cell suspensions were prepared as stated in the part of Preparation of spleen cell suspensions on day 30.   1 mL spleen cell suspension (3×106 cells/mL) was added into 24-well plate and cultured with 75 μL mitogen solutions or distilled water for control cultures. The plates were incubated at 37 °C with 5% CO2 for 68 h, then 0.7 mL supernatant in each well was discarded and 0.7 mL RPMI 1640 medium was added, in the meanwhile, 50 μL fresh prepared MTT solution (5 mg/mL, dissolved in PBS, pH 7.2) was added into each well, the plates were then incubated under the same conditions for an additional 4 h. Finally, 1 mL acid-isopropanol (4 mL of 1 mol/L HCl added to 96 mL isopropanol) was added into each well, the contents in each well were mixed thoroughly to make the purple crystallize fully dissolved and transferred to 96-well plates in triplicate, the absorbance at 570 nm was determined, and the results were calculated by subtracting the OD value of control well from OD value of sample well.

Delayed-type Hypersensitivity (DTH)

Mice were sensitized on day 25 by intraperitoneal injection of 0.2 mL of 2% (v/v) SRBC in sterile saline. Five days later, the thickness of left rear footpad of each mouse were determined with a vernier caliper (Changchun, Jilin Province, China). Then, the left rear footpads were injected with 20 μL of 20% (v/v) SRBC by subcutaneous injection. After 24 h, the thickness of left rear footpads were determined again. Swelling was expressed as the difference between two measurements before and after injection of SRBC to left rear footpad.

Mice Carbon-clearance Test

Mice were injected with diluted ink         (10 mL/kgbw) via caudal vein on day 30, at 2 min and 10 min after the injection, 20 μL blood was collected from the retro-orbital plexus of each mouse and added into 2 mL Na2CO3 solution (1 mg/mL), then the absorbance of the mixtures at 600 nm was determined. Mice were sacrificed and weight of liver and spleen were determined. Index of phagocytosis was calculated by the equation: Index of phagocytosis = [body weight/(liver weight + spleen weight)][(lgOD1-lgOD2)/(t2-t1)]1/3 (OD1: the absorbance at 600 nm of 2 min after injection of ink, OD2: the absorbance at 600 nm of 10 min after injection of ink).

Statistical Analysis

The values are expressed as means±SD (standard deviation). The data were analyzed using SPSS software (version 17.0). Comparisons between multiple groups were carried out using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test while equal variances assumed or Dunnett’s T3 post hoc test while equal variances not assumed. P-value less than 0.05 was considered statistically significant.

RESULTS

Body Weight, Organ Weight, WBC Count, and Histological Examination

A single dose of CY (200 mg/kg) induced a significant reduction in terminal body weight, spleen weight and thymus weight of mice when compared with animals in negative control group. No significant differences were observed on body weight, spleen weight or thymus weight of mice in the GM wheat group when compared with those in parental wheat group, common wheat group and negative control group (Figure 1, Table 1). The mean WBC value of mice in CY group was lower than that in negative control group, no significant difference on WBC count was observed in the GM wheat group when compared with parental wheat group, common wheat group and negative control group (Table 2). Histological examination was performed in all preserved organs, pathology changes were observed only in CY group, including mild atrophy of thymus (10/10), decrease of bone marrow hematopoietic cells, dilatation and congestion of sinusoid in marrow cavity of femur (10/10), lymphopenia in white pulp and red pulp of spleen, dilatation and congestion in medullary sinus of red pulp (2/10), unclear follicle structures of lymph nodes (4/8) and lymphopenia in lymph nodes (1/8). No pathological changes were observed in other groups.

 

Adobe Systems

Figure 1. Body weight of mice at different time points.


Table 1. Terminal Body Weight, Spleen Weight, and Thymus Weight of Mice (means±SD) (n=10)

Group

Terminal Body Weight (g)

Spleen Weight

 

Thymus Weight

Absolute (g)

Relative (%)

 

Absolute (g)

Relative (%)

Negative Control

21.0±0.3

0.085±0.010

0.407±0.047

 

0.049±0.006

0.234±0.032

Common Wheat

20.4±0.7

0.079±0.005

0.390±0.028

 

0.050±0.006

0.246±0.028

Parental Wheat

21.1±1.0

0.085±0.008

0.402±0.042

 

0.052±0.007

0.249±0.040

GM Wheat

21.2±0.8

0.087±0.008

0.410±0.042

 

0.050±0.005

0.239±0.030

Positive Control

19.4±1.0a

0.059±0.008a

0.304±0.048a

 

0.036±0.007a

0.185±0.039a

Note. aP<0.05 as compared with negative control group.

Table 2. White Blood Cell Count and Classification of Mice (means±SD) (n=10)

Group

WBC (×109/L)

LYM (%)

MO (%)

GRN (%)

Negative Control

7.99±2.21

82.77±2.51

8.47±1.82

8.76±1.73

Common Wheat

7.33±2.61

81.90±2.39

8.13±1.66

10.11±3.82

Parental Wheat

6.41±1.66

85.27±2.32

7.13±1.31

7.60±2.57

GM Wheat

6.82±1.47

85.72±2.59

7.88±1.01

6.40±2.28

Positive Control

2.67±0.68a

80.83±4.26

9.50±2.66

9.67±6.34

Note. aP<0.05 as compared with negative control group.

 


Peripheral Blood Lymphocytes Phenotyping

A single dose of CY (200 mg/kg) induced a significant increase in percentage of T lymphocytes (CD3+CD19-), CD4+ T lymphocytes (CD3+CD4+, Th cells), and CD4+/CD8+ (Th/Ts) ratio when compared with those in negative control group, a reduction in percentage of B lymphocytes (CD3-CD19+) was observed compared with negative control group. There were no significant changes in percentage of T lymphocytes, B lymphocytes, NK cells (CD3-CD49+), Th cells, CD8+ T lymphocytes (CD3+CD8+, Ts cells) or Th/Ts ratio in the GM wheat group when compared with parental wheat group, common wheat group and negative control group animals (Table 3), respectively.

Serum Cytokine and Serum Immunoglobulin

There were no significant differences in serum IL-2, IL-4, IFN-γ, or TNF between positive control and negative control group, and no significant differences were found in serum IL-2, IL-4, IFN-γ, or TNF in the GM wheat group when compared with parental wheat group, common wheat group and negative control group (Table 4), respectively. A single dose of CY (200 mg/kg) induced a significant reduction in IgM level when compared with negative control group. A significant increase of IgG level in the GM wheat group was observed when compared with negative control group and common wheat group respectively, a significant decrease of IgA level was observed when compared with negative control group, similar changes of IgG level and IgA level were occurred in parental wheat group. There were no significant differences in concentrations of IgG,  IgM or IgA between the GM wheat group and parental wheat group. The IgM level in the GM wheat group was comparable to that in common wheat group and negative control group, the IgA level was comparable to that in common wheat group (Table 4).

Immune Function Assays

The PFC assay and hemolysis test were commonly used to detect humoral immunity, the mitogen-induced splenocyte proliferation and DTH reaction were commonly used to detect cellular immunity, and mice carbon-clearance test was commonly used to detect non-specific immunity. In this study, a single dose of CY (200 mg/kg) induced a significant reduction in PFC/106 splenocytes, HC50 and LPS-induced splenocyte proliferation when compared with negative control group. In the GM wheat group, the ConA-induced splenocyte proliferation was higher than common wheat group but comparable to that in negative control group and parental wheat group (Table 5). There were no significant differences in PFC/106 splenocytes, HC50, LPS-induced splenocyte proliferation, DTH or index of phagocytosis in the GM wheat group when compared with parental wheat group, common wheat group and negative control group (Table 5).


Table 3. Phenotypic Analysis of Peripheral Blood Lymphocytes of Mice (means±SD) (n=10)

Group

CD3+CD19- (%)

CD3-CD19+ (%)

CD3-CD49+ (%)

CD3+CD4+ (%)

CD3+CD8+ (%)

CD4+/CD8+

Negative Control

70.43±4.44

13.89±3.19

2.84±0.90

55.87±3.80

13.45±0.98

4.16±0.29

Common Wheat

68.33±5.37

15.34±4.84

3.22±2.11

55.24±4.60

12.08±2.37

4.73±0.96

Parental Wheat

73.04±2.68

13.06±4.22

2.93±2.18

57.92±3.70

14.12±1.75

4.19±0.78

GM Wheat

74.11±8.79

14.66±5.13

1.89±0.60

59.25±7.30

14.00±2.46

4.31±0.75

Positive Control

90.42±1.66a

3.01±0.96a

1.61±0.94

77.37±2.31a

12.31±1.04

6.34±0.73a

Note. a P<0.05 as compared with negative control group.

Table 4. Serum Cytokine and Serum Immunoglobulin of Mice (means±SD) (n=10)

Group

IL-2

(pg/mL)

IL-4

(pg/mL)

IFN-γ

 (pg/mL)

TNF

 (pg/mL)

IgG

 (μg/mL)

IgM

(μg/mL)

IgA

(μg/mL)

Negative Control

2.05±0.21

2.33±0.53

2.90±0.50

11.10±1.77

1046.38±210.67

333.83±18.97

51.69±10.10

Common Wheat

2.00±0.13

2.22±0.54

2.76±0.42

10.06±0.85

1065.49±297.22

327.73±27.72

42.40±8.35

Parental Wheat

1.94±0.14

2.15±0.20

2.61±0.29

9.88±1.07

1517.73±299.52a,b

367.47±27.18

32.11±4.22a

GM Wheat

2.06±0.33

2.39±1.01

2.63±0.26

9.42±3.02

1454.36±229.22a,b

360.19±43.80

38.54±7.90a

Positive Control

1.99±0.21

2.30±0.13

2.44±0.38

9.25±1.60

1155.88±167.05

278.71±24.42a

41.45±8.89


Note. aP<0.05 as compared with negative control group. bP<0.05 as compared with common wheat group.

Table 5. Results of Immune Function Assays in Mice (mean±SD) (n=10)

Group

PFC (/106 cells)

HC50

ConA-induced splenocyte proliferation

LPS-induced splenocyte proliferation

Increase in footpad thickness (mm)

Phagocytic index

Negative Control

29.2±14.6

82.3±6.5

0.37±0.16

0.21±0.10

0.07±0.04

4.48±0.27

Common Wheat

28.0±20.0

79.7±4.6

0.30±0.25

0.21±0.14

0.07±0.03

4.18±0.41

Parental Wheat

34.8±30.9

75.8±4.1

0.38±0.21

0.26±0.12

0.06±0.04

4.09±0.50

GM Wheat

26.8±18.3

75.5±2.6

0.56±0.19b

0.35±0.11

0.04±0.04

4.19±0.35

Positive Control

4.8±5.3a

70.8±2.1a

0.24±0.14

0.07±0.06a

0.04±0.02

4.37±0.50

Note. aP<0.05 as compared with negative control group. bP<0.05 as compared with common wheat group.

 


DISCUSSION

The immune system is very sensitive to a variety of chemical and physical stressors and as such can be used as a tool to examine the subclinical effects of chemical exposure[13]. The purpose of this research was to evaluate the immunotoxicological effect of the GM wheat in BALB/c mice. Five groups including negative control group, common wheat group, parental wheat group, the GM wheat group and positive control group were set, then parameters of body weight, immune organ weight, WBC count, peripheral blood lymphocytes phenotyping, serum cytokine and serum immunoglubulin were determined, histological changes of immune organ were examined, and assays reflecting the function of humoral immunity, cellular immunity and non-specific immunity were performed in each group.

Cyclophosphamide can reduce the number of circulating lymphocytes and impair the function of humoral and cellular immune response, and was commonly used as immunosuppressive agent in immunotoxicology study[14-16]. In this study, a single dose of CY (200 mg/kg) was administered by intraperitoneal injection as a positive control. In CY group, a significant reduction of terminal body weight, spleen weight and thymus weight, WBC value, %B cells, serum IgM, PFC/106 splenocytes, HC50 and LPS-induced splenocyte proliferation was observed, and a significant increase of %T cells, %Th cells and Th/Ts ratio was detected, some histopathology changes in thymus, spleen, femur and lymph nodes were also observed. These results indicated that a positive control of immunity suppressive animal model was successfully established in BALB/c mice.

Results from the current study showed no significant differences on body weight, spleen and thymus weight, WBC count, peripheral blood lymphocytes phenotyping, serum cytokine, histological examination of immune organ of BALB/c mice in the GM wheat group when compared with mice in parental wheat group, common wheat group and negative control group. For serum immunoglobulin determination, the serum IgG level of mice in the GM wheat group was higher than those in negative control group and common wheat group, the IgA level was lower than that in negative control group, these differences were not considered to be related to the consumption of the GM wheat, because the same changes of IgG and IgA levels were seen in parental wheat group, and no statistical differences of IgG or IgA level were observed between the GM wheat group and parental wheat group. In addition, serum immunoglobulin changes could be indicative of some kind of humoral immunity damage, however, no significant differences on humoral immunity function were observed through the PFC assay and hemolysis test. For immune function assays, an increase of ConA-induced splenocyte proliferation was seen in the GM wheat group when compared with common wheat group, similarly, the difference was not considered to be related to the consumption of the GM wheat, because no statistical differences were observed between the GM wheat group and parental wheat group, and no significant differences were observed in other cellular immunity assays.

The proportion of the three kind of wheat incorporated into diet was up to 76%, which was the highest ratio could be achieved while the feed nutrients balance could be ensured, the nutritional content of protein, carbohydrates, etc. was adjusted to be the same level as AIN93G diets, the composition and nutritional analysis was performed in our laboratory and no significant differences were found in all diets. Therefore the variations stated above were considered to be attributed to the difference in nutrition source of the feed, these results demonstrate that different nutritional source could have different influence on an organism, similar findings have been reported before in 90-day feeding studies and not considered as indication of adverse effects[17-21].

In conclusion, there were no immunotoxicological effects observed in BALB/c mice consuming diets formulated with 76% of the GM wheat compared with mice consuming parental wheat diet, common wheat diet and AIN93G negative control diet. From the immunotoxicological point of view, results from this study demonstrate that the GM wheat with TaDREB4 gene is as safe as the parental wheat.

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