A 90 Day Safety Assessment of Genetically Modified Rice Expressing

Mar 30, 2015 - A 90 Day Safety Assessment of Genetically Modified Rice Expressing ... For 90 days, froglets were fed a diet with 30% HH1 rice, 30% par...
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A 90 Day Safety Assessment of Genetically Modified Rice Expressing Cry1Ab/1Ac Protein using an Aquatic Animal Model Hao-Jun Zhu,†,‡ Yi Chen,‡ Yun-He Li,‡ Jia-Mei Wang,†,‡ Jia-Tong Ding,† Xiu-Ping Chen,*,‡ and Yu-Fa Peng*,‡ †

College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, People’s Republic of China State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2 West Yuanmingyuan Road, Haidian District, Beijing 100193, People’s Republic of China

‡

ABSTRACT: In fields of transgenic Bt rice, frogs are exposed to Bt proteins through consumption of both target and nontarget insects. In the present study, we assessed the risk posed by transgenic rice expressing a Cry1Ab/1Ac fusion protein (Huahui 1, HH1) on the development of Xenopus laevis. For 90 days, froglets were fed a diet with 30% HH1 rice, 30% parental rice (Minghui 63, MH63), or no rice as a control. Body weight and length were measured every 15 days. After sacrificing the froglets, we performed a range of biological, clinical, and pathological assessments. No significant differences were found in body weight (on day 90: 27.7 ± 2.17, 27.4 ± 2.40, and 27.9 ± 1.67 g for HH1, MH63, and control, respectively), body length (on day 90: 60.2 ± 1.55, 59.3 ± 2.33, and 59.7 ± 1.64 mm for HH1, MH63, and control, respectively), animal behavior, organ weight, liver and kidney function, or the microstructure of some tissues between the froglets fed on the HH1-containing diet and those fed on the MH63-containing or control diets. This indicates that frog development was not adversely affected by dietary intake of Cry1Ab/ 1Ac protein. KEYWORDS: transgenic rice, Bt protein, frog, safety assessment, nontarget effect

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INTRODUCTION Rice is an important crop that feeds half of the world’s population. However, it is subject to severe pest damage, with ∼2−10% of Asia’s annual rice yield being lost to insect pests such as Lepidoptera.1 The rapid development of transgenic technologies provides new strategies for pest control in rice. Bacillus thuringiensis (Bt) is a Gram-positive soil bacteria that, during sporulation, produces the crystal insecticidal protein δendotoxin, which has a strict specificity to target insects.2 To control insect damage to rice, many transgenic lines expressing Bt genes have been successfully developed worldwide.3 In 2000, the Cry1Ab/1Ac fusion gene from Bt was transformed into the elite rice indica restorer line Minghui 63 (MH63), generating the Bt rice line Huahui 1 (HH1), which has a high resistance to stem borers.4 In August 2009, China’s Ministry of Agriculture issued a safety certificate for this Bt rice line, but it has not yet been approved for commercial cultivation because detractors argue that Bt rice may pose potential food safety and environmental issues.5 To assess food safety, a number of animal feeding studies using Bt rice have been conducted, primarily in rats,6−9 broilers,10 pigs,11 and carp.12 All of the above studies showed that the safety of Bt rice lines was comparable to that of their nontransgenic counterparts. To assess environmental safety, many field and laboratory experiments have also been performed, mainly focusing on terrestrial organisms. Bt rice, protected from the damage by Lepidoptera, had no obvious negative impacts on the individual fitness, population abundance, or diversity of nontarget organisms.13−17 However, the entry of Bt proteins into stream ecosystems was initially so insignificant that it was not considered in the assessment of risks associated with the cultivation of Bt crops.18 Moreover, because rice, unlike dry© 2015 American Chemical Society

land crops, requires water during most stages of development, the risk for aquatic organisms in and around Bt rice fields cannot remain ignored. To date, only a few studies have assessed the effects of Bt rice on aquatic organisms such as Daphnia magna19 and Chlorella pyrenoidosa.20 Frogs are commonly found in rice fields and play an important role in maintaining the biodiversity and stability of the paddy field ecosystem. In recent decades, frog populations have declined sharply worldwide.21 Frogs might be affected by Bt rice in two ways. First, frogs could ingest Bt proteins directly by consuming insects that have fed on Bt rice.22 Second, because of the relatively high permeability of frog skin, frogs could take up Bt proteins that are released into the water through exudation from roots, pollen dispersal, and disposal of postharvest detritus.18 Although some studies demonstrated that Bt rice releases detectable amounts of Bt protein into irrigation water,23,24 it remains unclear whether and how this affects frog development. Therefore, it is important to assess the potential nontarget effects of Bt rice on the development of frog species. Xenopus laevis is a model animal widely used in environmental toxicology because it is easy to feed, readily induced to lay eggs, and very sensitive to external contamination.25 Additionally, unlike wild frogs, which usually prefer moving prey, it will consume static food. Thus, in the present study, X. laevis froglets were fed a nutritionally balanced diet containing HH1 or its nontransformed parental rice line to assess the food Received: Revised: Accepted: Published: 3627

November 19, 2014 March 30, 2015 March 30, 2015 March 30, 2015 DOI: 10.1021/jf5055547 J. Agric. Food Chem. 2015, 63, 3627−3633

Article

Journal of Agricultural and Food Chemistry and environmental safety of Bt rice. Results from this study will provide important information concerning the environmental safety of genetically modified (GM) strains of rice.

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Table 1. Composition of Nutritionally Balanced Test and Control Diets for X. laevis Froglets amt of ingredient (%)

MATERIALS AND METHODS

Chemicals. Human chorionic gonadotropin (hCG) and methanesulfonate (MS-222) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Commercial frog feed was obtained from Cargill Feed Co., LTD (Nanjing, People’s Republic of China). Liver and kidney function detection kits and Bt-Cry1Ab/1Ac protein detection kits were purchased from JianCheng Bioengineering Institute (Nanjing, People’s Republic of China) and EnviroLogix Inc., (Portland, OR, USA), respectively. Test Materials. The HH1 transgenic rice line expressing the fusion gene Cry1Ab/1Ac exhibits resistance to stem borers, such as Chilo suppressalis, Scirpophaga incertulas, and Cnaphalocrocis medinalis.4 The insecticidal activity of HH1 seeds were previously confirmed at the seedling stage.26 The parental nontransformed control line (MH63) is an elite Indica restorer line for cytoplasmic male sterility commonly grown in China. These lines were simultaneously planted in two adjacent plots at a scientific research base at Jiangxi Academy of Agricultural Sciences (Nanchang, Jiangxi Province, People’s Republic of China). Transgenic rice had not been previously planted in these plots. Crops were managed in accordance with regulations of the Agricultural Genetically Modified Organism (GMO) Safety Management of China. Rice was harvested at the end of October 2013, and grains were collected and stored at −20 °C until use. The conventional nutrient components of each rice line were analyzed by the Beijing Research Institute for Nutritional Resources, People’s Republic of China. Animals. Mature female and male X. laevis were maintained separately in glass tanks containing dechlorinated water at 21 ± 2 °C on a 12 h light/12 h dark cycle and were fed chopped pork liver once per week. One pair of parent frogs was chosen and injected with 100 IU hCG to induce breeding. After eggs were laid, the mating pair was removed from the breeding tank. Fertilized eggs were incubated at 22 ± 2 °C on a 12 h light/12 h dark cycle. Tadpoles were started on a daily diet of green algae and D. magna on day 5 after fertilization and switched to commercial frog feed when they completed metamorphosis. Approximately 2 months after metamorphosis, froglets with uniform body weight (∼4.30 g) and body length (∼32.0 mm) were used for experimentation. Frogs were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Diet Formulation. Synthetic frog diets were produced based on the nutrient composition of a commercial frog feed. Two test diets contained 30% rice flour, whereas the control diet was formulated using cornstarch and soybean meal without rice. The detailed diet compositions are shown in Table 1. Experimental Design. A total of 96 froglets were equally divided into three experimental groups of 32 according to body weight, and their sexes were not considered. Each group was randomly divided into four glass jars (20 × 34 × 24 cm) to give four replicates. Froglets were fed daily (∼3% weight/body weight). Fresh dechlorinated water was replenished every 2 days and monitored daily to stay at 20−22 °C. A 12 h light/12 h dark cycle was maintained throughout the course of the experiment. Animals were observed twice daily, and body weight and length (from the tip of the snout to the tip of cloaca) were measured every 15 days for 90 days. Animals were fasted overnight before being sacrificed. Gross Necropsy and Histopathology. At the end of the experiment, the frogs were anesthetized by immersion in 1% MS-222. Before dissection, body weight and length were measured, and the gender was determined on the basis of gross gonadal morphology. A complete necropsy was then performed, and the following organs were excised and weighed: heart, liver, spleen, lung, kidneys, body fat, ovaries or testes, and intestines. Paired organs (lungs, kidneys, ovaries and testes) were weighed as a total of left and right. Additionally, the tissues (stomach, intestines (divided into duodenum, ileum, and rectum), liver, spleen, and gonad) of one male and one female from

ingredient

HH1

MH63

control

transgenic rice nontransgenic rice pregelatinized starch corn soybean meal soybean oil fish meal wheat bran meat and bone meal rock flour additivea total

30.0 0.00 8.00 0.00 6.00 3.00 52.0 0.00 0.00 0.00 1.00 100

0.00 30.0 8.00 0.00 6.00 3.00 52.0 0.00 0.00 0.00 1.00 100

0.00 0.00 8.00 17.0 21.0 3.50 38.0 5.00 6.00 0.50 1.00 100

a

Contents in mg/kg of diet: iron, 70; copper, 11; manganese, 70; zinc, 65; iodine, 0.49; selenium, 0.3; vitamin A, 8000 (IU); vitamin D, 2400 (IU); vitamin E, 20 (IU); vitamin K, 0.5 (IU); vitamin B1, 2; vitamin B2, 8; vitamin B6, 3.5; vitamin B12, 0.01; calcium pantothenate, 20; niacin, 35; folic acid, 0.75; biotin, 0.26.

each glass jar were fixed for a minimum of 24 h in 4% phosphatebuffered formaldehyde before histological processing. Tissue samples were embedded in paraffin, and ∼4 to 6 μm thick sections were cut and stained with hematoxylin and eosin for light microscopy. Determination of Liver and Kidney Function. The liver and spleen from the remaining six froglets in each glass jar (a total of 24 samples per treatment) were used to determine the following parameters: alkaline phosphatase (AKP) activity, albumin (ALB), alanine aminotransferase (ALT) activity, aspartate aminotransferase (AST) activity, urea (BUN), cholinesterase (CHE) activity, total protein (TP), creatinine (CR), glutamic acid (GLU), total cholesterol (TC), and triglycerides (TG). All parameters were detected using kits provided by the JianCheng Bioengineering Institute. All of the detection kits used in this study use a corresponding standard substance to show assay validity prior to analysis. Moreover, these kits are used strictly according to the recommendations and protocols set by the manufacture. Determination of Cry1Ab/1Ac Content. The Cry1Ab/1Ac protein content in rice grains, frog diets, frog tissues (stomach and intestine), and frog feces (∼0.20 g each) were determined using a BtCry1Ab/1Ac protein kit with a detection limit of 0.1 ng/g total protein. Solid feces were collected immediately after the renewal of rearing water at the later stage of the present study. Before analysis, animal tissues were washed in phosphate-buffered saline/Tween-20 to remove Bt toxins from the outer surface, lyophilized, and then homogenized in 1 mL phosphate-buffered saline/Tween-20 using a micropestle and mortar on ice. After centrifugation and appropriate dilution of the supernatants, an ELISA was performed following the manufacturer’s protocol. Optical density values were read using a microplate spectrophotometer (PowerWave XS2, BioTek, Winooski, VT, USA). A standard curve derived from purified Cry1Ab/1Ac samples was used to calculate Cry1Ab/1Ac levels. Statistical Analyses. All data are presented as mean ± standard deviation (SD) unless indicated otherwise. Statistically significant differences in conventional nutrient compositions between HH1 and MH63 rice grains were analyzed by Student’s t test. Body weights and lengths were analyzed using repeated measures analysis of variance (ANOVA), whereas a one-way ANOVA, followed by least significant difference (LSD) multiple comparison tests, was used to analyze differences in organ weight, as well as liver and kidney function among the three treatment groups. Differences were considered significant at p < 0.05. 3628

DOI: 10.1021/jf5055547 J. Agric. Food Chem. 2015, 63, 3627−3633

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Journal of Agricultural and Food Chemistry Table 2. Nutrient Composition of Rice Grains and Diets for X. laevis Froglets rice grain (n = 3) component

amt of componenta in MH63b

p valuec

amt of componenta in HH1

amt of componenta in MH63

amt of componenta in control

6.63 ± 0.24

6.85 ± 0.12

0.22

38.1

38.2

42.2

± ± ± ± ±

0.18 0.17 0.89 0.71 0.25

8.40 3.30 11.2 3.50 1.81

8.80 3.20 11.4 3.50 1.84

8.10 3.00 10.7 2.40 1.73

crude protein (%) crude fat (%) crude fiber (%) crude ash (%) calcium (g/kg) phosphorus (%) a

diet (n = 1)

amt of componenta in HH1b

2.83 6.97 11.8 0.66 0.36

± ± ± ± ±

0.58 0.06 0.26 0.16 0.01

2.90 6.65 11.9 0.61 0.37

0.00 0.50 0.06 0.13 0.01

Units for the amount of component are given in column 1. bData are presented as mean ± SD. cStudent’s t test, p < 0.05.

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RESULTS Nutrient Composition of Rice Grains and Froglet Diets. Levels of conventional nutrients (crude protein, crude fat, crude fiber, crude ash, calcium, and phosphorus) were very similar in HH1 and MH63 rice grains (Table 2), and no statistical differences were observed between the two (p > 0.05). Moreover, the measured values of these same nutrients in the HH1 diet were comparable to those in the MH63 and control diets (Table 2). General Health. Animal wellbeing was observed twice daily, and body weight and length were measured every 15 days for 90 days. No adverse effects on animal behavior were observed during the experiment. The mean body weights and body lengths of animals from each treatment are shown in Tables 3 and 4, respectively. Statistically significant differences

(p = 0.81 for body weight, p = 0.98 for body length). Additionally, the only significant difference in organ weight among the three dietary groups was the relative weight of fat body, which was significantly higher in the HH1 group in comparison to control (p < 0.05, Table 5). Liver Function, Kidney Function, and Fat Metabolism. Parameters for liver and kidney function, as well as fat metabolism, are given in Table 6. There were no statistically significant differences among the three groups (p > 0.05). Cry1Ab/1Ac Protein Content in Diets and Frogs’ Digestive Tracts. Cry1Ab/1Ac was present in HH1 rice grain at an average concentration of 752 ± 78.0 ng/g and was not detectable (ND) in the nontransformed control strain MH63 (Table 7). The measured Cry1Ab/1Ac contents in the HH1, MH63, and control diets (221 ± 21.2 ng/g, ND, and ND, respectively; Table 7) were close to the predicted values (226, 0, and 0 ng/g, respectively). To investigate the degradation of Cry1Ab/1Ac protein in the frog digestive tract, intestinal contents and feces were collected, and the Cry1Ab/1Ac protein content was determined by ELISA. Cry1Ab/1Ac protein was detected in the intestinal contents (249 ± 16.9 ng/g) and in feces (160 ± 34.7 ng/g) of froglets fed on the HH1 diet but not in those fed on the MH63 or control diet (Table 7). Gross Necropsy and Histopathology. Histological examinations of stomach, intestine (ileum), liver, kidneys, spleen, testes, and ovaries are shown in Figure 1. None of the three sections of intestines showed apparent pathological abnormalities; only representative images of ileum are shown in Figure 1. Overall, there were no gross pathological findings during the necropsies, and no group-related histopathological abnormalities were observed.

Table 3. Mean Body Weight over Time in X. laevis Froglets Fed Test versus Control Diets (n = 32) body weight (g)a time (days) 0 15 30 45 60 75 90

HH1 4.35 6.02 8.85 12.0 16.7 21.2 27.7

± ± ± ± ± ± ±

0.01 0.23 0.25 0.40 0.39 0.34 2.17

MH63 4.36 6.13 9.33 12.3 17.2 22.2 27.4

± ± ± ± ± ± ±

control

0.06 0.30 0.57 1.01 1.46 2.11 2.40

4.32 5.85 8.97 12.2 16.8 22.5 27.9

± ± ± ± ± ± ±

0.04 0.21 0.27 0.38 0.56 1.12 1.67

Data are presented as group mean values ± SD. Repeated measures ANOVA, p < 0.05.

a

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Table 4. Mean Body Length over Time in X. laevis Froglets Fed Test versus Control Diets (n = 32) body length (mm) time (days) 0 15 30 45 60 75 90

HH1 31.6 34.8 39.2 43.2 47.8 52.5 60.2

± ± ± ± ± ± ±

0.44 0.60 0.27 0.39 0.42 0.78 1.55

MH63 31.7 34.7 39.5 43.3 48.3 52.0 59.3

± ± ± ± ± ± ±

0.42 0.48 0.68 0.98 0.50 1.74 2.33

control 31.8 33.9 39.5 43.7 48.5 52.2 59.7

± ± ± ± ± ± ±

DISCUSSION

An integral part of the safety evaluation of GM plants is testing for substantial equivalence to the unmodified parent strain, which provides a starting point for the overall assessment.27 In the present study, we analyzed the differences in nutrient composition between HH1 and MH63 before evaluating the effects of the GM rice strain on development of froglets. No significant differences in composition, including crude protein, crude fat, and crude ash levels, were found between the two rice lines or between diets containing them (Table 2). Schrøder et al. found significant differences in some nutritional composition indices between brown rice material from transgenic Cry1Ab and its corresponding parental line, but all differences were within the normal reference intervals.7 Indeed, most studies have shown that transgenic Bt rice lines have nutrient compositions similar to those of their parental lines,6,8,28 which is consistent with our results.

a

0.19 0.39 0.51 0.54 0.53 1.38 1.64

Data are presented as group mean values ± SD. Repeated measures ANOVA, p < 0.05.

a

were observed among the seven repeated measures within each treatment group from day 0 to day 90 (p < 0.01 for body weight and body length), but not among the three treatments 3629

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Journal of Agricultural and Food Chemistry

Table 5. Absolute and Relative Organ Weights and Intestinal Lengths in X. laevis Froglets Fed Test versus Control Dietsa HH1 ± 2.17 ± 0.01 ± 0.16 ± 0.00 ± 0.01 ± 0.03 ± 0.14 0.02 (16) 0.01 (16) ± 1.11

body (g) heart (g) liver (g) spleen (g) lung (g) kidney (g) fat body (g) ovary (g) testis (g) intestinal length (cm)

27.7 0.14 1.70 0.03 0.16 0.21 1.84 0.12 ± 0.05 ± 15.1

heart liver spleen lung kidney fat body ovary testis intestinal length

0.51 ± 0.05 6.06 ± 0.14 0.10 ± 0.01 0.56 ± 0.04 0.74 ± 0.05 6.56 ± 0.22b 0.44 ± 0.06 (16) 0.18 ± 0.03 (16) 0.56 ± 0.02

MH63

statisticsc

control

Absolute Measurements 27.4 ± 2.40 0.14 ± 0.02 1.60 ± 0.16 0.03 ± 0.00 0.16 ± 0.01 0.21 ± 0.03 1.82 ± 0.21 0.11 ± 0.01 (13) 0.04 ± 0.01 (19) 15.0 ± 1.30 Relative Valuesb 0.49 ± 0.04 5.75 ± 0.20 0.11 ± 0.01 0.58 ± 0.05 0.76 ± 0.07 6.49 ± 0.39ab 0.42 ± 0.06 (13) 0.16 ± 0.02 (19) 0.58 ± 0.08

± 1.67 ± 0.02 ± 0.07 ± 0.00 ± 0.02 ± 0.01 ± 0.06 0.02 (19) 0.01 (13) ± 1.76

t t t t t t t t t t

= = = = = = = = = =

0.07; 0.41; 0.17; 0.02; 0.07; 0.16; 0.20; 0.11; 0.95; 1.94;

p p p p p p p p p p

= = = = = = = = = =

0.93 0.66 0.84 0.98 0.94 0.85 0.82 0.90 0.39 0.15

0.52 ± 0.06 5.70 ± 0.15 0.10 ± 0.01 0.56 ± 0.04 0.77 ± 0.04 6.00 ± 0.26a 0.48 ± 0.09 (19) 0.17 ± 0.01 (13) 0.63 ± 0.02

t t t t t t t t t

= = = = = = = = =

0.60; 2.00; 0.75; 0.27; 0.63; 3.99; 0.73; 0.60; 2.47;

p p p p p p p p p

= = = = = = = = =

0.55 0.14 0.48 0.76 0.54 0.02 0.49 0.55 0.09

27.9 0.15 1.64 0.03 0.16 0.22 1.73 0.12 ± 0.05 ± 16.2

Data presented as group mean values ± SD (n = 32, except for ovaries and testes, where n is given in parentheses). Different Roman lowercase letters in the same row indicate a statistical difference of p < 0.05. bRelative values expressed as g or cm per 100 g of body weight. cOne-way ANOVA, p < 0.05.

a

Table 6. Liver Function, Kidney Function, and Fat Metabolism in X. laevis Froglets Fed Test versus Control Diets (n = 24) indices

HH1

MH63

control

195 ± 12.6

Liver Function 193 ± 31.1

167 ± 12.1

7.31 ± 0.55

7.22 ± 0.58

7.04 ± 0.72

ALT (U/g protein) AST (U/g protein) BUN (mg/L) CHE (U/mg protein) TP (g/L)

860 ± 181

902 ± 224

813 ± 36.7

443 ± 66.5

432 ± 65.1

405 ± 22.0

1.82 ± 0.08

1.79 ± 0.08

1.72 ± 0.08

1.41 ± 0.15

1.40 ± 0.10

1.34 ± 0.12

9.27 ± 0.48

8.90 ± 0.21

9.10 ± 0.25

BUN (mg/L) CR (μmol/L) GLU (μmol/ g protein)

3.49 ± 0.11

Kidney Function 3.32 ± 0.18

3.36 ± 0.48

23.9 ± 4.07

25.1 ± 3.30

20.3 ± 5.56

30.6 ± 4.34

28.25 ± 3.74

29.75 ± 0.82

0.66 ± 0.05

Fat Metabolism 0.69 ± 0.10

0.67 ± 0.16

2.32 ± 0.34

2.11 ± 0.10

2.06 ± 0.68

AKP (U/g protein) ALB (g/L)

TC (mmol/L) TG (mmol/L) a

Table 7. Cry1Ab/1Ac Protein Content in Rice Grain, Diet, and Digestive Tract of X. laevis Froglets Fed Test versus Control Diets (ng/g)a

statisticsa t= p t= p t= p t= p t= p t= p t= p

0.81; = 0.45 0.73; = 0.49 0.55; = 0.58 0.75; = 0.48 2.71; = 0.08 0.06; = 0.94 1.28; = 0.28

t= p t= p t= p

0.47; = 0.63 2.82; = 0.07 0.74; = 0.48

t= p t= p

0.19; = 0.83 0.79; = 0.46

rice grain (n = 6) diet (n = 6) intestinal tract (n = 24) feces (n = 6) a

HH1

MH63

control

± ± ± ±

ND ND ND ND

NA ND ND ND

752 221 249 160

78.0 21.2 16.9 34.7

ND, not detectable; NA, not applicable.

comparable.30 The food and feed safety of Bt rice has been tested in many animals, including rats,6−9 broilers,10 pigs,11 and carp,12 and all of the above studies showed that the safety of the GM line and that of the parental line were comparable. For example, TT51 rice, the same Bt rice line as in the present study, showed no significant differences on reproduction performance of rats in comparison with MH63 and control.9 Moreover, Xu et al. found that Cry1Ab/Ac fusion protein produced in Escherichia coli has no adverse effects in mice by gavage at a high dose level of 5 g/kg body weight.31 Our results using X. laevis froglets were consistent, as there were no significant differences among the three dietary groups with respect to general health, including animal behavior, body weight (Table 3), and body length (Table 4). In addition, we assessed organ weight and relative organ weight, which are important indicators reflecting organ development. A change in organ weight can signal adverse effects from the external environment, including food.32 Our findings show that the diet containing HH1 rice resulted in no significant differences in absolute or relative organ weights in comparison with the MH63 and control diets, except for an increase in the relative weight of fat bodies in the HH1 group in comparison to the control group (Table 5). Furthermore, pathology exams revealed no obvious lesions in any group (Figure 1), and

One-way ANOVA, p < 0.05.

The consequences and potential risks associated with expressed insecticidal proteins are of particular concern in any safety assessment.29 Numerous experimental studies have consistently indicated that the health and performance of animals fed GM crop lines and isogenic non-GM crop lines are 3630

DOI: 10.1021/jf5055547 J. Agric. Food Chem. 2015, 63, 3627−3633

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Journal of Agricultural and Food Chemistry

Figure 1. Histopathological staining of tissues from X. laevis froglets after consuming the HH1 test diet (a), MH63 test diet (b), or control diet (c) for 90 days. For intestine, only representative images of the ileum are shown.

DNAs and proteins could not be detected in the gastrointestinal contents or feces of animals fed diets containing GM crops,11,33,34 others were able to detect foreign transgenic proteins in the gastrointestinal contents and/or feces of pigs,35 bovines,36,37 deer,38 and insects39 that were fed transgenic crops or diets containing transgenic crops. The amount of transgenic protein ingested by livestock depends on the concentration of the protein in the feed, the amount of feed intake, and the species.29 From previous studies we can conclude that Bt proteins can survive passage through an animal’s gastrointestinal tract but are not transferred to the visceral organs or their products.33,36 In the current study, Cry1Ab/1Ac

liver function, kidney function, and fat metabolism (Table 6) were not significantly different among the groups. Taken together, these results indicate that Cry1Ab/Ac protein had no significant adverse effect on organ development. The planting of GM crops brings huge economic and environmental benefits to humans; however, it also raises questions and concerns regarding their safety. Some people fear that foreign proteins will enter the food chain and eventually enter the human body, where they may cause potential harm to humans. They also fear that foreign proteins may be present in the feces of animals fed GM crops, where they may affect the environment. Whereas some researchers found that transgenic 3631

DOI: 10.1021/jf5055547 J. Agric. Food Chem. 2015, 63, 3627−3633

Article

Journal of Agricultural and Food Chemistry

(2) Hofmann, C.; Vanderbruggen, H.; Hofte, H.; Van Rie, J.; Jansens, S.; Van Mellaert, H. Specificity of Bacillus thuringiensis delta-endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midgets. Proc. Natl. Acad. Sci. U. S. A. 1988, 85 (21), 7844−7848. (3) Chen, M.; Shelton, A.; Ye, G. Y. Insect-resistant genetically modified rice in China: from research to commercialization. Annu. Rev. Entomol. 2011, 56, 81−101. (4) Wang, Y.; Zhang, G.; Du, J.; Liu, B.; Wang, M. Influence of transgenic hybrid rice expressing a fused gene derived from cry1Ab and cry1Ac on primary insect pests and rice yield. Crop Prot. 2010, 29 (2), 28−33. (5) Lu, C. The first approved transgenic rice in China. GM crops 2010, 1 (3), 113−115. (6) Wang, Z. H.; Wang, Y.; Cui, H. R.; Xia, Y. W.; Altosaar, I.; Shu, Q. Y. Toxicological evaluation of transgenic rice flour with a synthetic cry1Ab gene from Bacillus thuringiensis. J. Sci. Food Agric. 2002, 82 (7), 738−744. (7) Schrøder, M.; Poulsen, M.; Wilcks, A.; Kroghsbo, S.; Miller, A.; Frenzel, T.; Danier, J.; Rychlik, M.; Emami, K.; Gatehouse, A.; Shu, Q.; Engel, K. Z.; Altosaar, I.; Knudsen, I. A 90-day safety study of genetically modified rice expressing Cry1Ab protein (Bacillus thuringiensis toxin) in Wistar rats. Food Chem. Toxicol. 2007, 45 (3), 339−349. (8) Cao, S.; He, X.; Xu, W.; Luo, Y.; Yuan, Y.; Liu, P.; Cao, B.; Shi, H.; Huang, K. Safety assessment of transgenic Bacillus thuringiensis rice T1c-19 in Sprague-Dawley rats from metabonomics and bacterial profile perspectives. IUBMB Life 2012, 64 (3), 242−250. (9) Wang, E. H.; Yu, Z.; Hu, J.; Jia, X. D.; Xu, H. B. A two-generation reproduction study with transgenic Bt rice TT51 in Wistar rats. Food Chem. Toxicol. 2014, 65, 312−320. (10) Qin, H. F. Safety assessment of rice genetically modified with Cry1Ac and sck by feeding studies on broilers. Dissertation, Chinese Academy of Agriculture Science, 2012 (in Chinese with English abstract). (11) Cao, Z. H.; Wang, Z. B.; Gu, X. H. Residues and organ damage of exogenous gene and protein from transgenic Bt brown rice in growing pigs. Chin. J. Anim. Nutr. 2014, 26 (7), 1908−1915 (in Chinese with English abstract) . (12) Xu, Z. Y. Safety assessment of rice genetically modified with Cry1Ac and sck by feeding studies on carp (Cyprinus carpio). Dissertation, Chinese Academy of Agriculture Science, 2011 (in Chinese with English abstract). (13) Bernal, C. C.; Aguda, R. M.; Cohen, M. B. Effect of rice lines transformed with Bacillus thuringiensis toxin genes on the brown planthopper and its predator Cyrtorhinus lividipennis. Entomol. Exp. Appl. 2002, 102 (1), 21−28. (14) Liu, W.; Lu, H. H.; Wu, W. X.; Wei, Q. K.; Chen, Y. X.; Thies, J. C. Transgenic Bt rice does not affect enzyme activities and microbial composition in the rhizosphere during crop development. Soil Biol. Biochem. 2008, 40 (2), 475−486. (15) Akhtar, Z. R.; Tian, J. C.; Chen, Y.; Fang, Q.; Hu, C.; Chen, M.; Peng, Y. F.; Ye, G. Y. Impacts of six bt rice lines on nontarget rice feeding thrips under laboratory and field conditions. Environ. Entomol. 2010, 39 (2), 715−726. (16) Li, Y.; Zhang, X.; Chen, X.; Romeis, J.; Yin, X.; Peng, Y. Consumption of Bt rice pollen containing Cry1C or Cry2A does not pose a risk to Propylea japonica (Thunberg) (Coleoptera: Coccinellidae). Sci. Rep. 2015, 5, 7679. (17) Li, Y.; Chen, X.; Hu, L.; Romeis, J.; Peng, Y. Bt rice producing Cry1C protein does not have direct detrimental effects on the green lacewing Chrysoperla sinica (Tjeder). Environ. Toxicol. Chem. 2014, 33 (6), 1391−1397. (18) Viktorov, A. G. Transfer of Bt corn byproducts from terrestrial to stream ecosystems. Russ. J. Plant Physiol. 2011, 58 (4), 543−548. (19) Li, G.; Wang, Y.; Liu, B.; Zhang, G. Transgenic Bacillus thuringiensis (Bt) rice is safer to aquatic ecosystems than its nontransgenic counterpart. PLoS One 2014, 9 (8), e104270.

protein was detected in the intestinal contents and feces of froglets in the HH1 group (Table 7), indicating that they were exposed to Bt proteins by consuming a diet containing the Cry1Ab/1Ac protein. However, the microstructure of the digestive tract (Figure 1) and the froglets’ overall development were not adversely affected. Our results, combined with those of others, demonstrate that expressed foreign Bt proteins do not produce harmful effects in the digestive tract or elsewhere after ingestion by animals.29 Consumption of Bt rice fed arthropods is the main route through which frogs are exposed to the rice-produced Cry proteins. Zhang et al. found that the Cry2Aa protein content in most of the arthropods collected from the Cry2Aa rice field were much lower than 200 ng/g dry weight.22 Studies of Bt soybean and Bt maize showed that the plant-produced Cry proteins are diluted when moving through the foodweb.39,40 Additionally, Wang et al. did not detect Cry1Ab/1Ac protein in the stomach and intestine of frogs collected from HH1 rice field.26 In the present study, the Cry1Ab/1Ac protein concentration in the HH1 diet was 221 ± 21.2 ng/g (Table 7), which was much higher than the likely exposure under field conditions. In this 90 day study, X. laevis froglets were exposed to Bt proteins by consuming a diet containing HH1 rice that carries the gene encoding Cry1Ab/1Ac. Froglet development was not adversely affected in comparison with froglets fed a diet containing the parental rice (MH63) or no rice. Combining this laboratory study with our previous field experiment,26 we conclude that the planting of transgenic Cry1Ab/1Ac rice will not adversely affect the development of frogs.

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AUTHOR INFORMATION

Corresponding Authors

*X.-P.C.: tel, +86-10-62815947; fax, +86-10-62896114; e-mail, [email protected]. *Y.-F.P.: tel, +86-10-62815947; fax, +86-10-62896114; e-mail, [email protected]. Funding

This work was supported by the National GMO New Variety Breeding Program of the PRC (2012ZX08011-002 and 2014ZX08011-001) and a Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor Yongjun Lin (Huazhong Agricultural University, Wuhan, People’s Republic of China) for kindly providing transgenic rice seeds.

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ABBREVIATIONS USED AKP, alkaline phosphatase activity; ALB, albumin; ALT, alanine aminotransferase activity; AST, aspartate aminotransferase activity; BUN, urea; CHE, cholinesterase activity; TP, total protein; CR, creatinine; GLU, glutamic acid; TC, total cholesterol; TG, triglycerides

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DOI: 10.1021/jf5055547 J. Agric. Food Chem. 2015, 63, 3627−3633