Effects of genetically modified T2A-1 rice on the GI health of rats after 90-day supplement Yanfang Yuan 1, Wentao Xu 1,2, *, Xiaoyun He 2, Haiyan Liu 2, Sishuo Cao 1, Xiaozhe Qi 1, Kunlun Huang 1,2,* and Yunbo Luo 1,2 1 Laboratory of Food Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China 2 The Supervision, Inspection & Testing Center of Genetically Modified Food Safety, Ministry of Agriculture, Beijing 100083, China * Corresponding author
Figure legends Fig S1. Growth curves and daily feed intakes. (A) Growth curves for both male and female rats. Curves based on weekly measurements of body weights are shown with means based on six rats/sex/group; (B-1) Mean relative food intake of male rats (n=6); (B-2) Mean relative food intake of female rats (n=6). : Male rats; : Female rats. FigS2. Effects of diets on intestinal tight-junction proteins Tight-junction protein changes in ileum was measured with Zo-1 in (A) AIN93G group, (B) genetically modified (GM) group, (C) nongenetically modified (NM) group, and Occludin in (E) AIN93G group, (F) GM group, (G) NM group, and (D) experimental control without the addition of secondary antibody. Table S1. Organ weights. Table S2. Results of hematology analysis. Table S3. Bacteria composition of content from different parts of the gastrointestinal (GI) tract. Table S4. ph and water content in fecal and cecum contents. Table S5. Effect of cry2a rice on short-chain fatty acid (SCFA) concentration (umol/g) in cecum and feces of rats. Table S6. Primers used in this study.
Supplementary Tables Table S1 Absolute weights Male Female CK NM GM CK NM GM Body weight 519.33±37.75 518.5±20.75 521±24.97 311.83±19.34 312.67±15.65 309.67±24.79 Adrenals 0.092±0.013 0.108±0.029 0.11±0.024 0.097±0.008 0.092±0.02 0.102±0.023 Brains 2.25±0.09 2.25±0.12 2.16±0.06 2.03±0.08 1.94±0.06 * 1.89±0.09 * Heart 1.93±0.26 2.09±0.09 1.91±0.11 1.15±0.14 1.09±0.10 1.08±0.11 Kidneys 3.67±0.30 3.68±0.25 3.65±0.37 2.06±0.20 2.10±0.19 2.06±0.07 Liver 13.12±0.84 12.33±0.67 12.3±1.49 7.63±0.72 7.91±0.50 7.96±0.79 Lung 1.66±0.09 1.92±0.12 * 1.78±0.15 1.62±0.16 1.80±0.30 1.74±0.59 Spleen 0.74±0.08 0.82±0.13 0.88±0.08 * 0.16±0.02 0.19±0.04 0.17±0.03 Testis 3.43±0.23 3.28±0.28 3.41±0.24 0.54±0.09 0.56±0.03 0.60±0.08 Thymus 0.38±0.05 0.34±0.08 0.41±0.16 0.29±0.06 0.35±0.1 0.31±0.05 Relative weights Adrenals 4.58±0.66 5.42±1.46 5.50±1.18 4.83±0.41 4.58±1.02 5.08±1.16 Brains 0.44±0.04 0.43±0.01 0.42±0.03 0.65±0.05 0.62±0.04 0.62±0.07 Heart 0.37±0.06 0.40±0.03 0.37±0.03 0.37±0.05 0.35±0.04 0.35±0.04 Kidneys 0.71±0.06 0.71±0.04 0.70±0.07 0.66±0.08 0.67±0.04 0.67±0.07 Liver 2.54±0.19 2.38±0.17 2.38±0.40 2.46±0.30 2.53±0.17 2.60±0.44 Lung 0.32±0.02 0.37±0.03 * 0.34±0.04 0.52±0.05 0.58±0.08 0.56±0.18 Spleen 0.14±0.02 0.16±0.02 0.17±0.02 * 0.05±0.01 0.06±0.01 0.05±0.01 Testis 0.66±0.07 0.63±0.03 0.66±0.04 0.17±0.03 0.18±0.02 0.19±0.04 Thymus 0.08±0.01 0.07±0.02 0.08±0.03 0.09±0.02 0.11±0.03 0.10±0.02 Results are showed in mean values ± SD. *:Statistically significant differences from the CK group (P < 0.05) CK: AIN93 G group; NM: non-genetically modified group; GM: genetically modified group.
Table S2 Routine analysis of blood Male Female CK NM GM CK NM GM WBC (*10 9 /L) 8±1.19 9.72±2.53 7.57±1.31 7.25±0.93 7.25±1.78 7.62±2.38 RBC (*10 12 /L) 7±0.27 6.69±0.55 6.76±0.55 5.97±0.31 5.67±0.47 5.9±0.44 PLT (*10 9 /L) 229.33±21.87 198.67±29.82 189.5±22 138.67±47.13 133.67±36.95 157.6±25.61 HGB (g/l) 203±6.48 200.17±10.94 196.83±7.78 186.33±12.61 184.17±18.1 166.17±37.69 HCT (%) 52.27±2.4 51.17±3.51 51.33±2.19 47.83±3.96 45.87±4.34 47.2±3.77 MCV (fl) 74.72±2.56 76.55±1.74 76.2±4.06 80.12±4.17 80.85±2.73 79.98±1.02 MCH (pg) 29.02±0.91 29.98±1.16 29.25±2.02 31.2±1.09 32.48±1.95 30.72±0.98 MCHC (g/l) 388.67±5.92 391.67±9.54 383.67±6.86 390.33±17.22 401.67±13.06 384.2±12.48 Differential count Lymphocytes (%) 75.17±11.02 66.67±17.14 75.33±14.8 66±10.33 76±12.1 69.17±12.88 Neutrophils (%) 23±10.71 32.17±16.28 22.5±14.22 32±11.14 22±11.52 29±12.73 Eosinophils (%) 1.17±0.75 1.17±1.47 1.67±1.86 1.67±1.63 2±1.67 1.33±1.75 Basophils (%) 0±0 0±0 0±0 0±0 0±0 0±0 Monocytes (%) 0.67±0.82 0±0 0.5±0.84 0.33±0.52 0±0 0.5±0.84 Blood biochemistry TP (mmol/l) 7.03±0.33 7.10±0.44 7.40±0.78 9.67±0.61 9.32±0.74 9.26±0.92 ALT (U/L) 80.78±12.67 67.4±12.6 * 58.77±7.45 * 77.92±16.74 66.52±18.39 68.68±19.74 AST (U/L) 326.37±54.85 308.77±43.56 354.57±29.63 376±47.24 376.8±85.46 331.75±45.84 ALP (U/L) 106.65±10.91 91.03±16.71 87.77±12.29 94.38±9.72 92.95±13.76 99.98±5.40 ALB (g/l) 39.71±1.18 40.37±1.78 41.91±1.31 * 53.82±4.40 52.23±3.15 51.54±4.97 BUN (mmol/l) 7.06±0.89 6.79±1.05 7.05±1.02 9.01±2.65 8.82±0.84 8.14±2.09 CREA (μmol/ml) 29.41±2.42 30.96±6.41 32.73±3.74 33.99±5.09 37.02±2.33 33.62±2.91 T-CHO (mmol/l) 2.32±0.43 1.83±0.23 1.81±0.56 2.23±0.52 1.83±0.50 1.73±0.40 * TG (mmol/l) 1.23±0.44 1.03±0.24 1.40±0.35 1.06±0.35 1.00±0.25 1.17±0.41 GLU (mmol/l) 8.18±1.31 8.50±1.02 7.99±1.03 6.51±0.86 6.48±0.78 6.65±1.19 P (mmol/l) 2.87±0.16 3.16±0.51 2.89±0.36 2.57±0.35 2.42±0.33 2.65±0.19 Ca (mmol/l) 2.83±0.15 2.91±0.19 2.76±0.16 2.97±0.08 2.93±0.13 2.93±0.10 Results are showed in mean values ± SD. *:Statistically significant differences from the CK group (P < 0.05) :Statistically significant difference from the parental line (P < 0.05) CK: AIN93 G group; NM: non-genetically modified group; GM: genetically modified group; WBC:white blood cells; RBC: red blood cells, PLT: platelets, HGB: hemoglobin, HCT: hematocrit, MCV: mean corpuscular volume; MCHC: Mean Corpuscular Hemoglobin Concentration; TP: total protein; ALT: alanine transaminase; AST: aspartate transaminase; ALP: alkaline phosphatase; ALB: albumin; BUN: blood urea nitrogen; CREA: creatinine; T-CHO: total cholesterol; TG: triglyceride; GLU: glucose; Ca: calcium, P: inorganic phosphorus.
Table S3 Lactobacilli Bifidobacteria Bacteroides Coliforms Enterococci Clostridium Total bacteria B/E bacteria-day 30 CK 8.38±0.40 8.61±0.53 9.05±0.30 6.85±0.41 6.59±0.37 6.11±0.35 9.59±0.34 1.26 NM 8.87±0.28 * 8.86±0.55 9.14±0.19 7.2±0.59 7.22±0.57 * 6.16±0.26 9.86±0.62 1.23 GM 8.82±0.34 * 8.71±0.53 8.93±0.40 7.29±0.64 7.21±0.31 * 6.13±0.29 9.71±0.26 1.19 CK 8.45±0.38 8.54±0.52 8.97±0.41 6.80±0.50 6.57±0.39 6.08±0.25 9.56±0.38 1.26 NM 8.90±0.37 * 8.60±0.60 9.12±0.36 7.25±0.62 7.34±0.48 * 6.24±0.37 9.74±0.48 1.19 GM 8.93±0.41 * 8.76±0.47 9.03±0.25 7.31±0.65 7.21±0.55 * 6.10±0.35 9.84±0.59 1.20 bacteria-day 60 CK 8.61±0.43 8.36±0.43 9.09±0.34 6.65±0.47 6.80±0.38 6.20±0.37 9.61±0.46 1.26 NM 8.82±0.31 8.52±0.55 9.33±0.38 6.95±0.47 6.86±0.47 6.10±0.47 9.91±0.36 1.22 GM 8.87±0.46 8.45±0.50 9.16±0.31 6.80±0.44 6.94±0.43 6.28±0.33 9.86±0.29 1.24 CK 8.51±0.22 8.34±0.52 9.18±0.36 6.81±0.44 6.98±0.51 6.38±0.49 9.63±0.29 1.22 NM 8.81±0.34 8.43±0.54 9.29±0.51 6.81±0.52 7.04±0.44 6.36±0.41 9.89±0.43 1.24 GM 8.90±0.34 8.36±0.49 9.34±0.42 6.73±0.27 6.91±0.52 6.14±0.60 9.97±0.46 1.24 bacteria-day 90 CK 9.12±0.45 8.53±0.59 9.38±0.43 6.60±0.08 6.79±0.33 6.46±0.38 10.01±0.44 1.29 NM 9.20±0.40 8.47±0.65 9.44±0.33 6.49±0.54 6.89±0.41 6.37±0.32 10.12±0.21 1.31 GM 9.15±0.34 8.46±0.56 9.27±0.37 6.50±0.51 6.92±0.32 6.33±0.41 10.03±0.48 1.30 CK 9.18±0.41 8.59±0.52 9.50±0.41 6.56±0.26 6.79±0.38 6.38±0.34 10.13±0.41 1.31 NM 9.08±0.36 8.32±0.39 9.25±0.49 6.60±0.31 6.95±0.35 6.39±0.36 9.94±0.39 1.26 GM 9.06±0.34 8.49±0.46 9.28±0.45 6.38±0.49 6.87±0.37 6.32±0.46 10.00±0.38 1.33 Duodenum CK 6.24±0.46 6.71±0.34 7.51±0.42 4.60±0.35 4.98±0.46 5.11±0.50 8.20±0.34 1.46 NM 6.42±0.52 6.22±0.42 7.40±0.36 4.39±0.48 5.22±0.56 4.96±0.30 8.08±0.21 1.42 GM 6.84±0.61 6.65±0.39 7.49±0.48 4.50±0.35 5.24±0.42 5.00±0.44 8.32±0.32 1.48 CK 6.53±0.47 6.65±0.36 7.32±0.28 4.36±0.51 4.93±0.34 4.97±0.34 8.14±0.36 1.53 NM 6.76±0.43 6.36±0.40 7.39±0.41 4.42±0.47 4.98±0.55 5.14±0.36 8.15±0.74 1.44 GM 6.53±0.47 6.20±0.38 7.29±0.44 4.23±0.41 5.14±0.60 5.14±0.27 8.04±0.53 1.47 Ileum CK 7.70±0.49 7.68±0.33 8.04±0.42 5.26±0.35 6.38±0.54 5.58±0.32 8.92±0.32 1.46 NM 7.58±0.39 7.29±0.40 8.04±0.14 5.71±0.67 6.62±0.52 5.35±0.28 8.87±0.25 1.28 GM 7.77±0.41 7.31±0.28 7.94±0.45 5.69±0.64 7.04±0.26 5.25±0.43 8.88±0.44 1.28 CK 7.61±0.40 7.65±0.26 8.03±0.39 5.19±0.31 6.25±0.29 5.29±0.35 8.92±0.36 1.47 NM 7.52±0.43 7.22±0.40 8.09±0.51 5.67±0.48 6.41±0.36 5.44±0.32 8.71±0.36 1.27 GM 7.86±0.32 7.43±0.48 8.1±0.35 5.68±0.56 6.72±0.55 5.45±0.42 8.87±0.38 1.31 Caecum CK 8.76±0.33 8.96±0.30 9.22±0.43 6.75±0.36 6.50±0.37 7.10±0.49 9.97±0.50 1.33 NM 9.31±0.49 * 9.16±0.40 9.53±0.43 6.68±0.43 7.12±0.57 * 7.15±0.28 10.29±0.45 1.37 GM 9.36±0.34 * 9.19±0.33 9.52±0.41 6.71±0.39 7.31±0.51 * 6.98±0.61 10.34±0.24 1.37 CK 8.61±0.51 8.89±0.28 9.24±0.47 6.68±0.40 6.53±0.19 7.12±0.44 9.90±0.34 1.33 NM 9.17±0.57 * 9.28±0.34 9.54±0.59 6.71±0.42 7.28±0.58 * 6.99±0.43 10.27±0.46 1.38 GM 9.20±0.38 * 9.16±0.44 9.66±0.49 6.8±0.32 7.23±0.6 * 1 7.01±0.29 10.25±0.35 1.35 Data are shown as log10 cfu g-1 ± SD. *:Statistically significant differences from the CK group (p < 0.05) CK: AIN93 G group; NM: non-genetically modified group; GM: genetically modified group.
Table S4 Feces Cecum Wet weight (g/d) Water content % ph Wet weight (g) ph CK 5.05±0.34 43.88±3.37 7.13±0.23 3.11±0.38 7.32±0.14 NM 5.36±0.36 47.45±3.29 7.08±0.17 3.14±0.37 7.17±0.09 * GM 5.28±0.35 46.72±3.85 7.11±0.18 3.13±0.25 7.16±0.11 * CK 4.27±0.31 43.88±2.71 7.03±0.2 2.63±0.37 7.33±0.15 NM 4.31±0.27 44.67±3.77 7.08±0.24 2.68±0.34 7.06±0.18 * GM 4.44±0.38 46.35±2.74 7.08±0.16 2.75±0.24 7.10±0.18 * Values are means ± SD for 6 rats per dietary group. *:Statistically significant difference from the CK group (p < 0.05) CK: AIN93 G group; NM: non-genetically modified group; GM: genetically modified group.
Table S5 Cecal content Male Female CK NM GM CK NM GM Acetate 33.96±4.19 38.75±5.62 * 38.99±4.31 33.42±3.51 40.1±4.75 39.38±5.69 Propionate 10.76±2.28 10.15±2.27 10.71±2.24 10.22±2.39 9.5±2.03 9.37±2.82 Butyrate 9.15±1.86 8.48±1.38 8.53±0.49 8.87±1.88 8.36±1.25 8.71±1.46 Total SCFA 53.62±5.23 58.53±6.64 58.44±2.14 52.51±4.47 57.96±5.56 57.46±5.78 Fecal content Acetate 19.21±3.41 18.85±4.71 17.96±3.5 18.74±3.05 17.91±3.43 17.65±2.91 Propionate 5.38±2.22 5.09±1.46 4.98 ± 2.10 5.42 ±1.65 5.11±1.55 5.21±1.89 Butyrate 3.21±1.46 3.15±1.41 2.89±1.02 3.00±0.92 3.32±1.38 3.02±1.22 Total SCFA 27.8±4.98 27.1±6.54 25.84±5.9 27.16±4.52 26.34±3.76 25.88±5.42 Values are mean ± SD of eight rats from each group. *:Statistically significant difference from the CK group (p < 0.05) CK: AIN93 G group; NM: non-genetically modified group; GM: genetically modified group; SCFAs: short-chain fatty acids.
Table S6
Target bacteria Amplicon size (bp) Annealing temperature ( C) Sequence (5 3 ) Reference Lactobacillus group 341 58 Bifidobacterium genus 437 60 Escherichia coli subgroup 340 61 Enterococcus genus 144 61 Clostridium perfringens 120 57 Bacteroides Prevotella group 418 59 Total bacteria 466 58 GC-PRBA338F& 518R 77 60 PRBA338F & 518R 77 58 β-actin 150 60 ZO-1 108 59 occludin 155 60 Bar 75 58 Bar 74 59 Cry2A-1 74 60 Cry2A-2 74 60 Cry2A-3 74 61 Cry2A-4 76 62 Cry2A-5 76 59 Cry2A-6 77 58 Flank sequence 79 61 Flank sequence 107 59 NOS 180 61 35s 195 60 SPS 277 60 Ubiquitin 314 60 Chloroplast 180 60 F: AGCAGTAGGGAATCTTCCA R: CACCGCTACACATGGAG F: GGGTGGTAATGCCGGATG R: TAAGCCATGGACTTTCACACC F: GTTAATACCTTTGCTCATTGA R: ACCAGGGTATCTAATCCTGTT F: CCCTTATTGTTAGTTGCCATCATT R: ACTCGTTGTACTTCCCATTGT F: ATGCAAGTCGAGCGAKG R: TATGCGGTATTAATCTYCCTTT F: GAAGGTCCCCCACATTG R: CAATCGGAGTTCTTCGTG F: TCCTACGGGAGGCAGCAGT R: GGACTACCAGGGTATCTAATCCTGTT F: CGCCCGCCGCGCGCGGCGGGCGGGGCGGGG GCACGGGGGGACTCCTACGGGAGGCAGCAG R: ATTACCGCGGCTGCTGG- F: ACTCCTACGGGAGGCAGCAG R: ATTACCGCGGCTGCTGG- F: CCCATCTATGAGGGTTACGC R: TTTAATGTCACGCACGATTTC F: GAGGCTTCAGAACGAGGCTATTT R: CATGTCGGAGAGTAGAGGTTCGA F: GCCTATGGAACGGGCATCTT R: GCCAGCAGGAAACCCTTTG- F: GCACCATCGTCAACCACTACA R: GTCCACTCCTGCGGTTCCT F: ACTGGGCTCCACGCTCTACA R: ATGACAGCGACCACGCTCTT F: ACGGACCACAGCCTCTACGT R: TGAGGCTACCCACCTTCTTCA F: ACCCTCGCTCGTGTCAATG R: GTTGTCCACCTGTTGATTGAACTC F: CATGCTTGAGTTCCGCACAT R: TGGTACTTGAAGAGGCTCCAGAT F: AGTTCCAGATCCAAGGCTACCA R: GATGAAGCTCAGGTGCATGTTG F: ACAGCGCCAGGGTGAACTAC R: GTTGAAGTTGTGGTTGAGATTGGT F: TGGCCCCCGAAGACTACA R: GAAAGTGCGGGTTTGATTGTTC F: CGCAATGTGTTATTAAGTTGTCTAAGC R: GAGATATGATGATACTCCGTTTGCA F: TGCAATGCAAACGGAGTATCAT R: CGTATCTCGTCAATTCCAATTCAG F: GAATCCTGTTGCCGGTCTTG-3 R: TTATCCTAGTTTGCGCGCTA-3 F: GCTCCTACAAATGCCATCA-3 R: GATAGTGGGATTGTGCGTCA-3 F: TTGCGCCTGAACGGATAT-3 R: GGAGAAGCACTGGACGAGG-3 F: AACACTGGCAAGTTAGCAAT-3 R: CCGTAATAAATAGACACCC-3 F: CGAAATCGGTAGACGCTACG-3 R: TTCCATTGAGCTTCTGCA CCT-3 Walter et al. 2001; Heilig et al. 2002 Bartosch et al. 2004 Malinen et al. 2003 Rinttila et al. 2004 Rinttila et al. 2004 Bartosch et al. 2004 Nadkarni et al. 2002 Lee, et al.2005 Lee, et al.2005
Supplementary Figures Fig Sl
Fig S2