Toxicity assessment of modified Cry1Ac1 proteins and genetically modified insect-resistant Agb0101 rice

1 Division of Biosafety, National Academy of Agricultural Science, Rural Development Administration, Jeonju, Republic of Korea. 2 Division of Central Area Breeding, National Institute of Crop Science, Rural Development Administration, Jeonju, Republic of Korea. 3 Division of Molecular Breeding, National Academy of Agricultural Science, Rural Development Administration, Jeonju, Republic of Korea. 4 School of Applied Biosciences, Kyungpook National University, Daegu, 702-701, Korea.


INTRODUCTION
Genetically modified (GM) crops are becoming an increasingly important feature of the agricultural landscapes.In 2013, approximately 175 million hectares of GM crops were planted by 18 million farmers in 27 countries.Due to the unprecedented 100-fold increase between 1996 and 2013, biotech crops are now the *Corresponding author.E-mail: tataby@korea.kr.Tel:  Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, urea nitrogen; TG, triglyceride; A/G Ratio, albumin/globulin ratio; IP, inorganic phosphorus.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License fastest-adopted crop technology in the history of modern agriculture.Products with insect-resistant traits were the fastest growing group between 2009 and 2013 (James, 2013).Rice (Oryza sativa) is an important crop and staple food that used to main energy source in Korea.Not only the major food but also economical impact, Rice production affects all around of Korea economy.In Korea, 1 million people are cultivating rice in about 1 million ha of rice field.Various factors limit rice productivity, including pest which annually destroy 20 to 30% of rice crops (Estruch et al., 1996).Pest managements are very important factor to consider when attempting increasing rice production.In Korea, Cnophalocrocis medinalis guenee is the one of major problem in rice cultivation especially.Generally, this insect can be managed using several kinds of pesticides (Warren et al., 1997).However, many farmers and consumers want to use less pesticide due to environmental and economic concerns.Insect-resistant rice was developed to prevent pest explosions and seek ecofriendly purposes.Many GM Crops for pest managements are developed and commercialized.Almost 32% of commercial GM crops are insect-resistant due to prevent pest damage.Almost all GM Crops use the gene as commonly known as Bt gene from the bacterium Bacillus thuringiensis that code for insecticidal crystal proteins were engineered into plants in the mid-1980s in order to develop the insect-resistant genetically modified plant.Bt corn was the first genetically modified by introducing the Bt gene since 1987 to combat the crop damage.Bt products have been used as insecticides for more than 40 years without documented evidence of adverse effects.Additionally, a number of toxicological studies on insecticidal Cry proteins have not identified any safety concerns for using Bt proteins (McClintock et al., 1995).
Genetic modification using insect-resistant genes is one of the most common modifications used to generate transgenic crops, including rice (Bajaj and Mohanty, 2005;Maqbool et al., 2001;Ramesh et al., 2004;Tu et al., 2000;Ye et al., 2003).Commercializing GM rice lags behind other cereals such as maize.One reason is that rice is cultivated more than 100 countries around the world and is a staple for about a half of the world's population; thus its safety must be strictly evaluated prior to its release to the market (Jiao et al., 2010).The cry1Ac gene can be isolated from B. thuringiensis and encodes the Cry protein, which exhibits toxic effects by forming pores in the cell membrane, thereby injuring epithelial cells in the midgut of insect.The effect is highly specific to target insects such as Lepidoptera and Coleoptera species, but is harmless to plants and mammals including humans (Bravo et al., 2007).We recently developed insect-resistant Agb0101 rice that contains the mcry1Ac1 gene, a modified synthetic and truncated version of the cry1Ac1 gene that expresses the same toxic protein with Cry1Ac1.Agb0101 rice contains a single copy of the truncated cry1Ac gene, and the toxic protein expressed by this transgene targets the chloroplast (Lee et al., 2009).
Furthermore, research has revealed that Agb0101 demonstrates high resistance to rice leaf folder, rice green caterpillar, and rice skipper under laboratory conditions and to rice leaf folder under field conditions (Kim et al., 2009).
This study was performed to assess the toxicity of insect-resistant rice, develop scientific methodologies assess the safety of GM crops.These results could be used to further commercialization of insect-resistant Agb0101 rice.Although, this study described toxicity only, our results could help elucidate the food safety of Agb0101 rice.

Test materials
The modified cry1Ac1 gene (mcry1Ac1: GenBank accession no.AY126450) used in this study was derived from the truncated cry1Ac1 gene (GenBank accession no.AAA22551).This mcry1Ac1 gene encodes the same amino acid sequences with the truncated Cry1Ac1 protein.For transformation into rice, some nucleotides were changed for optimal use in plants.Because the expression level of the mCry1Ac1 protein is extremely low in transgenic Agb0101 rice (typically less than 100 mg/kg), it is impractical for use in animal studies.Therefore, we conducted safety assessments using mCry1Ac1 protein that was produced in Escherichia coli.Proper characterization of the equivalence between recombinant and rice expressed protein is a necessary pre-requisite for use in safety evaluations of specific transgenic events.Agb0101 and control rice (Nak-dong) were grown in the experimental field of the National Academy of Agricultural Science (Suwon, Korea).To purify the mCry1Ac1 protein from rice, transgenic rice leaves containing the mcry1ac1 gene were used as the plant material.Samples were collected, and the leaves were stored at -80°C until use.

Expression and purification of mCry1Ac1 protein from E. coli
The mCry1Ac1 proteins were produced and characterized.The pMAL-p2g/mcry1ac1 vector (BioLabs Inc., USA) was used to express the mCry1Ac1 protein.mCry1Ac1 referred to the modified cry1ac1 gene in order to be expressed in E. coli strain BL21 CodonPlus (DE3) RIPL (Stratagene, La Jolla, CA) as a fusion protein containing a maltose binding protein(MBP) tag and was purified using immobilized amylase resin chromatography.The MBP tag was cleaved from the affinity purified protein with cysteine protease from Tobacco Etch virus (TEV).And the fusion tag and TEV were removed by dialysis.For the acute toxicity study, the protein was lyophilized, mixed and stored at −80°C.The purity of the total protein was determined using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and scanning densitometry.The concentration of the total protein was determined using the Bradford method.The identities of both lots of proteins were characterized using amino acid analysis and western blot analysis using a proprietary mCry1Ac1-specific antibody.

Using bioinformatic analysis to determine similarities between toxic proteins and the mCry1Ac1 protein
Sequence similarity searches were conducted on the amino acid sequence of the mCry1Ac1 protein using the BLASTP algorithm of the NCBI protein database.Sequence similarities were manually inspected manually to identify known toxic proteins.Generally, a st atistically significant sequence similarity requires a match with an E score of <0.01; however, a threshold E score 1.0 provides greater assurance that proteins with even limited similarities will not be overlooked.The BLOSUM62 scoring matrix was used, low complexity filtering was turned off, and the number of alignments returned was set to a maximum value of 1,000 proteins.

Protein sequencing and In-gel trypsin digestion and protein identification by MS/MS
The mCry1Ac1 protein was electroporated and transferred to PVDF membranes.Targeted band were cut and amino acid sequencing was carried out by Edman Sequencer ABI494.MALDI-TOF mass experiments were carried out according to Sun et al. (2007), with minor modifications.Protein bands with mCry1Ac1 were carefully cut out from CBB R250-stained gels and subjected to in-gel trypsin digestion according to Sun et al. (2007), with minor modifications.MALDI-TOF/TOF-GPS Explorer™ software version 3.6 (Applied Biosystems) was used to create and search files with the MASCOT search program for peptide and protein identification.

Acute toxicity assessments of the mCry1Ac1 protein in ICR mice
Acute toxicity studies were conducted in accordance with OECD guidelines (OECD, 2001) and the Laboratory animal Act by Korea Food and Drug Administration (KFDA).60 ICR mice (3 weeks old, 18 to 22 g) were obtained from Hanlim Animal Experiment Institute, (Hwasung, Korea).After 5 day acclimatization period, the mice were randomly divided into 3 groups.Each group was related to mCry1Ac, Bovine serum albumin (BSA) and water as control.Each group contained 20 mice / group (10 males and 10 females per treatment).All mice were kept in stainless steel wire cages (2/cage) at 21 to 23°C, relative humidity 40 to 60%, 15 air change times per hour, and electric lights were turned on from 9 AM to 9 PM.Mice were allowed free access to both food and water.The mCry1Ac1 protein was dissolved in distilled water to 95% purity, and the mCry1Ac1 protein concentration was adjusted to 250 mg/mL.Each mouse received about 0.2 mL/kg protein (2,000 mg/kg mCry1Ac1 protein for each mouse) by gavage on the first day.BSA was used as the negative control and water was used as the blank control.Mice were given basal diet and tap water and observed for 14 days for any signs of morbidity or mortality.During the experimental period all animals were inspected twice daily (cage side).At study completion day 15, all animals were anaesthetized by carbon dioxide inhalation and sacrificed by exsanguinations for the subsequent gross and histopathological examinations.

Subchronic toxicity of Agb0101 rice in SPF rats
Subchronic toxicity study was conducted in accordance with OECD guidelines (OECD, 1998) and the Laboratory animal Act by KFDA.80 SPF rats (40 male and 40 female) were obtained from Orient Co. (Kapyung, Korea).All rats were 5 weeks old at study initiation.Following a 7 day acclimatization period, they were randomly divided into 4 groups, 20 mice/ group (10 males and 10 females per treatment).All diets were administered to rats for 90 consecutive days.Animals were housed pair wise in stainless steel wire cages at 23 ± 2°C, relative humidity 55 ± 5%, 10 to 15 air change per hour and electric light with 200 to 300 lux from 9 AM to 9 PM.Rats were allowed free access to both food and water.Insect resistant Agb0101 rice was mixed with the basal diet in order to take into account nutritional balance.We made 4 kinds of feed for this experiment.The composition of the feed administrated to each group is listed in Table 1.Diets were identically adjusted to assure an adequate supply of macronutrients and vitamins after substitution with 60% rice, but no adjustments were made to balance differences in the constitution of the rice (as observed by the compositional/chemical analyses).Rats were allowed free access to both food and water (KFDA, 2007).

Serum biochemistry and haematology
All animals were fasted overnight to minimize fluctuations in the measured parameters.Blood samples were taken from the eyeball veniplex and stabilized using heparin.The following biochemical parameters were measured: alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN), triglyceride (TG), albumin/globulin ratio (A/G ratio), inorganic phosphorus (IP), sodium, potassium, cholesterol, protein, albumin, creatinine and glucose.All analyses on blood plasma were performed on a Express plus (Bayer Diagnostics Inc., USA).Blood samples used to assess the hematology characteristics were stabilized using EDTA.The following characteristics were assessed using the Baker system 9118 Hematology Analyzer (Biochem Immunosystems Inc., Allentown, PA).White blood cells, Red blood cells, hemoglobin concentration, hematocrit, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration and platelet count.The differential count distinguishes between basophils, eosinophils, lymphocytes, monocytes and neutrophils.

Organ weights, gross necropsy and histopathology
Necropsy was performed and the following organs were sequentially excised: brain, thymus, heart, lungs, liver, spleen, stomach, duodenum, adrenals, kidneys, ovaries, and testes.The brain, heart, liver, spleen, lungs, kidneys, thymus, adrenals, ovaries and testes were immediately weighed, and the stomach, duodenum, kidneys and liver were immediately fixed in 4% buffered formaldehyde for histological processing.Tissue samples were embedded in paraffin, and sectioned to 3 to 5 μm thick, and then stained with standard hematoxylin-eosin for light microscopy.

Statistical analysis
Statistical comparisons were designed to determine if differences in the mentioned response variables between groups could be attributed to the mCry1Ac1 protein and Agb0101 rice.Data obtained from the mCry1Ac1 protein groups were compared with the values from the vehicle and BSA control groups.Similarly, each Agb0101-treated group was compared with the values from the control groups.Homogeneity variance was analyzed using one-way analysis of variance (ANOVA) using Statistical Product and Service Solutions (SPSS) v12.0 (SPSS Inc., Chicago, IL).Differences were considered significant when p<0.05 and then a step-down analysis was conducted using least squares differences (LSD).

BlastP comparison of the mCry1Ac1 proteins to other toxic proteins
BLASTP similarity searches were conducted using the amino acid sequence of mCry1Ac1 in order to identify the 1000 closest matches.The highest E score returned was 0.85, confirming that sequences with limited similarity were not overlooked by the search.95 of the accessions returned by the searches demonstrated complete significance (E = 0) and represent very closely related Cry proteins from various bacterial species.A total of 827 other sequences were identified as Cry proteins from various bacterial species.The remaining 173 hits represented a variety of proteins that are all functionally related by the possession of one or more well characterized conserved thiamine pyrophosphate binding domains.No information is available on the toxicity of these proteins returned by the BLASTP search identified similarities to proteins known toxic.Assessing the safety of a novel protein requires determining its amino acid sequence similarity with known toxic proteins that have potential safety concerns.Accordingly, amino acid sequences were compared as part of the current food safety decision tree strategy as recommended by FAO/WHO (2001) and the CODEX (2003).Amino acid sequence similarities between the mCry1Ac1 protein and known toxic proteins were determined according to published guidelines (CODEX, 2003).The results of the in silico analysis revealed no evidence for similarities between mCry1Ac1 and any known toxic protein.

Isolation and characterization of heterologously produced mCry1Ac1 protein
The recombinant mCry1Ac1 protein was expressed in E. coli and purified as a soluble protein.The mCry1Ac1 Lee et al. 1939 protein migrated as a major band and demonstrated a molecular weight of approximately 66 kDa according to our SDS-PAGE analysis.The purity of the mCry1Ac1 protein was greater than 95% according to densitometry analysis which was performed the SDS-PAGE analysis (Figure 1).After blotting to PVDF membrane, we performed N-terminal sequencing by Edman degradation.
The N-terminal sequence of the analyzed mCry1Ac1 protein was identical to deduced amino acid sequence of mCry1Ac1.MALDI-TOF MS analysis confirmed 71% sequence coverage for the mCry1Ac1 protein (data not shown).

Acute toxicity of mCry1Ac1 protein
The acute toxicity of the mCry1Ac1 protein was assessed in mice following the oral administration of the purified heterologously expressed protein at 2,000 mg/kg body weight of the test substance (corresponding to approximately 2,000 mg/kg of body weight of mCry1Ac1 protein) via oral gavage.Control groups were administered either vehicle (that is, water) alone or BSA at 2,000 mg/kg body weight.All mice survived the study period and no clinical signs of systemic toxicity were observed in any treatment groups (data not shown).All mice in all treatment groups gained weight relative to day 0 of dosing (Table 2), and no gross lesions were present in any of the mice at necropsy, thereby indicating that the mCry1Ac1 protein was not acutely toxic.The mCryAC1 protein demonstrates no special toxicity according to our acute toxicity tests.We did not observe animal death and special symptoms at 2,000 mg/Kg body weight.

Subchronic toxicity of genetically modified rice Agb0101 Mortality and clinical signs
There was no instance of treatment-related mortality in the animals treated with Agb0101 rice during this study.No significant clinical signs were observed in any other group.

Body weight and feed consumption
No reliable differences in body weight (Figure 2), feed consumption, weight gains, or food efficiency were observed between rats in the Agb0101-treated groups and those in the related control group (that is, rat that received Nak-dong rice).Also, no reliable changes in consumption between treated groups were observed during the test periods (Figure 3).

Hematology
As shown in Table 3, no significant changes were observed  in most of the hematology response variables between groups that consumed the different diets.However, certain hematology variables, such as reticulo-cytes in the male rats in the G4 group demonstrated significant decrease in comparison with G1 whereas female in the G3 and G4 groups demonstrated significant increases in comparison with G1, G2 groups.Reticulocytes are a known indicator of anemia, but related variables like red blood cells, hemoglobin, and hematocrit did not significantly changed in either males or females.These results indicate that the reticulocyte changes in males and females were not cause by Agb0101 rice.

Serum biochemistry
No differences were observed in most of the values of the serum response variables between the groups consumed different diets (Table 4).However, some differences were observed in total protein and some ion parameters.Total protein demonstrated a significant decrease among female in the G4 and G1 groups.However, this value difference was not observed in the G2 group females.Calcium ion also statistically and significantly decreases in G3 and G4 females, but not in males, though this finding was not significantly between the G1 and G4 groups.Chloride ion decreased in G3 and G4 males in comparison with G1 males, but increased in G4 female comparison with G1 females.These changes were not consistently demonstrated in males and females, and the increases/decreases were inconsistent.Therefore, the observed differences in these parameters are attributed to background variability and sporadic deviation.All differences are within the normal ranges for rats.Therefore, they are not considered related to Agb0101 rice.Significant differences as compared with G1: a P < 0.05.Significant differences as compared with G2 : b P < 0.05.

Necropsy findings and organ weight
There were no gross indications of adverse effect in any rat organs on necropsy.No statistically significant differences were observed in the mean relative organ weights between the different treatment groups and the control group (Table 5).Additionally, no adverse effects were noted on histopathological observation (Table 6).Insect-resistant Agb0101 rice was mixed with normal feed at two different concentrations, 5 and 20%.The amounts of protein incorporated into the diet of each groups were 92 and 132 mg/Kg, respectively.As control groups, normal Nak-dong rice also mixed with normal feed into the diet at 20%.Over the course of the in-life phase of this study, no statistically significant differences in body weights (Figure 2) or feed consumption were observed between the control groups and the groups that consumed Agb0101 rice-containing diets (Figure 3).The consumed doses of mCry1Ac1 protein averaged 0.42 and 1.62 mg/kg body weight/day in males and 0.3 and 1.3 mg/kg body weight/day in females that calculate from the results of Kim et al. (2013).

DISCUSSION
This study describes our toxicity assessment that was Significant differences as compared with G 1 : a P < 0.05.Significant differences as compared with G 2 : b P < 0.05.
conducted on the mCry1Ac1 protein and Agb0101 rice.This assessment evaluates the potential toxicity of transgenic proteins using weight-of-evidence and tiered approaches, respectively (CODEX, 2003;EC 2003).No evidence for potential toxicity was identified for the mcry1Ac1 gene according to the components of a first tier.The bt gene was obtained from Bacillus sp., which has a long history of safe use in agricultural pesticides.
Rice also has a long safe history as a common component of the human diet.Human exposure to the mCry1Ac1 protein is most likely extremely low because it is present at low concentrations in the entire genetically modified Agb0101 rice plant.The effects of the mCry1Ac1 protein determined by the 90-day toxicity study should be considered in comparison to the concentration determined in rice seeds obtained from genetically modified insectresistant Agb0101 rice as determined using mCry1Ac1specific ELISA (0.2 mg/Kg mCry1Ac1 protein in dry tissue).The average human (average body weight 60 kg) would need to consume about 60 kg/day of grain expressing the mCry1Ac1 protein to approximate the same daily dose consumed by the high dose group enrolled   0) 10( 0 in the 90-day toxicity study.This is an extremely conservative estimate of human exposure to the mCry1Ac1 protein because in vitro digestion studies indicate that mCry1Ac1 will not be absorbed intact because it will most likely be degraded within the gut.Dietary proteins are taken in as nutrients and typically demonstrate no relation to toxic effects.However, some proteins do cause acute toxicity (Metcalfe et al., 1996;Sjoblad et al., 1992).For this reason, guidance documents from CODEX and KFDA described procedures for assessing the potential toxicity of transgenic proteins.Most dietary proteins are non-toxic and absorbed for nutritional purposes.However, because some identified proteins are toxic to mammals and other species, new reliable recommendations were developed to assess transgenic proteins that could cause toxicity using a twotiered approach (Delaney et al., 2008).
The first tier assesses the history of use of the organism from which the gene is obtained.Generally, similarities between known toxic proteins and transgenic proteins can be determined using bioinformatical analysis, mechanism of action of the protein, in vitro stability to digestive enzymes were major research process (Delaney et al., 2008).A second tier analysis may be conducted if there have harmful results.Elements within this tier include acute toxicity studies that use purified transgenic proteins and 90-days oral toxicity (sub-chronic) studies with genetically modified organisms as requested by regulatory authorities.

Conclusion
Our present data demonstrate the safety of the mCry1Ac1 protein and insect-resistant Agb0101 rice for use in food and feed applications and indicate that the mCry1Ac1 protein and Agb0101 rice presents no risks for adverse health effects when used in the context of agricultural biotechnology.No evidence of toxicity was observed in mice or rats following acute or 90-day oral exposure to heterologously produced mCry1Ac1 protein in Agb0101 rice.Therefore, according to our current study findings, the no-observed-adverse-effect-level (NOAEL) for the mCry1Ac1 protein is more than 10 g/Kg body weight for both male and female mice.Results from these studies further support the use of a tiered approaches that include Tier II studies, such as including hazard characterizations and acute and sub-chronic oral toxicity studies, but do not provide additional information or results that contradict the results of the Tier I studies that do not identify evidence for potential toxicity of the mCry1Ac1 protein.

Table 1 .
Study design of feed composition for the sub-chronic study.

Table 2 .
Body weights changes of mice from acute toxicity study with mCry1Ac1 protein.
Body weight changes of rats orally treated with Agb0101 rice for 90 days.
Figure 3. Feed consumption of rats orally treated with Agb0101 rice for 90 days.

Table 3 .
Analysis of Blood components contents treated with Agb0101 rice.

Table 4 .
Biochemical values of SD rats orally treated with Agb0101 for 90 days.

Table 5 .
Organ weight in treated rat.

Table 6 .
Histopathological findings of rats treated with GM rice for 90 days.